The Sperm Cell 2017

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The Sperm Cell

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The Sperm Cell

Production, Maturation, Fertilization, Regeneration
Second Edition
Edited by
Christopher J. De Jonge
University of Minnesota, Minneapolis, MN, USA
Christopher L. R. Barratt
University of Dundee, Ninewells Hospital, Dundee, UK

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Contents
List of Contributors page vii
Foreword by Ryuzo Yanagimachi x
Preface xiii
1 Spermatogenesis: Clinical and 9 Proteomics of Capacitation 143

Experimental Considerations 1 Mark A. Baker
Ellen Goossens and Herman Tournaye
10 Current Concepts and Unresolved
2 Sperm Chromatin Stability and Questions in Human Sperm Cumulus
Susceptibility to Damage in Relation to and Zona Interaction 152
Its Structure 21 Christopher J. De Jonge and Christopher L. R.
W. Steven Ward Barratt
3 Sperm Ultrastructure in Fertile Men and 11 Sperm-Speciic WW-Domain-Binding
Male Sterility: Revisiting Proteins 157
Teratozoospermia 36 Richard Oko, Mahmoud Aarabi, Jiude Mao,
Hector E. Chemes Hanna Balakier and Peter Sutovsky
4 Sperm RNA and Its Use as a Clinical 12 Fundamental Role for Sperm
Marker 59 Phospholipase C ␨ in Mammalian
Meritxell Jodar, Ester Anton and Stephen A. Fertilization 177
Krawetz Michail Nomikos, Karl Swann and
F. Anthony Lai
5 Role of the Epididymis in Sperm
Maturation 73 13 Male Infertility and Assisted
Robert Sullivan and Clémence Belleannée Reproduction 193
Nigel Pereira, Queenie V. Neri, Tyler Cozzubbo,
6 Seminal Plasma Plays Important Roles in
Stephanie Cheung, Zev Rosenwaks and
Fertility 88
Gianpiero D. Palermo
Susan S. Suarez and Mariana F. Wolfner
14 The Genetic Basis of Male
7 Physiological and Pathological Aspects Infertility 208
of Sperm Metabolism 109
Amin S. Herati, Peter R. Butler and
Zamira Gibb and Robert John Aitken Dolores J. Lamb
8 Regulation of Sperm Behaviour: The 15 The Sperm Epigenome 230
Role(s) of [Ca2+ ]i Signalling 126
Timothy G. Jenkins and Douglas T. Carrell
Stephen Publicover

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16 Environmental Factors and Male 18 Mouse Genetics – How Does It Inform

Fertility 240 Male Fertility Research? 280
Tina Kold Jensen, Hanne Frederiksen, Katrine Laura O’Hara and Lee B. Smith
Bay and Niels E. Skakkebaek
17 Susceptibility of the Testis to Lifestyle
and Environmental Factors During the
Life Course 260 Index 297
Richard M. Sharpe Colour plates are to be found between pages 178
and 179.
vi

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Contributors
Mahmoud Aarabi, MD, PhD Clémence Belleannée, PhD

Postdoctoral Fellow, Department of Human Genetics, Associate Professor at the Department of
School of Medicine, McGill University, Montreal, QC, Obstetrics, Gynecology and Reproduction,
Canada Université Laval, and Reproduction, Mother
and Youth Health Division, CHU de Québec-
Robert John Aitken, PhD Université Laval Research Center, QC, Canada
Pro Vice-Chancellor, Faculty of Health and Medicine,
Laureate Professor of Biological Sciences, Priority Peter R. Butler, BA
Research Centre for Reproductive Science and Center for Reproductive Medicine, Baylor
President at the International Society of Andrology, College of Medicine, and the Scott Department
he University of Newcastle, Callaghan, NSW, of Urology, Baylor College of Medicine, Houston,
Australia TX, USA
Ester Anton, PhD Douglas T. Carrell, PhD
Aggregate Professor at the Department of Cell Department of Surgery (Urology) and
Biology, Physiology and Immunology, Universitat Department of Human Genetics, University of
Autònoma de Barcelona, Bellaterra (Cerdanyola del Utah School of Medicine, Salt Lake City, UT,
Vallès), Spain USA
Mark A. Baker, PhD Hector E. Chemes, MD, PhD
Head of Reproductive Proteomics, Discipline of Laboratory of Testicular Physiology and
Biological Sciences, University of Newcastle, Pathology, CEDIE-CONICET, Center for
Callaghan, NSW, Australia Research in Endocrinology, National Research
Council, Endocrinology Division, Buenos Aires
Hanna Balakier, PhD Children´s Hospital, Argentina
Laboratory Director, CReATe Fertility Centre,
Toronto, ON, Canada Stephanie Cheung, BS
Ronald O. Perelman and Claudia Cohen Center for
Christopher L. R. Barratt, PhD Reproductive Medicine, Weill Cornell Medical
Professor of Reproductive Medicine at the School of College, New York, NY, USA
Medicine, University of Dundee, Ninewells Hospital,
Dundee, UK Tyler Cozzubbo, BS
Katrine Bay, PhD Reproductive Medicine, Weill Cornell Medical
Scientiic Writer, Department of Growth and College, New York, NY, USA
Reproduction, Rigshospitalet, University of
Copenhagen, Denmark, and International Center for Christopher J. De Jonge, PhD
Research and Research Training in Endocrine Director, Andrology Program at the University of
Disruption of Male Reproduction and Child Health Minnesota Medical Center, and Adjunct Professor at
(EDMaRC), Rigshospitalet, University of the Department of Urology, University of Minnesota,
Copenhagen, Denmark Minneapolis, MN, USA
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Hanne Frederiksen, PhD Dolores J. Lamb, PhD

Senior Researcher at the Department of Growth and Center for Reproductive Medicine, Baylor College
Reproduction, Rigshospitalet, University of of Medicine, the Scott Department of Urology,
Copenhagen, and International Center for Research Baylor College of Medicine, and the Department
and Research Training in Endocrine Disruption of of Molecular and Cellular Biology, Baylor
Male Reproduction and Child Health (EDMaRC), College of Medicine, Houston, TX,
Rigshospitalet, University of Copenhagen, Denmark USA
Zamira Gibb, PhD Jiude Mao, PhD
Priority Research Centre for Reproductive Science Research Assistant Professor, Division of Animal
and Discipline of Biological Sciences, he University Sciences, University of Missouri, Columbia, MO,
of Newcastle, Callaghan, NSW, Australia USA,
Ellen Goossens, PhD Queenie V. Neri, MSc
Biology of the Testis Research Unit, Vrije Universiteit Ronald O. Perelman and Claudia Cohen Center for
Brussel, Brussels, Belgium Reproductive Medicine, Weill Cornell Medical
College, New York, NY, USA
Amin S. Herati, MD, PhD
Center for Reproductive Medicine, Baylor College of Michail Nomikos, PhD
Medicine, and the Scott Department of Urology, College of Biomedical and Life Sciences, Cardif
Baylor College of Medicine, Houston, TX, USA University, Cardif, UK
Timothy G. Jenkins, PhD Laura O’Hara, PhD
Department of Surgery (Urology), University of Utah Postdoctoral Research Fellow at the MRC
School of Medicine, Salt Lake City, UT, USA Centre for Reproductive Health, University of
Edinburgh, he Queen’s Medical Research Institute,
Tina Kold Jensen, MD, PhD Edinburgh, UK
Consultant at the Department of Growth and
Reproduction, Rigshospitalet, University of Richard Oko, PhD
Copenhagen, Denmark, and Department of Professor, Department of Biomedical and Molecular
Environmental Medicine, Institute of Public Health, Sciences, School of Medicine, Queen’s University,
University of Southern Denmark, Odense, Denmark Kingston, ON, Canada
Meritxell Jodar, PhD Gianpiero D. Palermo, MD, PhD
Postdoctoral Fellow, Molecular Biology of Ronald O. Perelman and Claudia Cohen Center for
Reproduction and Development Research Group, Reproductive Medicine, Weill Cornell Medical
Institut d’Investigacions Biomèdiques August Pi i College, New York, NY, USA
Sunyer (IDIBAPS), University of Barcelona,
Barcelona, Spain Nigel Pereira, MD
Stephen A. Krawetz, PhD Reproductive Medicine, Weill Cornell Medical
Associate Director at the C.S. Mott Center for Human College, New York, NY, USA
Growth and Development and Charlotte B. Failing
Professor of Foetal herapy and Diagnosis, Stephen Publicover, PhD
Department of Obstetrics and Gynecology, Center for Reader in Reproductive Physiology, School of
Molecular Medicine and Genetics, C.S. Mott Center Biosciences, University of Birmingham, Edgbaston,
for Human Growth and Development, Wayne State Birmingham, UK
University School of Medicine, Detroit, MI, USA
Zev Rosenwaks, MD
F. Anthony Lai, PhD Ronald O. Perelman and Claudia Cohen Center for
Professor, College of Biomedical and Life Sciences, Reproductive Medicine, Weill Cornell Medical
Cardif University, Cardif, UK College, New York, NY, USA
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Richard M. Sharpe, PhD Peter Sutovsky, PhD

Honorary Professor at the MRC Centre for Professor, Division of Animal Sciences, and
Reproductive Health, he Queen’s Medical Research Departments of Obstetrics, Gynecology & Women’s
Institute, he University of Edinburgh, Edinburgh, Health, School of Medicine, University of Missouri,
UK Columbia, MO, USA
Niels E. Skakkebaek, MD
Senior Researcher at the Department of Growth and Karl Swann
Reproduction, Rigshospitalet, University of College of Biomedical and Life Sciences, Cardif
Copenhagen, Denmark, and International Center for University, Cardif, UK
Research and Research Training in Endocrine
Disruption of Male Reproduction and Child Health Herman Tournaye, MD, PhD
(EDMaRC), Rigshospitalet, University of Centre of Reproductive Medicine, University
Copenhagen, Denmark Hospital UZ Brussel, Brussels, Belgium
Lee B. Smith, PhD
Professor, Chair of Genetic Endocrinology and Head W. Steven Ward, PhD
of Male Health Research, MRC Centre for Professor and Director at the Department of
Reproductive Health, University of Edinburgh, he Anatomy, Biochemistry and Physiology, Institute
Queen’s Medical Research Institute, Edinburgh, UK for Biogenesis Research, and Chief, Research
Division and Lakshmi Devi and Devraj Sharma
Susan S. Suarez, PhD Endowed Chair, Department of Obstetrics and
Department of Biomedical Sciences, Cornell Gynecology and Women’s Health, John A. Burns
University College of Veterinary Medicine, Ithaca, School of Medicine, University of Hawaii at Manoa,
NY, USA Honolulu, HI, USA
Robert Sullivan, PhD
Professor at the Department of Obstetrics, Mariana F. Wolfner, PhD
Gynecology and Reproduction, Université Laval, and Department of Molecular Biology and Genetics,
Reproduction, Mother and Youth Health Division, Cornell University, Ithaca, NY, USA
Centre de recherche du CHU de Québec-Université
Laval, QC, Canada
ix

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Foreword
Eggs are made for sperm. Sperm are made for eggs. spring. What is found in one species must be extrapo-
All other (body) cells are made to support, directly or lated to other species with caution.
indirectly, the development of eggs and sperm and the Today, it is theoretically possible to reproduce any
survival of their united product: the zygote – the next mammals without males. In fact, hundreds of cows
generation. he prime function of spermatozoa is to have already been produced by somatic cell nuclear
deliver the male genome safely into eggs. Any errors transfer. Clearly, males are not essential for ani-
during sperm formation, maturation and union with mal and human reproduction. Why are there males?
eggs will result in serious problems in the male’s fertil- At the beginning of life on Earth, there were no
ity and in the wellbeing of the ofspring. males. Females reproduced by themselves. During the
his book covers our current knowledge of (1) the course of evolution, a bisexual mode of reproduction
formation of spermatozoa, (2) the preparation of sper- emerged, and it has been maintained in most animals,
matozoa for fertilization, (3) the union of spermatozoa including humans. Compared with animals propagat-
with eggs, (4) the awakening of ‘sleeping’ eggs by sper- ing unisexually (females only), animals using a bisex-
matozoa leading to embryo development, (5) genomic ual mode of reproduction seem to be less vulnerable to
and nongenomic (e.g. environmental) factors afect- extinction in the face of constantly changing, compet-
ing the development and fertility of spermatozoa, and itive environments. Technically, human cloning (non-
(6) the challenges of overcoming male (sperm) fer- sexual reproduction) is possible today. In other words,
tility problems. Information compiled in each chap- humans can reproduce without males. Is this what we
ter should be considered a stepping stone to better desire? A few years ater the birth of Dolly (a cloned
understanding and better control of male fertility and sheep) and many cloned mice, I gave talks to groups of
infertility. people about animal and human cloning. At the end of
he very irst chapter of this book mentions the my talk I asked the audience if they wanted to live in
possible production of ‘artiicial human spermatozoa’ a world without men. With no exception, women did
from pluripotent stem cells such as human iPSCs. not want to live in the world without men. ‘It would be
Obviously, it is not appropriate to use live animals or boring. We cannot use men? hat would be horrible.’
get assistance from live animal cells to achieve this Men are needed by women, and we will stay that way.
goal. To eliminate or minimize the stress and risks When I started research as an undergraduate stu-
these cells would face during their transformation into dent, I thought everything written in books and
haploid cells, we must learn much more about what is research papers was a fact. I now know that what is
really happening in the natural environment of sper- written is authors’ interpretations or just a part of
matogenic cells, within the testes. he last chapter con- the whole story. Many things written in books and
siders the value of the mouse as a model for the study reported in original papers will be modiied and even
of mammalian fertility and infertility. Is the mouse a discarded during the next 40–50 years. Science pro-
perfect animal model to use for the study of fertil- gresses that way.
ity and infertility of all mammals, including humans? he comprehensive collection of topics that com-
Although the mouse is certainly one of the most heav- pose this new edition of he Sperm Cell provide read-
ily used model animals for studying mammalian fer- ers with a map and compass to chart a course for
tility and reproduction, we must remember that each future investigations. It is the readers’ task ater reading
animal uses species-speciic tactics to produce its of- these highly topical research areas to determine what

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Foreword
subjects are let unclear and compelling, what next

courses might be important to follow and what bur-
geoning questions are yet to be studied.
Ryuzo Yanagimachi, PhD

Professor Emeritus, Department of Anatomy,
Biochemistry and Physiology, Institute of Biogenesis
Research, John A. Burns School of Medicine, University
of Hawaii, Honolulu, Hawaii
xi

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Preface
Ten years have quickly passed since the publication of structure and function of the cell that are having an
he Sperm Cell – Production, Maturation, Fertilization, impact on clinical diagnoses.
Regeneration. When published in 2006, this unique here has been breathtaking progress in our
book provided a comprehensive introduction to the knowledge base of the human spermatozoon, yet there
formation, generation and function of the human male is still much to learn, and many areas remain relatively
gamete. Over the past 10 years science and technol- poorly explored. For example, ICSI is still regarded as
ogy have advanced remarkably and so similarly has the primary treatment option for men with presumed
advancement in understanding and characterizing the sperm dysfunction. Insights provided in these chapters
sperm cell. hus, it is now very timely that we present a will hopefully stimulate investigations that will make
completely revised and much expanded second edition less uncertain the structural and functional potential
of he Sperm Cell. of sperm for fertilization and embryogenesis.
In he Sperm Cell, second edition, we have again he remarkable cover art for he Sperm Cell, bears
focused on providing the reader with a tapestry of top- some similarity to the cover art of the irst edition.
ics that reveal a more comprehensive characterization However, a diference between the images can clearly
into the generation and function of the spermatozoon be seen. For the latter, a somewhat foggy, less dis-
and that encompasses both basic and clinical aspects. tinct cross-sectional image of the seminiferous tubule
Up-to-date information on subjects where there has was used – relecting, in essence, the ‘scratching at the
been very recent and rapid progress in our understand- surface’ knowledge base of the ield at the time. he
ing – sperm cell epigenetics, proteomics and basic present cover shows an image of a seminiferous tubule
genetics and the consequences of such as potential that is sharp and distinct, relecting greater clarity –
markers of sperm function – is included. New topics clarity in our understanding and characterization of
have been added where novel data have revealed fasci- this most unique cell, the spermatozoon.
nating insights into the biology of reproduction, such Our hope is that the collective contributions in this
as the role that seminal plasma may play in modify- book will inspire and encourage the next generation
ing both the female tract and the fertilising potential of research and clinical scientists to the ield and, per-
of sperm. Additionally, the book provides two chapters haps, reinvigorate older and experienced scientists to
that present competing mechanisms for the process in think anew from the fresh perspectives ofered in he
which a sperm activates an egg. Importantly, a chap- Sperm Cell, second edition.
ter on sperm ultrastructure is included. he applica-
tion of electron microscopy for scrutinizing ultrastruc- Christopher J. De Jonge
tural components provides amazing insights into the Christopher L. R. Barratt
xiii

Chapter
Spermatogenesis
1 Clinical and Experimental Considerations

Ellen Goossens and Herman Tournaye
Introduction can be obtained through testicular biopsies, how-

ever; when no sperm are retrieved, there are currently
Spermatogenesis is a complex process that starts in
no therapeutic options available for these patients to
early foetal development and continues through a
father a biological child. However, several therapeu-
man’s entire lifespan. The process involves cell speci-
tic approaches are under investigation. In cases where
fication, cell migration, mitotic and meiotic cell divi-
undifferentiated cells are the only germ cells present
sion, differentiation and eventually maturation. Only
in the testis, in vitro or in vivo strategies aiming to
when all these events take place in a correct sequence,
generate sperm from spermatogonial stem cells (SSCs)
in a specific setting and without any errors, will enough
or their daughter cells are to be established. On the
mature haploid spermatozoa be produced to enable
other hand, if germ cells are lacking, induced pluripo-
both fertilization of an oocyte and embryonic devel-
tent stem cells (iPS) derived from the patient’s own
opment. A single error can hamper sperm produc-
somatic cells may be the only possible source to gen-
tion and render a man infertile. The severity of infer-
erate patient-specific gametes.
tility depends on the specific time point when the
In this chapter, we will summarize the main events
error occurs during spermatogenesis. Errors in the
during normal spermatogenesis, along with potential
first steps of establishing spermatogenesis in foetal life
errors that may arise in the specific stages. In addition,
cause more severe infertility than errors happening in
the resultant fertility problem(s) will be described,
later phases of spermatogenesis.
together with potential treatments either already avail-
Male infertility is a health problem with a dra-
able or still under investigation.
matic impact on both individuals’ and couples’ psy-
chosocial wellbeing, as well as a significant healthcare
cost. Worldwide, at least 45 million couples are suf- Primordial Germ Cells
fering from infertility [1]. In about 50% of couples, a
male factor is involved, either alone or in combination Physiology
with a female-related problem [2]. Since the cause for The primordial germ cells (PGCs) are the bipoten-
infertility cannot be identified in all patients, especially tial ancestors of the germ line. These cells can dif-
in men, most infertile men suffer from unexplained ferentiate to either spermatozoa or oocytes. In the
infertility. Yet in recent years, a genetic background for mouse embryo, around 6.25 days postcoitus (dpc), six
male infertility conditions is being established more PGC precursors are specified in the posterior prox-
and more in these patients. imal epiblast cells near the region where the primi-
In patients with oligozoospermia or obstructive tive streak will form. This specification is induced by
azoospermia, spermatozoa can easily be retrieved the transforming growth factor ␤1 (TGF␤1) super-
either from the semen or by surgery from the epi- family, namely, bone morphogenetic protein (BMP)
didymis or the testis. This sperm can be used for 8a, BMP4 and BMP2. Like all other somatic cells,
intracytoplasmic sperm injection (ICSI). In about half these PGCs are diploid [3]. In human embryos, PGC
of patients with nonobstructive azoospermia, sperm precursors can already be observed in the primary
The Sperm Cell, Second Edition, ed. Christopher J. De Jonge and Christopher L. R. Barratt. Published by Cambridge
University Press.
C Cambridge University Press 2017.
1
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https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316411124.003
Chapter 1: Spermatogenesis: Clinical and Experimental Considerations
ectoderm (epiblast) in the second week after concep- Sertoli-Cell-Only Syndrome

tion. In the third week, PGC precursors migrate out-
Mouse PGCs lacking ␤1 integrins or the c-KIT recep-
side of the embryo proper into the extraembryonic
tor fail to migrate normally to the genital ridges. Muta-
ectoderm, where they become PGCs. A cluster of 30–
tions in the human c-KIT gene have not been reported.
50 PGCs can be observed near the dorsal wall of the
However, some reports suggest that variants within
yolk sac at the basis of the allantois. During the fourth
the nucleotide sequences of the c-KIT and SCF genes
week, when the embryonic disc undergoes a folding
are associated with Sertoli-cell-only (SCO) syndrome
process, PGCs incorporate passively into the embryo
(Figure 1.1A), also known as Del Castillo syndrome,
and are set aside as single cells among the endoder-
after the author who first described this condition
mal cells of the primitive hind- and midgut epithelium.
characterized by germ cell aplasia [9].
By this time, their number has increased, reaching up
A significant group of men with this syndrome
to 100 PGCs. From week 5 onward, the PGCs travel
have microdeletions in the azoospermia factor (AZF)
along the developing nerve fibres from the wall of the
region of the Y chromosome. This region contains
hindgut via the dorsal mesentery to the midline of the
three important genetic domains (AZFa, AZFb and
dorsal wall and laterally into the gonads [4]. The mech-
AZFc). Though a definitive genotype/phenotype cor-
anisms by which PGCs migrate to the gonadal ridges
relation does not exist, deletions spanning multiple
include contact guidance with somatic and/or extra-
AZF regions or deletions restricted to AZFa usu-
cellular matrix molecules and chemotactic and repul-
ally result in patients with SCO, whereas deletions
sive signals. It has been shown that mouse PGCs may
restricted to AZFb or AZFc can result in patients
use various types of integrins for dynamic adhesive
with phenotypes ranging from SCO to moderate oligo-
interactions with extracellular matrix molecules such
zoospermia [10]. AZFa contains three genes: USP9Y,
as fibronectin, laminin and collagen IV [5]. Stromal-
DBY and UTY. Deletions or mutations in USP9Y
derived factor 1 (SDF1) and stem cell factor (SCF) have
may cause severe oligozoospermia. DBY is frequently
been proposed as chemoattractants for human PGCs
deleted in infertile patients, and its absence leads to
[6, 7]. During migration, and after their arrival in the
severe oligozoospermia or azoospermia due to SCO.
gonadal ridges, PGCs keep on proliferating. It has been
Moreover, all patients in whom both the USP9Y and
estimated that the total number of PGCs increases
the DBY gene are deleted show a testicular histology
from 1000 (in week 5) to 150,000 (in week 9) [7]. Once
of SCO [11].
the PGCs arrive in the genital ridges, the somatic cells
(early Sertoli cells in a male embryo) will enclose the
PGCs to form primitive seminiferous sex cords. From Artificial Gametes
this moment onward, PGCs are called “gonocytes”. Currently, couples with the man suffering from SCO
While migratory PGCs can be identified by can undergo TESE for retrieving testicular sperma-
their expression of the pluripotency markers OCT4, tozoa eventually to be used for ICSI. However, sper-
NANOG, SSEA1 and c-KIT, the PGCs that have matozoa can be observed after TESE only in about
reached the gonadal ridges lose these pluripotency half of these men [12]. These men represent a sub-
markers and start to differentiate into sex-specific group referred to as “incomplete SCO”. In the other
gonocytes. half, no spermatozoa can be found, even after multiple
During their development and migration, PGCs biopsies. At present, these men with “complete SCO”
undergo extensive epigenetic reprogramming. While can have children only via gamete donation. However,
the majority of the genes are demethylated in migra- most couples prefer to raise their genetically own child.
tory PGCs, a number of CpG islands (short stretches Therefore, several investigators address the question
of DNA in which the frequency of the CG sequence of whether artificial gametes could become a possi-
is higher than in other regions) in imprinted genes, X- ble alternative. In order to produce gametes for these
linked genes and genes involved in meiosis and gamete patients, induced pluripotent stem cells (iPSCs) have
generation become demethylated once the PGCs enter to be developed from the patient’s own somatic cells.
the gonadal ridge. At this point, the epigenome has Subsequently, these iPSCs should be differentiated into
reached its most “naive” state. During later stages functional gametes (Figure 1.1B).
in gamete development, new epigenetic marks and The most promising strategy for the generation of
genomic imprints will be acquired [8]. patient-specific human iPSCs is the reprogramming of
2
Somatic cell
Pluripotent cell
Spermatogonial
stem cell
A B
Figure 1.1 Fertility restoration in SCO patients. The testes of patients with SCO lack germ cells (A). Gametes might be produced from
patient-specific somatic cells (B). The somatic cells (e.g. skin) are reprogrammed to pluripotent cells by overexpression of Sox2, Oct4, cMyc
and Klf4. The pluripotent stem cells are then differentiated to spermatozoa in vitro, which can be used in assisted reproduction. Alternatively,
pluripotent stem cells are differentiated to spermatogonial stem cells, which can be transplanted to the testis to further differentiate in vivo.
(A black and white version of this figure will appear in some formats. For the colour version, please refer to the plate section.)
differentiated somatic cells by forced overexpression cells, but the efficiency of the process needs further
of the pluripotency genes Sox2, Oct4, cMyc and Klf4. improvement, and eventually quality and safety tests
However, one must be aware that these induced PSCs have to be conducted.
may retain their somatic epigenetic memory, which In addition to the many scientific hurdles that must
could affect their eventual differentiation into gametes. be overcome before this method may become clinically
So far, production of artificial gametes from PSCs available, many ethical concerns associated with this
has been achieved only in mice [13]. These gametes procedure need to be addressed, e.g. what kind of pre-
were able to fertilize oocytes, resulting in viable off- clinical safety studies have to be conducted, and which
spring, although some of the pups showed tumour for- results will be considered safe enough to make the step
mation in the neck region, which could be related to towards clinical trials.
imprinting problems.
Another group also succeeded in obtaining live off-
spring from haploid cells produced from iPSCs, but the Germ Cell Tumours
offspring died prematurely, probably due to aberrant As PGCs follow the sympathetic nerve fibres on
imprinting [14]. their way towards the gonads, PGCs failing to exit
To date, complete in vitro spermatogenesis from the nerve branches at the gonadal site may continue
human iPSCs has not been demonstrated. Panula et al. along the sympathetic trunk, ending up in other
found that 5% of human iPSCs can differentiate into organs where, under normal circumstances, they are
PGCs after stimulation with bone morphogenetic pro- eliminated through apoptosis [17]. However, PGCs
teins. In response to the overexpression of DAZ fam- that have migrated aberrantly might survive in the
ily proteins, germ cells entered meiosis and differen- ectopic location if they overexpress c-KIT [18]. If
tiated into haploid cells [15]. Recently, human iPSCs that is the case, these PGCs may form germ cell
were shown to differentiate directly into haploid sper- tumours. Germ cell tumours have been described
matidlike cells when cultured for 10 days under con- in the head, neck, mediastinum, pelvis and testis.
ditions used for culturing spermatogonial stem cells These tumours can be benign (teratoma) or malignant
[16]. These results may indicate that human iPSCs (teratocarcinoma). Two main germ cell cancers have
derived from adult somatic cells can develop germ line been described: seminomas and nonseminomas. The
3
finding that seminomas show OCT4 expression cent germ cells in the same seminiferous cord section,
emphasizes the hypothesis that these germ cell while only a small percentage of gonocytes are simul-
neoplasms may result from a failure of the PGCs to taneously in S phase [21]. Moreover, the migration of
differentiate properly. the gonocytes towards the basement membrane of the
Germ cell cancers can be treated by surgery, seminiferous cord occurs randomly, as these cells can
chemotherapy and/or radiotherapy. Although the be found located at both the periphery and the centre
majority of patients with germ cell cancer are fertile, of the cord [22]. Whereas cells in the centre of the cord
certain treatments for testicular cancer can cause long- are still dividing and premigratory, the cells located
term sterility. As these tumours are mostly diagnosed at the basal membrane are already in the process of
in adolescents and young adult men (⬍35 years of age), becoming SSCs.
it is recommended that these patients be offered the During this phase in germ cell development, the
possibility to store a semen sample before starting any DNA methylation patterns that had been erased dur-
cancer treatment [19]. ing PGC migration are now being remethylated. It
was hypothesized that most of the DNA involved in
From Gonocyte to Spermatogonial paternal imprinting and transposons is methylated
in quiescent gonocytes [23]. Correct establishment of
Stem Cell paternal imprints is of major importance, as studies
Germ cell stages between PGCs and spermatogo- have shown that aberrations in the DNA methylation
nial stem cells (SSCs) are usually named gonocytes, pattern and inactivation of proteins involved in this
suggesting that these cells represent a single devel- process can lead to embryonic lethality in rodents.
opmental stage. Nevertheless, several studies have In humans, defective DNA methylation of imprinted
indicated that rodent and human gonocytes in fact genes has been associated with oligozoospermia (see
encompass a number of consecutive stages, described the section on Oligozoospermia under Spermiogenesis
as mitotic (M), quiescent (Q) and transitional migra- and Epididymal Sperm Maturation) [24].
tory (T) gonocytes. During the first trimester of ges-
tation, gonocytes are mitotically active, but during the
second trimester, most but not all gonocytes progres- Spermatogonial Stem Cell
sively lose mitotic activity, together with their pluripo- Proliferation and Differentiation
tency and PGC markers. In rodents, there is a sec-
ond mitotic phase in the early neonatal period, but Physiology
in humans, the gonocytes remain quiescent until two
or three months after birth. By that time, the gono- Spermatogonial Stem Cell Proliferation and
cytes start to express more advanced germ cell mark- Differentiation
ers such as melanoma antigen-A4 (MAGE-A4), and SSCs are single triangle-shaped cells located on the
they reexpress c-KIT, which mediates their migration basement membranes of the seminiferous tubules, in
from the centre of the seminiferous cord towards the close contact with the Sertoli cells. The population
basal membrane. When the gonocytes attach to the of SSCs is a small subpopulation of the spermatogo-
basal membrane, they start differentiating into sper- nia. In rodents, the prevailing model is the As -model
matogonia. The only clear differences between neona- [25]. The As or single undifferentiated type A sper-
tal gonocytes and spermatogonia are their morpho- matogonium is considered to be the “true” SSC. If As
logical appearance (large spherical gonocytes versus spermatogonia divide completely, they usually migrate
smaller half-moon-shaped spermatogonia) and their separately and retain stem cell activity. If, upon divi-
different locations within the seminiferous cord [20]. sion, they remain connected to each other by a cyto-
Although M, Q and T gonocytes express different plasmic bridge, they become paired type A (Apr ) sper-
levels and combinations of proteins, at a given time, matogonia. The production of type Apr spermatogonia
subsets of cells positive and negative for specific mark- is the first step towards differentiation. Type Apr sper-
ers do exist. In mice, the various germ cell subsets are matogonia divide once more to produce groups of four
not restricted to specific time frames in development. aligned type A (Aal ) spermatogonia, also connected
Rather, there seems to be an overlap of subpopulations one to each other. The Aal cells proliferate, resulting in
in time, with the presence of both mitotic and quies- chains of 8, 16 and occasionally 32 cells. As , Apr and
4
As
Apale Adark
Undifferentiated
spermatogonia
Undifferentiated
spermatogonia
Apr
Aal
spermatogonia
Differentiated
A1
A2 B
spermatogonia
Differentiated
A3
A4
Spermatocytes
In
B Spermatids
Spermatozoa
Spermatocytes
Spermatids
Spermatozoa
Figure 1.2 Model of spermatogonial stem cell proliferation in rodents (A) and humans (B).
Aal spermatogonia have the same morphology and can their gene expression. Inhibitor of DNA binding 4
be distinguished only according to their topographi- (ID4), for example, has the most restricted expres-
cal arrangement on the basement membranes of the sion pattern observed to date, but not all single sper-
seminiferous tubules. Most of the Aal spermatogonia matogonia express this marker [27]. Others showed
will undergo a morphological change and transform that NGN3 expression was heterogeneous, since only
into type A1 spermatogonia. These A1 spermatogonia 11% of transplantable SSCs were NGN3-positive. The
are the first generation of differentiating spermatogo- implications of this heterogeneity for SSC function are
nia. Next generations include A2 , A3 , A4 , intermedi- largely unknown. However, recent findings have elu-
ate and B spermatogonia. A lot of research has been cidated that the NGN3 positive subpopulation is des-
done to characterize rodent SSCs. The combination of tined for differentiation, implying that not all SSCs
fluorescence-activated cell sorting with SSC transplan- act equivalently as stem cells. Moreover, Apr and Aal
tation has revealed that As spermatogonia express ␤1 - spermatogonia were found not to be committed uni-
integrin (CD29), ␣6 -integrin (CD49f), THY1 (CD90), directionally to differentiation but to be capable of
CD9, GFR␣1 and E-cadherin, but do not express ␣v - reverting to shorter chains by fragmentation [28]. As a
integrin (CD51), MHC-I, C-KIT and CD45. Cells consequence of the progress made by these characteri-
expressing OCT4 showed higher stem cell activity than zation studies, a revision of the As model was neces-
the OCT4− cells. Also, PLZF, SOX3, NGN3, NANOS2 sary. Stem cell activity is not limited to SSCs (or As
and STRA8 were determined in undifferentiated sper- spermatogonia), but Apr and Aal -spermatogonia also
matogonia. Other genes that are expressed in sper- have the potential to self-renew (Figure 1.2A).
matogonia, but not in somatic cells, are MAGE-A4, In primates, two morphologically different classes
UBE1Y, USP9Y, RBMY, OTT, DDX4, TEX14, USP26, of type A spermatogonia are observed: the dark Ad (or
PIWIL2 and PRAMEL1 [26]. New evidence shows “reserve” stem cells) and the pale Ap spermatogonia
that the As population and spermatogonial chains (or “renewing” stem cells). When Ap spermatogonia
of the same length are heterogeneous in respect to divide, they usually remain connected to each other
5
by cytoplasmic bridges, forming doublets of Ap sper- stimulates the proliferation of Sertoli cells. As a result,
matogonia. The production of these Ap doublets is the the number of SSC niches increases, enabling the sup-
first step towards differentiation. Although most of the port of more stem cells, since each niche houses one
type Ap spermatogonia appear in clones of two, four stem cell. Within the seminiferous tubules, Sertoli cells
and eight cells, single Ap cells also may exist. Further- are the main regulators of SSC proliferation. Sertoli
more, Ad and Ap cells can transform into each other. cells have large areas of contact with the differen-
Because Ad spermatogonia are often found in clusters, tiating germ cells through desmosome-gap junction
it was hypothesized that these Ad clusters are the result complexes, but these intercellular contacts are rarely
of a transformation of Ap into Ad at low renewal fre- seen on type A spermatogonia. Therefore, Sertoli
quency. Conversely, after cytotoxic injury, the Ad may cells regulate spermatogonial proliferation by secret-
transform into Ap and start to proliferate. Spermato- ing paracrine factors. After stimulation with FSH, Ser-
genesis is initiated by two divisions of pairs or quadru- toli cells produce and secrete the key regulator of SSC
plets of Ap cells: a first division, after which clones self-renewal, glial-cell-line-derived neurotropic factor
of Ap separate, and a second division, which leads to (GDNF), which acts on undifferentiated spermatogo-
clones of B spermatogonia as well as pairs or quadru- nia through the RET/GFR␣1 receptor complex [32].
plets of Ap cells. These latter cells are responsible for Another factor that stimulates self-renewal is fibro-
the maintenance of the original size of the type A pop- blast growth factor 2 (FGF2). This factor is secreted by
ulation. Because the Ap , which are found in clones of the Sertoli cells in response to testosterone and acts on
two or four cells, cycle continuously, the “true” stem both undifferentiated spermatogonia (paracrine) and
cells are probably the rarely dividing single Ap and Ad Sertoli cells (autocrine). As SSCs are in close contact
spermatogonia [29] (Figure 1.2B). During the last few with the basement membrane, this allows the SSCs to
years, a lot of progress has been made in the char- respond also to diffusing paracrine factors secreted by
acterization of human SSCs. Human spermatogonia Leydig cells, myoid cells or macrophages in the inter-
express many markers equivalent to those of rodent stitial space. When stimulated by luteinizing hormone
spermatogonia, e.g. ␣6-integrin, GFR␣1 and THY1, (LH), Leydig cells produce testosterone, which in turn
although other markers are not shared. For example, stimulates Sertoli cells to produce either self-renewal
human SSCs do not express ␤1-integrin but are posi- (FGF2) or differentiation (ABP, oestradiol) factors. LH
tive for TSPY1, CD133 and SSEA4 [30]. From a clinical action on Leydig cells is also responsible for the pro-
viewpoint, this model including “reserve” stem cells duction of colony-stimulating factor 1 (CSF1), a self-
may explain why both the degree of recovery of sper- renewal factor acting directly on spermatogonia. The
matogenesis and the time for eventual recovery after influence of peritubular myoid cells on germ cell regu-
gonadotoxic treatment depend on the number of sur- lation has long been questioned, but the interest in this
viving stem cells in the different compartments. still poorly known cell type is growing. Its role in SSC
maintenance was suggested by the fact that CSF1 and
Spermatogonial Stem Cell Niche GDNF were detected in peritubular myoid cells [33].
SSCs are located in specialized niches. The niche can Very recently, the niche has been extended with testic-
be defined as the microenvironment that regulates tis- ular macrophages. In particular, one subset of testicu-
sue homeostasis by controlling the balance between lar macrophages, those that are located on the surface
SSC self-renewal and differentiation. The SSC niche in of the seminiferous tubules, close to areas enriched
the mammalian testis is mainly located on the basal for undifferentiated spermatogonia, are found to be
membrane of the seminiferous tubules, but part of the involved in the regulation of SSC proliferation and
SSC niche is thought to lie outside the seminiferous differentiation. These macrophages express spermato-
tubules. One might assume that all germ cells located gonial proliferation- and differentiation-inducing fac-
at the basal membrane are SSCs, but this is not the case. tors, such as CSF1 and enzymes involved in retinoic
Stem cell niches are not distributed randomly along the acid (RA) biosynthesis [34] (Figure 1.3).
tubule but are thought to be localized in areas near the
vasculature, implying an important regulatory func-
tion for specific factors transported through the blood Gonadotoxic Treatment During Puberty
or produced by the vascular endothelial cells [31]. In One child in every 600 is diagnosed with cancer. As
the neonatal testis, follicle-stimulating hormone (FSH) the majority of childhood cancers are systemic cancers
6
Differentiation Self-renewal ity [36]. For radiotherapy, recovery depends on the

received dose, the scattered radiation and the frac-
Sertoli cell
tionation of the radiation bundles. Some patients need
treatment for testicular cancers, and therefore the
FGF2
gonads are irradiated directly. Radiotherapy applied
spermatogonia to other sites in the body can cause a scatter dose
ABP GDNF
GDNF
CSF1
that damages the testes. Whereas, in adults, low doses
E2 Peritubular
myoid cell only cause transient infertility, both low and high
doses are harmful to prepubertal SSCs, because these
T FSH T stem cells have a much higher mitotic rate. Also, their
CSF1
shorter stature causes a greater risk for scattered radi-
LH
LH ation in children compared with larger (adult) indi-
viduals. Hence, children are much more vulnerable
blood vessel Leydig cells
to the effects of chemotherapy and irradiation than
Figure 1.3 Spermatogonial stem cell niche. Effects of FSH (line) adults (see section on Gonadotoxic Treatment During
and LH (dotted line) on spermatogonial proliferation and Adulthood).
differentiation.
As gonadotoxic treatments affect the prolifer-
(leukaemia, lymphoma, tumours of the central ner- ating Ap -spermatogonia, restoration of the SSC
vous system), high (and multiple) doses of chemother- pool depends on the survival of quiescent Ad -
apy and/or total body irradiation have to be admin- spermatogonia. As soon as niches become available
istrated to cure the patient. These treatments are now due to SSC loss, these Ad -spermatogonia start dividing
very efficient, and therefore 80% of the children even- to replenish the stem cell pool. After a first mild treat-
tually will survive their disease. Unfortunately, when ment with a gonadotoxic agent, most of the patients
these patients grow up they may encounter an impor- show a higher germ cell number than untreated
tant life-quality-threatening problem: sterility. Since individuals [37]. However, when these patients have
spermatogonia are also extensively proliferating before to be treated for a second time, the risk of losing
puberty [35], the prepubertal testicular tissue is highly their SSCs is even higher, as these cells are now more
sensitive to damage by chemo- or radiotherapy, which proliferative. Other testicular cell types are much less
can lead to destruction of the SSC pool. Some dis- susceptible to damage by chemo- and/or radiotherapy
eases such as acute lymphoblastic leukaemia even than germ cells. Sertoli cells, peritubular myoid cells
cause decreased gonadal function independent of can- and Leydig cells have a lower turnover rate. To cause
cer therapy. Besides cancer patients, children affected damage to the Leydig cells of prepubertal boys, doses
by non-malignant diseases such as sickle cell disease of at least 2400 cGy are needed, potentially causing
or drepanocytosis may require gonadotoxic treatments delay or arrest of pubertal maturation and problems
as a conditioning therapy for their curative bone mar- in developing secondary sexual characteristics. Once
row transplantation. As these patients are treated with the SSC pool is completely lost, fertility cannot be
high doses of chemotherapy and sometimes also total restored. Therefore, although still experimental,
body irradiation to destroy their bone marrow, their several fertility centres have started a preventive
chances for spontaneous fertility restoration are virtu- fertility preservation program for prepubertal boys
ally nonexistent. [38]. Preferably before the start of any gonadotoxic
If the SSC pool is not completely destroyed, sper- treatment, testicular tissue containing SSCs has to be
matogenesis can recover from the surviving SSCs. biopsied. During the time of therapy and recovery, the
After chemotherapy, the chance of spermatogenic testicular tissue is stored in liquid nitrogen.
recovery mainly depends on the received dose and the Several cryopreservation protocols are in use, but
gonadotoxic agent. All alkylating agents (cyclophos- slow freezing using dimethyl sulphoxide as cryopro-
phamide, ifosfamide, nitrosoureas, chlorambucil, mel- tectant is preferable. Two different protocols were
phalan, busulphan, procarbazine) are gonadotoxic. developed in animal models to restore fertility after
On the other hand, antimetabolite agents such as thawing: transplantation of cell suspensions contain-
methotrexate and cisplatin-based regimens such as ing SSCs into the seminiferous tubules (aka SSC trans-
bleomycin do not have a long-term effect on fertil- plantation) and testicular tissue grafting. Although
7
Digestion Organ culture
Flow cytometry
SSC propagation Differentiation culture
culture Cryopreservation
Cryopreservation
Testicular Testicular
biopsy biopsy
Patient needing gonadotoxic treatment
Figure 1.4 Fertility preservation strategies depend on indication. According to the patient’s disease, the ultimate use of the banked tissue
will be different. If the patient was suffering from a non-malignant disease, testicular tissue will be biopsied and cryopreserved during the
patient’s treatment. Once cured, the patient might return for intratesticular tissue grafting (open arrows). If, however, the patient has a risk of
malignant cell contamination in the testis (filled arrows), the frozen–thawed biopsy has to be enzymatically digested to enable the removal
of malignant cells by flow cytometry. To increase the chance for fertility restoration, SSCs should be propagated in vitro. SSCs will be
transplanted to the testis by the spermatogonial stem cell transplantation technique. For Klinefelter patients whose testes might be atrophied
at adult age, the only option would be in vitro differentiation of SSCs to spermatozoa through organ or cell cultures. The spermatozoa thus
produced could be used for ICSI (dotted arrows). (A black and white version of this figure will appear in some formats. For the colour version,
please refer to the plate section.)
in mice fertility can be re-established in 50% of trans- from certain malignant disorders because of the risk
planted animals, the colonization efficiency of trans- of reintroducing malignant cells back into an other-
planted SSCs is rather low (12%), probably because the wise cured patient [42]. Although, in rodents, it was
natural niche contacts have been lost. Since a very large reported that sorting malignant cells out of a testicular
number of SSCs are required to recolonize an adult cell suspension did not reintroduce tumour formation
testis, increasing the amount of SSCs by in vitro culture after transplantation, it remains unknown whether the
could solve this problem. Although it was described same strategy could be used in humans [43]. The cul-
that in vitro SSC culture could increase SSC numbers ture system developed to propagate SSCs in vitro could
18,000-fold, these findings have not been widely con- be an alternative strategy for depleting malignant cells,
firmed [39; 40]. It might be more efficient to trans- since a pilot study showed that this culture system does
plant the tissue as a whole, as the grafting technique not support the growth of leukemic cells [44].
retains the natural SSC niche and was reported to yield Whereas at present transplantation of SSCs or tis-
a 100% success rate in mice [41]. On the other hand, sue is not yet ready for clinical application in patients
this technique cannot be used in patients suffering because of unresolved safety issues, patients who
8
suffered from a non-malignant disease will probably likely to occur as the result of exposure to a mixture
be the first to have their stored testicular tissue trans- of environmental chemicals.
planted back given the absence of the risk for malig- Foetal exposure to dioxin, which is a highly toxic
nant contamination (Fig 1.4). byproduct of incineration, and polycyclic aromatic
hydrocarbons (PAHs) that are constituents of exhaust
Environmental Factors Reduce fumes, smoke and cooking processes are also hypoth-
esized to result in a lower sperm concentration in the
Niche Numbers adult. These molecules interact with the aryl hydrocar-
Apart from chemotherapeutic agents and irradiation, bon receptor. The activation of this receptor can antag-
other environmental and lifestyle factors can influence onize androgen-mediated action, leading to a reduc-
the number of SSCs. Our lifestyle may indeed affect tion in Sertoli cell number and lower sperm counts in
sperm quality in a negative way, both during foetal and adulthood [48; 49]. Dramatically lower sperm counts
neonatal life and in adulthood (see section on Environ- were found in men whose mothers had smoked heavily
mental and Lifestyle Effects During Adulthood). During during pregnancy, probably caused by the interaction
foetal and neonatal life, testosterone levels increase, of PAHs in the cigarette smoke with the aryl hydro-
promoting important early events in spermatogenesis: carbon receptor [50]. A similar pathway might explain
testicular development is initiated, PGCs differentiate the significant reduction in sperm counts in sons of
into spermatogonia and Sertoli cells proliferate. As the mothers who consumed a lot of beef during pregnancy.
number of Sertoli cells is related to the number of SSC However, this could also be due to saturated fats or
niches, and hence the number of SSCs, factors that anabolic steroids [51].
interfere with androgen function may influence Ser- In general, any environmental compound that
toli cell proliferation and via the indirect effect on SSC affects testosterone production and/or function in the
numbers future sperm output. Sertoli cells stop prolif- foetus may theoretically have consequences in terms of
erating shortly after birth, followed by another prolif- reduced number of SSC niches, and therefore reduced
eration phase at puberty. However, the effects that arise sperm production and reduced sperm counts later in
during foetal and neonatal life are indirectly important life [for review, see 52].
in determining their final number at adulthood [45].
Together with changes in lifestyle, our exposure Niche Deficiencies
to a wide range of environmental chemicals (PCBs,
phthalates, pesticides) has increased during the last Mumps Orchitis
decades. Several of these chemicals accumulate in the Thanks to international vaccine programs, mumps has
fat and have anti-androgenic activity. During preg- become less common in children. However, if postpu-
nancy and lactation, fat cells are broken down in bertal men get infected, mumps may be complicated by
response to a higher energy need. This process may orchitis in about 20–30% of patients [53]. The mumps
cause the release of accumulated PCBs, which are virus infects the testicular glands within the first
passed on to the foetus. In the male foetus, androgen days of infection, leading to parenchymal inflamma-
activity may be hampered, resulting in lower prolifer- tion, separation of seminiferous tubules and perivas-
ation rates in Sertoli cells and thus a reduced sperm cular interstitial lymphocyte infiltration. As the tunica
output in adulthood. The impact of these environmen- albuginea forms a barrier against oedema, the intra-
tal chemicals may be even higher among obese women testicular pressure rises, resulting in pressure-induced
(more fat, thus higher accumulation) and women who testicular atrophy.
become pregnant later in life (longer exposure to these Mumps orchitis may also affect Leydig cell func-
compounds) [46; 47]. Several research groups inves- tion, as low testosterone levels, elevated luteinizing
tigated the associations between maternal exposure hormone levels and an increased pituitary response
to environmental chemicals present in cosmetics, toi- to luteinizing-hormone-releasing hormone have been
letries or medications during pregnancy and the risk reported. Testosterone concentrations return to nor-
for testicular dysgenesis syndrome in their sons, but mal levels after several months, but follicle-stimulating
little consistency was found. Therefore, it was hypoth- hormone and luteinizing hormone concentrations
esized that effects on the developing foetal testis are remain significantly increased until at least one year
9
later [54]. Azoospermia is a rare possible consequence ronment or altered X-linked gene expression. Unravel-
of mumps orchitis and mainly linked to severe cases ling the mechanisms playing a role in KS-related infer-
of bilateral orchitis with testicular atrophy. However, tility could provide important insights into preventing
even azoospermia is not necessarily associated with sterility in these patients. While some reports claim
the complete absence of spermatozoa in the testes. A that 47,XXY germ cells are able to complete meio-
testicular biopsy often yields a few spermatozoa and sis, it is also hypothesized that spermatogenesis in KS
fertilization and pregnancy can thus be achieved by patients exclusively arises from 46,XY germ cells [61].
intracytoplasmic sperm injection [55]. If this were supported by further evidence, the feasi-
bility of in vitro differentiation would become ques-
Klinefelter Syndrome tionable. Another question that needs to be elucidated
Klinefelter syndrome (KS) is a sex chromosomal syn- is whether the 46,XY germ cells eventually generating
drome, affecting 1600 newborn males. This syn- spermatozoa in adulthood are already present at birth
drome is characterized by the presence of one or more or arise during development, as this has important
extra X chromosomes and is among the most com- implications for future fertility preservation strategies.
mon genetic causes of human infertility. It is estimated Detailed histological study on testes from peripuber-
that less than 10% of the cases are diagnosed during tal KS patients (aged 13–16 years) showed that all dis-
childhood; the majority are diagnosed during infertil- played a significant loss of SSCs and no meiotic differ-
ity counselling or remain undiagnosed [56]. Infertil- entiation, with widespread fibrosis and hyalinization
ity in KS patients is caused by the loss of germ cells, of the seminiferous tubules [62].
which starts in early infancy and accelerates at the
onset of puberty. Before puberty, the testicular archi-
tecture is normal, but some studies claim that germ In vitro Spermatogenesis
cell numbers are already reduced during childhood. The first studies on in vitro spermatogenesis (IVS)
The adult KS testis often shows extensive fibrosis and date back to the beginning of the twentieth century.
hyalinization of the seminiferous tubules and hyper- Although simple culture techniques were employed,
plasia of the interstitial tissue [57]. Only in a minority promising results were obtained that formed the basis
of KS patients can sperm cells be found in the ejacu- for later research. Several attempts were undertaken
late; most patients are azoospermic. However, intrates- to improve incubation parameters, feeders or coat-
ticular residual foci of spermatogenesis can be present ings of dishes, but all of these failed to accomplish
in adult azoospermic KS patients. The introduction complete IVS [for review – 63]. The most successful
of intracytoplasmatic sperm injection provided hope study was performed by Cremades et al., who cul-
for fertility in Klinefelter patients. Successful recov- tured immature germ cells from infertile men with
ery of spermatozoa by testicular sperm extraction is arrested germ cell development [64]. Only when start-
reported in about half of the azoospermic KS patients ing from postmeiotic germ cells (round and elongat-
referred to centres specializing in assisted reproduc- ing spermatids) could fertilization-competent gametes
tive techniques [58]. Currently, the other half of the be derived. Studying spermatogenesis in situ has led
patients do not have any option to father their geneti- to the understanding that the spatial arrangement of
cally own children. As it is impossible to predict which the testicular cells is very important for the regu-
patients will be fertile at adulthood, the banking of pre- lation and completion of germ cell maturation. In
pubertal tissue containing SSCs is an attractive strategy line with this, Stukenborg et al. hypothesized that
[59]. Whenever azoospermia with failed retrieval of under improved culture conditions (low tempera-
testicular spermatozoa is a fact at adulthood, frozen– ture, appropriate endocrine and paracrine milieu, 3D-
thawed SSCs could hypothetically be differentiated in structures supporting cell-to-cell contacts), germ cells
vitro and subsequently used for ICSI (Figure 1.4). would be able to enter and pass through meiosis and
While the cause of the germ cell loss in KS patients spermiogenesis in vitro. Eventually, morphologically
is still unexplained, it is also not clear from which normal spermatozoa from mouse spermatogonia were
SSCs the testicular spermatozoa (that are found in half obtained in such a 3D culture system [65]. A few
of adult men) originate. It is still a matter of debate years later, in vitro sperm production was accom-
whether degeneration of the testis is a consequence of plished in neonatal mouse testes using an organ cul-
meiotic errors, the changed testicular/endocrine envi- ture method [66]. These sperm were able to fertilize
10
oocytes by microinsemination, resulting in healthy fer- mosomes remain tightly bound to each other at the
tile offspring. chiasmata, the synaptonemal complex degrades so
To date, complete in vitro maturation from diploid that the chromosomes can separate slightly (diplotene
SSCs to haploid spermatozoa has never been reported spermatocytes). Next, the nucleoli disappear, the
in humans. Recently, we designed a study in which nuclear membrane disintegrates, and the meiotic spin-
we aimed at translating the mouse 3D culture sys- dle begins to form (diakinesis). The paired homolo-
tem to the human by developing a protocol to isolate gous chromosomes align along the metaphase plate
the human testicular matrix. This matrix maintained (metaphase I), after which the microtubules of the
the native 3D structure as well as important tissue- spindle shorten. The homologous chromosomes, con-
specific extracellular matrix components. Since the de- sisting of pairs of sister chromatids, are attracted to
cellularized testicular matrix exhibited good cytocom- opposite poles (anaphase I) and reach the poles during
patibility, it may prove a valuable tool for the further telophase I. The spindle disappears and a new nuclear
development of human in vitro spermatogenesis [67]. membrane forms around each haploid set of chro-
mosomes. The chromosomes decondense into chro-
matin, and cytokinesis completes the creation of two
Meiosis secondary spermatocytes. No DNA replication occurs
during interphase.
Physiology The second meiotic division is similar to mitosis.
The seminiferous tubule is divided into a basal and an In prophase II, the nucleoli and the nuclear envelope
adluminal compartment by polarized columnar Ser- disappear again, the chromatids condense, and the
toli cells. The two compartments are separated by tight spindle starts to form. The centromeres attach to the
junctions between Sertoli cells, the so-called blood– spindle (metaphase II), which is followed by the sister
testis barrier. The basal compartment comprises chromatids moving towards opposing poles (anaphase
mainly spermatogonia, while the adluminal compart- II). Telophase II is similar to telophase I, in which the
ment houses the more advanced germ cells. From chromosomes decondense and the spindle disassem-
puberty onward, Ap spermatogonia will mature and bles. Meiosis ends when the nuclear membrane is cre-
become B spermatogonia, which move on to the adlu- ated and the cells have cleaved. This special process
minal compartment. While crossing the blood–testis of cell reduction-division results in the formation of
barrier, the primary spermatocytes will start the first four spermatids, all containing haploid sets of chromo-
meiotic division. As the opening of the blood–testis somes [69].
barrier, being under the control of A-kinase anchoring
protein 9 [68], permits the passage of preleptotene and Meiotic Errors: Sperm Aneuploidy and
leptotene spermatocytes, the differentiation process
from leptotene spermatocytes up to mature sperm is Maturation Arrest
separated from the systemic circulation. As such, Ser- Aneuploidy in gametes is closely associated with mei-
toli cells can supply developing germ cells with all nec- otic errors. While aneuploidy can be present in 20%
essary nutrients and establish an immune-privileged of human oocytes, about 2% of sperm can be affected
environment for haploid germ cells. too [70]. During the first meiotic division, three events
The first phase of meiotic division is prophase I. can cause aneuploidy: a failed disconnection of chi-
During this phase, individual chromosomes condense asmata (true nondisjunction), an early disappearance
and form visible strands within the nucleus (leptotene of (or failure to form) chiasmata (achiasmatic nondis-
spermatocytes). Homologous chromosomes position junction) and a premature separation of sister chro-
side by side and connect to each other by the synap- matids. In the second meiotic division, only a nondis-
tonemal complex, forming homologous chromosome junction of sister chromatids can lead to aneuploidy.
pairs (zygotene spermatocytes). In pachetene sperma- The sex chromosome pair is particularly susceptible to
tocytes, nonsister chromatids of homologous chro- nondisjunction, since only a small region of homology
mosomes can form chiasmata, where exchange of exists between the X and Y chromosomes [71].
homologous segments can take place. This so-called Aneuploid spermatozoa are capable of fertiliz-
homologous recombination of DNA lies at the foun- ing oocytes, but eventually the embryos will either
dation of genetic variation. While homologous chro- not implant or result in miscarriage or even in
11
aneuploid offspring. Approximately 50% of individu- and on the cumulative dose. Whereas germ cell prolif-
als with Klinefelter syndrome (47,XXY) have resulted eration is severely disturbed by alkylating agents, other
from paternal nondisjunction [72]. In cases where agents are less harmful. If alkylating agents are used
spermatozoa from males with meiotic abnormalities in combination with other chemotherapeutic drugs,
were used for ICSI, a large number of chromosomal their germ cell toxic effects increase dramatically. High
abnormalities (42.5%) were found in the embryos [73]. doses of scatter irradiation (⬎200 cGy) and alkylating-
On the other hand, it is also plausible that aneu- based chemotherapy can also cause Leydig cell dys-
ploid germ cells will not complete meiosis. Such a mat- function, resulting in raised plasma concentrations of
uration arrest can occur at any stage of spermatogene- luteinizing hormone and low levels of testosterone.
sis (spermatogonia, primary spermatocyte, secondary Other somatic cells are less susceptible to chemo- and
spermatocyte or spermatid), and can result in oligo- radiotherapy [19].
zoospermia, if only some germ cells are affected, or Gonadotoxic-treatment–induced maturation arrest
azoospermia, if all germ cells are affected. But again, often arises in the first six months after the start of
even in about half of men azoospermic because of mat- chemotherapy. Although spermatozoa keep on being
uration arrest, testicular spermatozoa can be recov- produced in these first months, the DNA in these germ
ered after TESE for ICSI. These men are referred to cells might be damaged. Therefore, during this mat-
as having an incomplete maturation arrest vs. a com- uration depletion period, patients are advised to use
plete maturation arrest in those men where even mul- contraception to avoid a pregnancy. If the seminif-
tiple testicular sampling fails to show any spermatozoa erous epithelium is depleted by cytotoxic agents or
[12]. In general, a meiotic arrest is caused by synaptic irradiation, restoration of spermatogenesis must occur
defects, which are due to a reduced number of chias- from surviving SSCs. Although SSCs are less sensitive
mata between homologous chromosomes in prophase then differentiating spermatogonia, they still can be
I. This can cause misalignment of chromosomes on the lost. In this situation, SSCs prefer self-renewal above
metaphase plate and subsequently induce abnormal differentiation. The time until spermatogenesis is re-
chromosome segregation in the first meiotic division, established depends on the amount of SSC loss. It can
resulting in secondary spermatocytes with an abnor- take up to five years before fertility recovers. In 30–50%
mal chromosome complement [70, 71]. Meiotic arrest of men with long-term azoospermia, spermatozoa can
can have a genetic cause (see chapter 14) or can be also be retrieved by testicular sperm extraction [38]. A
acquired during life (see sections below on Gonado- large registry-based study showed that children born
toxic Treatment During Adulthood and Environmental from fathers who had chemotherapy before impreg-
and Lifestyle Effects During Adulthood). nating their partners showed a slight, but significant
increase in congenital malformations [75].
The best option for preserving the fertility of men
Gonadotoxic Treatment During Adulthood facing germ cell loss due to chemotherapy or radio-
Since chemotherapy interferes with cell division, the therapy regimes is the cryopreservation of semen
adult SSCs are less susceptible to gonadotoxic treat- samples before any cancer treatment starts. Sufficient
ment than those from children, because of their lower sperm need to be collected to optimize the success of
mitotic rate. Nevertheless, radio- and chemotherapy later assisted reproduction techniques [19].
can induce fertility problems. Doses as low as 10 cGy
can cause oligozoospermia and a dose of 35 cGy Environmental and Lifestyle Effects
can cause azoospermia. Effects of such low doses are
generally transient, as the slowly dividing SSC pool During Adulthood
is not completely destroyed and full fertility can be Although it is believed that exposure to environmen-
restored over time. However, higher doses can lead to tal chemicals can impair spermatogenesis in adult men
spermatogonial abnormalities, testicular atrophy and and lead to reduced sperm counts, hard evidence to
infertility. When 200 to 300 cGy doses are applied, irre- support this belief has not been reported. Only a few
versible azoospermia can occur [74]. Chemotherapy compounds have been shown to affect spermatogen-
mainly affects the differentiating spermatogonia, caus- esis in men with an exposure profile. Occupational
ing a maturation arrest. The effect of chemotherapy on exposure to the pesticide dibromochloropropane can
spermatogenesis depends on the combination of drugs cause severe impairment of spermatogenesis, resulting
12
in infertility. Other studies investigating the impact of ated with a 25% reduction in sperm count and motil-
pesticides in general did not find any evidence. Sim- ity. Reduced sperm production in obese men can occur
ilar effects on spermatogenesis were seen when men for several reasons. The most plausible explanation is
were occupationally exposed to volatile glycol ethers. the alteration in hormone levels, as obese men have
Nowadays, glycol ethers have been replaced by other reduced blood testosterone levels. Alternatively, the
compounds, which do not impair spermatogenesis and reduced spermatogenesis could be due to fat deposi-
fertility in men. Other occupational exposures that tion around the scrotal blood vessels, which can ham-
reduce sperm count and quality include heavy met- per the blood cooling and increase the testicular tem-
als, e.g. lead, cadmium and mercury, which are found perature. Also, the more sedentary life of obese men
in gasoline, paint and fish [76]. In the general pop- might enhance testicular warming [79].
ulation, evidence points towards a high exposure to Although it could be expected that smoking, mod-
persistent pollutants such as PCBs, ozone and insec- erate alcohol consumption or the use of recreational
ticides. Such pollutants were found to reduce sperm drugs has a negative impact on sperm production in
quality, but without affecting fertility [77]. Other envi- adult men, strong evidence that supports this idea is
ronmental chemicals that have been shown to have not available. Nevertheless, in chronic alcoholics and
a negative impact on spermatogenesis are phthalates. in men using cocaine for a long time, reduced sperm
Although men are less exposed to phthalates in cos- counts are reported. On the other hand, the use of
metics and body creams than women, the use of such anabolic steroids as a medicinal or sport drug (by ath-
products is becoming more popular among young letes, weightlifters, bodybuilders) is not without risk.
men. Also, sun creams and basic toiletries are com- These steroids suppress luteinizing hormone secretion
monly used. These products have an adverse effect on from the pituitary gland, leading to reduced intrat-
testosterone production, however, only when exposure esticular testosterone levels and lower sperm counts.
reaches extremely high levels [52]. Other commonly prescribed drugs such as H1 recep-
More important is the impact of lifestyle. Sper- tor antagonists (for allergy relief), anti-epileptics and
matogenesis proceeds at an optimal temperature about antibiotics are associated with adverse effects on sperm
2–4°C lower than the actual body temperature. This number, morphology or motility [52].
lower temperature is achieved by the location of the While environmental factors and lifestyle choices
testes in the scrotum, by heat loss through the scrotal can impair spermatogenesis in adult life, these effects
vasculature and by the plexus pampiniformis, which may not be as severe as during foetal life. While their
cools the arterial blood coming from the body. Any- effect is theoretically plausible, hard evidence to prove
thing that hampers scrotal heat loss will affect tes- that these factors affects a man’s fecundity is still
ticular temperature and may thus have a negative scarce. Furthermore, as already reported in women,
effect on spermatogenesis. The scrotal temperature a man’s genetic background may render him more or
will increase in fever or by exposure to an exoge- less susceptible to the potential gonadotoxic effect of
nous heat source, for example putting a laptop on many lifestyle factors [80].
the lap or taking a hot bath or sauna. In animals, a
30 min hot bath (40–42°C) impaired spermatogenesis Spermiogenesis and Epididymal
and induced germ cell apoptosis. In general, the longer
the testicular temperature is elevated, the greater will Sperm Maturation
be the effect on spermatogenesis. Similarly, spending
a long time in a sedentary position is associated with Physiology
lower sperm counts. Many men working in Western During spermiogenesis, the large plump haploid
countries today spend the whole day seated. When a round spermatids undergo complex cell differentia-
man is seated, air does not circulate so easily around tion to transform into streamlined smaller cells that
the scrotum and therefore there is less efficient cooling. are capable of moving, i.e. spermatozoa. This process
Wearing tight underpants or trousers exacerbates this takes place without further cell divisions. The imma-
effect [78]. ture spermatid first creates lysozyme vesicles from the
Obesity is another important lifestyle factor that Golgi apparatus. These granules, which are full of pro-
adversely affects spermatogenesis. In 2008, 35% of teolytic enzymes, group together to form the acro-
adult men were obese (BMI ⬎ 25) and this was associ- some vesicle and eventually develop into the acrosome
13
during further differentiation. The acrosome plays a and/or morphological abnormalities. Oligozoosper-
crucial role in the recognition and penetration of the mia can be the result of a variety of causes, includ-
egg at the time of fertilization. Furthermore, the centri- ing genetic defects, environmental factors and lifestyle
ole relocates to the opposite cell pole, where it initiates choices. However, most cases are idiopathic. The use of
the growth of the flagellum. The mitochondria that are artificial reproduction techniques (IVF and ICSI) can
responsible for the energy supply to support motility circumvent the sperm defect and substantially improve
rearrange around the flagellum and form the middle the fertility prognosis of oligozoospermic men. In
piece or neck of the spermatozoon. The nucleus also case of extreme oligozoospermia or cryptozoosper-
undergoes important structural changes: the nucleo- mia, with only very few spermatozoa detected in the
somal chromatin condensates into compacted chro- semen, the chances to obtain a natural pregnancy are
matin fibres; the nucleus compacts and achieves high minimal, but ICSI can be an option [83].
density; and the shape changes into the species-specific Here again, sperm cells may contain chromosomal
head shape. These transformations are only possi- anomalies, which could lead to inherited problems in
ble when histones are replaced by transition proteins, the offspring. Available data indicate that IVF-ICSI is,
and eventually by protamines. As the transcription however, associated only with a slightly higher risk for
machinery no longer has access to the DNA, the sper- congenital malformations [84; 85].
matid stops active gene transcription. That is why
mRNAs that are necessary for spermiogenesis during Globozoospermia
maturation in the epididymis or just before fertiliza-
Globozoospermia is diagnosed when all sperma-
tion are transcribed earlier in round spermatids, and
tozoa are round-headed and lack acrosomes. This
are inhibited from translation by miRNAs until the
defect is genetic in origin and originates in spermio-
protein is required. Unnecessary organelles (e.g. endo-
genesis, specifically during acrosome formation and
plasmic reticulum, Golgi apparatus) are disposed of
sperm head elongation. The chromatin compaction
as “residual bodies” and phagocytized by Sertoli cells.
appears to be disturbed and, in some cases, cells with
Mature elongated spermatids are ultimately released in
DNA fragmentation or aneuploid cells are observed.
the seminiferous lumen via a process known as “sper-
Affected males suffer from reduced fertility or even
miation” [81].
infertility. ICSI can be proposed, although fertiliza-
Most testicular spermatozoa are non-motile and
tion rates might be severely reduced after ICSI with
incapable of fertilizing an oocyte. They gain their
sperm from globozoospermic men. On the other hand,
fertilizing capacity during their passage through the
globozoospermia does not seem to have an effect on
epididymis, where they undergo sequential modifica-
the number of spontaneous abortions nor on the pro-
tions. Only at the end of their transit between proximal
portion of children with congenital defects. Neverthe-
and distal epididymis, do male gametes acquire pro-
less, the question remains of whether assisted repro-
gressive forward motility and the capability to capac-
duction with round-headed sperm cells has long-term
itate, migrate through the female tract, bind to the
consequences for children, as the abnormal sperm
oolemma and fuse with the oocyte to create a viable
head morphology might be associated with genetic
embryo. As epididymal spermatozoa are transcrip-
deviations in the sperm cells [86].
tionally inactive, their maturation requires interaction
with proteins that are synthesized and secreted by the
epididymal epithelium. Each epididymal region fulfils Complete Asthenozoospermia
its own characteristic functions and contributes to the Spermatozoa acquire motility during epididymal
generation of functionally mature spermatozoa [82]. transit, unless genetically inherited defects cause
ultrastructural deficiencies in the sperm flagellum.
Although the spermatozoa are viable, this condition
Oligozoospermia leads to reduced numbers or even total absence of
According to the new guidelines of the WHO, men are motile sperm in the semen. Selecting viable sper-
diagnosed with oligozoospermia if their sperm counts matozoa for ICSI improves the fertilization rate
are below 15 million per ml. A reduced sperm count significantly. Diagnostic tests that can be used to
is observed in 5% of men and goes often together with detect viable sperm are the eosin-Y and the eosin–
other altered sperm parameters, such as poor motility nigrosin tests. To select viable sperm for ICSI, the
14
mechanical touch technique or hypoosmotic swelling Side effects might be avoided or minimized by
test (HOST) can be applied. These techniques are methods making use of nonhormonal factors. Sev-
easily implemented in the IVF lab, whereas LAISS or eral drug targets for nonsteroidal suppression of sper-
birefringence tests need expensive equipment. Men matogenesis are under investigation. Potential strate-
with absolute asthenozoospermia (15000), i.e. 100% gies involve the targeting of testicular and epididymal
immotile spermatozoa, have a poor fertility prognosis. processes with the objective of reducing sperm counts
Complete asthenozoospermia may be caused by or impairing sperm function. At the testicular level, the
necrozoospermia, i.e. the absence of live sperm in the first compound that was tested was gossypol, a natu-
ejaculate (1500–1200). The most common form is ral phenol in cotton plants. Although the use of gossy-
epididymal necrozoospermia, whereby sperm become pol was very effective, resulting in severe oligozoosper-
apoptotic and lose their viability during epididymal mia, it also appeared to be irreversible in 20% of
transit, but testicular necrozoospermia also exists. The men and was therefore prohibited. Antispermatogenic
two main causes of epididymal necrozoospermia are a effects have been attributed to commonly used medic-
hostile environment in the epididymis or a structural inal drugs such as the antihistaminicum indenopyri-
instability in the spermatozoa. Degeneration can dine (CDB-4022), which was found to disrupt germ
be due to genital infections, metabolic disorders cell adhesion. In primates, severe oligozoospermia
affecting ATP production, antisperm antibodies, was observed after one week of treatment, and this
oxidative stress, environmental chemicals or hin- was reversible. The anti-cancer drug lonidamine had
dered epididymal transport. Even though ejaculated similar effects by disrupting Sertoli–germ cell junc-
spermatozoa are nonviable, assisted reproduction is tions and releasing immature germ cells, but it caused
still possible, as viable testicular spermatozoa may be unwanted side effects. The analogues Adjudin (AF-
retrieved by TESE. Therefore, in cases of persistent 2364) and 2-gamendazole turned out to be more effi-
necrozoospermia, testicular sperm should be favoured cient and safer in rats. Studies using larger animals are
over ejaculated or epididymal sperm [87–89]. needed before trials in men can be initiated. Other
potential testicular targets are the molecules associ-
ated with retinoic acid (RA) metabolism. Since RA
Contraception is a promoting factor for spermatogonial differenti-
While medical research and the pharmaceutical indus- ation and meiosis, compounds targeting this path-
try have long focused on female contraception, inter- way will cause infertility. BMS-189453 inhibits all
est in new male contraceptive methods is grow- three RA receptors, leading to reversible infertility in
ing among men and women of different races, reli- mice. However, because of concerns with potential
gions and ethnicities. Current research aims at devel- side effects, RAR␣-specific antagonists are currently
oping a non-invasive and reversible pharmaceuti- under development. The bisdichloroacetyldiamine
cal contraceptive that rapidly and consistently causes WIN18446 prevents the conversion of Vitamin A to
azoospermia without adverse effects. The currently RA by inhibiting aldehyde dehydrogenase 1a2 activ-
best-developed pharmaceutical option is hormonal ity. Unfortunately, this compound also affects alcohol
contraception with progestin and testosterone. This metabolism and causes unpleasant side effects when
strategy down-regulates luteinizing hormone and combined with alcohol consumption. The search for
follicle-stimulating hormone secretion by the pituitary a testis-specific aldehyde dehydrogenase inhibitor is
gland resulting in decreased testosterone levels in the ongoing.
testis and impaired spermatogenesis. However, this Chromatin remodelling during meiosis and spe-
option will not be available shortly, as clinical trials rmiogenesis has also been the target in contraceptive
have revealed that a significant percentage of men did studies. JQ1 is an inhibitor of the testis-specific bro-
not achieve sufficient suppression of spermatogene- modomain, which regulates epigenetic modifications.
sis for contraception. Moreover, some undesirable side The use of this small molecule impaired sperm count
effects were reported, such as weight gain, increased and motility, resulting in complete and reversible infer-
serum glucose levels, acne, decreased libido, reduced tility in mice in a safe way. In postmeiotic sper-
testis size and mood changes. Also, the thought of fre- matids, serine threonine kinase 1 and 2 are subjects
quent drug injection encounters disapproval by many of investigation, as knocking these proteins out causes
men [90]. infertility.
15
In addition to testicular targets for male contra- Concluding Remarks

ception, epididymal targets are being studied as well.
Nowadays, ICSI is the solution for many couples
The G-protein coupled receptor HE6, which is specif-
when dealing with oligozoospermia or azoosper-
ically expressed in the epididymis, plays a role in the
mia. Fertilization and pregnancy rates after ICSI are
reabsorption of testicular fluids by the epididymal duc-
good in patients with oligozoospermia and/or incom-
tules. In mice, HE6 inhibition resulted in reduced fer-
plete asthenozoospermia. However, ICSI outcomes are
tility without side effects. In rats and other mammals,
reduced in patients with complete asthenozoospermia,
the glycoprotein CRISP1 suppresses sperm capacita-
globozoospermia and nonobstructive azoospermia. At
tion and sperm–egg fusion. EPPIN is an epididymal
this moment, the only infertile patients for whom
protease inhibitor bound to the surface of the sperma-
no treatment is yet available are those nonobstructive
tozoon. Because of its modulating function in sperm
azoospermic men in whom no testicular spermatozoa
motility, it was identified as a key target for contra-
can eventually be retrieved by TESE. Their only option
ceptive development. Another compound that inter-
is gamete donation or adoption. Novel in vitro and ex
feres with sperm motility is N-butyldeoxynojirimycin
vivo strategies are being developed and the first results
(miglustat), which inhibits glycosphingolipid biosyn-
seem to be promising. These methods may use either
thesis, resulting in reversible infertility in mice. In
adult SSCs or pluripotent stem cells such as embryonic
humans, on the other hand, this compound did not
stem cells (ESCs) or induced pluripotent stem cells
lead to changes in sperm count, morphology or motil-
(iPSCs) as starting material for the generation of arti-
ity. The sperm-associated cation channel (CatSper)
ficial gametes.
seems to be a better target. CatSper is involved in
However, many important questions remain unan-
sperm capacitation, hyperactivation, and egg pene-
swered. Despite the fact that most therapeutic inter-
tration. Disruption of this gene results in decreased
ventions in the field of fertility have been implemented
sperm motility and inability to fertilize oocytes. In
in the clinic without prior safety considerations, exper-
mice, the CatSper inhibitor HC056456 was reported
imental techniques such as SSC and testicular tis-
to block CatSper channel activity specifically and
sue cryopreservation and transplantation need to be
inhibit hyperactivation. Targeting glyceraldehyde 3-
validated as efficient and safe methods before they
phosphate dehydrogenase-S, the enzyme essential for
can be fully implemented in routine treatment. The
the sperm-specific glycolysis, renders male mice infer-
potential clinical use of artificial gametes is associated
tile by reducing ATP levels in spermatozoa. Similar
with even more concerns (e.g. epigenetic memory and
effects might be expected for the sperm-specific lac-
reprogramming) and should therefore be proceeded
tate dehydrogenase-C4. A final excellent target for
by extensive safety assessments as well as reflections on
reversible contraception might be the sperm Na+ /H+
the societal and ethical implications of using artificial
exchanger, which is essential for sperm motility. Mice
gametes in the clinic.
knocked out for the sperm-specific isoform Na,K-
ATPase ␣ produce sperm with abnormal morphol-
ogy and reduced motility. Although many potential
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20
Chapter
Sperm Chromatin Stability and
2 Susceptibility to Damage in Relation to

Its Structure
W. Steven Ward
Introduction sperm DNA is packaged, what we do know has many

direct implications for the clinical analysis of sperm
In this chapter we will describe how the three feet (1 m)
DNA damage.
or so of DNA is packaged into the 10 µm long, 0.5 µm
wide sperm nucleus (Figure 2.1). This engineering feet
must be accomplished in such a way that the precious Protamines
cargo retains its original sequence information in pris- Sperm chromatin can be packaged so tightly in sper-
tine condition, because it must be copied more than a matozoa because the DNA is coated with special-
trillion times during the life of the progeny to which it ized proteins, the protamines, that evolved solely for
will contribute half the embryo’s genetic material. The the purpose of condensing sperm DNA. The two
evolutionary pressure to protect the paternal genome human protamines, P1 and P2, are the product of
while it is on its relatively long journey during repro- an evolutionary stepwise progression that can still be
duction has selected the chromatin to make fantastic inferred from the wide range of sperm-binding pro-
efforts in condensation – almost to the limit of what is teins throughout the phylogenetic tree (for an excel-
possible for DNA. However, this must be accomplished lent review on protamine evolution, see [1]). The
in a manner that allows the unpacking of the DNA DNA in all eukaryotic cells, except for sperm, is pack-
in a relatively short time span after fertilization into aged mainly by histones, the well-known basic pro-
a functional paternal genome that can replicate itself teins that serve multiple roles in chromatin structure
and even begin transcription. as well as gene regulation [2]. Two copies of each
It would be extremely gratifying to be able to state of the four canonical histones, H2A, H2B, H3 and
in this Introduction that we have a very clear idea of H4, bind to about 140 bp of DNA as heterodimers
how nature has solved this difficult structural prob- to form a nucleosome, and successive nucleosomes
lem. But the reality is that while we do know a lot are spaced relatively evenly along the length of the
about sperm chromatin, our current models are just DNA [3]. Figure 2.2 compares how histones and pro-
that, models based on work from many different labo- tamines differ in folding DNA, to scale [4]. His-
ratories that are bound to change as we discover more. tones wrap the DNA into relatively small coils, induc-
The models, as flawed as they might be, are neces- ing negative supercoils into the DNA. Protamines
sary to build future experiments to test the ideas that wrap much larger segments of the DNA, reducing
further modify them. In this chapter, we have tried the level of supercoiling to almost zero. The transi-
to bring together all the data in the field and have tion from histone-bound DNA in the round spermatid
put forth our best model for sperm chromatin struc- to protamine-bound DNA in the mature spermato-
ture. We have also endeavoured to make clear how zoa occurs during spermiogenesis. This transition is
strongly the data support each part of the model and not well understood, but requires transition proteins
where more work needs to be done to clarify the dif- to effect the change. Histone H1 binds to the DNA
ferent structural levels of packaging. Some aspects are that is between the nucleosomes, and it plays various
well established, while others lean more towards spec- roles in condensing the chromatin to heterochromatin
ulation. However, even with the uncertainties of how [5, 6]. Most species have sperm nuclear basic proteins
University Press.
21
Chapter 2: Sperm Chromatin Stability and Susceptibility to Damage in Relation to Its Structure
Mouse Sperm Chromatin Folding
A. DNA
Length Packing
Ratio
1.02 m 1
B. Loop Domains
C. Protamine Toroids
1.5 mm 700
D. Unknown
Chromatin
Structure
E. Chromosomes
10 μm 102,000
Figure 2.1 Packing ratios of DNA inside the sperm nucleus. (A) There is a little over 1 m of DNA in a mouse sperm nucleus. (B) This DNA is
folded into loop domains that are attached at their bases to a sperm nuclear matrix. (B,C) Each loop domain is coiled into a protamine toroid.
Depending on how much DNA is in one loop/protamine toroid and whether it is stacked in single or double file, this arrangement brings the
packing ratio to about 700. (D) Given that the packing ratio of the protamine toroids when stacked together as shown in (D) is still short of
the estimated 102,000 needed to fold all the chromatin into the sperm cell, the protamine toroid chromatin (D) must be folded into another
type of tertiary structure to fit into the sperm cell. (E) The fully condensed chromosomes are organized into U-shaped structures with the
centromeres pointed towards the centre and the telomeres on the sperm nuclear periphery.
22
Solenoid Schematic Intermediate Doughnut
(Side)
Protamine-DNA
Complex
Histones Chromatin
replaced Condensation
by
Protamines
MAR MAR MAR MAR
Nuclear Matrix Filaments
Figure 2.2 Comparison of sperm DNA folding by histones and protamines. In round spermatids, and in all somatic cells, small segments of
DNA are coiled by histones that create negative supercoiling (left). During spermatogenesis, the histones are replaced by protamines that fold
the DNA into a much smaller overall space but do so by coiling much larger segments of DNA into toroids (right). In this diagram, one loop
domain, anchored at its bases by the nuclear matrix, is shown folded with histones (far left) or protamines (far right). The intermediate diagram
is not representative of what happens during spermatogenesis but is shown to depict the change in coiling. In the far left and centre diagrams,
only the DNA is shown for clarity. The histones would fill the double circles of DNA in the far left. This figure is reproduced from reference [4].
(SNBPs) that have all evolved from histone H1 with In nucleosomes, the DNA is wrapped twice around
varying degrees of similarity to the progenitor [7, 8]. the histone octamer, which leaves one side of the
Mammalian protamines are the least similar to histone double helix exposed to the immediate environment,
H1, having evolved a unique feature that enables very no matter how tightly the nucleosomes are subse-
tight DNA binding – a high concentration of arginine quently packed by tertiary chromatin structures [3].
residues, whose positive guanidinium residues bind Protamines have capitalized on a feature that is inher-
tightly to the negative phosphates on the DNA. Human ent in the DNA molecule – when the negative charges
sperm contains two distinct protamines that occur in of the phosphates are neutralized by a divalent cation,
roughly a 1:1 ratio in sperm chromatin [9], although large portions of the DNA coil into tightly packed
this can vary widely in fertile men [10]. Both are small toroids [13, 14] (Figure 2.3). Much of the DNA in
proteins, with P1 having 50 amino acids [11] and the the toroids is packed in uniform hexagonal arrays, so
fully processed form of P2 having 57 amino acids [12]. that every DNA molecule is surrounded by six DNA
P1 has 22 arginine residues, including one stretch of 6, fibres. The toroids are not perfectly hexagonal, as they
and P2 has 27. need some crossing to fill the volume, but the DNA is
23
Protamine-DNA
Toroid
Protamine DNA
Protamine DNA Fibers

External
Salt
Internal
extraction
DN ase Sensitive
Sperm Nuclear Matrix Attachment Toroid Linker Region
Matrix Region
Figure 2.3 Donut loop model for sperm chromatin structure. This model was modified from [17] and reveals the internal structure of the
protamine–DNA fibres within the toroid (inset).
packaged into a state as nearly crystalline as possible that develops into a mature pup [18]. The only func-
for long fibres. Protamines fold the DNA into toroids tion of protamines is to condense the sperm DNA,
that are very similar to those that can be artificially presumably to protect this precious cargo during the
induced by divalent cations [15]. In most mammalian relatively long journey the sperm cell must take to
DNA, this condensation is stabilized by intramolecu- fertilize the oocyte. This condensation, of course, nec-
lar disulphide bonds between the protamines. It is also essarily shuts down transcription and DNA replica-
important to consider that when DNA binds to pro- tion, and these may be secondary functions of the
tamines it is far less supercoiled than when it binds to protamines.
histones [4, 16]. Supercoiling means coiling the dou- Because of its primary role in sperm chromatin
ble helix DNA strand upon itself. Histones coil the formation, several laboratories have examined aber-
DNA once every 100 bp to wind the DNA into tight rant protamine deposition in human samples. Two
packages. But protamines coil the DNA an estimated types of aberrations have been studied. The first is the
once every 600 bp or so [4, 16], so the DNA is pack- ratio of P1 and P2 protamines, but this has not proven
aged more tightly but coiled less. This will become very diagnostic [9, 10]. The second is the incom-
important later in this chapter when we discuss the role plete replacement of histones by protamines during
topoisomerase may play in the generation of single- spermiogenesis [9, 19]. The first haploid progenitor of
stranded nicks in sperm DNA. sperm, the round spermatid, contains only histones
The protamine toroid is the defining element in its chromatin. As spermiogenesis progresses, these
of sperm chromatin condensation and is the best- histones are gradually replaced by transition proteins
studied. Of all the elements of DNA packaging in the and then protamines [20]. Errors in this process can
sperm that are discussed in this chapter, the protamine result in sperm that are not fully condensed and may
toroid is the most firmly accepted. This is the feature not be functional. Several assays have been used to
that makes sperm chromatin the most stable form of quantify the level of sperm condensation, including
eukaryotic chromatin known. This structure is resis- CMA3 (chromomycin A3) staining [21] and the HDS
tant to nucleases, for example [17]. Protamines also (high DNA stainability) assessment of the sperm chro-
condense the DNA so tightly that sonication, which matin structure assay (SCSA) [22]. These assays are
would normally destroy somatic histone-bound chro- based on the ability of fluorescent dyes to penetrate
matin, does not affect mouse sperm DNA – sonicated the sperm chromatin, and DNA bound into protamine
mouse sperm are still capable of fertilizing an oocyte toroids is much less accessible to any molecule than
24
histone-bound DNA. Because, as described above, Gatewood et al. in 1987 [27]. That study and all sub-
sperm condensation is almost entirely defined by pro- sequent studies take advantage of the facts that his-
tamine condensation of the chromatin, these assays are tones are much more easily extracted from DNA than
a measure of the completeness of protamine deposi- protamines and that histone-bound DNA is much
tion during spermiogenesis. The results suggest that more nuclease-sensitive than protamine-bound DNA.
errors in protamine deposition play an important role Since that first publication, several laboratories have
in human infertility. assessed the genomewide positioning of histones in
mouse and human sperm with varying results. Ham-
moud et al. [28] suggested that histones where pref-
Histones erentially located on expressed genes and, in the
In addition to protamine deposition, the major com- human, histone-coated genes that would be expressed
ponent of sperm chromatin structure, other well- in the developing embryo. Arpanahi et al. [29] mapped
known aspects have important functions. One of these nuclease-sensitive regions in both human and mouse
is the small proportion, 2% to 15%, of sperm DNA sperm and found that these tended to be localized
that remains bound to histones. Two different lines of to promoter regions of genes and to CCCTC-binding
evidence suggest that the histones in sperm serve spe- factor (CTCF) sites. Another genomewide study pro-
cific functions related to embryogenesis after fertiliza- posed that transcriptional activity during spermiogen-
tion, and that they are not simply residual because of esis directed the deposition and retention of histones
imperfect protamine deposition. The first was that his- to specific regions in the sperm nucleus, and this was
tone modifications and specific histone variants that related to DNA methylation [30]. If this is true, then
occurred in fully condensed sperm chromatin could histone deposition may have more to do with what
be found in the paternal pronuclei of fertilized oocytes occurred prior to the final protamine deposition than
[23, 24]. This suggested the possibility that these his- as a set of inherited epigenetic instructions for the
tone modifications and/or variants helped to prepare zygote. Finally, a recent study using a different con-
the paternal genome for its function in early embryo- centration of nuclease to release the histone-bound
genesis. To date, no direct evidence for the function chromosomal segments mapped histone-associated
of histones modified in the sperm cell on the devel- regions to gene-poor sites, in apparent contradiction
oping embryo have been found. However, there is one to earlier studies [31]. Two things are clear from
intriguing coincidental finding that offers some sup- these studies. First, most researchers that have looked
port for this idea. Mouse sperm chromatin contains for sequence-specific association of histones to sperm
the histone variant H3.3 [25]. After fertilization, all DNA have found evidence to support it. This sug-
the protamines are replaced by histones, and H3.3 gests that the histones that remain in sperm chro-
is loaded onto the paternal DNA, while the mater- matin are positioned by a specific mechanism and are
nal pronucleus contains a different variant, H3.1 [26]. not random. Second, the maps of histone-associated
During early mouse development, H3.3 is specifically sperm DNA are not in full agreement. Most labs sug-
modified in the male pronucleus by methylation at gest that there are enrichments of histones at cer-
K27 (H3.3 K27me), and if this modification is genet- tain sites, but not full penetrance, indicating that the
ically inhibited, embryogenesis halts before develop- data now support a preference, but not an absolute
ment to the blastocyst. The sperm cell cannot sup- requirement, for sequence-specific histone position-
ply all the H3.3 for the paternal pronucleus, but the ing. However, this conclusion may change with further
small amounts of H3.3 in the sperm cell may signal the data.
oocyte to populate the sperm chromatin with that par-
ticular histone variant.
The second line of evidence that the small histone Loop Domains and Nuclear Matrix
population in the sperm cell contributes to embryo- The next level of DNA packaging, recognized by sev-
genesis comes from several laboratories that have eral different laboratories, is controlled by the sperm
mapped the histones in the sperm cell to specific loca- nuclear matrix that organizes the DNA into large (25
tions on the chromosomes. The idea that the resid- to 50 kb) topologically constrained loop domains [32–
ual sperm histones either remain on or are deposited 35] (Figs. 2.1B and 2.2). The sites of DNA attachment
to specific genomic locations was first addressed by to the matrix (matrix attachment regions or MARs)
25
appears to be sequence-specific or at least shows donut loop model (Figure 2.3) predicts at least three
preferences for certain sites [35–37]. This attachment separable levels of chromatin accessibility. The first and
to the nuclear matrix is not well understood. Several most open is the toroid linker region that contains
laboratories have tried to identify the proteins that the nuclear matrix attachment region (MAR). The
are involved, and topoisomerase II may play a signif- toroid linkers are more sensitive to DNAse I than the
icant role. However, one thing is very clear, that this protamine-bound DNA [17]. The chromatin structure
attachment survives 2 M NaCl extraction and is there- of the toroid linkers is not known, but work by Krawetz
fore much tighter than either histone or protamine and colleagues has shown that histone-bound DNA
binding to DNA. The relationship between the sperm is associated with the nuclear matrix in human sper-
nuclear matrix and the compaction of sperm DNA matozoa [37, 48]. This suggests that the toroid linkers
into toroids by protamines appears to be that each may be in the more open histone-bound configuration.
DNA loop domain is coiled into a single protamine The second level of accessibility would be the chro-
toroid [17, 38]. In somatic cells, DNA is replicated matin fibres that are on the surface of the protamine
on the nuclear matrix, and many authors have sug- toroid (Figure 2.3). These fibres would be expected
gested that MARs also originate in replication [39– to be accessible to enzymes, but the protamines
41]. We have suggested that the sperm nuclear matrix that bind so completely to this DNA would proba-
organizes the DNA so that the replication origins are bly severely, if not completely, inhibit the activity of
attached to the sperm nuclear matrix, and that this DNA-binding enzymes. The third level of sperm chro-
structural organization will manifest in the paternal matin accessibility would be the majority of the chro-
pronucleus of the embryo [42]. If so, the organization matin fibres that are inside the toroid and completely
of DNA by the sperm nuclear matrix would represent a covered by neighbouring protamine–DNA strands
type of epigenetic structure that can be inherited by the (Figure 2.3). These DNA strands are not accessible
embryo. to any exogenous proteins while the toroids remain
compact.
Sperm DNA Damage Assays and Let us now consider the different assays that are
available to test for sperm DNA damage with respect
Human Fertility to these three levels of DNA accessibility. One of
Many assays for sperm chromatin structure have been the most common assays for the presence of DNA
developed in an attempt to determine whether cases breaks in human spermatozoa is the TUNEL assay,
of idiopathic male infertility could be explained by the or terminal deoxynucleotidyl transferase (TdT) medi-
sperm DNA being damaged. The rationale is that DNA ated dUTP nick end labelling [49]. In this assay, the
damage may not be reflected in the sperm morphol- enzyme TdT is used to add labeled nucleotides to
ogy, its motility or even its ability to fertilize the oocyte. free 3’OH groups at the ends of DNA strands, result-
However, damaged sperm DNA would be expected ing in single-stranded poly-U extensions. Therefore,
to destroy or reduce the spermatozoon’s potential to the TUNEL detects both single-stranded and double-
fertilize and/or inhibit embryo development. Several stranded breaks. The labeled nucleotides that are thus
excellent reviews have recently been published that incorporated into the DNA are detected with fluo-
describe how the various techniques relate to each rescent antibodies. Most current protocols for sperm
other and how they correlate with human fertility [43– TUNEL assays do not include an extensive extrac-
47], and we will not attempt to assess these clini- tion procedure that would be expected to remove pro-
cal aspects. Instead, we will explore these assays in tamines or histones, except for the fixation proce-
the context of our current model of sperm chromatin dure, which includes ethanol and acetic acid that might
structure. remove some or all of the histones [50, 51]. Indeed,
There are two features we would like to con- this assay often depends on sperm nuclei remaining
sider: whether the assay detects single-stranded (ss) condensed so that they can be sorted by fluorescence-
or double-stranded (ds) DNA breaks, and what level activated cell sorting, or FACS [52, 53], confirming that
of accessibility to the sperm chromatin the assay pro- most of the nuclear proteins remain in situ. The in
vides. The differences between ss and ds DNA breaks situ translation assay also uses an enzymatic method to
are clear, but the latter point deserves some considera- detect single-stranded DNA breaks but cannot detect
tion with relation to our view of sperm chromatin. The double-stranded breaks. These two assays have the
26
least accessibility to sperm chromatin of all the assays A. TUNEL Assay

described in this section. TdT
These facts suggest that most of the sperm DNA Poly dU
remains inaccessible to the TdT in the TUNEL assay
because the protamine toroids remain largely intact
and most of the DNA is localized within the toroid.
Thus, we would predict that the major type of sperm
chromatin that would be accessible to the TUNEL
assay would be the toroid linker regions. We have
shown that these regions are accessible to DNAse I
[17], and they should be accessible to the TdT that is B. SCSA
used in the TUNEL assay. The second type of chro-
matin in which damage may be detected by this assay
is the DNA that happens to be on the surface of the
protamine toroids. We view this as less likely because
the very high affinity that protamines have for DNA
would probably prevent TdT binding to DNA. How- Acid Acridine Orange
ever, since the TdT only adds to free 3’ OH DNA ends, Denaturation ds DNA
TUNEL may be able to detect these breaks. The third ss DNA
type would be severely damaged chromatin that has
few or no protamines due to some type of chromatin
defect (Figure 2.4A). Thus, the TUNEL assay is lim-
ited to those areas of sperm chromatin that remain
accessible to enzymatic modification. Because these
ssDNA
areas may, in fact, be the most active sites during the Nick
first hours of fertilization when sperm chromatin is
decondensed, this aspect may be why the TUNEL assay
is more closely correlated with human infertility [45] Figure 2.4 The TUNEL and SCSA assays considered with respect to
than, for example, the COMET assay, which detects the donut loop model. (A) The TUNEL assay uses the enzyme TdT
virtually all DNA breaks, as described below. to add uridine residues to 3’OH ends of nicked and broken DNA.
(B) The SCSA assay denatures DNA that has nicks and then uses
Another popular method of examining sperm acridine orange to detect single- and double-stranded DNA.
DNA stability is the sperm chromatin structure assay
(SCSA) developed by Don Evenson [54]. In this assay,
sperm samples are treated with mild acid to denature only if there is a DNA break. Therefore, the SCSA can
DNA that contains ss or ds nicks but are otherwise also infer ss or ds DNA breaks.
not treated with conditions that are strong enough to In the light of our model for sperm chromatin
extract the protamines. As in the TUNEL assay, the structure, we predict that most of the DNA breaks
sperm nuclei remain condensed enough to be sepa- the SCSA identifies are located in the toroid linker
rable by flow cytometry. However, in the SCSA, no regions, but a substantial number may also occur in
enzymes are required. The acid-extracted sperm nuclei the protamine toroids (Figure 2.4B). Acridine orange
are stained with acridine orange, a DNA-intercalating is a relatively small molecule when compared to the
dye that stains single-stranded DNA red and double- enzymes TdT and DNA polymerase, so access to the
stranded DNA green. The ratio of red to total stain- sperm chromatin should not be limiting. However,
ing (red/red+green) is called the DFI, or DNA frag- Evenson has suggested that condensed chromatin does
mentation index. The DFI is therefore a measurement not bind acridine orange well [55], and it may be that
of the amount of sperm chromatin that can be dena- the protamines prevent the actual intercalation of the
tured by mild acid or heat treatment. Because chromo- dye into the ds DNA. Intercalating agents extend the
somal DNA is tethered by attachments to the nuclear DNA and can distort it so much that histones can no
matrix every 50 kb or so [16], and by the tight bind- longer bind it. It is possible that protamines, which
ing of the protamines (Figure 2.2), it can be denatured are linked by covalent intermolecular disulphides,
27
cannot be displaced by intercalators and actually halos, in which naked loops of DNA of about 50 kb
inhibit their binding. Regardless of whether acridine in length, are attached at their bases to the sperm
orange can bind to the protamine-bound DNA, it is nuclear matrix [58] (Figs. 2.1–2.3). The DNA is then
clear that protamine-bound DNA will not denature denatured by base (alkaline COMET assay) or kept
even if ss DNA nicks are present. Given these facts, at neutral pH (neutral COMET assay) and subjected
we predict that the SCSA would have accessibility in to an electric current. This means that the alkaline
detecting ss and ds DNA breaks similar to that of COMET assay can detect single- and double-stranded
the TUNEL assay (Figure 2.4B). More recent evidence DNA breaks, since the DNA is denatured, while the
from both of our labs suggests that some ss DNA neutral COMET assay only detects double-stranded
breaks can be detected in the protamine toroids [56] breaks. This allows researchers to differentially assess
(discussed below). single- and double-stranded breaks in sperm. Those
The last assay that we will discuss is the COMET fragments of DNA that are free of the sperm nuclear
assay. This assay is unique in that all the protamines matrix migrate toward the positive electrode, creat-
and histones are extracted by high salt and disulphide ing a cometlike appearance (Figure 2.5). According to
reducing reagents [57]. Spermatozoa are embedded this view of the COMET assay, ssDNA breaks would
in agarose on a glass slide so that when the proteins only be freed from the sperm nucleus if two ssDNA
are extracted the chromosomal DNA remains local- breaks were present on the same strand of DNA within
ized. The extraction procedures used with the COMET one loop (Figure 2.5). Likewise, dsDNA breaks would
assay are consistent with the formation of nuclear be detected only if two dsDNA breaks occurred in
COMET Assay +
2 M Nacl
Alkaline
+ DTT COMET Assay
Alkaline
Denaturation
and
Electrophoresis −
+
Electrophoresis
Nuclear Matrix
Only
Neutral
COMET Assay
ds DNA Breaks
ss DNA Breaks
−
Figure 2.5 The neutral and alkaline COMET assays and the nuclear matrix. Both COMET assays extract the histones and protamines with high
salt, so that the donut loop model is not relevant in this assay. The attachment sites of the DNA to the nuclear matrix, however, would remain
intact in this assay. Note that DNA double helices depicted in the extracted loop diagrams are not drawn to scale but are shown as shorter
segments for clarity.
28
one loop. Finally, if some patients had sperm nuclear during chromatin remodelling, fragmentation due to
matrix aberrations [59], the COMET assay might iden- reactive oxygen species (ROS) [67, 68], endogenous
tify more DNA breaks. nucleases that can be activated under certain condi-
If the models shown in Figs. 2.4 and 2.5 for under- tions [17, 69], and actual apoptotic DNA cleavage. And
standing SCSAs are correct, we may conclude that the while some apoptotic markers positively correlate with
TUNEL, SCSA, and in situ translation assays primar- infertility in humans, a causative effect has yet to be
ily detect the toroid linker regions, while the COMET seen [70].
assays detect many more DNA breaks per sperm cell, As mentioned above, there exists a major topo-
but without any distinction for their chromosomal logical problem in going from histone-bound to
localization. Thus, if these different assays are shown protamine-bound DNA. Histones coil DNA more than
to predict different results for ART, analyses of which protamines, and thus, when protamines replace his-
assays predicted which result would lead to an under- tones, these supercoils must be removed. To make this
standing of the chromosomal aberration that was asso- possible, nicks must be introduced into the DNA by
ciated with the defect. a nuclease. Topoisomerase has been implicated in this
process [71], as it allows controlled nicking, increase
in linking number and subsequent DNA relaxation,
Sperm Apoptosis and religation of the DNA. DNA nicks can be seen
Apoptosis is a strictly regulated programmed cell in situ using the TUNEL assay to detect DNA strand
death [60] utilized by somatic cells for proper develop- breaks in the early stages of spermatogenesis. DNA
ment, homeostasis, and removal of damaged or dan- nicks are maximally seen during the transition from
gerous cells. It is marked by chromatin condensa- round to elongated spermatids in the testis and visu-
tion, plasma membrane blebbing, nucleosome-sized alized in close to 100% of the cells [51]. These nicks
as well as high-molecular-weight (50–100 kb) DNA are nearly absent once packaging is complete. A posi-
fragmentation, the externalization of certain inner- tive correlation has been seen between topoisomerase
membrane constituents, and cell fragmentation into presence and DNA strand breaks; topoisomerases have
compact membrane-enclosed structures termed apop- been identified in spermatogonia, spermatocytes, and
totic bodies which contain cytosol, the condensed round and early-elongating spermatids [72], while few
chromatin, and organelles. Apoptosis has also been people report their localization in mature spermato-
suggested to play key roles in adjusting the appropri- zoa [73]. Some spontaneous DNA fragmentation has
ate number of proliferating germ cells associated with been observed postejaculation, and it increases with
Sertoli cells, removing abnormal sperm, and other glutathione peroxidase inhibitor treatment. This sug-
normal spermatogenic processes [61–63]. The apop- gests an involvement of ROS as a possible cause of
totic machinery is also implicated in the selective DNA fragments in mature sperm [67]. Finally, nucle-
depletion of unneeded portions of cytoplasm during ase have been found in the lumen of the caudae epi-
Drosophila [64] and rat [65] spermiogenesis into cyto- didymides of bulls, boars, rabbits, and rats [reviewed
plasmic masses dubbed ‘residual bodies’. And while in 74].
apoptotic markers including DNA nicks, caspases and Apoptotic markers seen in ejaculated spermato-
other proteins, and phosphatidylserine (PS) translo- zoa include Bcl-xl [50, 75], caspase-3, -8, and -9, Fas
cation have been observed in ejaculated spermatozoa, receptors [50,76], and PARP [77], as well as the exter-
it is unclear whether they are residues of an abortive nalization of the normally membrane-internal PS by
apoptotic process started before ejaculation [66], an Annexin V binding [76, 78] and DNA strand breaks
anomaly of sperm production, or signs of apoptosis [50, 79–82]. An increase in caspase enzyme activity has
initiated postejaculation. also been seen in men with decreased motility, but also
Do mature spermatozoa have the ability to go in motile fractions from subfertile patients [83]. Sam-
through apoptosis? This is a more difficult question ples with lower sperm concentration and poor mor-
to answer. The presence of DNA strand breaks and phology correlated with increased TUNEL staining
spontaneous DNA fragmentation in ejaculated sper- and Fas and p53 expression [50] and with increased
matozoa has led to much speculation. Hypotheses as to immature sperm concentration and Bcl-xl expression
the reasons for their existence in mature spermatozoa [50, 75]. However, TUNEL positive cells and apop-
include the failure to repair naturally induced nicks totic markers are not always found together [50]. The
29
prevailing idea is that apoptosis does not occur in part of this process. We have demonstrated that mouse
mature spermatozoa; rather, apoptotic markers exist in and human sperm can be induced to degrade their
immature spermatogenic cells that were not selected own DNA when treated with divalent cations in a pro-
against during spermiogenesis. DNA fragmentation cess we have termed sperm chromatin fragmentation
as detected by the TUNEL assay can be caused by (SCF) [92]. Sperm isolated from mouse epididymides
topoisomerase during the change from histone- to that are induced to undergo SCF cleave their DNA to
protamine-bound DNA or by reactive oxygen species loop-sized fragments that can be reversed with EDTA,
(ROS) [67, 68, 84]. The protein markers can be left suggesting that a topoisomerase 2-like protein medi-
over from abortive apoptosis, cells that should have ates the degradation. Topoisomerase 2 is present in
been removed from the mature sperm population but mature sperm, not only during spermatogenesis, and
were not, or normal spermiogenic occurrences such as SCF suggests that this enzyme can be activated in
cytoplasmic depletion. Membrane phosphotidyl ser- mature sperm in response to specific conditions. It
ine translocation, while seen in sperm under differ- may be that the topoisomerase 2 in mature sperm is
ent experimental conditions, could be related to pos- different from that at earlier stages of spermatogene-
itive functional changes in sperm such as capacitation sis, but this still needs to be studied. Sperm from the
[85, 86], and in cryopreserved sperm is likely due to vas deferens degrade their DNA much further, and
membrane damage [87]. In fact, in experiments where the degradation is not reversible, indicating the pres-
mature sperm are incubated for 24 h, necrosis rather ence of a nuclease. This is very similar to the way
than apoptosis is detected, even though there is a sig- that somatic cells degrade their DNA during apopto-
nificant decrease in cell motility [88]. In an experi- sis [93, 94], except that in mouse sperm, the two steps,
ment where apoptosis was activated in sperm cells, reversible degradation by topoisomerase 2 followed by
betulinic acid was used to induce apoptosis and it was irreversible degradation of DNA by a nuclease, can be
detected by mitochondrial transmembrane potential separated.
disruption and activation of caspase-9 and -3 [89]. We have proposed that the first, reversible step
Nevertheless, naturally induced apoptosis has yet to of SCF occurs on the sperm nuclear matrix at the
be fully described or accurately detected in mature MARs. We used the Comet assay to assess single- and
spermatozoa. double-stranded breaks in SCF and to assess the role
More recently, a series of publications from the lab- of the nuclear matrix in generating the breaks [95].
oratory of John Aitken have established that sperm are Using a modified, neutral Comet assay, we found that
highly responsive to reactive oxygen species (ROS) and the DNA at the initial double-stranded DNA breaks
that the generation of ROS by mitochondria is associ- that occur during SCF remains associated with the
ated with sperm apoptosis. This group has proposed a nuclear matrix. As the degradation proceeds, the DNA
model [90] in which sperm are normally predisposed is released from the matrix. This supports our model
towards apoptosis, and it is only the presence of pro- for SCF, in which the first double-stranded breaks are
survival factors, notably prolactin and insulin [91], created by topoisomerase located at the MARs on the
that keeps them alive. According to the model, pronuclear matrix. This study also revealed that in addi-
survival factors maintain phosphoinositide 3– (PI3-) tion to the double-stranded DNA breaks, SCF also
kinase in the phosphorylated active state, and this creates many single-stranded DNA breaks that appear
keeps another kinase, AKT1, active. It is this latter occur throughout the genome. This is supported by the
enzyme that maintains the survival status of the sperm fact that sperm release ROS during apoptosis [91].
cell. AKT1 maintains several prosurvival factors, such SCF, then, is a model for testing sperm DNA dam-
as Bad, in the phosphorylated, and therefore active, age assays and how they relate to sperm chromatin
state, actively inhibiting apoptosis. structure. SCF-induced epididymal sperm have minor
double-stranded and single-stranded DNA breaks,
Mouse Models to Test for DNA while SCF-induced vas deferens sperm have much
higher levels of both types of breaks. We used this
Degradation model to compare the alkaline and neutral Comet
The work cited in the previous section suggests that assay (as mentioned above) and the sperm chromatin
sperm are capable of initiating apoptosis. It is possible, structure assay (SCSA test) and the TUNEL assay [56].
therefore, that sperm can also degrade their DNA as For the TUNEL assay, the results were consistent with
30
our prediction that signal detection would be limited function occur at the sites where the DNA is attached
to the toroid linker regions. For the SCSA assay, how- to the nuclear matrix [100]. Thus, we have suggested
ever, the data suggested that single-stranded breaks that these same sites in sperm nuclei serve as the nucle-
can be detected even within the protamine toroids. ation sites for pronuclear DNA replication [101]. If
these hypotheses are correct, it seems predictable that
sperm chromatin structure assays that focus on these
Conclusions toroid linker sites would be more clinically useful than
There is now no doubt that the chromatin of mature assays that are not able to distinguish them from the
mammalian spermatozoa, which we once viewed as rest of the sperm chromatin.
almost impenetrable to DNA-damaging agents, can, Of course, it is not yet clear how much of what we
and often does, contain both ss and ds DNA breaks. have proposed in this work will be modified as research
Understanding the mechanisms surrounding the gen- on sperm chromatin structure progresses. However,
eration of sperm chromatin breaks has clinical sig- considering the current sperm DNA assays in the con-
nificance in humans, as the presence of this DNA text of our donut-loop model predicts the clinical cor-
damage often correlates with male infertility. Several relations that have been observed to date. We antici-
recent reviews have presented hypotheses as to the pate that further research on how sperm chromatin is
generation of these breaks. The possibilities include packaged will contribute to a better understanding of
abortive apoptosis of spermatogenic cells [44], incom- both the clinical assays that detect sperm DNA damage
plete repair of topoisomerase-induced nicks during and the biological significance of these DNA breaks.
histone replacement by protamines [51, 96], and gen-
eration of ROS that cause DNA nicks in mature sper-
matozoa [97]. Our recent work has demonstrated that Acknowledgements
mammalian spermatozoa also contain endogenous The author wishes to thank Andrei O. Zalensky,
nucleases that are capable of digesting all the sperm Ph.D., D.Sci., Professor Emeritus of the Jones Institute
chromatin [17, 98]. Much work remains to be done to for Reproductive Medicine, for helpful discussion in
understand the origins of DNA damage in the tightly preparing this revised edition of the chapter.
packed sperm chromatin, but it is clear that it has a sig-
nificant clinical impact.
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35
Chapter
Sperm Ultrastructure in Fertile Men and
3 Male Sterility
Revisiting Teratozoospermia
Hector E. Chemes
Introduction matozoon to be microinjected. It then became clear

that abnormal and immotile spermatozoa could suc-
Knowledge of the structure of spermatozoa can be cessfully fertilize oocytes, and the question was raised
traced back to the seventeenth century, when Antoni of the convenience of using them in ART procedures.
van Leeuwenhoek communicated for the first time the Some andrologists stressed the importance of differ-
existence of numerous animalcula in the seminal fluid ent tools to characterize sperm pathologies and estab-
of animals and men. He reported his findings in a letter lish a diagnosis; still others were more inclined to use
submitted to the Royal Society of London in Novem- them for assisted reproduction without much atten-
ber 1677. In his morphological rendering of sperma- tion paid to diagnosis. Recent evidence has indicated
tozoa, he reproduced with precision the main sperm that in many of these patients a genetic component is
components and documented a striking heterogeneity present and that, depending on the nature of sperm
that, beyond the accuracy of his observations, is the pathologies, the outcome of IVF-ICSI may change
first account of teratozoospermia. Intensive research dramatically.
during the eighteenth and nineteenth centuries estab- When studied in sperm smears, abnormal forms
lished the testicular origin of spermatozoa and their are defined solely on the basis of atypical sperm shapes
fundamental role in fertilization. The introduction of that do not identify the cellular basis of their func-
modern morphological, biochemical, and molecular tional incompetence because of technical limitations
techniques, together with advancements in reproduc- of light microscopy. Ultrastructural evaluation of ter-
tive medicine during the twentieth century, resulted in atozoospermia, coupled with immunocytochemistry
the characterization of various distinct sperm abnor- and molecular techniques, allows precise characteri-
malities of infertile males. It was soon realized that zation of sperm abnormalities, including their struc-
there were a limited number of abnormal, immotile tural, molecular and functional aspects. This approach
and dead spermatozoa in the ejaculates of fertile indi- goes beyond descriptive morphology of the appear-
viduals and that these percentages were pathologi- ance of spermatozoa. Sperm pathology is therefore a
cally increased in numerous cases of male infertility. special example of the general concept of cell pathol-
From these observations evolved the concepts of tera- ogy coined by the German pathologist Rudolph Vir-
tozoospermia, asthenozoospermia and necrozoosper- chow. who, in the mid-nineteenth century, introduced
mia,1 all conditions negatively influencing the fertility the idea that all disease originated with injury to the
prognosis in spontaneous conditions or with the use cell and in particular to the structure and function
of various assisted reproductive techniques including of cell organelles. It may seem outdated to claim the
IVF. In all these circumstances, the quality of the sin- application of a nineteenth-century concept to cur-
gle fertilizing spermatozoon could not be established rent reproductive pathology, but the fact is that nor-
with certainty. The introduction of ICSI allowed exam- mal spermatozoa have been characterized in relatively
ination of motility and morphology of the very sper- recent times, and their pathological alterations can
University Press.
36
Chapter 3: Sperm Ultrastructure in Fertile Men and Male Sterility: Revisiting Teratozoospermia
only now be understood in their physiopathological to a particular observation. The golden rule is that an
complexity. extraordinary finding should make sense and be con-
Following the concept of sperm pathology, two sistently present to be considered an original struc-
main forms of abnormal spermatozoa can be dis- ture or a pathologic trait. An interesting feature present
tinguished. In the first and more common variety, only in a limited sample from a single patient or ani-
a heterogeneous combination of different alterations mal would probably lack significance or be the result of
is found randomly distributed in each individual processing artefacts. These comments are not meant to
and among different patients. These alterations can discourage the search for new features that constitutes
be referred to as nonspecific or nonsystematic sperm the basis of scientific advancement. When such results
defects. These anomalies are usually secondary to var- are being considered, their importance as promising
ious pathologies that affect the normal function of findings should be analysed in the context of current
the testis or the seminal pathway, such as varicocele, structural and physiological knowledge.
immune factor, or seminal infections. The second vari-
ety presents with a characteristic anomaly that involves
the vast majority of spermatozoa in a semen sample.
Spermatogenesis and Spermiogenesis
These alterations may be called systematic in the sense Spermatozoa arise within testicular seminiferous
that there is a common sperm phenotype that pre- tubules by spermatogenesis, the series of cell processes
dominates in a given patient and resembles similar by which undifferentiated germ cells give rise to
defects in other individuals suffering from the same mature spermatozoa. It comprises three sequential
condition. Systematic alterations tend to show fam- phases:
ily clustering and have proven or suspected genetic (1) Mitotic multiplication/differentiation of
origin. spermatogonia, resulting in a geometric
In the present chapter, we will describe in detail numerical expansion, loss of their stem cell
ultrastructural studies of human semen in health and properties and further commitment to advance in
disease that are now available for basic or translational spermatogenesis. This phase yields a large
research in reproductive biology and medicine. The number of spermatogonia ready to undergo
content is not meant to be addressed particularly to meiotic division.
electron microscopists, but to all researchers and clin- (2) Meiosis, a series of two coupled cell divisions
icians interested in exploring the utility of basic ultra- leading to genomic recombination and
structural studies of spermatozoa, their application to haploidization. These processes occur during the
the understanding of the biological basis of abnor- prophase of Meiosis I by pairing and genetic
mal cell function and their use as a diagnostic tool in recombination between homologous
andrology. chromosomes and are followed by reduction of
Since electron microscopy allows the visualization chromosomal numbers. Each resulting secondary
of the interior of the cell and its organelles, a word of spermatocyte, a unique combination of the male
caution in relation to subcellular dimensions is appro- paternal and maternal genomes, goes into
priate for all those not currently experienced in ultra- Meiosis II and generates two haploid spermatids
structural studies. It is important to understand that ready to go through the last phase of
the microscopic field under electron microscopic eval- spermatogenesis.
uation is extremely small. This means that cell sam- (3) Spermiogenesis, through which newly formed
pling is of paramount importance in evaluating how postmeiotic round spermatids undergo a complex
significant a finding may be. The final areas selected for cell differentiation leading to the production of
ultrastructural studies should be chosen after careful elongated spermatids that leave the germinal
light microscopic evaluation of semithin sections from epithelium to become free mature spermatozoa in
different areas to guarantee that all important features the lumen of seminiferous tubules.
are represented.
The validity of an ultrastructural observation While spermatogenesis is highly conserved through
requires consistency of the findings throughout a tis- evolution, it has individual species variations. In par-
sue or cell type. This is particularly true if the goal ticular, human spermiogenesis depicts particular fea-
is to assign biological or physiopathological meaning tures related to the uniqueness of human spermatozoa,
37
Figure 3.1 Graphic representation of the eight steps of spermatid differentiation during spermiogenesis, according to Holstein and Holstein
and Roosen Runge [1, 2]. See full description in the text. A: acrosome, An: annulus, Ax: axoneme, C: centriole, F: flowerlike structures, Fs:
flagellar substructures, M: mitochondria, Mp: middle piece, Mt: manchette microtubules, N: nucleus, Ne: neck (connecting piece), PP: principal
piece, R: ribs of the fibrous sheath, Sb: spindle-shaped body. The acrosome of STEP 1 spermatids is oriented toward the seminiferous tubule
lumen. When progressing to step 2, spermatids rotate 180° so that the acrosome faces the seminiferous tubule basement membrane.
Reproduced from Holstein (Andrologia, 1976; 8:157–65; reproduced with permission from John Wiley and Sons).
Figure 3.2 Electron micrographs of human spermatids at different steps. A: Early step 1 (previous phase to that depicted in Figure 3.1). Very
near the spermatid nucleus [N] the Golgi complex contains parallel membrane stacks [G] and numerous vesicles, some of them containing
dense core proacrosomic granules [PA] that will later fuse and apply to the nuclear envelope to form the acrosome. B: Step 2 spermatid [cap
phase]. The Golgi complex [G] is close to the nucleus [N]. The acrosome [Acr] extends peripherally as a dense acrosomal cap covering the
cranial aspect of the nucleus. C: Step 6. The nucleus [N] is elongated, the chromatin in advanced condensation and the acrosome fully
developed [Acr]. The connecting piece [CP] contains a cross section of the proximal centriole close to the nucleus, and the distal centriole that
gives rise to the axoneme [distal to the nucleus]. This complex is lodged in the concavity of the nuclear implantation fossa [IF]. Note the
ring-shaped annulus [An] and the microtubules of the manchette [Ma] inserting in the postacrosomal perinuclear ring. Bars in A–C represent 1
µm. Panel C was previously published by Chemes et al. (Anat. Rec. 1978; 192: 493–512, reproduced with permission from John Wiley and Sons).
including specific modifications of nuclear shape and triole migrates and lodges at the caudal nuclear pole
chromatin structure, acrosome development, flagellar and the axoneme of the future sperm cell grows from
growth, and formation of the mitochondrial sheath. the distal centriole and emerges into the extracellular
Round euchromatic nuclei of early spermatids elon- space. These changes have been summarized in a series
gate while their chromatin progressively condenses, of eight steps, as shown in Figs. 3.1 and 3.2 [1, 2].
the Golgi-derived acrosome locates at the cephalic pole Steps 1–2: Spermatid nuclei have finely granu-
and spreads flat over the nucleus, the proximal cen- lar chromatin with small nonprominent nucleoli. The
39
Golgi complex, in a juxtanuclear location, has given The Spermatid Nucleus and the Golgi
rise to proacrosomic vesicles that contain dense gran-
ules and fuse into a large acrosomic vesicle that Complex: Nuclear Remodelling,
attaches to the nuclear membrane at the spermatid cra- Chromatin Compaction and Acrosome
nial pole. This vesicle flattens and spreads as a dense
acrosome that will eventually cover between half and
Development
two-thirds of the nuclear surface. At these stages the During spermiogenesis, the organization of germ cell
centrioles migrate first to the cell membrane (where DNA undergoes remarkable modifications. In early
the axoneme grows from the distal centriole toward round spermatids, DNA is associated with nuclear his-
the extracellular space), and later to the caudal nuclear tones, forming a protein complex that organizes into
pole, where the proximal centriole lodges in a shallow globular octamers by the association of a pair of each
concavity (the implantation fossa) defining the bipo- of the four histone components [H2A, H2B, H3 and
lar nature of the spermatozoon. Mitochondria, first H4]. These core particles are encircled by 146 DNA
located at the cell periphery, become randomly dis- base pair filaments, forming nucleosomes, the basic
tributed on the caudal spermatid cytoplasm in step 2. structure of loosely bound DNA–histone complexes
At the neck region, around the proximal centriole, a linearly assembled as beads in a string. This loose asso-
dense striated structure develops that will become the ciation is characteristic of euchromatin, prevalent in
connecting piece, a structure that fastens the tail to the transcriptionally active somatic cells and germ cells
sperm head [2–4]. up to early spermatids, and results in a dispersed
Steps 3–5: The spermatid nucleus progressively microgranular and filamentous nuclear substructure.
elongates and flattens at the area covered by the acro- During spermiogenesis chromatin condensation starts
some while chromatin starts to condense by increas- when histones are removed and replaced by intermedi-
ing the size and density of its granular components. ate proteins and ultimately by protamines, smaller and
Chromatin condensation starts at the tip of the head structurally very different proteins that accommodate
and progresses caudally, accompanied by a significant into minor DNA grooves and establish a strong bond
reduction of nuclear size and further flattening of the that is further stabilized by cross-linking of disulphide
acrosome-covered area. The axoneme elongates and bonds, resulting in very stable, highly compacted DNA
thickens by the addition of periaxonemal structures: [5, 6]. This spatial macromolecular organization ren-
the outer dense fibres and the fibrous sheath. The sper- ders DNA transcriptionally silent, but at the same
matid cytoplasm now occupies a distal position that time shields it and ensures its stability and resiliency
anticipates the formation of the residual cytoplasm to external influences during sperm transit. The pro-
that will eventually disengage from the main sperm cell cess of DNA protamination–condensation is visual-
body at spermiation. ized through spermiogenesis as the evolution of dis-
Steps 6–8: Nuclear elongation/flattening and chro- persed chromatin into a progressively denser granu-
matin condensation proceed to their mature stage. lar structure that leads to the highly compact chro-
Mitochondria assemble around the first part of the matin of mature spermatozoa (Figure 3.3). Still, chro-
axoneme to form the sperm midpiece. The residual matin condensation does not affect all the genome, and
cytoplasm further separates from the main cell body even in mature spermatozoa there are small lighter
but remains attached to it by a slender stalk until granulofilamentous areas of no more than 0.1 to 0.2
mature spermatids are ready to be released into the ␮m in diameter (Figs. 3.2, 3.3 and 3.6). By means
lumen of seminiferous tubules as free spermatozoa. of ultrastructural immunohistochemistry, Haraguchi
Electron micrographs in Figure 3.2 depict various et al. [7] localized polyubiquitinated [pUb] proteins
steps of human spermiogenesis. The following sections throughout sperm nuclei and proteasomes in the
will cover in detail the development and structure of clear areas just described and proposed a mecha-
sperm components, the significance of the annulus and nism through which nuclear proteins are degraded
transient structures such as the manchette, and patho- and removed by the action of proteasomes on pUb
logical modifications giving rise to distinctive sperm substrates (possibly nuclear histones and/or interme-
phenotypes. Structure–function correlations and the diate proteins). Proteins would further degrade and
prognostic significance of sperm pathologies in human be eliminated by migrating and leaving the nucleus
male infertility will also be discussed. through a nuclear pore-rich area (the ‘nuclear pocket’
40
Figure 3.3 The upper four panels depict progressive stages in chromatin condensation during spermiogenesis: A: round spermatid, B:
elongating spermatid, C: spermatid in advanced maturation. Mature spermatozoa (D) have complete chromatin condensation and clear small
uncondensed areas [∗ ]. The two bottom drawings illustrate the relationship between DNA strands and histones (left) or protamines (right).
Note the closer packing of DNA–protamine complexes. Bars in A–D represent 0,5 µm. Previously published by Chemes and Alvarez Sedo
(Asian J. Androl. 2012; 14: 14–23, reproduced with permission from SIM, SJTU and Wolters Kluwer – Medknow).
or redundant nuclear envelope), a clear area under- with ‘a structural framework that includes molecular
neath the pore-rich nuclear membrane situated at the regulatory factors that are required for proper embry-
caudal pole of the nucleus and credited with being ‘the onic development’ [9]. The emerging figure is that of
degradation site for temporarily functioning proteins a very complex DNA organization that may render
generated during condensation of chromatin in late the sperm nucleus particularly vulnerable to structural
spermatids’ [7] (Figure 3.4). The result of chromatin disruptions [see below].
condensation and nuclear remodelling is a sperm
nucleus with most of its DNA tightly packaged by its
association with protamines. In addition to this tran- Chromatin Abnormalities
scriptionally silent DNA, there is also a small frac- Alterations in chromatin condensation are among the
tion [2–15%] that remains associated with histones most frequent pathologies in teratozoospermia, more
and is transcriptionally active. This allows male pronu- frequent in spermatozoa with irregular nuclear pro-
clei to retain the activity of genes necessary for the reg- files (‘amorphous’), but not necessarily associated with
ulation of early steps in zygote development. Sperm specific nuclear shapes. Their distinguishing feature is
DNA is not distributed randomly but organized into the presence of large chromatin rarefactions (1–3 µm),
loop domains associated with cytoskeletal structures, where the uniformly dense chromatin is replaced with
including the nuclear matrix and the ‘annulus’ near the granulofibrillar or ‘empty’ areas sharply demarcated
site of head–tail coupling [8, 9]. Therefore, besides car- from normally condensed chromatin [10, 11]. These
rying the male hemigenome that will be sequentially rarefactions may be single, occupy as much as 20–50%
expressed during embryo growth and organogenesis, of the nucleus and locate near the cranial pole, but
fertilizing spermatozoa provide newly formed zygotes sometimes appear as multiple hypodense areas, larger
41
Figure 3.4 Pathologies of the sperm head: anomalies of chromatin maturation and compaction. A: Irregularly shaped (amorphous) sperm
head with large hypodense chromatin rarefaction (asterisk), surrounded by densely packed chromatin. The hypoplastic acrosome is partially
detached from the nucleus (arrow). B: Immature, granular chromatin (about step 5 of spermiogenesis) with four smaller hypodense areas. The
acrosome is hypoplastic. C: High-magnification detail of a rarefaction, showing fine granulofibrillar substructure surrounded by granular
chromatin. D: Amorphous head with uncondensed chromatin (asterisk) and hypoplastic acrosome (arrow). There are two peripheral
uncondensed nuclear pockets (NP) limited by the nuclear envelope. E: High-magnification detail of an uncondensed area (asterisk) in
continuity with a nuclear pocket (arrow). F: nuclear rarefaction (NR) opening into the subacrosomal space at the tip of the sperm head
([arrow-limited asterisk). Acr: acrosome. Bars in A–E represent 1 µm. F: 0.5 µm. Panels A, C and D were previously published by Chemes and
Alvarez Sedo (Asian J. Androl. 2012; 14: 14–23, reproduced with permission from SIM, SJTU and Wolters Kluwer – Medknow).
than the small ones that may be present in normal originate by apoptosis, but no relationship has been
spermatozoa (Figure 3.4). They have been character- found to sperm DNA fragmentation [13–15].
ized by light microscopy as ‘nuclear vacuoles’, which The ubiquitin–proteasome system is involved
is incorrect, because they lack a limiting membrane. in the process of protein removal and replacement
Their likely origin is an abnormal spermiogenesis, as during the histone–protamine transition. Polyu-
confirmed by their presence in immature spermatids biquitinated proteins distributed throughout the
found in testicular biopsies or precociously desqua- nucleus bind and are incorporated into proteasomes,
mated in semen [12]. It has been suggested that they barrel-shaped multisubunit proteolytic complexes that
42
remove redundant or damaged proteins. According ratios. Some associations with abnormal sperm head
to Haraguchi et al. [7], proteasomes are located in morphology have been reported, but protamine alter-
multiple small areas of uncondensed chromatin in ations or polymorphisms–mutations in the protamine
the heads of normal spermatozoa, where proteolysis genes are rarely found in populations of infertile males.
begins. Chemes and Alvarez Sedo [16] have proposed Furthermore, the possible relation between protamine
that the larger rarefactions of abnormal chromatin alterations and the nuclear lacunae here described has
condensation here described are their pathological not been documented [23–33].
counterparts, probably resulting from increased
proteolysis of histones and transition proteins by
an overactive or dysregulated ubiquitin proteasome Pathologies of the Acrosome
system. The frequent connections between chromatin The sperm acrosome develops by the interaction
hypodense areas and unusually large pore-rich nuclear between early round spermatid nuclei and the adja-
pockets (Figure 3.4) tends to confirm this suggestion, cent Golgi complex. An assortment of Golgi-derived
since nuclear pockets are proteolytic centres and vesicles containing hydrolytic enzymes migrates and
the exit site from which protein residues leave the incorporates into the main acrosomic vesicle and con-
nucleus. Furthermore, the close association between tributes to form the dense acrosomal granule. The
interphase chromosomes, the nuclear matrix and acrosomic vesicle and granule spread on the ante-
peripheral nuclear lamina may render chromatin rior nuclear aspect to cover 50–75% of its surface
particularly susceptible to disruptions of the nuclear (Figures 3.1 and 3.2). Alterations in this process result
cytoskeleton such as those derived from the presence in a lack of or insufficient acrosomal development,
of large nuclear rarefactions (‘vacuoles’) in abnormal two head anomalies compromising fertility. Spermato-
spermatozoa of infertile men. zoa lacking acrosomes usually display spherical nuclei
The presence of chromatin abnormalities of the due to the absence or insufficiency of the Sertoli
kind described here represents a serious challenge cell F-actin hoops that embrace spermatid nuclei and
for male fertility [10, 12, 17]. They are the ultra- of the acrosome–acroplaxome–manchette complex,
structural counterpart of amorphous spermatozoa cytoskeletal structures that model sperm head shape
highly increased in severe teratozoospermia (values by flattening the acrosome-covered area [34]. Even
below the 4% limit). Low fertilization, abnormal or though the lack of acrosome is usually accompanied by
delayed embryonic development, diminished implan- spherical head shape (‘globozoospermia’), oval, irreg-
tation and high abortion rates have been reported ular or amorphous sperm heads can also occur in
when abnormal ‘vacuolated’ spermatozoa are used this condition [16]. This pathology was one of the
in microinjection techniques [ICSI, 18–20]. These first to be recognized when electron microscopy was
authors and others have communicated significant applied to characterize abnormal sperm phenotypes
improvements with selection techniques using high- [10, 11, 35–39]. Earlier reports had attributed this con-
resolution light microscopy in the embryology labora- dition to lack of acrosome formation, but this erro-
tory, but their results have not been confirmed by oth- neous interpretation was modified when numerous
ers [reviewed in 16]. studies demonstrated that it derives from an abnor-
Chromatin rarefactions are possibly the result of mal development of Golgi proacrosomic vesicles that
diverse testicular pathologies that result in abnor- fail to attach and/or spread over the spermatid nucleus
mal spermiogenesis. Numerous genetic studies have [reviewed in 40]. As a result, detached acrosomes are
tried with little success to find a genetic cause for seen either unattached to sperm nuclei or absent from
these defects. Haplo insufficiency of protamine-1 or mature spermatozoa because they have been elimi-
-2 genes causes abnormal chromatin packaging and nated with residual bodies before sperm release. We
male infertility in mice, and targeted disruption of now believe this to be the most common mecha-
Camk4 and transition protein 1 genes or transgenic nism to explain the origin of acrosomeless spermato-
models expressing heterologous avian protamines in zoa, and not acrosomal ‘aplasia’ or ‘agenesis’ (absent
mice result in diminished fertility potential [21, 22]. In formation), as we and others had previously pro-
view of these results, men seeking infertility treatment posed [11, 19, 41, 42]. Ultrastructural studies in globo-
have been investigated for mutations in protamines 1 zoospermia have revealed a mixture of acrosome-
and 2 genes, P1 polymorphisms and abnormal P1/P2 less sperm nuclei, abnormal multivesicular structures
43
*
A B
C D
Figure 3.5 Pathologies of the sperm head: acrosome anomalies. Panels A and B show round head-
acrosomeless spermatozoa (globozoospermia) with either normal chromatin maturation (A) or partial granular immaturity (B). Acrosome
hypoplasia is associated with spheroidal (C) or round (D) head shapes. Panels A–C show local detachment of the nuclear envelope (asterisks).
In D there are lacunar defects of the chromatin and immaturity. All bars represent 1 µm. Panel C was previously published by Chemes and
Alvarez Sedo (Asian J. Androl. 2012; 14:14–23, reproduced with permission from SIM, SJTU and Wolters Kluwer – Medknow).
replacing the acrosome or dense well-developed acro- acrosomes with diminished content, which are easily
somes in the residual cytoplasm, detached and away overlooked in patients with unexplained fertilization
from the nucleus (Figure 3.5). failure (Figure 3.5). The real prevalence of acrosomal
In addition to acrosomeless spermatozoa, ultra- hypoplasia is difficult to determine because it remains
structural studies carried out in patients with usually unrecognized. It should be actively searched
increased numbers of abnormal ‘amorphous’ sper- for by immunofluorescence or transmission electron
matozoa have revealed hypoplastic small acrosomes microscopy in cases of ‘unexplained’ fertilization
with more frequency than usually thought. Acrosomal failures. In the frequent event that acrosomal patholo-
hypoplasia is characterized by small and detached gies associate with chromatin abnormalities, the
44
sperm fertilizing capacity is further compromised (see uclear theca along the inner acrosomal membrane.
below). Interestingly, defective chromatin compaction during
Patients suffering from acrosome anomalies are spermiogenesis was also reported in Dpy19l2 KO mice
infertile because spermatozoa are unable to penetrate due to abnormal kinetics in the interchange of his-
the oocyte. The introduction of ICSI had been antici- tones, transition proteins and protamines. This leads
pated to offer a solution for these men, but this was not to sperm DNA damage and defective early embryo
always the case. Subsequent research pointed to insuf- development [56, 57] and provides an explanation for
ficient activity of perinuclear theca proteins, which the association of acrosome and chromatin anomalies
were found to be involved in triggering Ca2+ oscil- reported in previous studies.
lations responsible for oocyte activation [43–45]. In In summary, absence and deficient development
vitro stimulation by electric pulses and oocyte incuba- of the acrosome are two pathologies that significantly
tion with Ca2+ ionophores or strontium has been tried affect sperm structure and fertilizing potential. Dur-
with variable success [46, 47, reviewed in 16]. These ing the last decade, numerous studies have delineated
conflicting results suggest a more intricate regulation phenotypes and diagnostic characteristics and stressed
of oocyte activation and fertilization than previously the importance of the acrosome, perinuclear theca and
anticipated and highlight the complexity of alterations postacrosomal area in the molecular mechanisms reg-
in the basic mechanisms of sperm functioning. ulating sperm penetration, oocyte activation and early
In recent years, the study of the spermatid embryonic development. In particular, the association
cytoskeleton has uncovered the presence of a fam- of acrosomal and chromatin pathologies has revealed
ily of protein components of the perinuclear theca, the close interaction between acrosome development
a structure that is believed to fasten the develop- and molecular mechanisms of chromatin compaction
ing acrosome to the nuclear membrane [48, 49]. in achieving proper sperm functioning.
Proteins of the perinuclear theca act as a trigger
of Ca2+ fluxes that activate the oocyte, allowing
the initiation of embryonic development. Proteomic, The Sperm Neck and the Connecting
ultrastructural and immunocytochemical studies have
identified significant diminutions in six protein sub-
Piece: Development of the Head–Tail
units of the perinuclear theca in spermatozoa and Coupling Apparatus
immature germ cells from five men with globo- The remarkable architectural complexity of mature
zoospermia and localized these proteins to the sub- spermatozoa is the result of an evolutionary process
acrosomal region [40, 50]. Insufficient oocyte activa- that generates a properly condensed male genome
tion is now generally attributed to deficiencies in phos- that will be carried by the sperm tail and be pro-
pholipase C ␨ (PLC␨ ), widely considered to be the vided with an oocyte recognition and penetration sys-
sperm oocyte activation factor through the stimula- tem (the acrosome and sperm head plasma mem-
tion of Ca2+ oscillations in the oocyte. Other reports brane) that allow specific binding and entry into the
have suggested that a sperm postacrosomal protein oocyte.
may be the key factor in the stimulation of pulsatile cal- The spermatozoon is a very polarized cell, with
cium release. A PLC␨ mutant with reduced binding to an apical pole marked by the position of the acro-
its target has been shown to abolish Ca2+ oscillations some and a caudal pole determined by the connect-
within the oocyte [51, 52]. ing piece (CP), which ensures head–tail attachment
Family incidence has been reported in cases of [2, 58, 59]. The CP organizes around the pair of sper-
acrosomal hypoplasia or globozoospermia [38, 53], matid centrioles as they move towards the nucleus
and a homozygous deletion in chromosome 12 has and attach to the shallow concavity of the implan-
been found in 4 of 5 brothers and 15 out of 20 unre- tation fossa (Figure 3.1). The proximal centriole is a
lated globozoospermic men [54, 55]. Moreover, recent cylinder perpendicular to the sperm axis with its wall
publications have shown that the DPY19L2 gene is formed by nine longitudinal triplet microtubules in
the main genetic cause of human and mouse globo- a circular arrangement (Figs. 3.6 and 3.7). The dis-
zoospermia. In DPY19L2-associated globozoosper- tal centriole, parallel to the sperm axis, is also formed
mia, PLC␨ , the inducer of Ca2+ oscillations, is absent by nine circularly arranged triplet microtubules. In
or highly reduced. PLC␨ was localized in the perin- most mammals, including humans, the distal centriole
45
Figure 3.6 Neck region of a normal human

spermatozoon. The caudal part of the sperm head
shows a shallow concavity, the implantation fossa (IF),
where the nuclear envelope is locally reinforced (basal
plate BP). The sperm connecting piece articulates with
the implantation fossa through a dense plate, the
capitulum (arrow, C). The cross section of the sperm
proximal centriole [asterisk] depicts nine
circumferentially arranged microtubular triplets. The
distal centriole has disintegrated (double asterisk).
Centrioles are enclosed by nine segmented columns
(SC) that are in continuity with the corresponding
outer dense fibres (ODF), surrounded by midpiece
mitochondria (Mi). Note the beginning of axonemal
microtubules (Mt) originally derived from the distal
centriole. The bar represents 0.25 µm. Originally
published in Chemes et al. (Hum Reprod 1999; 14:
1811–8 reproduced with permission from Oxford
university Press) and with modifications in Chemes
(The Centrosome, H. Schatten ed., chapter 2,
reproduced with permission from Humana Press).
disintegrates after giving rise to the tail axoneme. Dur- sperm aster that bring about pronuclear approach and
ing the organization of the CP, dense protein compo- syngamy. Disruptions of this elaborate organization
nents gather around the centriole–axoneme complex, are the basis of specific sperm pathologies character-
assembling a longitudinal conico-cylindrical sleeve ized by increased fragility of the head–tail junction and
formed by nine striated columns and cranially closed acephalic spermatozoa.
by the capitulum. This is a concave disk that anchors
the connecting piece to the caudal pole of the sperm
head at the implantation fossa (Figs. 3.6 and 3.7). All
Abnormalities of the Head–Tail Junction
these structures form a sort of black box that encases and Acephalic Spermatozoa
the sperm centriole until the critical moment, after Spermatozoa displaying normal tails with very small
sperm penetration into the oocyte, when the whole thickenings at their cephalic end had been known for
structure disassembles to release the proximal cen- some time. They were initially believed to carry very
triole that becomes the zygote centrosome and the small heads and referred to as pinheads [60]. It was
46
PC
DC
DC
Ax
Ax
A B
Cap
C D
Figure 3.7 Schematic three-dimensional reconstruction of a human sperm connecting piece. For clearer visualization, extra space is allowed
between the proximal centriole (PC) and the nine segmented columns. The series of four panels facilitates understanding the structural
complexity of the connecting piece by building up successive partial views of its constitutive parts. (A) Four longitudinal segmented columns
(marked by an asterisk in each of the top segments) form a hemicylindrical concavity that lodges (as shown in panel B) the proximal centriole
(PC), the remains of the degenerating distal centriole (DC) and the beginning of the tail axoneme (Ax). In panel (C) five additional longitudinal
segmented columns (each marked by an asterisk) complete the conico–cylindrical sleeve that encloses the centrioles–axoneme complex.
The proximal centriole (PC), perpendicular to the sperm axis, exits the connecting piece through the wall formed by the segmented columns
(panels C and D). In panel (D) the capitulum (Cap), a concave disk that articulates with the sperm nucleus, closes the cranial end of the
connecting piece.
later recognized that they were headless spermato- istics of these loose tails were carefully described, not-
zoa; hence the name decapitated or acephalic, which ing that normal connecting pieces and midpieces were
led to the description of a human syndrome charac- present at the tail cranial end, sometimes accompanied
terized by the presence of abundant, predominantly by small globular drops of residual cytoplasm. It was
headless spermatozoa, in men suffering from infertil- considered that the most common plane of head–tail
ity [61–65]. Light and electron microscopic character- separation was between heads and proximal centrioles,
47
although cleavages between both centrioles or even at the last few years, various animal models of acephalic
the midpiece level were also reported [66–68]. These spermatozoa have been reported, including mutations
early publications concentrated on headless tails, in the Hook1 gene [72] or in the Centrobin gene
while little attention was paid to some patients with that codes for two sperm-centriole-related proteins
abundant spermatozoa displaying centriolar implan- [59]. Targeted deletion of Odf1 with abnormal widen-
tations away from the sperm axis, with midpieces at ing of the head-to-tail linkage was also related to
varying angles with respect to heads. The presence of increased fragility of the head–tail junction [73]. In
these two forms indicated that headless spermatozoa addition, inactivation of Spata6 disrupts development
and misalignments of heads and tails expressed differ- of the connecting piece and causes acephalic forms
ent severities of the same condition, produced by an [74]. Even though these models had similarities to the
abnormal migration of the centriole–tail complex that human phenotype, there were important differences,
either fails to join the spermatid nucleus or attaches to and to this day no gene mutations or deletions have
it in a lateral position. Therefore, the more inclusive been reported in men with acephalic spermatozoa and
denomination of alterations of the head-neck attach- defects of the head–neck attachment.
ment was proposed [68–70]. Because of these abnor- Ultrastructural studies of ejaculated spermatozoa
malities, there is increased fragility of the head–tail and testicular biopsies showed that acephalic sperma-
connection, as shown by additional in vitro decapita- tozoa display, in the majority of cases, normal tails and
tion of these spermatozoa when subjected to mechan- midpieces with the plasma membrane directly cover-
ical stress [68, 71]. Family clustering was reported in ing the connecting piece and capitulum (Figure 3.8).
various publications, suggesting that this syndrome In some acephalic spermatozoa, a nuclear-envelope-
was a consequence of abnormal spermiogenic pro- derived vesicle is seen directly underneath the
gramming of possibly genetic origin [66, 68, 70]. In plasma membrane (Figure 3.8). The same vesicle is
Figure 3.8 (A) Acephalic spermatozoon. The plasma membrane covers a small connecting piece. The midpiece is well formed. Cranial to the
proximal centriole there is a nuclear-envelope-derived vesicular structure (asterisk). The inset shows a loose head (asterisk, upper left corner)
and two acephalic spermatozoa (the one to the right with two tails). (B) Abnormal head–midpiece connection. Head and tail are misaligned
and form a 90º angle. In the inset there is one spermatozoon with lateral insertion of the head in the midpiece. Bars in A and B represent 1 µm.
Previously published in Chemes and Rawe (Cell Tissue Res 2010; 341: 349–57, reproduced with permission from Springer).
48
sometimes present in maturing spermatids separating The Midpiece and Sperm Flagellum:
the connecting piece from the nucleus, indicating
that, in these cases, the plane of separation involved Structure and Development of the
the caudal end of the nuclear envelope. The mor- Sperm Midpiece and Tail
phogenesis of the sperm defect can be followed in
testicular biopsies where the abnormal migration Axoneme and Periaxonemal
of the centriolar pair, its abaxial attachment to the
nucleus and the orientation of sperm flagella at various
Structures: Outer Dense Fibres and
angles with respect to the sperm axis can be clearly the Fibrous Sheath
identified [68]. In most cases, head–tail separation The sperm tail begins to form very early in spermio-
occurs at spermiation, and the heads are phagocytized genesis, as the axoneme grows from the distal centri-
by Sertoli cells, which explain their absence in semen. ole by recruitment of tubulin heterodimers organized
Just before spermiation, midpieces and tails are mostly over the template of the nine triplet centriolar micro-
in the lumina of seminiferous tubules, while heads are tubules. In some species, this happens when the distal
still embedded in the apical cytoplasm of Sertoli cells. centriole has migrated and anchored to the cell mem-
This is the reason that only heads are phagocytized brane opposite the acrosome [58]. In humans, Hol-
after spermiation of acephalic spermatozoa, while stein [1] has documented that the tail axoneme starts
tails are released free. development before centriolar migration (Figure 3.1,
Men suffering from alterations of the head–neck step 1). In both cases, when the distal centriole is
attachment are infertile. After the advent of ICSI anchored to the cell membrane, the axoneme extends
(intracytoplasmic sperm injection into the oocyte), toward the extracellular space, while dense mate-
numerous failed attempts were reported. In one par- rial collects around the centrioles to form the first
ticular patient, after various ICSI failures, it was anlage of the connecting piece (see the previous sec-
noted that there was oocyte activation and pronu- tion). At this stage the centrioles approach the sper-
clear development, but syngamy never took place matid nucleus, invaginating with them the cell mem-
and all zygotes degenerated [68]. This outcome was brane attached to the distal centriole, which ensures
confirmed by later reports [75–77] and suggested that the sperm axoneme remains in the extracellular
deficient aster formation that prevented pronuclear space (Figure 3.1, steps 2 and 3). The structure of the
approach and fusion. Since Wojcik et al. [78] had pro- axoneme consists of nine microtubular doublets cir-
posed that the release of the sperm centriole after cularly arranged around two centrally located micro-
fertilization probably involved the action of protea- tubules, defining the classical 9+2 pattern (Figure 3.9).
somes in the neck region of human spermatozoa, we The buildup of tubulin heterodimers is achieved by
hypothesized that the ICSI failures were due to pro- an intraflagellar transport system that carries axone-
teasome proteolytic deficiency and failure to liber- mal constituents and delivers them to the distal assem-
ate the proximal centriole from the dense proteina- bly site. Each axonemal doublet consists of two closely
ceous black box of the connecting piece. This was associated microtubular subunits, A and B. Subunit
confirmed by findings of decreased proteasome enzy- A is a complete microtubule formed by 13 protofila-
matic activity in acephalic human spermatozoa and ments, while subunit B is incomplete and contains 10
failure of these spermatozoa to develop sperm asters microfilaments. Two dynein arms (outer and inner)
when injected into bovine oocytes [76, 79]. Further- are anchored to the A subunit and reach out to the
more, immunologic or pharmacologic blockage of B subunit of the neighbouring doublet (Figure 3.9).
proteasome activity of normal spermatozoa resulted Dynein arms are motor proteins with ATPase activity,
in failed sperm aster formation when injected into responsible for the generation of the sliding force that
bovine oocytes [76]. Since then there have been var- gives rise to axonemal bending and flagellar motion.
ious reports of successful ICSI pregnancies [70, 80, Nexin links bridge the gap between peripheral dou-
81]. This indicates that in this syndrome there is blets, and radial spokes run between each of the nine
a wide pathological spectrum in different sperma- doublets and the central pair (Figure 3.9). Finally,
tozoa, including some with preserved centrosomal the two central microtubules are joined by a bridge
function. and surrounded by the inner sheath. Two additional
49
A B C
F G H
Figure 3.9 A: Transverse section of a normal flagellum at the distal principal piece. The axoneme is composed of 9 doublet microtubules
with two dynein arms each (arrow). Nine radial spokes project toward the central pair of singlet microtubules. The fibrous sheath is composed
of two lateral columns (asterisks) and two semicircumferential ribs (double arrow head). B and C correspond to transverse sections of flagella
with nonspecific anomalies. In B the central pair is out of centre (asterisk) and there are three translocated doublets (arrows). In C the axoneme
is ‘fractured’ and laterally displaced at the midpiece. D: Scanning electron micrograph of a DFS sperm (dysplasia of the fibrous sheath). Note
two thick, irregular and very short tails (length 10 µm, normal 50–60 µm). E: A longitudinal section of a DFS sperm showing absence of the
mitochondrial midpiece and very thick tail with supernumerary ribs of the fibrous sheath. F–H: Three transverse sections of DFS spermatozoa.
The FS forms thick disordered periaxonemal rings and the lateral columns are misplaced in F and G. The axoneme is preserved in F (9+2
structure), but in G there is a lack of the central pair and one doublet microtubule is missing [(8+0 configuration). In H the fibrous sheath is
disorganized and the axoneme completely distorted. One remaining doublet microtubule lacks dynein arms (arrow) Scale bars: 0.1 µm in A–C
and 1µm in D–E. Diameters of pathological flagella in F–H range from 1 to 1.2 µm (normal flagellar diameter 0.4 µm). Different panels in this
figure were originally published in Chemes et al. (Hum Reprod 1998; 13:2521–2526, reproduced with permission from Oxford University
Press), Chemes & Rawe (Human Reproduction Update, 2003; 9:405–428, reproduced with permission from Oxford University Press) or Linck
et al, J Assist Reprod Genet 2016 (DOI 10.1007/s10815–016–0652–1 reproduced with permission from Springer).
cytoskeletal structures organize around the axoneme, when microinjection techniques such as ICSI are
the nine outer dense fibres (ODFs) and the fibrous used [87].
sheath (FS). The ODF builds up in a proximal-to-distal
direction. At the level of the midpiece each ODF fibre
is closely associated to the corresponding axonemal Abnormalities of the Sperm Flagellum
doublet, but ODFs 3 and 8 do not continue to the Ultrastructural studies of the sperm tail in patients
principal piece. The FS assembles in a distal to proxi- with decreased motility have revealed high preva-
mal direction around the axoneme-ODF complex [82]. lence of alterations in the axoneme and periaxone-
At the distal end of the connecting piece, the annu- mal structures. The 9+2 microtubular organization of
lus forms a ring around the axoneme that migrates the axoneme and the correct arrangement of ODFs
distally, allowing spermatid mitochondria to gather and FS are essential for normal sperm motility. Two
around the axoneme to form the sperm midpiece. This main types of tail pathologies with different pheno-
is followed by the sperm tail principal piece, where typic characteristics and consequences for male fertil-
the axoneme/ODF complex is encircled by the ribs of ity have been reported in studies of large populations
the FS that run transversally between two longitudi- of infertile males with asthenozoospermia: the more
nal columns of the FS that occupy the place of ODF 3 common nonsystematic flagellar anomalies (NSFA)
and 8 (Figure 3.9). At the end of the tail (endpiece), the and those rather uncommon genetic disorders com-
FS vanishes and the disassembling axoneme is directly bining primary ciliary dyskinesia with periaxonemal
covered by the plasma membrane. anomalies of the dysplasia of the fibrous sheath [11,
90, 91], also referred to as pathologies of the sperm
cytoskeleton [92] or multiple morphological flagellar
Mitochondrial Structural Anomalies of the anomalies [MMAF, 93, 94].
The relative frequency of these alterations was
Sperm Midpiece reported in a series of 247 severely asthenozoospermic
Midpiece structural defects are exceedingly rare and patients with low motility values (3–4% rapid forward
usually associated with lower fertility potential caused progression) [95]. The most frequent finding (205
by asthenozoospermia (diminished or absent sperm patients, 83% of the population) was NSFA, alterations
motility). Probably owing to their infrequent occur- of unknown origin or secondary to different condi-
rence, they have received little attention in the lit- tions affecting fertility, such as varicocele, infections of
erature [83–87]. Different phenotypes have been the seminal pathway, immunologic factors or oxida-
reported, including absence or insufficient develop- tive damage. NSFA are characterized by random het-
ment with diminished mitochondrial numbers and erogeneous alterations that affect variable numbers of
tendency to acute bends at the midpiece level. There spermatozoa. When studied with the electron micro-
are also reports of abnormally long extension with the scope there are modifications in the number, topog-
number of mitochondrial gyres increased up to 30 (for raphy and organization of axonemal microtubules
a normal of 11–13). Shorter or abnormally long mid- resulting in disruption of the normal 9+2 axonemal
pieces result in mechanical weakness or stiffness, pro- architecture (Figure 3.9). These anomalies were only
ducing motility disorders, because they interfere with discernible at the ultrastructural level because micro-
transmission of the axonemal bending wave. Severe tubular alterations do not result in modifications in
asthenozoospermia may also result from abnormal flagellar diameter, with tails keeping their normal long,
oxidative phosphorylation and ATP generation such as thin and wavy appearance. The configuration of the
those described for mutation/deletions in mitochon- middle piece was usually normal. Longitudinal fol-
drial genes [88, 89]. A particular type of midpiece lowup revealed that NSFA patients who experienced
absence is observed in dysplasia of the fibrous sheath improved motility as a result of etiologic or empiri-
[90, 91], a genetic condition where the midpiece does cal treatments could expect reasonable fertility success
not assemble due to lack of distal migration of the utilizing conventional methods or IVF [91]. In severe
annulus. This unique phenotype will be dealt with in cases or when there is no response to treatment, ICSI
the next section. In spite of severe disruption in sperm has a good prognosis and does not pose additional
motility, fertility prognosis in patients carrying mito- risks in view of the lack of genetic component in this
chondrial sperm anomalies may be partially preserved pathology.
51
The rest of the population (42 patients, 17% of the of PCD is due to various autosomic recessive muta-
total) showed a typical sperm phenotype compromis- tions with extensive locus heterogeneity. Involved pro-
ing flagellar cytoskeletal components, with associated teins include heavy chain dyneins, microtubule bind-
respiratory pathology and familial incidence. Sperma- ing proteins, components of the central pair micro-
tozoa either were immotile or displayed up to 0.2–0.5% tubules, etc. [100–105]. Typical DFS phenotypes with
progressive motility and had short, thick and irregular FS disarrays have been found combined with lack of
tails referred to as ‘stump tails’ or ‘short tails’. However, dynein arms in respiratory cilia. This association was
this is a misnomer that either fails to provide insight first reported in two unrelated patients, suggesting
into the underlying nature of these tail abnormali- a close etio-pathogenic relationship between the two
ties or encompasses a heterogeneous array of sperm conditions [106]. Extensive family clustering and the
defects having a short tail as their common feature. fact that the DFS phenotype is present in most sper-
Chemes et al. [90, 91] introduced the more com- matozoa, is very stable in time and does not respond
prehensive denomination of dysplasia of the fibrous to any therapies have suggested a genetic origin, which
sheath [DFS], which identifies the main defect as a has more recently been confirmed by reports of vari-
developmental anomaly. The ultrastructural study is ous mutations in genes coding fibrous-sheath-related
necessary to distinguish DFS from nonspecific flagellar proteins such as AKAP4 and thioredoxins [reviewed
deterioration and fragmentation secondary to sperm in 11, 93, 94, 107–109]. The close relationship between
death or aging in cases of seminal pathway obstruc- abnormalities affecting axonemes, ODFs, the FS and
tion. Modifications of the fibrous sheath are present in the midpiece has led to the understanding that there
most affected spermatozoa and appear as superfluous is a wide range of phenotypic manifestations of PCD
and hypertrophic fibrous sheath constituents that form and DFS features. In fact, these observations have been
thick rings around the axoneme or distort the topog- acknowledged in papers dealing with sperm cytoskele-
raphy of axonemal microtubules (Figure 3.9). Lack of tal pathologies [92], in reports of combinations of cil-
central pairs (9 + 0 pattern) and missing dynein arms iary and flagellar phenotypes [106, 110], in the descrip-
in microtubular doublets are frequently observed in tion of complete and incomplete forms of DFS [11,
many, but not all cases. There is also an abnormal 91] and in recent publications on the existence of
extension of outer dense fibres 3 and 8, which con- MMAF2 [93, 94]. It appears that DFS constitutes a
tinue beyond the midpiece into the principal piece group of interrelated cytoskeletal pathologies affecting
(Figure 3.9). Moreover, as a consequence of its failed respiratory cilia and/or sperm flagella. A unification
caudal migration, the annulus remains close to the of current denominations that may synthesize clinical,
connecting piece and prevents normal mitochondrial ultrastructural and genetic findings would be highly
assembly and midpiece formation (Figure 3.9) [11, 92]. welcome.
Studies on testicular biopsies have demonstrated that There have not been reports of natural concep-
these abnormalities are a consequence of abnormal tions in DFS/MMAF and, due to the sperm immotility,
spermiogenic programming [91, 96]. classical IVF methods fail in achieving fertilizations
Association of DFS features with chronic respira- and pregnancies. Results of ICSI have been encourag-
tory disease has strongly suggested similarities with ing. Fertilization, embryo development and pregnancy
early pioneering findings of Afzelius et al. and Peder- may proceed normally, which has been attributed to
sen and Rebbe reporting that the lack of dynein arms in preservation of normal nuclear and chromatin features
the axonemes of spermatozoa and bronchial cilia was [reviewed in 11]. Since DFS and PCD have genetic
responsible for immotility of spermatozoa and respi- components, there is a potential risk of transmission
ratory cilia [97, 98]. These studies provided the bio- to the male offspring. In the limited experience so far,
logical basis for the understanding of the ‘immotile available male and female newborns have been healthy,
cilia syndrome’, a denomination later changed to ‘pri- but information on male fertility will be available only
mary ciliary dyskinesia’ (PCD) to acknowledge the when these babies become adults. It is important to
fact that partial motility was sometimes present [99]. make patients aware of the risks involved and pro-
Other axonemal anomalies were later described in ceed to ICSI only with informed consent. Apart from
PCD patients, including total or partial microtubu- the possible genetic risk, flagellar pathologies seem to
lar translocations and absence of the axoneme or the have the best prognosis with ICSI. Alterations in the
central pair [reviewed in [11]]. Family transmission chromatin, acrosome and neck region have variable
52
outcomes with lower success rates, which highlights rather common, but usually underdiagnosed, acro-
the characteristic role played by individual sperm some hypoplasia. Also, the presence and prognos-
components in the processes of fertilization, early tic significance of ‘nuclear vacuoles’ has elicited fre-
embryonic development and implantation [16]. quent debate, but their relationship to abnormalities
in the process of chromatin maturation and com-
paction is not generally recognized. The identifica-
Revisiting Teratozoospermia tion of acrosome and chromatin anomalies has impor-
In the preceding sections the ultrastructural character- tant prognostic and therapeutic implications in ART
istics of abnormal sperm phenotypes were described, because insufficient acrosomal function and oocyte
with special attention paid to the mechanisms of activation can be treated with methods that restore
abnormal spermiogenesis that generate such patholo- Ca2+ fluxes (see Pathologies of the Acrosome), and
gies. These studies have provided a deeper under- in cases of serious chromatin abnormalities (nuclear
standing of abnormal sperm function, but the rela- ‘vacuoles’), high-resolution light microscopy can be
tionship between these phenotypes and the light used to select unaffected spermatozoa. In both cases,
microscopic appearance of abnormal spermatozoa in EM studies have clarified the nature of subcellular
teratozoospermia is not always clear. The very con- anomalies beyond their (possible) recognition under
cept of teratozoospermia is something that needs to light microscopy. EM may not be necessary as a rou-
be revisited, because it is based on the identification tine diagnostic procedure, but the knowledge gained
of atypical sperm shapes but does not necessarily rec- through its use frequently explains the reasons for fer-
ognize the cellular basis of their functional incompe- tility failures and allows a rational election of the best
tence. road to take to help couples facing fertility problems.
Morphology correlates with sperm fertilizing Moreover, in severe teratozoospermia with repeated
capacity and has prognostic value in assisted repro- fertilization or implantation failures or early preg-
duction. But what hides behind a head-shape change nancy loss, the identification of sperm phenotypes
in amorphous or tapering spermatozoa? In other of genetic origin highlights the risk of transmitting
words, what is it that impairs sperm function in genetic conditions. An adequate diagnosis is required
morphologically abnormal sperm? Is it just abnormal not only for a rational therapeutic choice but also to
shape or is there something wrong with specific sperm help infertile couples make informed decisions.
components? Sperm pathology is the discipline of
characterizing structural and functional deficiencies in
abnormal spermatozoa. This is significant because it Acknowledgements
helps to explain the mechanisms of sperm inefficiency,
In the Introduction and Revisiting Teratozoospermia
identifies genetic phenotypes, suggests strategies to
sections and throughout the text there are quotations
improve fertilization and opens the door to molecular
from previous publications of the author: Chemes and
genetic studies that will probably lead to the design of
Rawe (Human Reproduction Update, 2003; 9: 405–28,
the therapeutic tools of the future.
reproduced with permission from Oxford University
The advent of ICSI has revolutionized the field of
Press), Chemes (Spermatogenesis, Methods and Pro-
assisted reproductive technologies and removed for-
tocols, D. Carrell and K. Ashton eds. 2013, chapter
merly challenging barriers to the treatment of patients
29, reproduced with permission from Springer) and
with severe male factor infertility. Before the intro-
Chemes and Alvarez Sedo (Asian J. Androl 2012; 14:
duction of ICSI, sperm immotility and tail abnormal-
14–23, reproduced with permission from SIM, SJTU
ities seriously compromised the ability of sperm to
and Wolters Kluwer – Medknow).
reach and penetrate the oocyte. Microinjection tech-
niques have demonstrated that, after penetration into
the oocyte, the tail does not play a significant role
in fertilization. Conversely, head and neck anomalies
are sperm characteristics that seriously challenge nor-
Notes
mal fertilization and early embryonic development. 1. Pathological increase in abnormal, immotile or dead sperma-
tozoa.
Amorphous heads, one of the most common find-
ings in routine semen analysis, may be associated with 2. Multiple morphological abnormalities of the sperm flagellum.
53
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58
Chapter
Sperm RNA and Its Use as a Clinical Marker
4 Meritxell Jodar, Ester Anton and Stephen A. Krawetz
Introduction ning as a primordial germ cell differentiates into a sper-

matogonium. The proliferation and differentiation of
Infertility affects 12–30% of childbearing-age couples
spermatogonia into spermatocytes, the progression of
[1], and it is expected that its prevalence will keep
spermatocytes through meiosis to form spermatids,
rising [2]. This likely reflects the influence of several
and final differentiation yield spermatozoa. Dramatic
lifestyle factors in developed countries, such as pro-
changes in the shape of the round spermatid and
gressive delay in maternal age, stress, obesity, expo-
nucleus, as well as the formation of new organelles,
sure to environmental hazards and the use of drugs.
typify the mature sperm. This is exemplified by the
It is suggested that upon presentation to an infertility
development of the flagellum and the acrosome, while
clinic half of these infertile couples involve a geneti-
most cytoplasm is removed during the final steps of
cally associated male factor. Nevertheless, a significant
spermatogenesis [8–10]. These last steps are also char-
proportion of male infertility (around 25%) remains
acterized by a general shutdown of transcription and
diagnosed as idiopathic [3].
translation, as the genome is compacted by the pro-
The current standard of practice in assessing male
gressive, albeit incomplete replacement of the majority
fertility in the andrology laboratory is usually through
of histones by protamines. Despite the resulting tran-
semen analysis. This includes a microscopic analy-
scriptional blockage and the loss of a large proportion
sis in which sperm concentration, motility and mor-
of RNAs during the final stages of spermatogenesis, a
phology are evaluated [4]. Although this test is widely
small but complex population of RNAs is preserved in
used in clinics to ascertain male reproductive poten-
mature sperm. Several studies have suggested that the
tial, it has been suggested that additional clinical mark-
retention of these transcripts may not be random, but
ers are needed for more accurate assessments [5, 6].
rather reflects residues of spermatogenic processes or
In this context, several parameters have been pro-
the selection of molecules that will display their func-
posed as suitable fertility biomarker candidates, some
tion in early embryogenesis [11–15].
of which have already been incorporated in clinics (e.g.
A single spermatozoon contains 50–90 fg of
sperm FISH studies, sperm DNA integrity) [7]. The
long RNA (⬎200 nt), including coding and noncod-
recent development of new molecular methodologies
ing RNAs [16]. This is about 200 times less than a
has been driven by genomics, transcriptomics, pro-
somatic cell [14]. An extensive and detailed catalogue
teomics and metabolomics. In this chapter, we present
of sperm RNAs has been generated with the recent
a synopsis of the current state of the art of spermato-
application of Next Generation Sequencing (NGS) as
zoal RNAs, with a glimpse to the future.
summarized in Figure 4.1, revealing the singularity of
RNAs retained in the sperm [14, 17]. The most abun-
Spermatozoal RNAs dant transcripts in sperm correspond to ribosomal
Spermatogenesis is a complex biological process that RNAs (rRNAs), but unlike those in somatic cells, these
requires a highly regulated genetic program to orches- rRNAs in sperm are selectively cleaved, most likely to
trate a successful differentiation program. This pro- ensure the translational quiescent state of spermatozoa
cess occurs over at least 64 days in humans, begin- [18]. A substantial portion of sperm coding RNAs also
University Press.
59
Chapter 4: Sperm RNA and Its Use as a Clinical Marker
Figure 4.1 Characteristics of spermatozoal RNAs. The spermatozoa contain a complex population of coding and noncoding long RNAs
and small noncoding RNAs. Whereas the majority of coding RNAs are in a biologically fragmented state showing significant 3’ end profile bias
(i.e. RN2), a small percentage of transcripts are intact where all exons are well represented (i.e. DDX3X). A high percentage of sperm RNAs
correspond to long noncoding RNAs including rRNAs, annotated long noncoding RNAs, natural antisense transcripts and sperm-specific
RNAs named intronic elements. High levels of specific intronic sperm RNAs are also observed, while the coding regions of this transcript are
absent in sperm (i.e. QRICH1). Several types of small noncoding RNAs are also observed in sperm, including repeat-associated small RNAs,
piRNAs, transcription start sites/promoter associated RNAs, miRNAs, small nucleolar RNAs and small nuclear RNAs.
appear fragmented as remnants of spermatogenesis. specific retention in all samples sequenced (more than
However, some transcripts are maintained intact, with 100 individuals) and the presence of the correspond-
potential roles in sperm transit through the female ing full transcript in testes suggests that they may be
reproductive tract, fertilization and early embryogen- part of a separate regulatory mechanism [17, 19].
esis (Figure 4.1) [17]. Additionally, sperm contain approximately 0.3 fg
A large proportion of human spermatozoal RNAs of small noncoding RNAs (sncRNAs) [14] which have
correspond to noncoding RNAs. Some are annotated been described as playing an important regulatory role
in other cell types as long intergenic noncoding RNAs throughout spermatogenesis [20, 21]. Their presence
(lincRNAs), transposable elements (LINE1, ERVL- in human spermatozoa was first described by Oster-
MaLR, etc.), natural antisense transcripts (NAT) meier et al. [22], and subsequently, several studies have
and chromatin-associated (CAR) and small-nuclear evaluated their content in fertile and infertile males. To
ILF3/NF30-associated (snaR) RNAs. Regulatory roles date, 2,588 mature microRNAs (miRNAs) have been
have been ascribed to these long noncoding RNAs, described in humans (Sanger miRBase v.21;1 ) [23]. It
functioning both at the transcriptional and post- is estimated that they regulate the expression of up to
transcriptional level. In addition to this group of 60% of the coding genes [24]. sncRNAs include sev-
known RNAs, sperm-specific noncoding RNAs are eral classes of RNA transcripts with sizes ranging from
also observed [14, 17]. Typically they range in size 21 to 30 nucleotides (nt) for miRNAs [25] and up
from 100 to 300 nt and overlap either the coding, to 35 nt for piRNAs and tRNA fragments. The best-
intronic or untranslated (UTR) regions of an otherwise characterized are the miRNAs, which are considered
low-expressed or absent transcript (Figure 4.1). Their to be highly relevant to regulating important processes
60
that encompass germ cell development, differentiation observed [40–42], elimination may not be complete.
and proliferation [26]. The study of sperm sncRNAs Recent data describe the presence of several piRNA
has been directed primarily towards miRNAs as new species in human sperm from fertile individuals [43,
potential fertility biomarkers [27]. This is reflected in 44], along with a population of different intact mRNA
the enrichment of categories of processes modulated transcripts, in mature sperm cells [14]. The reduc-
by miRNAs. The canonical pathway of miRNA bio- tion in piRNA abundance parallels that observed for
genesis is highly regulated through their transcrip- the population of mRNAs as the elongating spermatid
tion, processing, loading into effective ribonucleopro- matures and the cytoplasm is expelled [45, 46].
tein complexes and turnover [28]. For example, the In the human spermatozoa, the major classes of
polymerase II pri-miRNAs transcripts can be as large sncRNAs identified by the first RNA-seq study [43]
as 3–4 kb, encoding multiple miRNAs [29]. The pri- included repeat-associated small RNAs (65%), piRNAs
miRNAs are then processed by the RNase III Drosha (17%), transcription start sites/promoter-associated
into shorter 41–180 nt (mode 83 nt) pre-miRNAs [30]. RNAs (11%), miRNAs (7%), small nucleolar RNAs
When exported to the cytosol by exportin 5, they are (0.3%) and small nuclear RNAs (0.1%) [43]. Het-
primed for processing by the RNase III Dicer, yield- erogeneity among the three sperm samples reflected
ing semicomplementary double-stranded structures. 20–60% donor specificity. Interestingly, only 35 miR-
These are unwound through a helicase to yield two 16– NAs were consistently present in the three samples
27 nt (mode 22 nt) mature miRNAs [30] denoted as sequenced. This included several epi-miRNAs (miR-
-5p and -3p, respectively, for their strand of origin. NAs that specifically target effectors of the epige-
Each mature miRNA is capable of regulating the trans- netic machinery) and miRNAs with a potential exclu-
lation of several target mRNAs by forming imperfect sive paternal origin for early embryonic development
3’UTR complementary stem–loop structures, subse- (not detected in mouse oocytes but present in the
quent to their assembly as part of an Argonaute (AGO) zygote). The most abundant miRNA was hsa-miR-34c,
family protein complex. which has been described as playing an important
Another major class of sncRNAs is the piwi- role in spermatogenesis [47] and early embryogene-
interacting RNAs (piRNAs). They have been described sis [48], although species-specific conflicting data have
as essential for mammalian spermatogenesis [31], been reported [49]. Interestingly, most potential tar-
although their function and biogenesis are still not get mRNAs of these ubiquitous miRNAs have not been
fully understood. To date, ⬎30,000 piRNAs have been detected in sperm [50] supporting their potential role
described in humans (piRNABank2 ) [32]. piRNAs can in translational suppression by degradation. In direct
be distinguished from miRNAs using several criteria. contrast, 1,137 piRNAs were detected (the most abun-
These include their larger size (24–32 nt), their gen- dant of which was has-piR-020548), which preferen-
esis from single-strand precursors through an RNase tially target MER, LINE1 and LTR elements.
III-independent mechanism, and the formation of More recently, Pantano et al. [44] used the same
effector complexes with PIWI proteins (germ-line- technology to evaluate the sncRNA in two other nor-
specific subfamily members of AGO) [33, 34]. The pri- mozoospermic individuals. They observed 182 miR-
mary functions of piRNAs are transposable element NAs present in both samples with predicted tar-
silencing [35], epigenetic programming [36] and post- gets among sperm-specific genes. The most abundant
transcriptional regulation of gene expression [37]. miRNA detected by these authors was hsa-miR-1246.
Recent studies have shown the existence of a turnover Discordance with respect to this new study and that
mechanism that promotes the active degradation of Krawetz et al. [43] primarily reflects the exclusion
of the ribonucleoprotein complex piRNAs-MIWI of miRNA with multiple alignments to the genome
(mouse PIWI protein) in the late stages of spermatoge- by Krawetz et al. [43] and the rapid progress in tech-
nesis through the ubiquitin–proteasome pathway [38]. nology. The similarity between hsa-miR-1246 and U2
This turnover has been proposed to be part of transpo- small nuclear RNAs is notable. However, the cor-
son silencing to avoid transmitting paternal piRNAs responding immature form, pri-miR-1246, has been
to the embryo, where they could function in a sim- identified in sperm, confirming its presence. Consid-
ilar manner and may impede the zygote-to-embryo ering only the unique aligned miRNAs, hsa-miR-34c
transition [39]. Although the depletion of piRNAs in was also found as the most abundant. In their study,
the later stages of germ-cell development has been Pantano et al. [44] also observed piRNAs as the most
61
abundant sncRNAs molecules (some of them pro- or 39 million sperm cells, respectively [4], but exclud-
cessed from pseudogenes), including 408 small RNA ing the absence of spermatozoa (azoospermia). There
clusters containing ⬎1 known piRNA (34 clusters in are many known causes for oligozoospermia, includ-
particular accounted for 13,509 piRNAs). The prefer- ing hormonal and chromosomal disorders, single
ential targets predicted for these piRNAs also included genetic defects, testicular and post-testicular factors,
LINE1 transposons, concordant with the study of such as varicocele and hypogonadism, and environ-
Krawetz et al. [43]. ment insults [56]. However, even with this multitude
of correlative presentations, the primary causative fac-
tor of oligozoospermia remains to be identified. The
Discovering Potential Causes of observed reduction of sperm density in unexplained
Male Infertility oligozoospermia suggests that altered spermatogene-
The diagnosis of male infertility is currently based on sis could reflect an altered transcript profile.
the study of seminal parameters such as sperm con- Gene knockout technology has shown that about
centration, motility and morphology. To date, a lim- 388 genes are critical for murine spermatogenesis
ited number of causes associated with altered param- (Mouse Genome Informatics3 ). Down-regulation of
eters have been identified [51]. This is exemplified some of these genes has been shown to lead to
by the general class of chromosomal abnormalities significantly decreased RNA levels in infertile men
that are detected in approximately 5% of patients presenting oligozoospermia. Examples include the
with fertility problems. Examples include numerical or DEAD (Asp-Glu-Ala-Asp) Box Polypeptide 4 (DDX4)
structural chromosomal aberrations, as in Klinefelter [57], Ubiquitin-Conjugating Enzyme E2B (UBE2B)
syndrome (47,XXY), structural chromosome reorga- [58] and some heat-shock proteins (HSPA4, HSF1and
nizations (e.g. balanced translocations or inversions) HSF2) [59]. These transcripts encode proteins that
and Y chromosome deletions. All can have a direct play an essential role in the early stages of sper-
negative influence on spermatogenesis, thereby affect- matogenesis, e.g. DDX4, which is key to the differ-
ing sperm production, probably disrupting meiotic entiation of primordial germ cells and spermatogo-
pairing. As an adjunct, the study of ejaculated sperm nia [60] and UBE2B is involved in chromatin organi-
RNAs has emerged. It has provided insight into the zation of meiotic cells [61]. Microarray studies have
basic molecular mechanisms that regulate production, shown that the transcript profiles of oligozoosper-
maturation and transit of sperm, as well as the patho- mic patients also display a massive down-regulation
genesis of male infertility. of transcripts involved in germ cell development and
spermatogenesis [54].
Coding RNAs and Male Infertility Asthenozoospermia
The initial approach to assessing male fertility by Asthenozoospermia is characterized by reduced
spermatozoa RNAs relied on RT-PCR for quantifi- sperm motility or the absence of motile sperm in
cation of sperm coding RNAs known to be essen- greater than 35% of the spermatozoa examined.
tial during spermatogenesis. This was followed by Although a high percentage of oligozoospermic
the first-generation high-throughput techniques, i.e. patients also present low sperm motility, some infer-
microarrays, which revealed specific biological path- tile patients have a normal sperm count but very poor
ways affecting the seminal parameters from the dif- sperm motility. This can be caused by the presence
ferent subtypes of male infertility [52–55]. The rela- of ultrastructural anomalies, seminal infections and
tionship of spermatozoal coding RNAs to human male antisperm antibodies. In addition, different habits
fertility is presented in Table 4.1. The corresponding such as smoking, alcohol intake or a poor diet have
summary of the published data highlighting the poten- also been associated with asthenozoospermia [62–64].
tial causes of different infertility phenotypes associated In contrast to oligozoospermia, RT-PCR stud-
with semen parameter alterations follows. ies revealed that asthenozoospermic patients present
alterations of RNAs associated with sperm matura-
Oligozoospermia tion or sperm metabolism in the latter stages of sper-
Oligozoospermia is characterized by a sperm concen- matogenesis. For example, some of the transcripts
tration or total number of ejaculate sperm below 15 encode for sperm nuclear proteins such as protamines
62
Table 4.1 Summary of studies showing altered spermatozoa transcripts and disrupted pathways associated with altered seminograms.
Male infertility GO biological altered

phenotype (microarray) Transcripts altered (RT-PCR) References
Oligozoospermic DDX4 Guo et al., 2007 [57]
HSPA4, HSF1, HSF2 Ferlin et al., 2010 [59]
Protein–lipid modification TPD52L3, PRM2, JMJD1A, NIPBL Montjean et al., 2012 [54]
mRNA transcription
Spermatogenesis and motility
Protein targeting and localization,
phospholipid metabolism
Nucleoside, nucleotide and
nucleic acid metabolism
Asthenozoospermic UBE2B Yatsenko et al., 2013 [58]
PRM1, PRM2 Kempisty et al., 2007 [65]
HILS1, TNP1, TNP2 Jedrzejczak et al., 2007 [66]
VDAC2 Liu et al., 2010 [68]
NFE2L2 Chen et al., 2012 [70]
Spermatid development
Ubiquinone biosynthesis pathway ANXA2, BRD2, OAZ3, PRM1, PRM2 Jodar et al., 2012 [53]
Metabolic processes Bansal et al., 2015 [52]
Translation
Cell cycle
Localization
Protein transport
Meiosis
DNA repair
Oligoasthenozoospermic CABYR Shen et al., 2015 [69]
NTRK1 Li et al., 2010 [79]
BDNF Zheng et al., 2011 [80]
Teratozoospermic Ubiquitin–proteasomal pathway Platts et al., 2007 [76]
Apoptotic pathway
MAP kinase signalling pathway
Oligoteratozoospermic HSPA2 Cedenho et al., 2006 [78]
(PRM1 and PRM2) [65], transition nuclear proteins and in different metabolic processes were also empha-
(TNP1 and TNP2) and Histone Linker H1 Domain, sized [52, 53].
Spermatid-Specific 1 (HILS1) [66]. Changes in the Different antioxidant trials have attempted to
expression of sperm nuclear proteins are associated negate the effects of reactive oxygen species known to
with abnormal sperm chromatin condensation and contribute to male infertility. It was hoped that seminal
higher DNA damage, which might trigger apopto- parameters would improve, reflecting on male fertility.
sis, inactivating mitochondria and thereby affecting Consistent with the hypothesis, nuclear factor NFE2L2
sperm motility [67]. The expression of other tran- RNA was down-regulated in asthenozoospermic
scripts representative of processes associated with the patients. Interestingly, this transcription factor reg-
regulation of energy metabolism and sperm motility, ulates the expression of several antioxidant enzymes
e.g. Voltage-Dependent Anion Channel 2 (VDCA2) [70]. This is consistent with the beneficial effect of
located in the mitochondrial outer membrane [68] antioxidant intake therapy on sperm motility [71].
and Calcium-Binding Tyrosine-(Y)-Phosphorylation
Regulated (CABYR) [69], are also detected as altered. Teratozoospermia
Microarray-based discovery studies have confirmed Teratozoospermia is diagnosed when less than 4%
some of the trends brought forth by RT-PCR. In this of sperm have normal morphology evaluated using
case, disturbances of later events of germ cell pro- Kruger’s strict criteria [72]. However, sperm morphol-
duction such as spermatid development were also ogy has not been consistently predictive of fecun-
observed. Alterations in the energy production with dity, and its etiology essentially remains unexplored
perturbations in ubiquinone biosynthesis pathways by conventional approaches. The one exception is
63
globozoospermia, which is characterized by altered regulated and 42 up-regulated miRNAs in sperm from
differentiation of the Golgi apparatus/acrosome. Dis- oligoasthenozoospermic patients. Among these differ-
eases such as Hodgkin’s disease and celiac disease, entially expressed miRNAs, five were selected (hsa-
lifestyle factors and habits may also be contributing miR-34b∗, hsa-miR-34b, hsa-miR-34c-5p, hsa-miR-
factors [73–75]. 429 and hsa-miR-122) and evaluated in a larger popu-
A single microarray-based study has been pub- lation of infertile males [82]. Based on these results, the
lished which examined the teratozoospermic sperm authors suggest this panel of five miRNAs as potential
RNA profile [76]. As in asthenzoospermia, tera- biomarkers of impaired spermatogenesis in affected
tozoospermic patients also present a deficiency of individuals.
spermatocyte and spermatid transcripts, indicating a Recently Salas-Huetos et al. used TaqMan arrays
disruption of the later stages of spermatogenesis. to evaluate the miRNA profiles in sperm from fertile
Specifically, the proteasome is broadly down- (n = 10) [83] and infertile individuals with a single
regulated, likely affecting sperm capacitation, thereby seminal parameter affected: oligozoospermia (n = 10),
impeding the hyperactive motility of spermatozoa teratozoospermia (n = 10) and asthenozoospermia
and the ability to undergo the acrosome reaction [77]. (n = 10) [84]. The results showed a stable population of
ontologically related miRNAs corresponding to sper-
Combined Phenotypes matogenesis and embryogenesis in sperm from fer-
The three seminal parameters, sperm count, motility tile individuals. In comparison, each group of infertile
and morphology, are usually interrelated and appear individuals presented a differential pattern of miRNAs.
coordinated in the individual. Oligozoospermia is These ‘altered’ profiles included 18 differential miR-
often accompanied by poor motility (asthenozoosper- NAs in the oligozoospermic group, 19 in the terato-
mia) and abnormal morphology (teratozoospermia), zoospermic group and 32 in the asthenozoospermic
which leads to even lower fertility. Alterations of group. Interestingly, ontological analysis of predicted
sperm RNAs involved in early spermatogenesis, but targets of these differential miRNAs showed a direct
not in later events, are observed in these collec- relationship with biological processes involved in the
tive phenotypes. For example, a transcript encoding specific seminal alterations present in each population.
heat-shock protein (HSPA2), essential for the mainte- Interestingly, certain miRNAs were correlated with
nance of the synaptonemal complex during meiosis, specific demographic parameters such as age (miR-
is down-regulated in oligoteratozoospermic patients 34b-3p), sperm motility (hsa-miR-629–3p) and sperm
[78]. Additionally, a receptor (Neurotrophic Tyrosine concentration (hsa-miR-335–5p, hsa-miR-885–5p and
Kinase, Receptor, Type 1; NTRK1) [79] and a nuclear hsa-miR-152–3p), indicating that these miRNAs may
factor (Brain-Derived Neurotrophic Factor; BDNF) act as biomarkers for these attributes. Unfortunately,
[80], involved in the paracrine regulation of spermato- studies describing the sperm piRNA cargo in infertile
genesis, are down-regulated in sperm from oligoas- individuals have yet to be published, and their func-
thenozoospermic individuals. These results suggest tional significance potential as biomarkers of fertility
that alterations of sperm RNAs resulting in reduced remains to be determined.
sperm production are more obvious than these affect-
ing sperm motility and/or morphology. Integrated Analysis of mRNAs and sncRNAs
Altered in Patients with Altered Sperm
sncRNAs and Male Infertility Parameters
Few studies have been published describing the Differential patterns of mRNAs and miRNAs in
sncRNA content of infertile individuals. Abu-Halima patients presenting altered seminal parameters (oligo-
et al. [81] used microarrays to analyze the sperm zoospermia, asthenozoospermia and teratozoosper-
miRNA content in infertile males with astheno- mia) suggests a possible functional relationship
zoospermia (n = 9) and oligoasthenozoospermia between different RNA molecules. It is well known
(n = 9) and compared the results with those of nor- that miRNAs regulate gene expression through
mozoospermic individuals (n = 9). They identified translational repression and/or mRNA deadenylation
27 down-regulated and 50 up-regulated miRNAs in and degradation (Figure 4.2A). Integrated analysis
sperm from asthenozoospermic patients and 44 down- is complex because each miRNA can target different
64
A
AGO
3′ 5′ TRANSLATION INHIBITION/ mRNA DEGRADATION/ mRNA DEADENYLATION

5′ Protein-coding region AAAA
3′UTR
B
Spermatogenesis
Pathways altered in oligozoospermia
has-miR-139-5p
Mitosis
Mitosis
2n GMPS spermatogonia
Meiosis
n
Pathways altered in teratozoospermia and asthenozoospermia
hsa-miR-143-3p
Spermiogenesis
Spermatid
TNP1 development
hsa-miR-370 Sperm motility
Sperm function
(motility and VDAC2
capacitation)
hsa-miR-198, hsa-miR-1305,
hsa-miR-432-3p
Sperm capacitation
PSMC5, PSMD14, GABRB1
Figure 4.2 Integrated analysis of mRNAs and sncRNAs altered in patients with altered seminal parameters. (A) The mature
miRNAs, in conjunction with Argonaute (AGO) proteins, form a complex able to regulate gene silencing by translational repression followed
by mRNA deadenylation and degradation. (B) Results of an integrated analysis of mRNA and miRNA altered in patients presenting altered
sperm parameters (oligozoospermia, asthenozoospermia and teratozoospermia). A correlation of some miRNAs and the corresponding
predicted mRNA targets is apparent. For example, guanine monophosphate synthase (GMPS), which is essential during mitosis of
spermatogonia, is post-transcriptionally regulated by hsa-miRNA-139–5p, and both RNAs are altered in oligozoospermia. Several miRNAs
(hsa-miRNA-143–3p, hsa-miRNA-370, hsa-miR-198, hsa-miR-1305 and hsa-miR-432–3p) and their corresponding targets Transition nuclear
protein 1 (TNP1), Voltage-Dependent Anion Channel 2 (VDAC2), Proteasome (Prosome, Macropain) 26S Subunit, ATPase, 5 (PSMC5) and
Proteasome (Prosome, Macropain) 26S Subunit, Non-ATPase, 14 (PSMD14) and Gamma-Aminobutyric Acid (GABA) A Receptor, Beta 1
(GABARB1) are affected in asthenozoospermia or teratozoospermia. These RNAs have important roles during spermatid development (TNP1),
sperm motility (VDAC2) and sperm capacitation (PSMC5, PSMD14 and GABARB1).
mRNAs and a single mRNA can be regulated by to asthenozoospermia and 1,531 were unique to
different miRNAs. The analysis showed that more teratozoospermia. Only one known altered tran-
than 50% of predicted targets from the differentially script in oligozoospermia (Guanine Monophosphate
abundant miRNAs in one altered sperm parameter Synthase (GMPS)) was predicted to be targeted by
were also potentially affected with other alterations of a miRNA known to be altered specifically in oligo-
seminal parameters. However, 278 predicted targets zoospermic individuals (hsa-miR-139) [54, 83, 84].
were unique to oligozoospermia; 2,346 were unique GMPS is crucial for purine synthesis. Alterations
65
in purine content could alter DNA synthesis and tion (IVF) or intracytoplasmic sperm injection (ICSI)
consequently, mitosis in spermatogonia (Figure 4.2B). are recommended. Less invasive treatments such as
Five transcripts associated with asthenozooospermia the combination of ovarian hyperstimulation with
(Voltage-Dependent Anion Channel 2 (VDAC2), controlled timed intercourse (TIC) and intrauterine
Gamma-Aminobutyric Acid (GABA) A Receptor, insemination (IUI) are typically indicated as the first
Beta 1 (GABARB1), Transition Nuclear Protein 1 treatment for couples with mild to moderate male or
(TNP1), C1D Nuclear Receptor Corepressor (C1D) female factor and unexplained infertility [85]. While
and Solute Carrier Family 25 (Mitochondrial Carrier; ART has proven to be invaluable for couples with
Phosphate Carrier), Member 3 (SLC25A3)) are pre- severely compromised semen parameters, the success
dicted targets of four differentially expressed miRNAs of TIC or IUI in infertile patients with normal semi-
(hsa-miR-143, hsa-miR-370 and hsa-mir-432 and nal parameters (normozoospermia) is unpredictable.
has-miR-615) in asthenozoospermic individuals After three or four unsuccessful cycles, idiopathic
(Figure 4.2B). hsa-miR-143 regulates both the nuclear infertile couples are usually shunted to ART. Some
protein TNP1 and C1D, which is involved in the studies suggest the primary use of ART, even if seminal
recruitment of RNA to the exosome [52, 66, 83, 84]. parameters are normal, thereby avoiding failed fertil-
Sperm motility is affected by and reflective of abnor- ization [86]. On one hand, the use of ART for treat-
mal chromatin condensation, but it is not known how ing some idiopathic infertile couples might be cost-
alterations of C1D could affect sperm function. Two effective when compared to providing IUI followed by
sperm channels involved in sperm metabolism and ART in those cases that fail [87]. On the other hand,
capacitation (VDCA2 and GABRB1) are predicted to a successful TIC or IUI cycle minimizes exposure of
be regulated by two differentially expressed miRNAs the female partner to invasive treatments such as egg
in asthenozoospermic individuals (hsa-miR-370 and collection.
hsa-mir-432, respectively) [53, 68, 83, 84]. Finally, one NGS of spermatozoal RNAs has revealed a set
differentially expressed miRNA in asthenozoosper- of sperm RNA elements that may indicate the best
mia, hsa-miR-615, regulates a solute mitochondrial fertility treatment for idiopathic infertile couples
carrier (SCL25A3) associated with sperm motility [19]. RNA-seq can provide a greater resolution than
as identified using a microarray [52, 83, 84]. Two microarrays, as the distribution of sequencing reads,
subunits of the proteasome (Proteasome 26S Subunit, not the individual probe, reflects transcription from
ATPase, 5 (PSMC5) and Proteasome 26S Subunit, that region. Each individual annotated or unannotated
Non-ATPase, 14 (PSMD14)) are highly disrupted in transcribed genomic region can be defined as a sperm
teratozoospermia and regulated by two altered miR- RNA element. This strategy allowed the identification
NAs in teratozoospermic individuals (has-miR-198 of 648 abundant sperm RNA elements (SREs) that
and hsa-miR-1305) (Figure 4.2B). Functional analysis were essentially at equivalent levels across seven fer-
of the potential targets of these seven miRNAs in the tile individuals, indicative of a natural conception. It
specific phenotypes could elucidate the mechanism by was observed that patients expressing all SREs were
which miRNAs affect sperm concentration, motility more likely to achieve live birth (LB) by TIC/IUI (73%;
and morphology. 22 of 30 individuals) than those with one or more
SREs absent (27%; 3 of 11 males; two-tailed Fisher’s
Spermatozoal RNAs in the exact test, P = 0.012). These findings suggested that in
those patients lacking at least one of the SREs, the ear-
Reproductive Clinic lier use of ART would avoid unsuccessful IUI cycles.
Reproductive treatments are typically recommended Approximately one-third of the idiopathic infertile
to couples unable to conceive after one year of unpro- males included in the study (19 of 56 infertile patients)
tected intercourse. The appropriate fertility treatment did not present the complete set of SREs, suggestive of
for each couple is established after both the female and a male factor underlying the couple’s infertility. In con-
the male partner are evaluated. If any severe female trast, the presence of all SREs in the remaining 37 idio-
or male factor is identified, e.g. an ovulatory or tubu- pathic infertile males could indicate the presence of an
lar disorder in females or a diagnosis of azoospermia unknown female or couple’s factor.
or severe oligoasthenozoospermia in males, assisted About 40% of the male fertility SREs were located
reproductive technologies such as in vitro fertiliza- within exonic regions of genes known to be involved in
66
Table 4.2 Sperm RNA elements (SRE) required for natural inherence are described in different animal models,
conception (described by Jodar et al., 2015) located within
transcripts altered in different infertile phenotypes (see e.g. altered metabolism of rodent offspring due to
Table 4.1). paternal diet (low protein or high fat diet) [90, 91]
or presence of cardiac malformations in zebrafish off-
Transcripts altered (number
spring after paternal exposure to bisphenol A similar
Altered seminogram of containing SRE)
to those after direct exposure [92]. Some authors sug-
Oligozoospermia DDX3X (1)
TAF10 (1)
gest that transgenerational epigenetic inherence could
TPT1 (1) reconcile the adaptation of species to new environ-
Asthenozoospermia OAZ3 (1) ments to which the parents were exposed. This is
TNP1 (2) exemplified in the case of the innate defence trau-
CANX (3) matic mechanism against predators transmitted by
BRD2 (7)
HSP90AB1 (1) olfactory receptors [93]. The concept is controversial,
RPS24 (2) as it implies transference of hereditary information
EIF4G2 (2) from soma to germline. A recent study from Cos-
HNRNPC (1)
TBC1D3C (4) setti et al. (2014) proposed that somatic-cell-derived
CABYR (2) RNAs can be transferred to epididymal spermato-
Teratozoospermia SKP1 (1) zoa, likely through exosomes [94]. Growing evidence
ODF2 (9) points to spermatozoal RNAs, e.g. miRNAs, as caus-
RERE (4) ing phenotypic variations in the progeny reflective of
the father’s life experience [95, 96]. One example is
spermatogenesis, sperm motility, fertilization and pro- the observation of a decreased fear response and the
cesses that precede implantation. As expected, some of presence of depressive symptoms in the offspring (F2)
the altered transcripts detected by RT-PCR or microar- of traumatized male mice [95]. Mice exposed to trau-
rays associated with alteration of one seminal param- matic early postnatal events showed a dysregulation of
eter (oligozoospermia, asthenozoospermia and tera- several sperm miRNAs that target genes involved in
tozoospermia; see Section 3.1) contain some of the DNA, RNA and epigenetic regulation. The injection
required SREs for natural conception (Table 4.2). of sperm RNAs from these stressed males into wild
type fertilized oocytes resulted in offspring with simi-
lar behavioural disorders, suggesting that sperm RNAs
New Perspectives participate in epigenetic transgenerational inheri-
The ability of sperm RNAs to inform male factor sta- tance. Although F3 also showed similar behavioural
tus in idiopathic infertile couples, together with the disorders, the population of sperm miRNA from F2
rapidly decreasing cost of NGS, suggests that deep did not present any alterations. This suggests that
sequencing of sperm RNA could change the coun- the information provided by altered miRNA in F1 is
selling approach for couples seeking reproductive care. reversibly rooted in the F2 genome, i.e. by an epige-
The routine use of this technique in reproductive clin- netic mechanism, possibly DNA methylation or chro-
ics might also make it possible to evaluate the qual- matin organization. Interestingly, it was observed that
ity of the genetic contribution from the male to the diet-induced paternal obesity in humans and rodents
embryo. With the use of suitable computational tools, could disturb metabolic processes in female offspring
it may be possible to identify genetic variants reflected [91, 97]. Obese males also exhibit changes in sperm
in the RNA-seq data [88]. This strategy may be useful miRNAs as well as altered sperm DNA methylation.
to evaluate the presence of single-nucleotide polymor- However, intervention through a diet and exercise
phisms associated with male infertility, as well as dif- program during two complete spermatogenic cycles
ferent monogenic diseases or allelic imbalance. appears to transmit a normal metabolic profile to
It is well known that environmental contaminants female offspring [98]. These findings suggest that the
and lifestyle factors influence human fertility, which use of spermatozoal RNAs in clinics may assist the
may be reflected to different degrees in the spermato- clinical care of male infertility and serve as a predic-
zoa [89]. These changes can be transmitted to future tor of childhood outcomes.
generations and may affect their health and fertil- All the studies reviewed in this chapter are based
ity. Several examples of epigenetic transgenerational on the study of sperm RNAs. However, spermatozoa
67
account for only 5% of the ejaculate, while the remain-

ing 95% corresponds to secretions from the epi-
didymis, prostate and seminal vesicles. These acces-
sory sex glands release a substantial number of exo- Extensive physical and Physical and seminal
somes, containing a large repertoire of RNAs and pro- molecular evaluation parameters evaluation
teins, into the seminal fluid [99]. The majority of sem- ART
FEMALE FACTOR OR SEVERE MALE FACTOR
inal fluid (70% by volume) is produced by the sem-
inal vesicles, with these secretions being rich in fruc- MILD OR NO MALE
Deep sequencing of
FACTOR
tose, a sugar essential for the nutrition of the sperma- spermatozoal RNAs and
miRNA may predict future
tozoa during the transit to the oocyte. The secretions Sperm RNA Elements health problems in offspring
analysis
from the prostate, which constitute approximately 20%
by volume, contain proteins required for the coagula- NO FEMALE FACTOR COMPLETE SET OF SRE TIC/IUI
tion and liquefaction of semen as well as immune com-
ponents. These classes of protein typically serve vary- NO FEMALE FACTOR
INCOMPLETE SET OF
SRE
ART
ing roles in intercellular interaction and determina-

tion of immune properties. Seminal fluid plays a much
greater role than simply being a medium to carry the
spermatozoa through the female reproductive tract. Figure 4.3 Proposed practice for reproductive counselling in
New perspectives suggest that the seminal fluid also couples displaying infertility. Reproductive counselling of
provides an optimal environment for the development infertile couples begins with an extensive physical and molecular
evaluation of female and a basic physical analysis and seminal
and successful implantation of the preimplantation parameters evaluation in males. If any severe female or male factor
embryo, and that its alteration may impact the success is discovered, assisted reproductive techniques such as in vitro
of the early pregnancy [100]. The integrative analysis of fertilization (IVF) or intracytoplasmic sperm injection (ICSI) are
recommended. In contrast, less invasive techniques such as
sperm and seminal fluid transcriptomic and proteomic controlled timed intercourse (TIC) and intrauterine insemination
high-throughput data has revealed the active transit of (IUI) are usually indicated as the first treatment for couples with mild
seminal fluid proteins required for sperm maturation male factor or unexplained infertility but with unpredictable results.
RNA-seq has revealed a set of 648 sperm RNA elements (SREs) that
[101], promoting the inclusion of seminal fluid RNAs are able to predict the success rate of TIC and IUI in idiopathic
in future studies of male infertility. Some seminal fluid infertile couples. The absence of at least one required SRE suggests
RNAs appear to provide additional molecular mark- the primary use of ART, thereby avoiding unsuccessful IUI cycles.
The future health of offspring may also be assessed with the use of
ers of foci of spermatogenesis in azoospermic patients RNA-seq based on the ability to evaluate the genetic contribution.
that may foreshadow the likelihood of testicular sperm
extraction [102].
as TIC or IUI may be successful. The current dilemma
faced by the couple is that there is no accepted clini-
Conclusion cal screen that avoids unsuccessful IUI cycles. Sperma-
tozoal RNAs including long and sncRNAs, in tandem
When a couple first visit a reproductive clinic, they ini-
with the seminal fluid RNAs, opens a window to coun-
tiate a well-defined clinical protocol with the goal of
selling idiopathic infertile patients (Figure 4.3). At
determining the etiology of their infertility and receive
present, the 648 sperm RNA elements required for nat-
advice on the treatment options, as well as the success
ural conception appears to discern which idiopathic
rate and costs. Currently, the evaluation of the male
infertile patients have a high likelihood of achieving
partner is principally based on the analysis of semi-
pregnancy using TIC or IUI while advising others to
nal parameters in order to exclude a severe male fac-
directly undergo ART [19]. However, it remains to be
tor, defined as the absence or immobility of sperma-
established whether sperm RNA can predict ART out-
tozoa. ART is recommended to those patients with a
come and provide information about the future health
severe male or female factor. The counselling of 15–
of offspring.
30% of the patients diagnosed with unexplained infer-
tility [103] presents a unique challenge. Despite ARTs
having a high success rate in cases of unexplained Acknowledgements
infertility, they present an increased risk to the female The authors apologize to other authors in the field
patient when the use of less invasive techniques such that because of space limitations we could not directly
68
reference their contributions to the field. This work was 13. Boerke A, Dieleman SJ, Gadell BM. A possible role
supported in part through a Charlotte B. Failing Pro- for sperm RNA in early embryo development.
fessorship to SAK. The authors thank Molly Estill of Theriogenology 2007; 68 Suppl 1: S147–55.
Wayne State University School of Medicine for her crit- 14. Jodar M et al. The presence, role and clinical use of
ical review of the manuscript. spermatozoal RNAs. Hum Reprod Update 2013; 19:
604–24.
15. Sendler E et al. Stability, delivery and functions of
human sperm RNAs at fertilization. Nucl Acids Res
2013; 41: 4,104–17.
16. Goodrich RJ, Anton E, Krawetz SA. Isolating mRNA
Notes and small noncoding RNAs from human sperm.
1. http://www.mirbase.org. Methods Mol Biol 2013; 927: 385–96.
2. http://pirnabank.ibab.ac.in/stats.html. 17. Sendler E et al. Stability, delivery and functions of
3. http://www.informatics.jax.org/ human sperm RNAs at fertilization. Nucl Acids Res
2013; 41: 4104–17.
18. Johnson GD et al. Cleavage of rRNA ensures
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72
Chapter
Role of the Epididymis in Sperm Maturation
5 Robert Sullivan and Clémence Belleannée
Introduction more information on epididymal functions obtained

by studying classical laboratory animal models.
The epididymis, from the Greek epi [1] and didymoi
[2], is an organ located on the surface of the testicle
in cartilaginous fishes, reptiles and mammals. The epi- Anatomy and Histology of the
didymis is a single long convoluted tubule connecting
the efferent ducts to the vas deferens. Epididymides Epididymis
are also found in birds, but in this class they are not The male reproductive tract in humans comprises
attached to the testis surface. In fact, the epididymis is the differentiated excurrent duct including the vasa
the hallmark of the male reproductive tract in verte- efferentia connected to the rete testis, followed by
brate species practicing internal fertilization [3]. Cop- mesonephric ducti derivatives: the epididymis, the vas
ulation is not always synchronous with ovulation in deferens, the seminal vesicles and finally the ejacula-
these species; thus, the epididymis may be responsi- tory duct. Described as early as 1668 by the anatomist
ble for generating a heterogeneous population of male Regnier De Graaf (cited by Turner [12]), the epi-
gametes with optimal fertilizing potential at different didymis is a single convoluted tubule. Classically, it
time points after sperm deposition in the female tract is divided into three anatomical regions: the proximal
in order to increase the fertilization window of a given caput, the elongated corpus, and the terminal and bul-
ejaculate [4]. Even though this hypothesis is attrac- bous cauda epididymis (Figure 5.1). During the second
tive with regard to comparative vertebrate anatomy, half of the eighteenth century, the physiologist William
the role of the epididymis in generating a heteroge- Hunter prepared a series of mercury-injected repro-
neous population of male gametes is not yet supported ductive organs that beautifully illustrated the testis and
by experimental evidence. epididymis from normal men; these are preserved at
The function of the epididymis remained puzzling Glasgow University, Scotland [13].
until 1967, when Marie-Claire Orgebin-Crist [5] and The vasa efferentia are small tubules that connect
J. Michael Bedford [6] independently demonstrated the rete testis to the epididymis. The number of these
the involvement of the epididymis in the acquisition linear tubules varies from one species to another. In
of fertilizing ability by transiting spermatozoa. Dur- humans, the organization of the vasa efferentia is much
ing this maturation process, the spermatozoa acquire more complex; some are branched or coiled and oth-
both forward motility properties and the potential to ers do not connect to the epididymal tubule. In fact,
interact efficiently with the oocyte and its surround- the proximal caput epididymis is mainly formed by
ing zona pellucida and cumulus oophorus. Studies on efferent ducts, a peculiarity of the human excurrent
human epididymal functions are scarce, and a number duct [14]. Evidence from different animal models has
of clinical observations challenge the importance of revealed the role of these tubules in water reabsorption
the excurrent duct in human sperm physiology [7–9]. under oestrogen control [15].
The status of the human epididymis will be reviewed In laboratory rodent species, an initial segment dis-
in this chapter. Readers are referred to [10, 11] for tinct from the caput forms the proximal region of the
University Press.
73
Chapter 5: Role of the Epididymis in Sperm Maturation
The epididymal epithelium in humans increases in

thickness from the proximal caput to the corpus, where
it reaches its maximum. It then decreases from the dis-
tal corpus to the distal cauda region; thus, the mid-
dle region of the epididymis is more active in pro-
tein secretion [21]. In parallel, the lumen area shows
a 100-fold increase from the proximal caput to the dis-
tal cauda (Figure 5.3) [22].
Whereas the principal cells constitute approxi-
mately 85% of the cell types forming the epididymal
epithelium, other cell types have been described: clear
cells play a role in intraluminal fluid acidification;
basal cells are sensors of the intraluminal compart-
ment involved in intercellular communications; and
halo cells have a possible involvement in immuno-
logical functions [10]. Although these cells have been
clearly described in animal models, their distribu-
Figure 5.1 Gross anatomy of epididymides of normal men. The tion in human epididymal epithelium and their func-
caput, corpus and cauda segments are indicated, as well as the tions remain to be further investigated. The epididy-
scrotal portion of the vas deferens. Note that these segments are
poorly distinguishable. Unpublished data from Sullivan lab.
mal tubule is surrounded by well-developed layers of
smooth muscle cells in the distal cauda region that play
a role in sperm progression along the excurrent duct.
epididymis. This segment is distinct from the rest of In general, the lumen of the human epididymal tubule
the epididymis, as its epithelium is formed of cuboidal is small when compared with those of other mammals.
cells. It is believed that the initial segment is highly Whereas the anatomy of the epididymis shows
active in protein synthesis and that it plays a major variability among mammalian species, the three seg-
role in sperm maturation. The proto-oncogene c-ros is ments of the epididymis can easily be visualized in
exclusively expressed in the initial segment of the epi- most. However, in humans, this organ appears to
didymis. In c-ros knock-out male mice the initial seg- be poorly differentiated; the proximal segment does
ment is absent, and males are infertile due to defects not have the characteristic bulbous appearance of the
in sperm osmoregulation [16]. However, in humans, epididymis in other species, and the cauda appears
there is no anatomical or histological evidence of a indistinguishable from the corpus and vas deferens
proximal epididymal segment distinct from the caput (Figure 5.1). With the knowledge that the transcrip-
segment. In fact, in normal men, the c-ros proto- tome and proteome show great variation along the epi-
oncogene is expressed along the length of the human didymis, this raises the question of how results regard-
epididymis, with the exception of the proximal caput ing epididymal physiology obtained in animal models
segment [17]. can be extrapolated to humans.
The epididymal lumen is delimited by a pseudos-
tratified epithelium formed of epithelial cells named
principal cells, with a large nucleus localized near Luminal Composition
the basal membrane (Figure 5.2). Highly specific The inorganic composition of the epididymal fluid was
tight junctions between these cells ensure an isolated determined using fluid collected from sections of the
intraluminal compartment that maintains the blood– vas deferens acquired during vasectomy procedures
epididymis barrier [18]. This is an important aspect of [23]. Potassium and sodium concentrations were eval-
epididymal function when it is considered that sper- uated at 111 and 30 mM, respectively, and calcium was
matozoa are subject to immune attack when exposed determined to be as low as 1.5 mM. The concentra-
to circulating immune cells [19, 20]. The thickness of tion of inorganic compounds found in the human epi-
the epididymal epithelium varies along the organ and didymis is similar to those reported in other mam-
corresponds to protein synthesis and secretion by the malian species. The osmolality increases dramatically
epithelium. along the epididymis and ranges from 280 mmol/kg
74
Figure 5.2 Histology of the epididymis of normal men. Haematoxylin–eosin stained longitudinal paraffin section of the epididymis of a
normal man. Higher magnifications of the epididymal tubule from each segment are illustrated in inserted micrographs. Unpublished data
from Sullivan lab. (A black and white version of this figure will appear in some formats. For the colour version, please refer to the plate section.)
75
A variable, ranging from 1 to 21 days [25]. More recent

500 reports estimate a 2–3-day sperm transit time along
the human epididymis [26–28].
Epididymal lumen area (X 103 µm2)
There are only a few reports describing morpholog-

400
ical changes undergone by human spermatozoa along
the epididymis. Bedford reported that intramolecular
300 disulphide bonds increase in spermatozoa during epi-
didymal transit to protect the sperm from postejacula-
200 tory stresses [29]. Sperm chromatin condensation also
occurs during human sperm transit of the epididymis
[30]. The dimensions of the head decrease during this
100
journey, and this is likely attributable to cell dehydra-
tion in response to osmolality increase [23, 24]. In con-
0 trast to what is known in nonprimate mammals, the
A B C D E F
cytoplasmic droplet does not migrate along the mid-
B piece during epididymal maturation, but remains in
100
the sperm neck region.
90 Testicular fluid, as deduced from measurements
80 performed with spermatocoele fluid, has a protein
Epithelial height (µm)
70 concentration of 3.4 mg/mL [31] and reaches nearly 30

60
mg/mL in the distal part of the epididymis [23]. This is
due to water reabsorption by the epididymal epithe-
50
lium and also to high protein synthesis and secre-
40 tion, mainly in the corpus segment of the human epi-
30 didymis [22].
20 In animal models, the epididymis is highly seg-
10 mented, as illustrated by the pattern of gene expres-
sion, which shows great variation from one segment
0
A B C D E F to another [10, 32]. As a consequence, the proteome
Epididymal region of the intraluminal compartment is also variable along
the excurrent duct (for review, see [33]). Thus, the tran-
Figure 5.3 Histomorphometric parameters of the epididymides of
normal men. Histomorphometric measurements of the lumen area siting male gamete will interact in a sequential man-
(A) and epithelial thickness [1] along the epididymides of normal ner with a proteomic environment that will sequen-
men. (a) Proximal and (b) distal caput, (c) proximal and (d) distal tially modify the spermatozoon in a manner that will
corpus, and (e) proximal and (f) distal cauda epididymidis. Redrawn
from Legare et al. [22]. confer its forward motility properties and its fertilizing
ability [11, 34]. As already mentioned, the anatomy of
the human epididymis does not show the hallmark fea-
in liquid collected from spermatocoeles [24] to 342 tures of the epididymides from other species, which are
mmol/kg in the vas deferens [23]. characterized by well-differentiated caput, corpus, and
Due to water reabsorption, the sperm concen- cauda segments. This suggests that the transcriptome
tration increases along the mammalian epididymis. and proteome characterizing the human epididymis
Although the sperm count increase along the human may not be as segmented as in nonprimate species.
epididymis has not been clearly described, it is believed
that a concentration of male gametes occurs in humans
similar to that in nonhuman primates. Epididymal Proteome
Upon leaving the testis, the human spermato- When analyzed by two-dimensional gel electrophore-
zoon is differentiated and undergoes additional mod- sis, the protein pattern found in the intraluminal com-
ifications while transiting the epididymis. Epididy- partment of the human epididymis does not show
mal transit was first estimated by tritiated thymidine major differences from one segment to another [21].
labelling of testicular spermatozoa and was found to be According to Dacheux et al. [21], albumin accounts
76
for approximately 50% of total human epididymal pro- in semen [43, 45], there is no direct evidence for the
teins, and less than 20 others account for 90% of all presence of epididymosomes in the human epididymal
proteins found in the epididymal intraluminal com- intraluminal compartment. One hundred forty-six
partment. The proteomics field is a rapidly evolving proteins are associated with extracellular vesicles pre-
discipline with an increasing number of techniques pared from fluid collected in the scrotal portion of the
that can be applied to characterize the proteomes of vas deferens during surgical vasectomy reversal. Some
different biological fluids or tissues samples. Recently, of these proteins were proposed to be associated to epi-
408 proteins were shown to be present in the human didymosomes, as they are encoded by mRNAs charac-
epididymal secretome; half of these are also detected terizing the human epididymal transcriptome [46].
on sperm cells and may thus be involved in sperm mat- Extracellular microvesicles isolated from the sem-
uration [35, 36]. The fact that some of these proteins inal plasma of normozoospermic, vasectomized and
may originate from the testis has to be kept in mind. vasovasostomized (surgical vasectomy reversal) men
Some of the identified proteins secreted by the were compared. With regard to miRNA content,
epididymis are known to play a major role in male microvesicles prepared from vasectomized men lacked
reproductive physiology and illustrate the function particular miRNAs that are present in normozoosper-
of the epididymis in humans. Niemann-Pick type C2 mic and vasovasostomized semen samples. Thus, it
disease protein, also known as HE1, is abundant in was concluded that some extracellular microvesicles
the epididymis and plays a role in sperm cholesterol present in human semen originated from the epi-
efflux occurring during sperm epididymal matura- didymis and that these were characterized by a spe-
tion; prostaglandin D synthase acts as a lipocalin that cific pattern of miRNAs [47]. These extracellular
transports hydrophobic molecules such as steroids and microvesicles (or epididymosomes) containing miR-
lipids; CD59 shown to be involved in oocyte fusion and NAs may be involved in the modulation of gene
immune-suppression; clusterin functions as an extra- expression along the male reproductive tract. Interest-
cellular chaperone; beta-defensins are small peptides ingly, an miRNA cluster, miR-888, is predominantly
with antimicrobial properties; HE4 secretory protein expressed in the primate epididymis [48], including
has sequence domains similar to protease inhibitors; that of humans [49], but is absent in other mam-
and P34H secreted by the corpus segment is involved malian species’ miRNA [50]. Micro RNAs are known
in sperm–zona pellucida binding ability (for review, to control gene expression by inducing mRNA degra-
see [10, 11, 34, 37]). dation or by inhibiting translation [51]. The presence
of miRNAs in extracellular microvesicles secreted by
the epididymal epithelium strongly suggests that they
Epididymosomes and miRNAs are involved in modulation of gene expression along
There is more and more interest in extracellular the epididymis [49] (Figure 5.4). The overall profil-
microvesicles in cell physiology. Such vesicles are ing of miRNA expression varies when newborn, young
found in all biological fluids and are thought to be adult and aged human epididymis are compared, sug-
involved in intercellular communication [38]. They gesting that their expression is, at least in part, under
have been classified according to their dimensions, androgenic control [52].
mode of secretion and composition [39]. Vesicles of
this type in the male reproductive tract were first
described as a constituent of human seminal plasma Transcriptome
[40] and were named prostasomes, as they are predom- During the last decade, microarray technologies have
inantly secreted by the prostate [41]. Similar vesicles, become a powerful tool for compare the transcrip-
visible by electron microscope, have been described tomes of different tissues at different steps of devel-
as constituents of the epididymal fluid, first in the opment, or to compare normal tissues with patholog-
hamster [42], followed by studies in other mammalian ical specimens. The transcriptome of the epididymis
species such as rat, mouse, ram and bull (for review, shows major differences along the organ; this has been
see [43]). These extracellular vesicles have been named well demonstrated in mice and rats [32, 53]. Around
epididymosomes and are described as an apocrine 500 genes expressed in both species are differentially
secretory product of epididymal principal cells [44]. expressed by more than fourfold between any two seg-
While extracellular vesicles have been characterized ments of the epididymis. Thus, the pattern of gene
77
A
miR-892a miR-892b
150 2500
2000
Expression intensity
Expression intensity
100
1500
1000
50
500
0 0
Caput Corpus Cauda Caput Corpus Cauda
B
7 Spag8 r = –0.7542 7.75 3.7 Esrrg r = –0.7542 7.75
miR-892a p = 0.0094 7.50 miR-891b p = 0.0094 7.50
6 7.25 3.5 7.25
7.00 7.00
Log 2 miRNA expression
Log 2 miRNA expression
6.75
Log 2 mRNA expression

Log 2 mRNA expression
5 3.3 6.75
6.50
6.50
6.25
4 3.1 6.25
6.00
6.00
5.75
3 2.9 5.75
5.50
5.50
5.25
2 5.00 2.7 5.25
4.75 5.00
1 4.50 2.5 4.75
4.25 4.50
0 4.00 2.3 4.25
Caput Corpus Cauda Caput Corpus Cauda
Figure 5.4 miRNAs in the epididymides of normal men. Mir-892a and miR-892b are highly expressed in the human epididymis and their
expression level correlates negatively with epididymal target genes. (A) Dot plots display the intensity level of miR-892a and miR-892b,
which increases in the distal regions of the human epididymis (n = 6 donors). (B) According to in silico studies, miR-892a and miR-892b are
predicted to target and regulate the expression of Sperm-associated antigen 8 (Spag 8) and of Estrogen-related receptor gamma (Esrrg),
respectively. MiRNA and transcript target expression levels negatively correlate along the human epididymis. r: Pearson correlation coefficient.
expression is highly regulated along the epididymides precise anatomical criteria for dissection of the epi-
of these rodent species. didymis, differences among the microarray platforms
To date, three groups have used microarrays to used, and variations in analysis criteria, it is diffi-
describe the transcriptome and its variation along the cult to establish a consensus from these three stud-
human epididymis [54–56]. Due to the absence of ies. These three transcriptome analyses still describe
78
A
Caput Corpus Cauda
0.5 1 3
B
Caput
1171
5 8
209 55 650
Corpus Cauda
Figure 5.5 Transcriptome along the epididymides of normal men. Differentially expressed genes in the human caput, corpus and cauda
transcriptome of the epididymis of normal men. (A) Unsupervised clustering algorithm applied to the 2,274 differentially expressed qualifiers
along the epididymis. Pseudo-colour represents the relative intensity of gene expression. (B) Venn representation of clusters of qualifiers
differentially expressed in the caput, corpus, and cauda segments of the epididymis in normal men. Reproduced with permission from
Thimon et al. [55]. (A black and white version of this figure will appear in some formats. For the colour version, please refer to the plate section.)
variations in gene expression patterns along the A limited number of researchers have reported
human epididymis; however, the differences from one on the outcome of artificial insemination performed
segment to another are less spectacular than those with human epididymal spermatozoa [57, 58]. With
described in rodent species (Figure 5.5). Gene ontolog- regard to animal models, a very low pregnancy rate is
ical analysis of clusters generated from each epididy- obtained with spermatozoa collected from the proxi-
mal segment revealed that the human caput segment mal segment, whereas the success rate increases when
transcripts act in cell–cell adhesion, and the corpus spermatozoa are collected more distally [59]. Dur-
region is characterized by immune defence and pro- ing the 1980s, microsurgical reanastomoses were con-
tease inhibitors, while the cauda is involved in tissue ducted along the male reproductive tract to restore
organization and muscular contractions [55]. These patency of the excurrent duct and the presence of
transcriptomic variations, possibly involving miRNAs sperm in the ejaculate. The vas deferens was reanas-
targeted by extracellular microvesicles named epididy- tomosed for surgical vasectomy reversal, or along the
mosomes, may reflect changes in the intraluminal length of the excurrent duct to overcome agenesis or
secretome that varies along the organ. Thus, the human occlusion. In a series of papers, Silber reported that
epididymis governs sequential changes undergone by the pregnancy outcome was not affected by the level of
the male gamete after testicular transit and is respon- reanastomosis of the vas deferens along the epididymis
sible for sperm maturation as defined using laboratory [60, 61]. This series was particularly controversial and
and livestock animal models. contradicted another study showing that the more dis-
tally the vas deferens is reanastomosed, the higher is
the pregnancy outcome [59] (Figure 5.6).
Although in vitro fertilization and embryo transfer
Epididymis and Sperm Maturation: technologies were originally developed to overcome
Clinical Observations Fallopian tube pathologies, male infertility has become
It is well recognized that the spermatozoa from ver- an indication for assisted reproductive technologies
tebrate species practicing internal fertilization have to over the years. These protocols now use intracytoplas-
transit a minimal length of the epididymis in order to mic sperm injection (ICSI) to overcome poor sperm
become fertile, i.e. to acquire forward motility proper- quality such as oligo- (low sperm count), astheno-
ties and the ability to encounter the egg and its vest- (poor motility) and even a- (absence) zoospermic
ments efficiently. This concept is based on artificial semen samples. In cases of oligo- and/or astheno-
insemination performed with spermatozoa collected zoospermia, spermatozoa with ‘normal morphology’
along the epididymis, or on the outcomes of in vitro are selected and microinjected into the oocyte cyto-
fertilization of intact oocytes. The point at which the plasm. Hundreds of thousands of children have been
first fertile spermatozoa appear along the epididymis born following these procedures, which are also
varies from one mammalian species to another, but in applied when semen samples are devoid of male
general they have to reach at least the proximal corpus gametes: azoospermia. Effectively, ICSI procedures
region in order to be functional. have been performed successfully with spermatozoa
Figure 5.6 Fertility following vas deferens

reanastomosis. Pregnancies established by
epididymovasostomy patients, displayed as a
function of the level of anastomosis, expressed as
the distance of upper point of anastomosis from
proximal border of caput epididymis (mm). In cases
of bilateral anastomosis, the lowest point is indicated.
Reproduced with permission from Schoysman and
Bedford [59].
80
collected all along the epididymis, even with spermato- and have been proposed to be involved in sperm–
zoa recovered in dispersed testicular biopsies [62]. The oocyte interaction. Other human sperm proteins of
success of these reproductive technologies may sug- epididymal origin have been described by other groups
gest that the epididymis plays no part in sperm fertil- [68–70]. In vitro fertilization assays in the presence
izing ability. However, it should be kept in mind that of interfering antibodies against two of these proteins,
ICSI procedures overcome all the natural processes P34H and FLB1, showed that these sperm proteins
of fertilization including cumulus dispersion, bind- acquired during maturation play a role in zona pellu-
ing and penetration of the zona pellucida and sperm– cida binding [71] and sperm/egg fusion, respectively.
egg fusion, which are abilities acquired by mammalian The role of P34H in the epididymal maturation of
spermatozoa during epididymal transit and collec- human sperm has been particularly well documented
tively referred to as sperm maturation. Furthermore, [72, 73].
surgeons performing sperm aspiration for ICSI proce-
dures recover sperm as distally as possible in order to
optimize the outcomes of these procedures. The Epididymis and Male Infertility
There is increasing evidence that sperm har- P34H, also known as DCXR (dicarbonyl L-xylulose
bour molecular information that is transferred to the reductase), is a moonlighting protein playing diverse
oocyte, with epigenetic (imprinting) consequences for roles in sugar metabolism, carbonyl detoxification, cell
the zygote [40]. The possible role of the epididymis in adhesion and fertilization/male fertility depending in
these imprinting processes cannot be excluded, since it which biological system it is expressed [74]. Sequenc-
is known that epididymosomes contain small noncoding of DCXR cDNA prepared from human epididymal
ing RNAs that can modulate gene expression postfer- tissues reveals the absence of a signal peptide [75], even
tilization and that these extracellular vesicles transfer though this protein is secreted into the intraluminal
macromolecules to transiting epididymal spermato- epididymal fluid. Orthologs of DCXR have been char-
zoa [43]. These new reproductive technologies should acterized in other mammalian species such as hamster
be used with more caution with regard to potential [76], mouse [77], Rhesus monkey [78] and bull [79].
transgenerational consequences. The DCXR protein is glycosylphosphatidylinositol-
anchored to the sperm plasma membrane and its dis-
Epididymis and Sperm Maturation: The tribution is restricted to the acrosomal cap. It is specif-
ically expressed in the corpus segment of the human
Physiological Evidence epididymis and is secreted by the epididymal princi-
Sperm undergo complex biochemical modifications pal cells via the apocrine pathway [43]. At ejacula-
during epididymal transit; these involve interaction tion, DCXR is masked by seminal plasma proteins,
between the male gametes and the epididymal prin- and it is unmasked during the capacitation process in
cipal cell secretions. Sperm epididymal maturation order to allow sperm–zona pellucida interaction medi-
includes removal, addition or modifications of sperm ated by this sperm protein [70]. Antibodies directed
surface proteins, relocalization of surface antigens and against DCXR inhibit sperm–zona pellucida binding
modifications to membrane lipids and sterol con- in vitro without affecting capacitation, sperm motil-
tents, including raft membrane domain remodelling. ity parameters or sperm/zona free oocyte interac-
Numerous studies using experimental and livestock tion [71]. Thus, DCXR is an epididymal secreted
species have exhaustively documented these biochem- protein clearly involved in the acquisition of sperm
ical processes necessary for the acquisition of sperm fertilizing ability in man and is a marker of sperm
functionality (for review, see [11, 63, 64]). Information maturation [72].
regarding human sperm modifications during transit One out of seven couples of reproductive age will
in the human epididymis is scarce. face infertility problems. It is generally accepted that
Tezon et al. [65–67] have established a human men contribute to 40% of infertility aetiology. Male fer-
epididymal organ tissue system. Under laboratory tility is evaluated by semen analysis in accordance with
conditions the epididymal epithelium responds to World Health Organisation [80] criteria for examina-
androgens by secreting a number of proteins that asso- tion of human semen [80]. A significant proportion
ciate with epididymal spermatozoa. These proteins of couples consulting for infertility include men pre-
remain associated with spermatozoa after ejaculation senting with normal semen analysis values according
81
to WHO criteria whose female partners have no with spermatozoa and in soluble form in the seminal
detectable pathology after complete clinical investiga- plasma. In humans, the epididymis is the only organ
tion. These cases of idiopathic infertility may involve of the urogenital system that secretes this protein [88].
one or both partners. When idiopathic infertility is It is a very reliable biomarker for the differential diag-
considered, it is clear that the testicular functions of nosis of obstructive and nonobstructive azoospermia
male gamete production and sex hormone secretion [89]. This has a significant clinical impact when one
are not involved in the absence of pregnancy. With the considers that to date, surgical exploration of the uro-
knowledge that the epididymis is involved in the acqui- genital system in conjuncture with histological anal-
sition of male gamete fertilizing ability, it is plausi- ysis of testis biopsies is the current diagnostic tool
ble that pathophysiological disorders affecting the epi- to determine treatment of men presenting with these
didymis may be implicated in some cases of human pathologies.
male idiopathic infertility. As an epididymal marker, In addition, since it is known that miRNAs asso-
the utility of DCXR for the diagnosis of idiopathic ciated with extracellular vesicles in seminal plasma of
male infertility has been investigated. normal and vasovasosotomized men are undetectable
The presence of DCXR on ejaculated spermato- in vasectomized men, these molecules are other poten-
zoa from couples presenting with idiopathic infertil- tial markers of vas deferens patency [47].
ity after at least 30 months of unprotected intercourse
was compared to that for male gametes from men
with proven fertility. Whereas all fertile samples have
Consequences of Vasectomy and
high levels of DCXR, 40% of semen samples from Vasovasostomy
idiopathic infertile couples lack this sperm protein. In Vasectomy consists of the interruption of the vas def-
parallel, sperm–zona pellucida binding assays in vitro erens permeability by ligation and cauterization of
show that sperm lacking DCXR were unable to bind the scrotal portion of the vas deferens [90]. Although
to the zona pellucida in vitro. This defect in sperm poorly reversible, it is now the most common male
maturation probably explains why a significant pro- contraceptive method in many countries [91]. A lim-
portion of idiopathic infertile men are unable to ited number of reports describe the consequences of
impregnate their partners [81]. Absence of DCXR is vasectomy for the epididymis. These include forma-
a powerful marker of failure for standard in vitro fer- tion of granuloma and production of antisperm anti-
tilization performed with gametes from infertile cou- bodies [92, 93]. In primates, antisperm antibodies pro-
ples [82]. Furthermore, DCXR is detected in all fertile duced in reaction to vasectomy result in circulating
couples, but is absent in up to 15% of semen from cou- immune complexes exacerbating atherosclerosis [94].
ples consulting at an infertility clinic for the first time Some reports also suggest an increase in cardiovascu-
[83]. Thus, the epididymis appears to be involved in lar diseases and prostate cancer frequency in vasec-
pathologies affecting male fertility, as the spermatozoa tomized men [95]. More recent epidemiological stud-
in some men do not undergo the post-testicular mod- ies demonstrate that such correlations do not exist
ifications necessary for fertilization in unprotected [96, 97]. These stress the fact that studies on the con-
intercourse. sequences of vasectomy conducted in animal mod-
els have a limited capacity for extrapolation to the
Markers of Epididymal Function human situation. Very few morphological or histolog-
ical studies have reported on the effect of vasectomy
and Patency on the human epididymis [92]. Following vasectomy,
Proteomic analyses of seminal plasma from normal thickness of the epididymal epithelium is diminished,
volunteers, postvasectomy patients [84] and patients whereas the surface of the intraluminal compartment
with nonobstructive azoospermia [85] have been per- is considerably increased [22].
formed to identify biomarkers of urogenital system Patterns of gene expression along the epididymis of
organs [86]. As an example, ECM1 and TEX101 have normal men have been compared to those characteris-
been shown to be powerful biomarkers (high speci- tic of postvasectomy epididymides. It appears that the
ficity and sensitivity) for the differential diagnosis epididymal transcriptome is greatly affected by vasec-
of azoospermia [87]. CRISP1 is secreted by the epi- tomy [98, 99]. Some genes undergo down-regulation,
didymal epithelium and is detected in association such as NPC2, which is known to be involved in
82
cholesterol transport and mediates sperm cholesterol

efflux during epididymal maturation [100]. Messen-
ger RNAs encoding the epididymal-originating sperm
protein DCXR undergo changes in their expression
patterns along the epididymis [22]. The delocalization
of DCXR synthesis along the human epididymis affects
acquisition of DCXR by the maturing spermatozoa.
In fact, similarly to the 40% of men presenting with
idiopathic infertility, the spermatozoa of some vasova-
sostomized men lack DCXR and consequently may be
unable to fertilize after intercourse [101]. The distri-
bution of miRNAs is also affected by vasectomy [47].
Some of these changes may not be reversible by vasova-
sostomy. This could explain the discrepancy between
the surgical success of vasovasostomy, as evaluated by
the presence of spermatozoa in the semen, and the
recovery of fertility, which is much lower [102]. Under-
standing the consequences of vasectomy for epididy-
mal physiology and the irreversibility of some sperm
sequelae following vasovasostomy could help in the
understanding of the involvement of the epididymis in
the pathophysiology of male infertility. Figure 5.7 Sperm reservoir in the human epididymis. Mean sperm
number in successive ejaculates produced over a 24 h period by
23–27 year-old men (N = 10). The sperm population in each
Sperm Reservoir Function ejaculate is expressed as a percentage of the total produces.
Reproduced with permission from Bedford [9].
The bulbous appearance of the cauda epididymis in
many mammalian species underlies the sperm reser-
voir function of the distal segment of the epididymis.
Protein synthesis and secretion in this segment are Acknowledgements
low, which correlates with the reduced thickness of The work from our laboratory described in this chapter
the epithelium lining the intraluminal compartment was supported by the Canadian Institutes for Health
and the large lumen diameter that permits stor- Research. Dr. Muriel Kelly is acknowledged for text
age of mature spermatozoa [28]. This function has editing and Christine Légaré for figure designs.
evolved in response to sperm competition and differ-
ent reproductive behaviour governing sperm deposi- References
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87
Chapter
Seminal Plasma Plays Important Roles
6 in Fertility
Susan S. Suarez and Mariana F. Wolfner
Most thinking about the mechanisms of fertility has believe it is important that physicians assessing male
focused on the eggs and sperm, which are, of course, fertility, or carrying out ART, be aware of the impor-
important. Another major component of fertility – tance of the components of seminal plasma.
the nonsperm components of semen – has often Rather than reviewing details of the many sem-
been assumed to be there simply to provide support inal plasma molecules and their effects in different
for sperm. Yet there are more than 2,000 proteins organisms, we present an overview of their major roles,
in human seminal plasma [1–4], as well as amino with examples. We discuss, and occasionally speculate
acids, polyamines, lipids, carbohydrates, RNAs and about, how seminal plasma components cause these
ions (reviewed in [5–7]). Many of these molecular effects. Where possible, we use examples from humans
components are in functional classes that are con- to illustrate these points (if no organism is indi-
served across organisms (e.g. [8]). It takes energy and cated, the molecule or effect under discussion is from
resources to make these molecules. If they were not humans). However, since information on humans is
important, why would they have persisted through limited, we also discuss insights that have been gained
evolution? by examining model systems with manipulable genet-
Genetic and physiological studies in several mam- ics or physiology; these likely reveal mechanisms that
mals and several insects have shown that, indeed, sem- might occur in humans. In addition to the mam-
inal plasma components are critical for fertility (e.g. malian models of mouse and bovine, we also con-
recent studies include [7, 9–13]). As we will review sider results from Drosophila fruit flies (and occa-
in this chapter, although some seminal plasma com- sionally other insects). There is striking congruence
ponents support/nourish the sperm, a role for sem- between the biochemical classes of seminal proteins
inal plasma that has been long posited and appreci- found in Drosophila and in mammals [8], including
ated, seminal plasma components are also critical to humans, even though the specific proteins are not
(1) ensure that sperm reach, persist at and leave stor- always orthologous. Because of these molecular and
age sites in females, (2) induce ovulation or fine-tune reproductive commonalities, the powerful and rapid
its timing and/or (3) induce changes in the female’s genetic, molecular, physiological and genomic meth-
immune response and other physiological functions ods and resources that are available for Drosophila have
to prepare her for pregnancy. It is likely that semi- led to insights into the fundamental features of sem-
nal plasma components will be found to significantly inal protein function. We integrate those here with
affect human fertility too, permitting diagnosis of pre- insights from studies on mammals. Although our main
viously undiagnosed infertilities and potentially being goal in including discussion of insect seminal pro-
worth considering for improving outcomes of assisted teins is to use findings from insect models to sug-
reproductive technologies (ART) (e.g. see [14]). For gest mechanisms for seminal plasma components in
example, the recent discovery that transfer of seminal all organisms, including humans, we note that under-
vesicle secretions is important for proper progeny standing functions of insect seminal proteins is also of
development in mice [9] is an additional reason that we biomedical importance in providing the potential to
University Press.
88
Chapter 6: Seminal Plasma Plays Important Roles in Fertility
control the reproduction of insects that transmit seri-

ous human diseases such as malaria and dengue fever.
For our purposes we posit that ‘seminal plasma
components’ are molecules (or organelles/structures) 3.
made by glands in the male’s reproductive tract and seminal vesicle
secreted so that they are transferred to the female
as part of the ejaculate, along with sperm. But it
is instructive to consider several possibilities. Some
secretions of male glands may be found entirely in
the seminal plasma (fluid), but others may bind to the 2.
sperm. The latter may thus appear to be part of the prostate gland
‘sperm proteome’, as well as (or instead of) being in vas deferens
the liquid part of the ejaculate, even though they were cauda epididymis
added to the sperm after spermatogenesis. In addition,
structures from the sperm (such as the cytoplasmic
droplet) which derive from the germline may also be
1.
within the seminal plasma. We focus this chapter on
molecules made by male reproductive glands, regard- bulbourethral gland
periurethral glands
less of whether they enter the female in the seminal
plasma or bound to sperm.
Seminal Plasma Components Are Made

by a Variety of Glands
In humans, sperm constitute only about 1–5% of the
volume of semen, while the seminal plasma constitutes
the rest. In mammals, seminal plasma is derived from a
variety of tissues: the testis, epididymis and male acces-
sory sex glands [2] (Figure 6.1).
Testis: Little is known about functions of testicular
components of seminal plasma. Some seminal plasma
components correspond to molecules that were identi-
fied as germ cell markers [2], indicating that they could
represent debris of germ cells, such as the cytoplasmic Figure 6.1 Sources of seminal plasma components in humans. In
droplet. An example of such a molecule is L-lactate this posterior view of the right side of the human male reproductive
dehydrogenase C chain (LDHC), a testis-specific gly- tract, organs are grouped and numbered in the order in which their
secretions enter the ejaculate. Note that the ampulla, which is the
colytic enzyme that is found in the cytoplasm of sperm expanded glandular portion of the vas deferens that can be seen
[15]. Other human testis-derived seminal plasma pro- where the vas enters the prostate, contributes its secretions when
teins associate with sperm in the ejaculate. Examples the vas contracts.
of such proteins include eppin (SPINLW1), lactotrans-
ferrin (LTF) and clusterin (CLU). These proteins are tions. Some epididymal secretions, particularly those
thought to form a protective complex on the sperm tail, packaged as extracellular microvesicles called epididy-
with microbicidal properties. They are also involved in mosomes, are secreted in excess so that they remain
trapping sperm in seminal gel shortly after ejaculation readily detectable in seminal plasma after ejaculation
[16]. (reviewed by [17]. It could prove highly informative
Epididymis: The main role of epididymal secre- to investigate whether epididymal secretions in sem-
tions is thought to be to mature sperm functionally inal plasma continue to affect sperm after they leave
by modifying their plasma membranes. We refer the the epididymis, and whether secretions directly exert
reader to chapter 5, where Dr. Sullivan discusses mat- effects on the female after deposition in the female
urational modifications of sperm by epididymal secre- tract.
89
Accessory sex glands: The ampullary glands of the mation and the activities of some ions channels [24,
vas deferens, the seminal vesicles (vesicular glands), 25]. The high concentration of spermine in human
the prostate gland, the bulbourethral glands, the peri- seminal plasma, along with its strong positive charge,
urethral glands (glands of Littre) and the preputial can reduce the acidity of vaginal fluid [26], thereby
gland all contribute components to seminal plasma. protecting sperm. Last, the prostate also secretes large
The secretions of these glands, and even the glands’ amounts of zinc, which can serve as a bacteriostatic
existence, vary greatly among species. For example, in factor as well as a cofactor for certain enzymes [19],
mice and humans, the seminal vesicles make major and citric acid, a metal chelating agent.
contributions to seminal plasma; however, dogs and Last, the seminal vesicles contribute the major por-
cats lack seminal vesicles altogether. Such major dif- tion of human semen (reviewed by [18]). These glands
ferences indicate that evolution of the accessory glands secrete fructose for use by sperm as a glycolytic sub-
has been rapid. strate. The most abundant proteins secreted by human
During ejaculation in humans, the bulbourethral seminal vesicles are semenogelins (known as SVS2
and periurethral glands contribute roughly 5% of the proteins in mice) and fibronectin, which coagulate to
semen volume [18]. These glands are stimulated to form a gel in semen shortly after ejaculation [27]. The
secrete their products just before the vas deferens and gel is liquefied in 15–60 min in vitro by the proteases
distal caudal epididymidis are stimulated to contract secreted by the prostate. Human seminal vesicles also
and expel the sperm-rich fraction of semen [19]. The secrete large amounts of prostaglandins, which were
secretions consist primarily of mucoid substances that misnamed because they were originally thought to
are thought to coat the urethra in order to lubricate be secreted exclusively by the prostate. As discussed
and protect it. Some, but not all, of the secreted mucoid below, functions of the prostaglandins include regula-
proteins have been detected in human semen samples, tion of smooth muscle contraction.
and thus could reach the female tract [20]. Insect seminal plasma components are also made
Next, prostatic secretions contribute 15–30% of by a series of glands that secrete their contents into the
human semen volume [18]. The human prostate is lumen of the male reproductive tract. Analogously to
stimulated to contract and release its secretions at the what was noted for mammals, the appearance, devel-
same time that the vas deferens is stimulated to expel opmental origin and number of these glands vary
sperm [19]. Some secretions are packaged as ‘prosta- between taxa [13]. Sperm from the testes are stored
somes’, small membrane vesicles that can carry lipids, in the seminal vesicles prior to ejaculation. Somatic
proteins, glycoproteins and RNAs (reviewed in [21]. cells of the testis sheath and cells of the seminal vesi-
The major protein contributions of the prostate gland cle likely contribute some seminal molecules, although
are a protease (prostate-specific antigen, PSA), a phos- this has not yet been determined. The majority of the
phatase (prostatic acid phosphatase, PAP) and fibri- 200-member Drosophila seminal proteome [7] and
nolysin, which act to liquefy semen after it has gelled small membrane vesicles analogous to prostasomes
[22]. Neuroendocrine cells are found within the pro- [28] are contributed by the pair of accessory glands in
static epithelium, where they secrete calcitonin and the male fly. These glands have two types of secretory
other neuropeptides into seminal plasma [23]; how- cells, which produce distinct but overlapping comple-
ever, little is known about the functions of these secre- ments of seminal plasma proteins [29, 30]. The acces-
tions. Polyamines, particularly spermine, are present sory glands and seminal vesicles open into the ejacula-
in large amounts in human semen [19]. Although tory duct of the male, a secretory tissue that itself con-
polyamines have long been known to be plentiful tributes some proteins to the ejaculate [31, 32]. At the
in human semen (spermine was discovered by van end of the ejaculatory duct, a further secretory tissue,
Leeuwenhoek in 1678), their functions are poorly the ejaculatory bulb, contributes proteins and small
understood [24, 25]. These small, positively charged molecules such as lipids to the ejaculate as well [33–
molecules are found in many types of cells and tissue 37]. Studies of interrupted matings in Drosophila indi-
fluids [24], where they are known to serve a wide vari- cate that, as in humans and other mammals, compo-
ety of functions. Due to their strong positive charges, nents of seminal plasma enter the female in a specific
they bind to membrane phospholipids and negatively order [32, 38]. The ejaculatory bulb products that will
charged proteins/glycoproteins on membranes [24], form the mating plug enter first; sperm and accessory
where they can affect membrane stability, domain for- gland proteins enter later.
90
Seminal Plasma Components Are Often sperm to begin moving up the female reproductive
tract.
Modified after They Leave Their Sites Some components of seminal plasma serve to pre-
of Synthesis vent backflow of sperm from the female tract and posi-
Seminal plasma molecules mix with sperm during tion sperm to ascend the tract. In mice, a copula-
transit through the male and during ejaculation, and tory plug forms inside the female after mating. This
can bind to (and modify) sperm. Seminal proteins plug is important in fertility: if its formation is pre-
themselves can be modified after secretion by the vented, for example, due to a transglutaminase (Tgm4)
male reproductive glands; they can also modify each mutation in the male that prevents the protein cross-
other. For example, seminal plasma of mammals and linking required to form the copulatory plug, the ejac-
insects is rich in proteases and inhibitors of proteases ulate does not appear to be retained well within the
(reviewed in [39]). Their presence in seminal plasma female [10]. Sperm from these mice, although of nor-
allows regulated cleavage of other seminal proteins, mal motility and fertilization ability, do not reach their
or potentially of sperm proteins. In primates, a pro- storage sites in normal numbers, suggesting a role for
teolysis cascade is initiated in ejaculated semen that the copulatory plug in helping sperm to ascend the
ultimately results in the cleavage of semenogelins and female tract.
thereby in dissolution of the semen clot; we will return A plug formed by coagulated male seminal secre-
to this briefly later in the chapter. In Drosophila, at tions also facilitates ejaculate retention and fertility in
least two seminal proteins (ovulin and Acp36DE) are insects. In Anopheles mosquitoes, a transglutaminase
proteolytically processed when they enter the female is also necessary for mating plug formation, and this
[40–42]. This processing, which might regulate their plug is essential for fertility and sperm retention within
activity or stability, is carried out by a metallopro- the female [47]. The plug also plays a role in trans-
tease derived from the male’s accessory glands [42]. fer of a hormone to females (see below) [48] and has
This protease is made as an inactive precursor and is been suggested to correlate with malaria transmission
activated upon cleavage by a seminal serine protease potential. In Drosophila, a mating plug forms in the
that is itself activated (by cleavage) while in transit mated female’s uterus [33, 49], although its coagula-
through the male. Although these male-derived pro- tion does not require a transglutaminase. This mating
teases can initiate the cleavage of the ovulin, efficient plug is essential for retention of the ejaculate within the
and complete cleavage requires an as yet unidenti- female [33]. If a mating plug cannot coagulate prop-
fied female contribution [43], indicating that for some erly, due to loss of a critical protein, the ejaculate is lost
seminal protein action (as for sperm–egg interac- from the female after mating, sperm are not stored in
tion), molecules from the two sexes must interact and proper numbers and fertility is compromised.
cooperate. In humans and other primates, semenogelin
secreted by the vesicular glands coagulates the semen
into a gel shortly after the semen is collected for in
vitro studies [50]. A cascade of proteases, including
Seminal Plasma Molecules Facilitate PSA, then degrades the semenogelin. In vitro, human
Retention, Storage and Activity sperm are reported to be trapped and immobilized in
of Sperm seminal gel through interaction between SEMG1 in
the seminal plasma and eppin on the sperm surface
Seminal Plasma Components Help Sperm [51]. In vivo, however, the semen may not be as well
mixed as samples studied in vitro. Examinations of
Ascend the Female Tract the human cervix, made within 3 min of ejaculation
As mammalian sperm are released from the epi- during coitus by human couples, revealed that many
didymis during ejaculation and come into contact with sperm had already penetrated into cervical mucus in
the secretions of the accessory sex glands, they become 39 out of 40 couples in which the woman was judged
exposed to increased levels of bicarbonate in the sem- to be in the fertile period of her menstrual cycle
inal plasma. This activates sperm flagellar motility and the man had motile sperm and normal semen
by stimulating the activity of soluble adenylyl cyclase coagulation [52]. In these cases, gelled semen could be
(SACY) in the sperm cytoplasm [44–46] and enables seen covering the portion of the cervix that projects
91
Sperm storage
Vagina
Fertilization
Uterus
Cervix
Ovary
Oviduct
Semen deposition
and pooling
Sperm passage
through cervical mucus
Figure 6.2 Lateral view of the human female reproductive tract, with the vagina, cervix and uterus shown in longitudinal section. Regions
are indicated where the semen is pooled immediately after coitus and where sperm may be held in storage in the oviduct.
into the vagina. Liquefaction of the seminal gel in [53]. A variant of the human orthologue of DEFB126,
the vagina occurred about 5 min after ejaculation in which produces abnormal mRNA and reduces glyco-
18 of the couples – that is, only a few minutes after sylation of the human sperm surface, has been associ-
coagulation. Given the in vivo observations, the first ated with reduced fertility in men [54].
few minutes after ejaculation must be crucial for
the passage of sperm into the cervix. The order of
contribution of the sperm and glands to the ejaculate
Seminal Plasma Proteins Facilitate Storage
suggests a mechanism by which a substantial number of Sperm
of sperm could enter the cervical mucus before In mammals, the main sperm storage site for many
the semen gels: the prostate secretes its liquefying species is the lower portion of the oviductal isthmus,
proteases into the seminal fluid at the same time as and sometimes also a part of the uterotubal junction.
sperm are propelled from the cauda epididymis and Seminal plasma proteins play major roles in holding
vas, yet before the seminal vesicles secrete coagulating sperm in the reservoir and maintaining sperm viabil-
proteins [18, 19]. This may prevent the sperm that ity and fertility during storage. These storage proteins
arrive closest to the cervix from becoming trapped in have been studied most extensively in farm species.
gel before they can enter the cervical mucus, while the In cattle, three proteins in the binder of sperm family
portion of semen gelling behind the liquid, sperm- (BSP1, BSP3, BSP5) have been implicated in holding
rich fraction maintains the sperm at the cervical os bull sperm in the reservoir. The proteins are secreted by
(Figure 6.2). the seminal vesicles [55] and coat the head of the sperm
Whereas semenogelins form clots of semen that predominantly in the acrosomal region, where they are
may serve to retard loss of sperm from the vagina adsorbed onto sperm by interacting with plasma mem-
after coitus and hold sperm at the entrance to the brane phospholipids [56, 57]. Subsequently, BSPs bind
cervix, another component of human (and other pri- sperm to receptors on the oviductal epithelium. When
mates’) semen, the glycoprotein beta-defensin 126 purified BSP proteins are added to epididymal sperm
(DEFB126), assists sperm in penetrating into cervical that have not been exposed to seminal vesicle secre-
mucus. DEFB126 is secreted in the macaque monkey tions, each BSP alone is able to coat the sperm and
epididymis and coats the surface of sperm. The coating stimulate binding to oviductal epithelium [58]. This
facilitates sperm penetration of cervical mucus from binding holds sperm in the reservoir.
oestrous females [53]. The facilitation of mucus pene- In addition to binding sperm to the oviductal
tration is primarily attributed to sialylated glycans on epithelium, each BSP prolongs the motile life of bound
DEFB126, which coat sperm with a negative charge sperm. This may be the result of the ability of BSPs to
92
stabilize membranes, either by reducing phospholipid capacity’ and involves a long and complex process in
fluidity [59, 60] or by inhibiting phospholipase A2 [61]. mammalian sperm that begins at ejaculation and may
Sperm of cattle are only held in the reservoir for up be completed shortly before fertilization [46].
to about a day; however, in the little brown bat, sperm When bull sperm were incubated under mild
are held in the reservoir for the entire winter hiberna- capacitating conditions, BSP5 was shed from sperm
tion period [62]. In humans, sperm may remain in the and some BSP3 on the sperm surface underwent prote-
reservoir up to five days, based on the finding that fer- olytic cleavage [72]. It would be interesting to learn the
tilization can occur if coitus occurs within about five mechanisms triggering these changes in the BSP coat-
days of ovulation [63]. Nevertheless, although human ing of sperm and how they promote sperm movement
sperm have been observed interacting with oviductal out of the reservoir.
epithelium in vitro [64], little is known about the time- Seminal proteins in Drosophila also contribute to
line of reservoir formation in humans or of ascent of the regulation of sperm within storage. In particular,
sperm up the oviduct to the site of fertilization. the release of sperm from storage is regulated, being
Even after coating sperm, the BSPs remain present very efficient, so that 1/3–1/2 of the 1,000 stored
in the seminal plasma in great excess. At 15–50 mg/ml, sperm fertilize eggs [73]. A seminal peptide called sex
BSP1 is the most abundant protein in bovine seminal peptide (SP) is necessary for this [74]. SP acts through
plasma; BSP3 and BSP5 are also quite abundant, but a G-protein coupled receptor (SPR [75]) in the ner-
at only about 10% of the concentration of BSP1 [65]. vous system and reproductive tract to mediate sperm
The abundance of these proteins strongly indicates release [76]; without SPs action, sperm are released too
that they promote reproductive success. Homologs slowly and fertility is decreased. Other than requir-
of the BSP genes have been identified in the mouse ing SPR, the mechanism of action of SP in mediat-
(mBSPH1, mBSPH2) and human (hBSPH1), where ing sperm release is unknown. However, it is known
they are expressed in the epididymis rather than the that the neuromodulators octopamine (an invertebrate
seminal vesicles [66]. Recombinant human BSPH1 analogue of norepinephrine) and tyramine are needed
binds to sperm [67]; however, its involvement in in females to modulate sperm release [71]. It is also
human sperm storage has not yet been reported. known that mating (and seminal proteins in aggregate)
DEFB126, which has been implicated in facilitat- regulate neuromodulator levels and secretion along
ing penetration of cervical mucus by macaque sperm, the reproductive tract [77], including the sperm stor-
has also been implicated in binding macaque sperm to age organs. It is possible that SP regulates sperm release
oviductal epithelium [68]; however, it is not yet estab- through regulating neuromodulator secretion, as it
lished that its human homolog serves to store sperm. does for a different postmating response [78].
In Drosophila, sperm storage is facilitated by uter- The SP has an interesting feature that allows it to
ine contractions (described in a later section) that continually regulate sperm release. Once inside the
are regulated by seminal proteins [69, 70]. One of female, SP binds to sperm, and some SP is detected
these proteins in particular, a novel glycoprotein called bound to sperm as long as sperm are in storage [79].
Acp36DE, facilitates part of the uterine contractions In an interesting parallel to the BSP3 situation noted
[71], but also binds to a region of the common oviduct above, a protease (of unknown source) cleaves the
just past the openings of the sperm-storage organs active portion of SP to release it from sperm. This
[41]. Its binding here and its presence in the uterus released portion of SP can then interact with SPR to
have been suggested to ‘corral’ sperm to keep them regulate sperm release.
near the openings to the storage sites.
Seminal Plasma Regulates Sperm
Seminal Plasma Components Help Regulate Functioning within the Female
the Release of Sperm from Storage Sites in In some mammalian species, particularly primates
and cattle, sperm quickly leave seminal plasma behind
the Female Tract in the vagina when they enter the cervix and swim
In mammals, capacitation of sperm has been associ- through cervical mucus. In other species, sperm
ated with release from the reservoir. Note that the term leave the seminal plasma behind when they pass from
capacitation is defined here as ‘acquiring fertilizing the uterus into the oviduct. Nevertheless, there are
93
seminal plasma components that bind to the surface of and also stimulate efflux of cholesterol and phospho-
sperm and are retained when sperm pass through the lipids from the plasma membrane [83]. In support of
cervix and/or uterotubal junction. These components this proposal, heparin-like glycosaminoglycans have
can regulate the capacitation state of sperm. been detected in bovine oviduct fluid [84].
In the mouse, sperm and seminal plasma directly
enter the uterus without having to swim through cer- Seminal Plasma Components Influence
vical mucus; however, semen also fills the vagina and
cervix, where seminal plasma protein seminal vesicle
the Female
secretion 2 (SVS2) forms the copulatory plug that fills While a role for seminal plasma components in sperm-
the vagina and cervix. However, SVS2 has also been related processes might not seem surprising, what may
reported to inhibit capacitation of mouse sperm [80]. be unexpected is that seminal plasma molecules also
SVS2 associates with the sperm surface by interact- exert major and profound effects on the female who
ing with the plasma membrane ganglioside GM1 [80]. receives them. These effects can be viewed as though
There is evidence that it inhibits capacitation by pre- the seminal plasma components are acting as hor-
venting cholesterol efflux from the sperm [80]. mones or other modulators to increase the efficiency
The human homologs of SVS2 are the semeno- of the female’s reproductive capacity, thereby benefit-
gelins, SEMG1 and SEMG2. When a purified mix- ting the male and in some cases also the female; see
ture of SEMG1 and SEMG2 was added to human the section Seminal Plasma Proteins Have Interesting
sperm prepared by centrifugation through Percoll, it Evolutionary Dynamics for further discussion. We dis-
inhibited capacitation in vitro [81, 82]. SEMG was cuss some examples here, both to present these find-
also detected on Percoll-prepared sperm without addi- ings and to raise the question of how our knowledge
tion of the purified proteins and was shed from the of the effects of seminal plasma on the female could be
sperm incubated under some capacitating conditions used to develop new methods for assessing and treat-
[81]. In vivo, human semen is deposited in the vagina ing infertility.
and sperm leave seminal plasma behind as they swim
through the cervical mucus; however, it seems likely Seminal Plasma Contains Hormones
that sperm carry some SEMG on their plasma mem- Seminal plasma can contain well-known hormones
branes as they ascend the tract, which could act to with stimulating effects on reproduction. By introduc-
delay capacitation until they reach the oviduct. The ing these hormones directly into the female, the male is
posited delay in semen gelation at the entrance to the modifying her hormonal milieu, presumably to accel-
cervix, as discussed above, could assist sperm not only erate or facilitate reproduction.
in passing into the cervix, but also in carrying a coating Prostaglandins are present in mammalian semi-
of SEMG with them. nal plasma and have been proposed to assist in draw-
The bovine BSP proteins, which, as discussed ing sperm up the female’s reproductive tract by stim-
above, have been shown to bind sperm to oviductal ulating smooth muscle contraction in the walls of the
epithelium, have also been implicated in sperm capac- tract [19]. Human seminal plasma contains 15 types of
itation. Bull sperm are exposed to excessive amounts prostaglandins, most of which are in the prostaglandin
of BSP proteins when they first come into contact E (PGE) group [19, 85]. In women, both excitatory
with seminal vesicle secretions during deposition of and inhibitory PGE receptors have been detected in
the ejaculate in the vagina. The great amounts of BSP smooth muscle of nonpregnant uteri [86]; however,
proteins in the fluid surrounding the sperm can pro- any direct local effects on the vaginal wall or cervix are
duce an efflux of cholesterol and phospholipids from as yet unknown. Effects of the contractions on sperm
the sperm plasma membrane, which can briefly stim- and fertilization could be positive or negative. On one
ulate capacitation [83]. Subsequently, the BSP proteins hand, excitation of peristaltic-type contractions could
that coat the sperm and are carried through the cervix draw sperm up into the female tract. This would be
can stabilize plasma membranes, as discussed above. analogous to the recently reported effects of semi-
However, at some point in the female tract (presum- nal plasma prostaglandin 2-alpha in facilitating sperm
ably in the oviduct), BSP proteins can further capac- storage and fertilization in mated quail females [87].
itate sperm when exposed to heparin and/or high- Alternatively, PGE-stimulated organized contractions
density lipoproteins, which interact with BSP proteins in the opposite direction could push sperm out of the
94
mammalian reproductive tract, and intense contrac- cytochemistry showed seminal molecules crossing a
tions could damage sperm. For example, in the rab- permeable part of the vaginal wall to access the cir-
bit, more than 90% of sperm that were found in the culatory system. Not all molecules can cross into
oviductal ampulla within 15 min of coitus were dam- the circulation, although peptides and some pro-
aged and immotile, presumably because of the con- teins have been shown to enter the circulation. In
tractions that must have drawn them rapidly through addition, in some insects, the male’s intromittent
the tract [88]. Absorption of small molecules, includ- organ can puncture the reproductive tract wall (e.g.
ing lipid prostaglandins, into the walls of the vaginal Drosophila: [70, 101]), leading to the possibility that
cavity can produce effects via the circulatory system. seminal plasma could be introduced directly into the
In fact, the vaginal cavity has been investigated as a circulation.
site for drug delivery, because of its high permeabil-
ity to drugs of low molecular mass [89]. Interestingly,
prostaglandin-synthesizing enzymes and PGE2-like Seminal Plasma Components Can Affect
material have been detected in reproductive tissues the Molecular Biology of the Female
of male crickets and in reproductive tracts of mated
(but not unmated) female crickets [90]. These data Reproductive Tract
suggest that the machinery to make prostaglandins, Seminal plasma components can cause changes in
as well as prostaglandins themselves, is transferred gene expression in the female’s reproductive tract. An
in seminal plasma. Injection experiments showed example was reported by Bromfield et al., who com-
that prostaglandins can stimulate oviposition in these pared gene expression patterns between female mice
crickets, suggesting a reason for their transfer during mated with control males and with males that lacked
mating. seminal vesicles [9]. Bromfield et al. showed that in
Introduction of other important reproductive hor- the absence of seminal vesicle secretions, the females’
mones in seminal fluid has also been observed in oviducts were down-regulated for growth-promoting
insects. The two best-known insect hormones, 20- cytokines and up-regulated for an apoptosis-inducing
hydroxy-ecdysone (20E) and juvenile hormone (JH), factor. Their results suggest that seminal vesicle secre-
both play positive roles in egg production [91–94]. tions can influence gene expression, and hence the
For example, in Anopheles gambiae mosquitoes, 20E is molecular output of the female’s reproductive tract tis-
introduced into females with the mating plug. Injec- sues. Their further results (see the next section) suggest
tion of 20E has been shown to mediate some post- that these changed outputs can actually affect the phe-
mating responses in these insects, consistent with the notype of the female’s progeny.
model that the 20E provided by the male during mat- Molecular changes are also triggered by semi-
ing can cause these changes in a normal mating [94]. nal proteins in female insects. In Drosophila (and in
Similar logic applies to the transfer of JH by male mosquitoes) there are many small-scale changes to
Aedes aegypti mosquitoes to their mates, where it also the transcriptome of the female reproductive tract,
enhances their reproductive output [95, 96]. In D. and in the whole female, shortly after mating [102–
melanogaster, transfer of these hormones has not been 109]. A few hours later, larger-magnitude changes are
reported after mating. However, the seminal peptide seen, though in a smaller number of genes [107, 108,
SP causes increased production of juvenile hormone 110]. One study compared transcriptomes of (whole)
in mated female Drosophila [97, 98], thus increasing female Drosophila after mating with normal males vs.
JH titres in mated females even though the hormone males that lacked seminal proteins from the acces-
was not directly introduced from the male; this is sory glands [102]. That study showed that some tran-
thought to contribute to increased oogenesis rates in scriptome changes were a response to seminal pro-
mated females [99]. JH can also down-regulate female tein transfer. Among these, a particularly striking set
pheromone synthesis, presumably contributing to the were genes that encode components of the immune
lower attractiveness of mated females to subsequent response, such as antimicrobial peptides, or specific
mates [100]. transcripts [102, 105, 109]. Seminal plasma was criti-
Again parallel to the situation in mammals, cal for the high induction of expression of these genes,
molecules introduced via seminal plasma can enter the suggesting a role for seminal proteins in inducing an
circulation of the mated insect female [38]. Immuno- immune effect in mated females. Some transcriptome
95
changes were not due to receipt of seminal plasma, and allow sperm to pass up towards the openings of the
may instead have responded to receipt of sperm, or to storage sites [70, 71]. In the absence of seminal pro-
the act of mating itself [102]. teins in aggregate, or of Acp36DE in particular, few
In mated female Drosophila, changes also occur sperm get into storage; those that do are stored and are
in levels and release of neuromodulators (non- able to fertilize eggs [112, 113].
neurotransmitter molecules that can increase or
decrease the effectiveness of neurotransmitters [77, Seminal Plasma Components Can
111]). Precise measurements in different parts of the
reproductive tract and at different times postmating Influence Ovulation
have shown that levels and release of octopamine, sero- Some mammals, such as humans, are spontaneous
tonin and other neuromodulators from vesicles change ovulators. A hormonal cycle in females of these taxa
with time postmating [77]. Each region of the repro- regulates the timing of ovulation. But in other mam-
ductive tract acquires a characteristic combination of mals, such as cats, rabbits and camelids, mating stim-
neuromodulators at any given time after mating. These ulates ovulation. The advantage of this is obvious: eggs
combinations are suggested to give each reproductive are released at exactly the optimal time for fertilization
tract region its characteristic function at that time. by sperm. The ovulation inducer varies in these cases
Some of these changes are caused by seminal protein (in rabbits, it is the physical act of mating) [114]. In
receipt. That a single event (mating, and in some cases camelids, a seminal plasma component was proven to
seminal plasma specifically) causes these local and induce ovulation ([115]; reviewed in [116]).
temporal effects provides a way to coordinate the tim- A role for a seminal plasma component in ovula-
ing of different postmating responses along the repro- tion by camelids was discovered when it was found
ductive tract. that intramuscular or intravascular injection of sem-
That seminal fluid components can cause changes inal plasma causes female Bactrian camels to ovu-
in gene expression and in neuromodulator levels and late [117]. Subsequent studies in alpacas and llamas
release in Drosophila suggest a general principle: sem- confirmed this phenomenon for them as well. The
inal plasma may affect females’ physiology, jump- ‘ovulation inducing factor’ was purified and identi-
starting events that will facilitate her overall fertility. fied as beta-nerve growth factor (ß-NGF) (115). Sub-
Recent results, in mammals as well as in Drosophila, sequent studies showed that ß-NGF induces luteiniz-
support this view. We illustrate this with several ing hormone (LH) synthesis and secretion in female
examples. camelids, promoting ovulation [118]. Interestingly, the
seminal plasma of other mammals, including males
Seminal Plasma Components Cause of species whose females are spontaneous ovulators,
such as cattle, also contains ß-NGF [116, 119]. This
Uterine Contraction raised the possibility that a seminal plasma compo-
Several prostaglandins are transferred in human male nent can regulate ovulation in spontaneous ovula-
seminal plasma. Their exact roles remain unknown, tors. Consistent with this idea, Tribulo et al. [120]
but findings in insects suggest possibilities. Specifi- found that bull seminal plasma affects the synchronic-
cally, in crickets, prostaglandins transferred by males, ity of ovulation and increases plasma progesterone lev-
or synthesized by females using enzymes transferred els in cows. Waberski et al. [121, 122] reported that
by males, affect female fertility and may induce uterine pig seminal plasma can advance the timing of ovula-
contractions. Studies in Drosophila have shown that tion in gilts. These findings raise the intriguing ques-
after mating, the uterus undergoes a reproducible set tion of whether a seminal plasma component might
of shape changes and contractions that appear to push be of therapeutic use in cases of human ovulatory
the mass of sperm ‘up’ towards the sperm storage sites disorders.
[69–71], and that components of seminal plasma are Mammals are not unique in showing stimula-
critical for inducing these contractions. Although the tion of ovulation by a seminal plasma component.
roles of prostaglandins in these contractions have not Insect females dramatically increase their rate of egg
been tested, a large seminal glycoprotein, Acp36DE, production and egg laying after mating; in some
has been shown to be essential: Acp36DE mediates the insects, females only start producing eggs after mat-
opening of a constriction near the top of the uterus to ing (reviewed in [7, 13]). The egg production/laying
96
process increases at all steps, including oogenesis, can regulate the ovulation, movement and laying of
ovulation, egg transit and egg deposition. In D. eggs, but this process needs to be activated by seminal
melanogaster, it has been possible to tease apart the proteins from males. It will be interesting to keep this
regulation of the component steps of this increase in paradigm in mind when considering possible effects
egg production. For example, the seminal peptide SP of seminal proteins on uterine contractions and repro-
increases egg production [123, 124], including oogen- ductive tract physiology in women, as well as in other
esis, by acting through its receptor SPR [75], whereas mammals.
another seminal protein, ovulin, specifically increases
the ovulation rate in mated D. melanogaster females
[125–127]. Seminal Plasma Affects Immune Responses
Studies of ovulin’s action in inducing ovulation in in the Mammalian Female
Drosophila have uncovered general mechanisms that It would not be unexpected for proteins in seminal
could translate into ways seminal plasma components plasma and on sperm to affect immune responses
could influence (or even induce) ovulation in mam- in the female. After all, sperm and male-specific
mals. As in mammals, insect ovulation involves cell seminal plasma proteins would certainly be identi-
signalling, and ovulin does so through a neuromod- fied as foreign by the female’s immune system. Also,
ulator signalling pathway [128]. In female Drosophila, microorganisms, including pathogens, are commonly
the neuromodulator octopamine, a tyrosine derivative introduced into the female during coitus. Elimina-
functionally analogous to vertebrate norepinephrine tion of these organisms can be enhanced by seminal
[129], is essential for ovulation [130, 131]: female flies plasma introducing immune modulators or stimulat-
that lack octopamine, or that lack one of its receptors ing the female’s immune response [136]. Indeed, mam-
in the reproductive tract [132–134], cannot ovulate. malian and insect seminal plasma contains antimicro-
Genetic studies [128] show that ovulin induces ovu- bial peptides, part of the innate immune response. In
lation in D. melanogaster females by turning up their Drosophila, mating also induces synthesis of antimi-
octopaminergic signalling and increasing the den- crobial peptides in the reproductive tract [107], and
sity of synapses of octopaminergic neurons along the at least one seminal protein, the SP, has been shown
female’s reproductive tract. The increased octopamin- directly to have this effect [105, 109]. One could imag-
ergic signalling relaxes the muscles around the oviduct ine that antimicrobial peptides provided in seminal
(and potentially could affect the muscle contractions plasma, or whose synthesis is induced by seminal
observed on the ovary by Middleton et al. [135]), per- plasma in females, could protect the female’s repro-
mitting ovulation. ductive tract, and the gametes, from microbes intro-
The small size and reproducible reproductive duced during mating. In addition, zinc, which is found
physiology of Drosophila permitted further, more in high levels in human seminal plasma, has bacterici-
detailed studies of how this oviduct relaxation can dal activity [19].
increase ovulation. Interior views of reproductive
organs in situ, obtained via microcomputed tomog-
raphy (micro-CT) scans of Drosophila females at spe- Seminal Plasma Effects on Mammalian
cific times after mating [70] showed that the oviducts
of unmated females contain tight loops that impede
Females’ Immune Responses Prepare Them
release of eggs by the ovary. These loops relax after for Pregnancy
mating, due to the action of ovulin. Once the oviduct A less expected effect of seminal plasma on the female
loop is relaxed, the oviduct can straighten, allowing an mammal’s immune response is an apparent role in
egg to be released. increasing the female’s tolerance for antigens. This is
As noted above, Drosophila seminal proteins also thought to prepare the female for pregnancy, the ges-
cause changes in uterine shape and contraction. The tation of a foetus that is antigenically different from
micro-CT scans [70] showed that those changes, in the female, thereby supporting reproductive success.
conjunction with the relaxation of the oviduct loop For example, there is evidence in the mouse that expo-
caused by ovulin, can coordinate the subsequent sure of females to male antigens in semen improves
movement, release and further ovulation of eggs. These tolerance of females to subsequent exposure to var-
experiments suggest that a female’s reproductive tract ious paternal antigens in the embryos [137]. This
97
tolerance is mediated in part by regulatory T cells, risk of preeclampsia during pregnancy [143], although
which can suppress inflammatory immune responses the role of sperm in this effect has not yet been
[138]. TGF␤ and prostaglandin E (PGE) in semi- determined.
nal plasma can stimulate proliferation and activa-
tion of regulatory T cells [136]. In mice, it has been
demonstrated that this modulation of female immune
responses does not require sperm in the seminal
Sperm Are (at Least Temporarily) Protected
plasma. Vasectomized males induced the same level from Female Immune Responses
of response as intact males [139]. In contrast, surgical If seminal plasma stimulates immune responses, how
removal of the seminal vesicles significantly reduced do sperm manage to move so far into the female
the female response [140]. tract? It is quite plausible to assume that the mas-
Most of the research on female immune responses sive numbers of sperm inseminated by a male mam-
has been done utilizing mouse models. Since much mal overwhelm the female responses for a time. Fur-
of the seminal plasma introduced at coitus fills and thermore, sperm move rapidly beyond the initial site
expands the mouse uterus, one can imagine that sem- of insemination, which would also be the epicentre
inal plasma components have a greater opportunity to of immune stimulation. Nevertheless, the sperm do
affect the female reproductive system than in humans, receive some protection from the female by a surface
in which seminal plasma is naturally inseminated into coat that is eventually shed as sperm ascend the tract.
the vagina and only components that are carried on the In the human epididymis, complement regulatory gly-
surfaces of sperm through the cervical mucus reach the coproteins (CD59 and CD55) are secreted into the
uterus. seminal plasma and attach to the sperm surface via
Nevertheless, work done in humans and with GPI anchors ([144]; reviewed in [145], 2015). Tecle
human tissue has indicated that seminal plasma and Gagneux [145] suggest that these glycoproteins
induces some of the same responses in women as could protect sperm against attack by the complement
in mice. Seminal fluid components induce cytokine system.
and chemokine production by the female, resulting Even more importantly, specific ␤-defensins have
in recruitment of various leukocytes into the cav- been implicated in masking sperm surface antigens.
ities of the female tract. In women, responses to DEFB126 is a highly glycosylated ␤-defensin secreted
coitus have been characterized in the cervix. Biop- by the epididymis in macaque monkeys, which blocks
sies of the ectocervix revealed increases in an array binding of various antisperm antibodies to sperm,
of proinflammatory cytokines and chemokines and particularly due to sialic acid moieties on its glycans
a highly significant influx of CD45+ cells, primar- [146].
ily macrophages and dendritic cells, as well as an Interestingly, even though most insects lay eggs
increase of CD3+ CD8+ CD45RO+ T lymphocytes rather than experiencing a pregnancy where progeny
[141]. Altogether, the increases in signalling molecules develop within their bodies, there is also a systemic
and cells would initiate adaptations of the immune decrease in the Drosophila female immune capacity
response that would promote fertility, particularly if after mating. Mated female Drosophila show decreased
this response also occurred in the uterus. In vitro, cul- immunity to bacterial pathogens injected into their
tured endometrial epithelium responded to seminal main body cavities [105, 147]. It is unclear why the
plasma by up-regulating mRNA production of trans- female’s capacity to fight systemic infection decreases
forming growth factor-␤1 (TGF-␤1), interleukin-6 after mating, but it may be due to the need to reallocate
(IL-6) and leukaemia inhibitory factor (LIF) [142]; resources to egg production (reviewed in [148]), or
however, this has not yet been examined in vivo after to protection of her reproductive tract from microbes
coitus. A local response in the uterus must require introduced during mating, or both [149, 150]. Seem-
that sperm carry the seminal stimulatory molecules ingly paradoxically, seminal proteins increase produc-
through the cervix into the uterus. Nevertheless, a tion of some antimicrobial peptides in mated female
response in the cervix could well serve to protect the Drosophila [102, 109]. However, this increase may not
female against invasion of the cervix by pathogens occur in a site that is relevant for systemic immunity
from the vagina. There is also mounting evidence (for example, it could primarily be in her reproductive
that exposure of women to semen, via coitus, reduces tract [107]).
98
Seminal Proteins Influence Progeny the oviducts of females that had mated with (vasec-
tomized) SV-deficient males, those embryos took on
Phenotype (in Mice) characteristics of embryos that had been sired by SV-
The actions of seminal proteins to affect sperm viabil- deficient males. Bromfield et al. [9] propose that the
ity and storage and female physiology have the likely effects of SV secretions on oviduct molecular biol-
result of improving fertility – increasing the likelihood ogy (see above) or perhaps on the sperm epigenome
of successful fertilization(s). A recent study showed result in changes that support normal development of
that, at least in mice, secretions of the seminal vesicles embryos.
(SV) also affect the quality of the progeny that result
from those fertilizations [9].
Bromfield et al. [9] examined the fertility of females
Seminal Plasma May Have Additional
mated to males whose seminal vesicles had been Effects on Females
ablated. Consistent with the roles of seminal plasma As described above, seminal plasma has dramatic
components in promoting sperm storage, mates of effects on gametes and on the reproductive physiology
SV-ablated males produced fewer progeny. Although of mated females. But studies in insects suggest that
these females produced and ovulated normal numbers seminal plasma components may have effects outside
of oocytes, there was a 60% decrease in the number the reproductive tract. To our knowledge such effects
of those oocytes that were fertilized and reached the have not been tested for in humans or other mammals.
two-cell stage (relative to control matings) and a 90% The insect work, described briefly below, suggests that
decrease in the number of blastocysts in mates of SV- it may be of value to examine such effects in humans
ablated males. and other mammals.
Strikingly, though, the embryos, pregnancies and
progeny resulting from matings with SV-ablated males
were abnormal [9]. Blastocysts from these matings
Drosophila Seminal Plasma Affects Gut
were small and showed abnormal morphologies. For Physiology and Digestion
blastocysts that did continue to develop, the placentas Seminal plasma, and the Drosophila SP seminal pep-
were larger than those in pregnancies sired by control tide in particular, affects the rate and output of diges-
males. The larger placentas were interpreted by Brom- tion in the mated Drosophila female [151, 152]. Specif-
field et al. as indicating abnormalities in placental effi- ically, SP (acting through its SPR receptor) causes
ciency. And the progeny from pregnancies sired by SV- altered rate of food transit and excretion characteris-
depleted males were not normal. Initially, after birth, tics and increased food absorption. These changes have
pups from those pregnancies grew more slowly than been suggested to assist the female in obtaining max-
normal; later, after puberty, those pups gained weight imal resources from the food she consumes, improv-
faster than controls. Male progeny were disproportion- ing her capacity to make eggs at a high rate. A recent
ately affected, showing significantly more adipose tis- study [98] also showed that the gut is remodelled after
sue than male progeny from control sires. Sons of SV- mating, and that this requires the action of the isoter-
ablated males also showed signs of insulin resistance penoid hormone juvenile hormone (JH). Although as
and had 15% higher systolic blood pressure than sons yet this remodelling has not been tied to seminal pro-
of control males; the phenotype of sons of SV-ablated tein action, the SP induces an increase in JH levels in
males resembled that of metabolic syndrome. Interest- females, suggesting that SP might be involved. Inter-
ingly, female progeny did not show such drastic effects, estingly, effects of mating on digestion are not confined
although their muscle mass was lower than that in the to Drosophila; for example, Aedes mosquito females’
progeny of control matings. ability to digest a blood meal is also increased by
Bromfield et al. [9] showed that these effects were mating [153].
the consequences of effects of SV secretions on the
reproductive tract of the female. They could partially
rescue some of the effects on early embryos by flushing
Insect Seminal Proteins Influence Females’
them from the pregnant females and incubating them Postmating Behaviour
in vitro. Even more convincing, when Bromfield et al. Insect females show dramatic changes in behaviour
[9] transplanted embryos sired by a normal male into after mating. Many of these changes are induced by
99
molecules transferred by the male in his seminal fluid seminal plasma; this has been reported in mammals
(reviewed in [7, 13]). Some of these changes are consis- and in insects (reviewed in [166–168]). In the latter
tent with assisting the female in egg production/laying: case, different members of a gene family appear to have
females eat more after mating [154] and have different been co-opted to provide analogous functions in the
food preferences [155]. Seminal plasma components seminal plasma of different taxa [165].
have been associated with some of these responses. For Several, not mutually exclusive, hypotheses have
example, the increased feeding by Drosophila females been proposed for why these seminal proteins might
is due to effects of the SP [154]. Activity by mated evolve rapidly. First, it is possible that this variation
females also changes after mating. Mated Drosophila could contribute to species isolation. It is advanta-
undergo less siesta sleep [156] – again a consequence geous for a seminal protein to improve the reproduc-
of the seminal peptide SP [156] – which may give them tive capacity of a mating pair of the same species. But
more chances to find and use egg-deposition sites. interspecies matings result in nonviable (or infertile)
Insect females also show a decreased willingness progeny, so it might be advantageous that seminal pro-
to remate (reviewed in [7, 13]). This change, which is teins differ enough between species to further decrease
due to receipt of seminal plasma in all cases tested, the chance of unproductive fertilizations. Second, in
is one that can be seen to be particularly beneficial taxa where females mate multiply, sperm competition
to the male, whose sperm are less likely to be out- [169] could drive rapid evolution of seminal proteins
competed by another male’s if his mate avoids remat- [170]. Since it is to each male’s advantage to garner
ing. Female insects are changed in several different the largest number of fertilization opportunities, selec-
ways to avoid remating. Insect females that normally tion will favour males with improved capacity for their
produce pheromones that call in males to mate stop sperm to be stored and used efficiently. This could lead
making those pheromones after mating (reviewed in to continual selection for new or better versions of
[7, 13]; see also [157, 158]). In other cases, including seminal proteins that favour or enhance a male’s pater-
Drosophila, mated females undertake active rejection nity. In insects, the existence of sperm competition
behaviour such as kicking suitors away and extruding may contribute to selecting for seminal proteins that
their ovipositors, which physically prevents mating. In prevent remating by females. In this context it is inter-
Drosophila, the SP has been shown to mediate some of esting that the Drosophila SP seminal peptide, which
these changes, acting through its receptor SPR in par- inhibits remating, remains bound to sperm [79], and
ticular neurons [159–161], but pheromones that rub thus remains in the female as long as the male’s sperm
off the male [162], and at least one seminal fluid com- are present. A third force that could drive rapid evo-
ponent from the ejaculatory bulb [163], also decrease lution of seminal proteins is the conflict between the
remating behaviour by mated females. reproductive strategies of males and females (reviewed
To our knowledge, no analogous studies have been in [171]). Females generally put more resources into
carried out in mammals to directly test for effects of each individual gamete than do males, and mam-
seminal plasma components on behaviours; however, malian females put further resources into pregnancy.
it would not be surprising to us if specific behavioural Thus, per gamete there is a larger energetic toll on the
effects were discovered in mammals – or even in female’s health and reproductive longevity than on the
humans. male’s. Females may therefore be advantaged by mat-
ing less and/or being choosy in terms of mates and
Seminal Plasma Proteins Have sperm. In contrast, it is to males’ advantage to mate
with many females and to donate large numbers of
Interesting Evolutionary Dynamics sperm to gain the highest chance of fertilization oppor-
Biochemical and structural classes of seminal proteins tunities, particularly in competitive situations. These
are well conserved [8]. The same basic biochemical different strategies can result in a conflict between the
processes are critical in seminal plasma in all ani- interests of male and female. For example, consider
mals. In addition, some seminal plasma proteins are the stimulation of females’ egg production by insect
conserved among related species [51, 164, 165]. Yet males’ seminal plasma. It is advantageous to both part-
within this background of conservation, other seminal ners that females increase egg production after mating,
proteins show remarkable variation in primary amino when the male’s sperm are present to fertilize those
acid sequence or in presence/absence of orthologues in eggs (indeed it may be beneficial for females to use a
100
seminal plasma signal to turn up egg production). But ing whether a seminal plasma component might be
resources within a female may be optimal only for a of therapeutic use in human ovulatory disorders. In
lower level of egg-production than would be ideal for another example, the finding that mouse progeny phe-
her mate. This situation could select for females that notype quality is improved by molecular changes in
resisted the effects of the male molecule to a level that the female that are induced by seminal vesicle pro-
limited the up-regulation of egg production to tolera- teins suggests the relevance of testing whether sem-
ble amounts. In response, males with seminal plasma inal plasma components could improve outcomes in
proteins that overcame this female resistance could assisted reproductive technologies: exposing women
be selected for. This in turn could drive selection for to seminal plasma (or specific seminal components)
female resistance to the new male modification, and so before in vitro-fertilized embryos are implanted could
on. Ultimately this can result in an arms race driving be beneficial. Furthermore, given the rapid evolu-
rapid evolution of the male’s seminal proteins. tionary change in seminal proteins’ sequences and
the importance of seminal proteins for fertility, we
wonder if there could be cases of incompatibility
Conclusions between the seminal plasma proteins of a particular
Rather than simply a vehicle for transmitting sperm man and his partner’s receptors for those proteins.
to females, seminal plasma is an important and active This could potentially result in couple-specific subfer-
contributor to reproduction. It contains proteins and tility or infertility. In conclusion, continued research
other molecules that alter females’ reproductive phys- into functions of seminal plasma components could
iology, enhancing the opportunities for egg–sperm reveal new ways to diagnose infertility, improve out-
interaction. From a basic research standpoint, seminal comes of ART and protect couples against unwanted
plasma is a delivery vehicle for known and currently pregnancies.
unknown agents that affect the female. It will be impor-
tant to determine what these agents are, how they
together modulate the female’s reproductive capacity
Acknowledgements
and how they may serve as signals from male to female We thank Chris Barratt and Chris DeJonge for the
that mating has occurred. Understanding these traits invitation and opportunity to write this article, and
will be important not only for understanding repro- Frank Avila, Soon Hon Cheong, Robert Gilbert,
ductive biology, endocrinology and chemical commu- Laura Harrington, Brian Lazzaro, Akanksha Singh and
nication, but also for determining the nature of the Nancy Tisch for helpful comments and advice on the
evolutionary pressures that can cause rapid changes manuscript. We apologize to authors whose papers we
in the specific components of seminal plasma while could not cite due to limitations on citation number
conserving the biochemical classes of protein in semi- for chapters. We thank NIH Grants R01-HD070038 to
nal plasma of animals as different as humans and fruit SSS and R01-HD038921 to MFW for support of this
flies. work.
As we reviewed, seminal plasma components in
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Chapter
Physiological and Pathological Aspects of
7 Sperm Metabolism
Zamira Gibb and Robert John Aitken
Physiological Aspects of Sperm as relatively depleted of intracellular enzymes and

energy reserves such as fat droplets, yolk granules and
Metabolism glycogen [3]. For this reason, spermatozoa are heavily
Spermatozoa are highly specialized cells, playing the dependent on their immediate extracellular environ-
vital roles of paternal DNA delivery and activation of ment for the energy substrates that drive metabolism,
the oocyte following fertilization. The site of sperm as well as a variety of specialized enzymatic activities
deposition (in the vagina or uterus in the mammal) is that would normally be conducted intracellularly [4].
physically removed from the site of fertilization (the For example, in somatic cells, the array of enzymes and
oviduct), and while a proportion of sperm transport low-molecular-mass scavengers involved in mediating
is facilitated by uterine contractions (in mammals), protection against oxidative stress is housed intracellu-
the spermatozoa must in themselves be sufficiently larly, largely within the cytoplasmic space. Spermato-
motile to traverse the uterotubal junction and ulti- zoa, on the other hand, largely depend upon the epi-
mately locate a single cell, the oocyte. In addition, dur- didymal and seminal plasmas to provide the richest
ing their sojourn within the female tract, spermatozoa and most diverse combination of antioxidants in the
must undergo a maturation process called capacitation body, including several that are unique to the male
in order to attain the competence to recognize the egg reproductive tract [5, 6].
and then engage in a complex cascade of cell–cell inter- In much the same way that economies trade using
actions in order to achieve union of the gametes at fer- a currency rather than a barter system, biological sys-
tilization. This process involves extensive remodelling tems have all evolved their own unique ‘currencies’ for
of the sperm plasma membrane as well as the induc- the exchange of energy. The most important of these
tion of hyperactivated motility and, as such, is a highly currencies is adenosine 5’-triphosphate (ATP), which
energy-dependent process [1]. provides the metabolic energy to drive activities in all
The process of spermatogenesis requires extensive living cells.
remodelling of a conventional spherical cell to become
one of the most highly specialized and morphologi-
cally differentiated cells in the body. During this trans- Cellular Respiration
formation, the DNA in the sperm nucleus reaches The generation of ATP may be achieved either in the
the physical limits of compaction to achieve a quasi- presence (aerobic) or in the absence (anaerobic) of
crystalline state [2]. This extreme compaction requires oxygen. At the advent of life on Earth, the atmosphere
the removal or resorption of most of the cytoplasm, at was almost if not entirely devoid of oxygen, and at this
the same time removing the majority of the organelles time anaerobic glycolysis presented the only metabolic
(such as the endoplasmic reticulum, ribosomes and pathway by which organic molecules might be broken
Golgi apparatus) that are intimately involved in the down with the release of energy. However, as glycoly-
regulation of metabolism in somatic cells. The result sis typically requires large quantities of carbohydrates
of this extensive remodelling is that spermatozoa are such as sugars, which would not have been available
left translationally and transcriptionally silent, as well to the earliest life forms, it has been suggested that
University Press.
109
Chapter 7: Physiological and Pathological Aspects of Sperm Metabolism
during this period of substrate famine, the phospho- fuse across membranes, and their transport is facili-
rylation of adenosine diphosphate (ADP) to produce tated by membrane-bound proteins, which were first
ATP was driven by the energy released during the described by Kasahara and Hinkle in 1977 [10]. These
cleavage of the carbon–nitrogen bonds of amino acids proteins may be broadly divided into two groups,
such as glycine [7]. the ATP-dependant sodium-coupled glucose trans-
Following the emergence of eukaryotic plant cells porters (SGLTs) and the facilitative glucose trans-
as a result of the association between glycolytic and porters (GLUTs), which allow passive transport of sug-
photosynthetic prokaryotes, the levels of atmospheric ars across the membrane [10, 11]. Of these, GLUTs
oxygen began to increase, giving rise to oxidative are significantly more abundant and have received a
metabolism and the endosymbiosis of the mitochon- great deal more attention than SGLTs. GLUTs are cat-
drion to form the first animal cells. The ability to egorized according to their relative ability to trans-
exploit the highly reactive oxygen molecule as the port hexoses (such as glucose, mannitol and fructose),
driving force behind ATP production results in an amino sugars and vitamins [12]. Since the discovery
extremely effective pathway for energy generation of the glucose transporter GLUT1, a great many addi-
which, while significantly more efficient than utiliz- tional GLUTs have been characterized [13, 14]. While
ing glucose, produces significant quantities of reactive GLUTs 1, 2, 3 and 5 appear to be the most abun-
oxygen species (ROS) as a by-product [8]. dant GLUTs expressed by spermatozoa (Table 7.1)
As with somatic cells, the predominant metabolic [12, 14–18], GLUTs 8, 9a and 9b have also been
pathways that spermatozoa use to produce ATP are described [19, 20]. The pattern of GLUT distribu-
glycolysis and oxidative phosphorylation (OXPHOS), tion, which is largely confined to the acrosome and
with the preferential pathway utilized by spermato- principal piece of the sperm cell, suggests that gly-
zoa of various species depending on a number of fac- colytic processes are involved in generating energy for
tors, including oxygen and hexose availability [9]. The the membrane modifications required for hyperacti-
enzymes necessary for glycolysis are primarily asso- vation and the acrosome reaction. Should this be the
ciated with the fibrous sheath located in the princi- case, the distribution of GLUTs would be expected
pal piece of the tail [9]. In contrast, OXPHOS occurs to change with the functional status of the cell (i.e.
in the mitochondrial gyres located in the midpiece. between noncapacitated and capacitated states), a phe-
OXPHOS is a significantly more efficient method of nomenon which has been reported in the dog, but
ATP production than glycolysis. Despite this, sperma- has not been observed in other species [14]. At this
tozoa from most heavily researched species, includ- stage, the significance of active glycolytic pathways
ing humans and laboratory rodents, depend predom- in OXPHOS-dependent spermatozoa (e.g. equine) for
inantly on glycolysis for ATP production [9]. During the production of ATP for either motility or capac-
spermatogenesis, DNA is condensed to a crystalline itation and the acrosome reaction remains poorly
structure which not only provides mechanical protec- understood.
tion from ROS damage, but also allows the sperma- Despite the fact that spermatozoa are able to take
tozoa to become streamlined for ease of movement up sugars and utilize them as energy sources, the
[2]. During this process, the majority of the cytoplasm extracellular glycolytic substrate availability in vivo is
is removed from the cell, and as a result, spermato- scarce. The concentration of glucose and other reduc-
zoa have limited intracellular space to store energy ing hexoses in epididymal fluid is in trace or non-
reserves in the form of glycogen, lipid droplets or yolk detectable amounts [21, 22], and in the oviduct is
granules and are almost entirely dependent on exter- generally in micromolar concentrations [23]. How-
nal substrates such as fructose for glycolysis and lac- ever, in the dog at least, the ability of spermatozoa to
tate, citrate or succinate for OXPHOS. engage in gluconeogenesis by utilizing stored glyco-
gen may be sufficient to provide the glucose necessary
for glycolysis [24, 25]. Interestingly, in the presence
Glycolysis of a sufficient concentration of glucose, dog sperma-
The role of glycolysis in driving the production of ATP tozoa actively store glycogen [24], providing a buffer
for motility has been well researched due to its rel- against periods of hexose starvation. Seminal plasma
ative importance in humans and laboratory species. contains a relative abundance of fructose and glu-
Large polar molecules such as glucose cannot dif- cose [26], and the ability to store energy efficiently
110
Table 7.1 Physical distribution of glucose transporters in mammalian spermatozoa
GLUT Species Immunocytochemistry (+; strong, ±; weak, −; absent signals) Reference

Acrosome Postacrosomal Midpiece Principal piece

GLUT1 Human + ± − +
Rat + − − + [12]
Bull + − − +
Boar ± − − +
Stallion ± − − + [14]
Dog + − − ±
Donkey + − − ± [15]

GLUT2 Human − − + −
Rat + − − − [12]
Bull ± ± ± −
Boar + − − −
Stallion + − − + [14]
Dog + − ± +
Donkey + − − ± [15]

GLUT3 Human − + + +
Rat − − + ± [12]
Bull + + + −
Boar + + − ±
Stallion ± ± − + [14]
Dog − − − +
Donkey ± − − ± [15]
GLUT4 Human − − − −
Rat − − − − [12]
Bull − − − −

GLUT5 Human + − + +
Rat + + + ± [12]
Bull + + + ±
Boar + − + ±
Stallion + − + ± [14]
Dog + − + ±
Bat − − − + [18]
Donkey ± − − + [15]
GLUT8 Mouse + − + + [19]
Human + − − − [20]
GLUT9a Mouse − − + − [19]
GLUT9b Mouse + − + + [19]
during the brief exposure that spermatozoa have to Although mitochondrial inhibition studies have
this nutrient-rich fluid following ejaculation may pro- highlighted the contribution of OXPHOS to energy
vide the energy required to ascend the female repro- production by spermatozoa [27], deciphering the
ductive tract and fertilize the oocyte. Although glyco- relative importance of glycolysis to sperm function
gen, glycogen synthetase and glycogen phosphorylase is somewhat more problematic. Many studies have
have also been described in spermatozoa of the ram, attempted to quantify the relative contribution of gly-
boar and horse [24], gluconeogenesis mechanisms are colysis by inhibiting this process using the biolog-
yet to be demonstrated in any species other than the ically unavailable isomer 2-deoxy-D-glucose. While
dog. However, the ubiquitous distribution of GLUT this method does indeed result in a decrease in ATP,
and the presence of enzymes associated with gluconeo- it is a somewhat blunt instrument in that the depletion
genesis and glycogen synthesis suggest that the utiliza- of ATP is due to both the absence of usable hexoses and
tion of stored glycogen is likely to play a role, at least in ATP exhaustion through the futile phosphorylation of
the mammal, in the maintenance of energy homeosta- the unusable 2-deoxy-D-glucose by hexokinases – a
sis and sperm survival in vivo. highly energy-dependent activity [28]. A more direct
111
approach to the quantification of glycolytic processes of elucidating the mode of ATP generation by sper-
would be to perform flux and product distribution matozoa may be achieved through the inhibition of
measurements using 14 C-labelled glucose and eval- OXPHOS using mitochondrial uncouplers such as car-
uating the production of 14 C-labelled pyruvate and bonyl cyanide m-chlorophenyl hydrazone, Antimycin
lactate in the presence and absence of mitochondrial A, rotenone or diphenylene iodonium. In the stallion,
inhibition. OXPHOS inhibition results in over a 70% reduction in
sperm velocity and a 75% reduction in sperm ATP lev-
els, while human sperm velocity and ATP remain unaf-
Oxidative Phosphorylation fected. In addition, the greater efficiency of OXPHOS-
An excellent example of a species with OXPHOS- mediated ATP production supports a higher veloc-
dependent spermatozoa is the horse. Despite the well- ity, and indeed stallion sperm velocity parameters are
characterized presence of GLUTs on stallion sperm, it around 60% faster than those of glycolysis-dependent
has become abundantly evident that these spermato- human spermatozoa [27].
zoa differ from those of other well-studied mammalian Ultimately, high ROS production by OXPHOS-
species, in that their energy demands are met not by dependent spermatozoa appears to be a physiologi-
glycolytic pathways, but by using OXPHOS [27, 29, cally normal scenario brought about by superoxide
30]; in the presence of mitochondrial inhibitors, they leakage from the mitochondrial electron transport
suffer a rapid loss of velocity and a dramatic decline in chain during OXPHOS [29], with a positive rela-
ATP content, while glycolytic human spermatozoa dis- tionship between mitochondrial ROS production and
play no significant decrease in motility parameters or sperm velocity, leading to increased rates of lipid per-
ATP levels [17]. This dependence results in an uncon- oxidation [27] and, following prolonged storage, a
ventional positive relationship between reactive oxy- loss of motility and vitality [33]. This phenomenon
gen species (ROS) production and fertility in the stal- has a number of implications for the in vitro storage
lion [27, 29, 30], with the source of ROS being the of OXPHOS-dependent spermatozoa, since the pro-
mitochondrial electron transport chain, within which longed generation of ROS in the absence of extracel-
about 1–3% of O2 reduced in the mitochondria during lular free radical and lipid aldehyde scavengers will
OXPHOS forms superoxide [8]. lead to irreversible oxidative damage, impairing DNA
While human clinical data consistently report neg- integrity and sperm functionality.
ative correlations between male fertility and sperm
oxidative stress [31, 32], a recent study has revealed a
paradoxical inverse relationship between fertility and The Translocation of ATP around the
the percentage of live cells without oxidative dam-
age in the OXPHOS-dependent spermatozoa of the Sperm Cell
stallion [27]. In addition, spermatozoa from matings In the majority of somatic cells, the mitochondria are
which resulted in a conception (therefore considered located within the cytoplasm and are therefore able
to be more fertile) had lower vitality and a higher per- to deliver ATP from the site of production to the
centage of cells displaying ROS-induced damage than sites of utilization in an effective and efficient man-
spermatozoa from matings which did not result in a ner. This system is rather more problematic for cells
conception upon arrival at the laboratory. During in such as spermatozoa, in which compartmentalization
vitro storage and transport of the samples, the more has resulted in mitochondria being physically discon-
metabolically active spermatozoa from the more fer- nected from the fibrous sheath of the tail where ATP
tile stallions were becoming exhausted at a higher rate, is most heavily required for motility. This anatomi-
so that by the time that the assays were performed cal anomaly has led to an unwillingness on the part
in the laboratory, the cells had suffered an accelerated of many researchers to acknowledge that for many
demise due to the accumulation of metabolic byprod- species mitochondrial ATP production plays a vital
ucts, such as ROS and cytotoxic lipid aldehydes. Essen- role in the energy homeostasis of spermatozoa. While
tially, OXPHOS-dependent spermatozoa ‘live fast and several plausible simple ATP diffusion models have
die young’. been postulated [34–36], these models do not account
To avoid the introduction of artefacts following for the need to remove and recycle ATPase byproducts
glycolytic inhibition [28], a relatively simple method such as ADP, Pi and H+ .
112
It has been suggested that in spermatozoa the do not tolerate the stresses associated with chilling or
movement of ATP and ADP between the sites of pro- cryopreservation particularly well [48–51]. Therefore,
duction (the mitochondria and the cytoplasm) and the there has recently been a concerted effort to develop
sites of utilization occurs through flux transfer chains media that will extend the longevity of spermatozoa
[37]. These depend upon the rapid transfer of the dis- without the need to chill or cryopreserve [52–56].
placement from equilibrium of an enzyme reaction The major advantage of chilling semen is a reduc-
down a chain of adjacent enzymes, such that an ATP tion in sperm metabolic rate that results in improved
added at one end could effectively be removed at the longevity during transport and storage and limits
other end. Such enzymatic shuttle systems not only the growth of harmful bacteria. Temperature-induced
facilitate the delivery of ATP to the sites of utilization, reduction of sperm metabolism is of particular impor-
but also remove and recycle ATPase byproducts [37– tance in the case of OXPHOS-dependent spermato-
39]. This equilibrium displacement process may pro- zoa [27]. If metabolism is not curtailed in these cells
ceed with either creatine kinase or glycolytic reactions, by temperature reduction, OXPHOS will produce sig-
and is far more rapid and efficient than the diffusion of nificant quantities of ROS [8], which invariably com-
reactants [39–42], a feature of particular importance to promise sperm function [6, 33]. Second, depletion of
species with extremely long sperm flagella, such as the ATP is known to compromise a wide range of ATP-
rat [43]. dependent functions in spermatozoa that are neces-
sary to maintain homeostasis and prevent premature
Modulation of Metabolism: In vitro Storage cell death [57]. Therefore, it is clear that in an ambient-
temperature storage medium, mitochondrial energy
of Spermatozoa production must be supported while unnecessary ATP
In vitro sperm storage is often necessary for a num- depletion is minimized, as a result of pressure placed
ber of reasons associated with assisted reproduc- on ATP–dependent pathways such as the regulation of
tive technologies such as artificial insemination (AI) ionic or osmotic flux [46, 58].
and in vitro fertilization (IVF). During the final Supplementation of ambient-temperature semen
phases of spermatogenesis, spermatozoa lose the abil- extenders with various antioxidants [55, 56], nutri-
ity to biosynthesize, repair, grow and divide, becom- ent substrates and osmolytes [58] has gone a long
ing remarkably simple in their metabolic functions way toward ameliorating the detrimental effects of
[44]. Typically, sperm ageing and the inevitable senes- ROS production and ATP depletion in sperma-
cence that follows can be delayed or even arrested tozoa during storage at ambient temperatures. A
through the implementation of temperature-induced temperature-independent mechanism for inhibiting
metabolic restriction by chilling or cryopreservation. sperm metabolism would provide the ideal solution
The phenomenon of cold-induced sperm preserva- for ART systems, avoiding the irreversible membrane
tion was first discovered by Spallanzani in 1776 [45]. damage induced by cooling and freezing, while avoid-
He observed that cooling of frog, stallion and human ing the accumulation of toxic metabolic byproducts
semen in snow did not kill all the ‘spermatic ver- and the depletion of ATP. The clue to such a strategy
micules’, but rendered them temporarily immotile and may be found by taking a closer look at the in vivo
induced a state of lethargy from which they could environments that are conducive to sperm longevity,
recover when returned to higher temperatures. By namely the epididymis and the oviductal isthmus,
restricting the metabolic rate of cells, the produc- where spermatozoa are stored in a quiescent state for
tion of toxic metabolic byproducts, such as hydro- considerable periods of time.
gen peroxide, lipid aldehydes and carbon dioxide, is
reduced and the depletion of ATP associated with Pathological Aspects of Sperm
the maintenance of homeostasis [44, 46, 47] is min-
imized. This temperature-induced metabolic restric- Metabolism
tion reduces the rates of both ROS production and In parallel with recent advances in our understanding
acidification of the storage medium through the accu- of the metabolic pathways that drive normal sperm
mulation of lactic acid and CO2 from glycolysis and function, we have seen significant advances in our
OXPHOS, respectively. However, the spermatozoa of understanding of the metabolic mechanisms responsi-
many stallions, human male patients and other species ble for defective sperm function. In this context there
113
is a general consensus that one of the major mecha- (ROO•) and alkoxyl (RO•) radicals that, in order to
nisms responsible for loss of sperm motility, fertiliz- stabilize, abstract a hydrogen atom from an adjacent
ing potential and DNA integrity is oxidative stress. In carbon, generating the corresponding acid (ROOH) or
many ways ROS are a two-edged sword when it comes alcohol (ROH). The abstraction of a hydrogen atom
to the regulation of normal sperm function. They are from an adjacent lipid creates a carbon-centred radical
required in small quantities to promote cellular pro- that combines with molecular oxygen to create another
cesses such as capacitation [59], but when generated in lipid peroxide. In order to stabilize, the latter must
excess, they can become extremely damaging because abstract a hydrogen atom from a nearby lipid, creat-
of their promiscuous reactivity. In the remainder of ing yet another carbon radical that on reaction with
this chapter we shall examine both aspects of these bio- molecular oxygen will generate more lipid peroxides.
logically important molecules in the context of sperm In this manner, a chain reaction is created that propa-
cell biology. However, in order to set the scene, we shall gates the peroxidative damage throughout the plasma
first overview the fundamental chemistry of ROS and membrane.
consider the difficulties encountered in detecting these Since the hydrogen abstraction process referred to
short-lived but highly reactive molecules. above is facilitated by the double bonds present in
unsaturated fatty acids, membranes that are rich in the
latter will be particularly vulnerable to oxidative stress.
What Are Reactive Oxygen Species? In this context, spermatozoa are especially susceptible
The term ‘reactive oxygen species’ covers a range of because their plasma membranes are extremely rich
metabolites that are derived from the reduction of oxy- in unsaturated fatty acids, notably 22:6 [60]. Such an
gen, including free radicals, such as the superoxide abundance of unsaturated lipids is necessary to cre-
anion (O2− •) or the hydroxyl radical (OH•), as well as ate the membrane fluidity required by the membrane
powerful oxidants such as hydrogen peroxide (H2 O2 ). fusion events associated with fertilization (acrosomal
The term also covers molecular species derived from exocytosis and sperm–oocyte fusion); however, their
the reaction of carbon-centred radicals with molecu- presence leaves these cells open to peroxidative attack.
lar oxygen, including peroxyl radicals (ROO•), alkoxyl Termination of such lipid peroxidation chain reactions
radicals (RO•) and organic hydroperoxides (ROOH). can be achieved with chain-breaking antioxidants such
The term ‘ROS’ may also refer to other powerful oxi- as vitamin E (␣-tocopherol). The latter is extremely
dants such as peroxynitrite (ONOO-) or hypochlorous effective in terminating lipid peroxidation cascades in
acid (HOCl) as well as the highly biologically active human spermatozoa in vitro [61] and has also been
nitrogen free radical nitric oxide (•NO). shown to improve the fertility of males selected on the
The specific term ‘free radical’ refers to any atom basis of high levels of lipid peroxidation in their sper-
or molecule containing one or more unpaired elec- matozoa, in vivo [62].
trons. As unpaired electrons are highly energetic, and The most commonly encountered ROS are O2− •
seek out other electrons with which to pair, they confer and H2 O2 . These molecules are capable of a range
considerable reactivity upon such radicals. Thus, free of rapid chemical reactions yielding a correspond-
radicals and related ‘reactive species’ have the ability ingly broad range of reaction products. When in aque-
to react with, and modify, the structure of many dif- ous solution, O2− • has a short half-life (1 ms) and is
ferent kinds of biomolecules including proteins, lipids relatively inert. The radical is more stable and reac-
and nucleic acids. The wide range of targets that can tive in the hydrophobic environment provided by cel-
be attacked by ROS is a critical facet of their chem- lular membranes. The charge associated with O2− •
istry that contributes significantly to the pathologi- means that this molecule is generally incapable of
cal importance of these molecules. As most chemi- passing across biological membranes, although there
cal species in biological systems have only paired elec- are reports of this molecule passing through voltage-
trons, free radicals are also likely to be involved in dependent anion channels [63]. As a result of its lack of
chain reactions, whereby new free radical products are membrane permeability, O2− • may be more damaging
formed. A classic example of such a chain reaction is if produced inside biological membranes than at other
the peroxidation of lipids in biological membranes. In sites. It is also important to note that while O2− • can act
this process, a ROS-mediated attack on unsaturated as either a reducing agent or a weak oxidizing agent in
fatty acids in the plasma membrane generates peroxyl aqueous solution, under the reducing conditions that
114
characterize the intracellular environment, O2− • acts The sum of these two reactions represents the iron-
primarily as an oxidant. Many of the effects of O2− • are catalyzed Haber–Weiss reaction:
believed to arise from its conversion to more reactive
oxidizing species [8, 64]. For example, protonation of H2 O2 + O2− • → O2 + OH− + OH•. [4]
O2− • forms the hydroperoxyl radical (HO2 •), a much Thus, O2− • also has a key role to play in the above
stronger oxidant: reaction by serving as a reductant and facilitating the
HO2 • H+ + O2− •. [1] regeneration of reduced metal ions in the extracellu-
lar space. It is also well recognized that other transi-
The pH at which this reaction reaches equilibrium tion metals may participate in these reactions. Thus,
(pKa) is 4.8, with the result that at physiological pH, while iron is the major player [69], copper is the other
HO2 • represents less than 1% of the O2− • present in major candidate and cobalt, aluminium, chromium,
a cell. However, given the considerable reactivity and nickel and titanium may also participate in such reac-
membrane permeability of HO2 •, this radical is still tions [70]. Moreover, in seminal plasma, both iron and
believed to be a significant contributor to oxidative copper are available in a free state and hence able to
damage in biological systems, with the potential to ini- take part in OH• production and the consequent pro-
tiate lipid peroxidation cascades [65, 66]. motion of oxidative stress in the ejaculate [71].
Conversion of O2− • to other ROS also occurs. An
important means by which this takes place is the dis-
mutation reaction, wherein O2− • reacts with itself (i.e. Detection of ROS in the Male Germ Line
superoxide is both oxidized and reduced). In this sit- Given the importance of ROS and the apparent vul-
uation, one molecule of O2− • is oxidized to molecular nerability of spermatozoa to oxidative stress, it might
oxygen, while the other is reduced to H2 O2 : be anticipated that sophisticated methods would have
been developed to detect these intermediate oxygen
O2− • + O2− • + 2H+ → H2 O2 + O2 . [2] metabolites for diagnostic purposes. In fact, this area
Superoxide dismutase (SOD) catalyzes this conver- has been severely compromised by the absence of sen-
sion. SODs are metalloenzymes thought to be present sitive, accurate analytical methods capable of confirm-
in all oxygen-metabolizing cells [67]. The reaction can ing the presence of specific ROS in biological systems.
occur spontaneously without SOD; however, in its The most commonly used method for detect-
absence, dismutation will proceed much more slowly ing ROS in an andrological context is chemilumi-
due to the electrostatic repulsion of the anions (rate nescence, using the probe lucigenin or luminol [72,
constant of about 5 × 105 M−1 s−1 at physiological pH). 73]. Lucigenin (N,N’-dimethyl-9,9’-biacridinium dini-
SOD is an efficient catalyst that will drive the above trate) carries a positive ionic charge; it is generally
reaction at a rate constant of about 1.6 ×109 M−1 s−1 thought to be relatively membrane-impermeant and to
over a wide pH range (5.3–9.5). In the human sperma- respond to O2− • in the extracellular space. However,
tozoon, there is sufficient SOD activity to account for the positive charge associated with this molecule may
all of the H2 O2 produced by these cells [68]. also favour its partition into mitochondria, as a con-
Superoxide formation can also lead to the genera- sequence of the electronegative mitochondrial mem-
tion of other types of highly reactive species apart from brane potential. Indeed, studies using rat spermatozoa
H2 O2 . Many transition metal ions are able to partici- as a model indicate that the lucigenin signal generated
pate in these processes, as they possess variable oxida- by these cells can reflect O2− • produced by the sperm
tion numbers, permitting them to change their redox mitochondria [74]. However, there are no data to sug-
status by either gaining or losing an electron. Conse- gest that the lucigenin signals generated by human
quently, transition metals act as very effective promot- spermatozoa are of mitochondrial origin, even though
ers of free radical reactions. For example, in the Fenton such signals are inversely correlated with sperm quality
reaction, H2 O2 undergoes decomposition in the pres- [75] and significantly elevated in cases of male infertil-
ence of ferrous ions to produce the pernicious hydroxyl ity [76, 77].
radical (OH•): One of the key features of lucigenin is that this
probe must undergo a one-electron reduction to the
H2 O2 + Fe2+ → Fe3+ + OH− + OH•, radical species, LH+ •, before it becomes sensitized
[3]
Fe3+ + O2− • → Fe2+ + O2 . to the presence of O2− • (Figure 7.1). In the case of
115
NAD(P)H
Cellular O2 -•
Lucigenin reduction
e.g., Cytochrome P450
reductase
+ +• = +
LucH+• LucH LucH2 Luc2+
Cytochrome b5 reductase
+1e- HO2-
LucH+• + O2_•
LucH+• Luc2+
Dioxetane formation
-1e- SOD
Chemiluminescence O2 O2-• H2O2
Figure 7.1 Schematic representation of the chemistry for lucigenin chemiluminescence; Luc2+ : lucigenin; LH+ •: a lucigenin radical created
by the one–electron reduction of Luc2+ . The reaction of LH+ • with oxygen generates O2− •. The latter then participates in an oxygenation
reaction with LH+ •, generating a dioxetane that decomposes with the generation of chemiluminescence. Any entity that can effect the
one-electron reduction of lucigenin can potentially create a redox cycle in the presence of oxygen that produces high levels of O2− • and
chemiluminescence. It is impossible to distinguish the relative contribution of such probe-dependent and cell-dependent
chemiluminescence. Hence data obtained with this probe should be interpreted with caution.
mitochondrial O2− • production, this reductive process great deal of data to link cytoplasmic retention with
is accomplished by the organelle’s electron transport defective sperm function [80–83], so such an explana-
chain. However, outside of the mitochondria, luci- tion would be fully compatible with our understanding
genin reduction can be induced by reductases such as of the etiology of male infertility.
cytochrome P450 reductase or cytochrome b5 reduc- It has also been argued that the reaction of LH+ •
tase [78]. This is particularly the case when exogenous with O2 is thermodynamically unlikely [84] and that
NAD(P)H is used to drive redox activity in popula- redox cycling of this probe does not occur in biologi-
tions of human spermatozoa [79]. The LH+ • generated cal systems. However, given the high dose of lucigenin
on reduction then combines with O2− • to produce the typically used to detect redox activity in human sperm
dioxetane that, in turn, decomposes with the genera- samples (250 µM), the possibility of spurious results
tion of light (chemiluminescence): generated by continuous cycling of the probe cannot
Although this chemistry seems straightforward, be excluded. As a consequence, we currently do not
complications may arise due to redox cycling reac- know the extent to which the elevated lucigenin sig-
tions whereby LH+ • combines with ground state oxy- nals detected in defective human spermatozoa reflect
gen (O2 ) to create O2− • and regenerate the parent luci- primary O2− • production or the superabundance of
genin molecule (Figure 7.1). The O2− • artificially cre- reductases due to the presence of excess cytoplasm.
ated in this manner will then combine with LH+ • to What we do know is that the activity of this probe cor-
generate additional dioxetane and further the chemi- relates well with defective sperm function whether the
luminescence response (Figure 7.1). If such redox activity is promoted by treatment with NAD(P)H or
cycling does occur, the particularly intense NADPH- phorbol ester [75, 77, 85, 86].
dependent lucigenin signals seen in defective human A similar argument may apply to luminol. This
spermatozoa may be as much an indication of exces- probe has to undergo a one-electron oxidation before
sive reductase activity as evidence for the overabun- it becomes sensitized to the presence of ROS. In a
dance of O2− • [75]. This explanation would provide a common form of this assay, horseradish peroxidase is
link between the high levels of redox activity detected used to promote luminol oxidation in the extracellular
in defective spermatozoa by lucigenin chemilumines- space. In this form, the luminol assay largely reflects
cence and enhanced reductase activity due to the pres- the presence of H2 O2 released to the outside of the cell.
ence of excess residual cytoplasm. There is certainly a In the absence of exogenous horseradish peroxidase,
116
NAD(P)H
Cellular O2−.
Lucigenin reduction
e.g., Cytochrome P450
reductase
LucH+
.
+ LucH+
.
= LucH2 + Luc2+
.
Cytochrome b5 reductase
+ +1e − HO2−
O2−.
.
LucH+
. .
LucH+ Luc2+
Dioxetane formation
−1e − SOD
Chemiluminescence O2 O2 −. H2O2
Figure 7.2 Schematic representation of the underlying chemistry for luminol-dependent chemiluminescence. L: luminol; L•: a luminol
radical created by the one-electron oxidation of L; L+ : azaquinone formed by the further one-electron oxidation of L• by oxygen, generating
O2− • as a byproduct. The reaction of L• with O2− • or L+ with H2 O2 generates an unstable endoperoxide whose decomposition leads to
production of the chemiluminescent species, an electronically excited aminophthalate. Redox cycling of the probe could result if human
spermatozoa possessed an appropriate reductase to convert L+ back to the parent L. Any reactant that can achieve the univalent oxidation of
luminol will generate chemiluminescence in this assay, including H2 O2 and ONOO_ .
the assay is dependent on the presence of intracellu- quinone (L+ ) and thereby contribute to the formation
lar peroxidases to activate the probe [72]. The one- of excited aminophthalic acid, the chemiluminescent
electron oxidation of luminol leads to the creation of species [87].
a radical species (L•). The latter then interacts with Fundamentally, luminol-based assays are mea-
ground state oxygen to produce O2− •, which induces suring redox activity characterized by the cellular
the oxygenation of L• to create an unstable endoper- generation of oxidizing species capable of creat-
oxide, which ultimately breaks down with the release ing L•. Notwithstanding the reservations that might
of light (Figure 7.2). According to this scheme, O2− • be expressed concerning the specificity of this probe,
is an essential intermediate in the creation of luminol- the luminol assay is robust and generates results that
dependent chemiluminescence and it is for this reason are strongly correlated with sperm function [31, 88].
that SOD is such an effective inhibitor of this reaction The clinical significance of this assay has also been
cascade. However, the activity of this scavenger should emphasized in a long-term prospective study of 139
never be taken to indicate the primary production of couples characterized by a lack of detectable pathol-
O2− • by human spermatozoa; O2− • is simply an artifi- ogy in the female partners. In this cohort of patients,
cially created intermediate that is essential for luminol- a negative association was observed between luminol-
dependent chemiluminescence. Indeed, any univa- dependent chemiluminescence and the incidence of
lent oxidant has the potential to generate O2− •, and spontaneous pregnancy [89]. Furthermore, within this
hence chemiluminescence, in the presence of lumi- data set, the conventional criteria of semen quality
nol, including ferricyanide, persulphate, hypochlorite, were of no diagnostic value whatsoever [89].
ONOO– and xanthine oxidase (Figure 7.2). Hydrogen A recent detailed analysis of these chemilumines-
peroxide lies upstream of O2− • in the reaction scheme cent probes confirmed that lucigenin is not a par-
depicted in Figure 7.2 and its involvement in the ini- ticularly useful probe for measuring ROS generation
tial oxidation of luminol accounts for the inhibitory by human spermatozoa [90]. Furthermore, while the
effects of catalase on this form of chemiluminescence. combination of luminol and peroxidase was found
In addition, H2 O2 will also react directly with aza- to be a sensitive means of detecting ROS in the
117
A C
DHE
O2• -
Oxid/Red
Ethidium
2OH-Ethidium
B
−2.40 Eth 2-OH Eth
−2.50
mV
−2.60
−2.70
−2.80
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00
212 Minutes, −2.7629 mV Minutes
Figure 7.3 Use of DHE as a flow cytometry probe for detecting the generation of O2− • by populations of spermatozoa. A, this probe can be
non-specifically oxidized to generate the parent ethidium (Eth); however, reaction with O2− • generates a ROS–specific DNA-sensitive
fluorochrome, 2-hydroxyethidium (2OH Eth). B, hplc analysis of the fluorochromes generated by human spermatozoa has demonstrated the
presence of both Eth and 2-OH Eth, confirming the generation of O2− • by these cells. C, the combination of DHE and Sytox green (green or
yellow where cells are nonviable) provides an extremely efficient means of demonstrating ROS generation by viable cells (nuclei stained red
with no trace of yellow or green staining). (A black and white version of this figure will appear in some formats. For the colour version, please
refer to the plate section.)
extracellular space, it was also found to be very molecule a positive charge that results in its concen-
susceptible to interference by free-radical-generating tration in the mitochondrial matrix. When run in
leukocytes [90]. Differentiation of the luminol signal conjunction with a vitality stain such as Sytox green
generated by human sperm suspensions into the com- (Figure 7.3), these probes provide a sensitive, effec-
ponent generated by leukocyte contamination and the tive and accurate means of assessing ROS genera-
component generated by defective spermatozoa still tion by spermatozoa [90]. A potential problem with
confounds the interpretation of studies employing this DHE and MSR is that they can be non-specifically
probe. oxidized to generate the parent ethidium molecule
The problem of cellular contamination has been and a positive signal in the assay. In order to be cer-
solved by the use of flow cytometry to focus on the tain that the activity probes are reflecting intracellu-
cellular generation of ROS by spermatozoa while gat- lar ROS generation, it is important to establish that 2-
ing out any contribution made by other cell types. The hydroxyethidium is being generated in the presence of
most commonly used flow cytometry probes are dihy- spermatozoa, since this reaction product is only pro-
droethidium and mitoSox red (MSR). These probes duced when these probes are oxidized by O2− •. Frac-
are both reduction products of ethidium bromide, tionation of the fluorochromes generated by human
but MSR has been chemically modified to give the spermatozoa, followed by mass spectroscopy, has
118
A 15 hour incubation in vitro B Xanthine oxidase system

y = −39.396x + 90.682, r = 0 .70 y = −63.633x + 110.083, r = 0.88
120 120
100 100
80
80
60
60
40
40
20
0 20
0
0 .5 1 1.5 2 2.5 3 0 .2 .4 .6 .8 1 1.2 1.4
MDA +4HA (µmol) MDA +4HA (µmol)
Figure 7.4 Sperm motility is negatively impacted by lipid peroxidation. Using an assay that records the generation of malondialdehyde and
4-hydroxyalkenals (MDA+4HA), it is possible to demonstrate a clear negative correlation between sperm motility and A, the generation of
lipid peroxides by spermatozoa following an overnight incubation at 37°C and B, the enzymatic creation of oxidative stress using xanthine
oxidase [96].
confirmed the generation of 2-hydroxyethidium and a correlation between the lipid peroxide content of
thus O2− • by these cells [91] (Figure 7.3). With MSR as human spermatozoa and severe motility loss. This rela-
a probe to assess the ability of human sperm mitochon- tionship between motility loss and oxidative stress
dria to generate O2− •, electron leakage from both com- is striking and has been repeatedly demonstrated in
plex I and complex III has been demonstrated [92]. independent studies [31, 32, 61, 68, 94, 95]. Thus expo-
When the generation of O2− • occurs on the matrix side sure of human spermatozoa to extracellularly gener-
of the inner mitochondrial membrane at complex I, the ated ROS induces a loss of motility that is directly
result is the induction of lipid peroxidation and motil- correlated with the level of lipid peroxidation experi-
ity loss, whereas ROS generation at complex III leads enced by the spermatozoa [96] (Figure 7.4). Similarly,
to the rapid formation of hydrogen peroxide, which the loss of motility observed when spermatozoa are
rapidly exits the cell and enters the extracellular space subjected to overnight incubation is highly correlated
[91]. MSR and DHE are excellent probes for mito- with the lipid peroxidation status of the spermatozoa
chondrial and total cellular ROS respectively, respond- at the end of the incubation period [96] (Figure 7.4).
ing readily to stimulants of ROS generation such The prognostic value of stress tests based on the loss
as lipid aldehydes, menadione and catechol oestro- of motility observed when spermatozoa are incubated
gens [90]. Recently, novel boronate probes have been for defined periods of time in the presence of transi-
reported for human spermatozoa that are more sen- tion metals [97] is probably another reflection of the
sitive for the detection of ROS than DHE, MSR importance of lipid peroxidation in the modulation of
and 2’,7’-dichlorohydrofluorescein diacetate (DCFH). sperm function. The ability of antioxidants to preserve
These reagents may well have clinical utility for the sperm motility in vivo and in vitro is yet more evi-
diagnosis of oxidative stress in the male germ line [93]. dence that lipid peroxidation is a major cause of motil-
ity loss in populations of human spermatozoa [62].
Impact of Oxidative Stress on Spermatozoa Recently, studies have been conducted using animal
The clinical significance of oxidative stress in the eti- models exhibiting infertility associated with oxidative
ology of defective sperm function was first indicated stress, such as the GPx5 knock-out mouse and testic-
by Thaddeus Mann and colleagues at the University of ular heating, that have clearly demonstrated the thera-
Cambridge, 37 years ago [60]. These authors observed peutic potential of antioxidants in vivo [98, 99].
119
The mechanisms by which lipid peroxidation leads tamine cross-linkage lead to imperfections in the state
to motility loss probably involve changes in the flu- of chromatin stabilization. Such deficiencies in chro-
idity and integrity of the plasma membrane and a matin packaging have, in turn, been associated with
subsequent failure to maintain membrane functions an increased risk of DNA damage [105]. The partic-
critical to flagellar movement. Disruption of mem- ular vulnerability of poorly compacted sperm DNA to
brane Ca2+ /Mg2+ ATPase activity as a consequence of oxidative attack results in high levels of the oxidative
decreased membrane fluidity would, for example, lead base adduct, 8-hydroxy-2’-deoxyguanosine (8OHdG),
to motility loss secondary to an increase in intracellu- being present in human spermatozoa [106, 107]. The
lar calcium. In addition, the electrophilic lipid aldehy- spermatozoon has a limited capacity to deal with this
des generated as a consequence of lipid peroxidation damage, possessing a truncated base excision repair
are known to form adducts with the nucleophilic cen- pathway characterized by only the first enzyme in this
tres of proteins involved in the orchestration of sperm process, 8-oxoguanine DNA glycosylase 1 (OGG1),
movement, such as the dynein heavy chain [100]. Of but none of the other downstream elements of this
course, lipid peroxidation will also disrupt all sperm DNA repair pathway [108]. As a result, spermatozoa
functions dependent on membrane activity, includ- are capable of responding to oxidative DNA damage
ing sperm–oocyte fusion and the ability to undergo a by creating an abasic site at the oxidized base posi-
physiological acrosome reaction [61]. tion, but cannot progress the DNA repair any fur-
Oxidative stress is also a major cause of DNA dam- ther (Figure 7.5). In contrast, the oocyte possesses
age in human spermatozoa. Using quantitative PCR to very low levels of OGG1 but does possess the down-
calculate lesion frequency, the mitochondrial genome stream components of the base excision repair path-
has been shown to be much more susceptible to DNA way [109]. The male and female germ lines therefore
damage than the nuclear genome [101]. As a con- collaborate in resolving any oxidative DNA damage
sequence, the integrity of the sperm mitochondrial brought into the egg by the fertilizing spermatozoon.
genome is an excellent marker of oxidative stress, even However, if the fertilizing spermatozoon has partic-
though this genome is of no biological significance ularly high levels of 8OHdG, as is often the case in
in its own right because sperm mitochondria do not the subfertile population [106], then these residues
generally replicate after fertilization. When quantita- will remain associated with the chromatin of the
tive PCR was used to compare the lesion frequencies sperm following fertilization and, because the oocyte
induced in spermatozoa and a variety of other cell is so poorly endowed with OGG1, the residues will
types following exposure to H2 O2 , the nuclear genome persist into the S-phase of the first mitotic division
of the male gamete was shown to be particularly resis- (Figure 7.5). At this point the high mutagenic poten-
tant to oxidative damage. This resistance is thought to tial of 8OHdG, particularly its ability to create GC–
mirror the unique manner in which nuclear chromatin AT transversions, will have a major impact on the
is packaged in spermatozoa, as reflected in the high mutagenic load carried by the embryo [110]. Some
levels of irradiation required to damage sperm DNA measure of protection against such an eventuality has
compared with somatic cells [102]. been recently indicated in a study [109] reporting an
Adequate compaction and stabilization of sperm increase in oocyte OGG1 activity following fertiliza-
nuclear DNA is therefore critical for protecting this tion as a consequence of post-translational modifica-
material from oxidative stress. Amongst Eutheria, tions to oocyte enzymes involved in the base exci-
spermatozoa of human origin appear to be more sus- sion repair pathway, causing nuclear localization and
ceptible to DNA damage than those of most other accelerated 8OHdG excision. Notwithstanding such
species. This is largely because the P2 protamine, char- changes within the fertilized egg, the low levels of
acteristic of human spermatozoa, has a limited num- OGG1 characteristic of the oocyte emphasize the vul-
ber of thiol groups for disulphide bonding [103]. Fur- nerability of embryonic development to high levels
thermore, the protamination of human spermatozoa of 8OHdG carried into the oocyte by the spermato-
is notably inefficient, with around 15% of the genome zoon (Figure 7.5). This phenomenon has major impli-
remaining histone-rich, even in normal fertile men cations for the assisted conception industry, where
[104]. Failures in either the ability of the testes to ade- potentially damaged spermatozoa are being used to
quately protaminate human sperm chromatin or the create embryos through the increasingly popular prac-
ability of the epididymis to support subsequent pro- tice of ICSI.
120
A
Base excision pathway
OGG1 Oxidatively
damaged base Damaged base
B
Abasic site
OGG1 glycosylase OGG1 APE1 XRCC1
Spermatozoa possess OGG 1

but none of the other enzymes APE1/XRCC1 AP endonuclease
in the base excision repair
pathway 3’ OH 5’ dRP Oocytes possess very little OGG 1 but do express
abundant APE 1and XRCC1, the downstream
contributors to the base excision repair pathway
Figure 7.5 Vulnerability of embryonic development to oxidative DNA damage in the sperm nucleus. A, spermatozoa possess only the
first enzyme in the base excision repair pathway, OGG1, but none of the other downstream elements of this repair process. As a result,
spermatozoa are capable of responding to oxidative DNA damage only by the generation of an abasic site. B, oocytes possess the
downstream elements of the base excision repair pathway, APE1 and XRCC1, in abundance. Thus under ideal conditions, male and female
gametes collaborate to repair oxidative DNA damage brought into the egg by the fertilizing spermatozoon. However, the lack of OGG2 in
oocytes means that any 8OHdG residues that have not been dealt with in the spermatozoon will persist into the S-phase of the first mitotic
division, with potential mutagenic consequences for the embryo. (A black and white version of this figure will appear in some formats. For the
colour version, please refer to the plate section.)
Conclusions 4. Bucci D, Rodriguez-Gil JE, Vallorani C, Spinaci M,

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Chapter
Regulation of Sperm Behaviour
8 The Role(s) of [Ca2+]i Signalling

Stephen Publicover
Introduction a
In almost every known animal species, sexual repro- 50 μm
duction requires the active movement of the sperm to c
the oocyte. During external fertilization, when male e
and female individuals shed large numbers of gametes
into the water, currents may assist gamete mixing by
stirring this ‘soup’, but motility of the sperm is essen-
tial to bring male and female gametes into contact. b
For sperm of internal fertilizers the task is apparently f
simpler, since the cells are introduced directly into the
female tract. However, the tract is an inhospitable envi-
ronment that does not favour migration of sperm and d
limits the number of sperm that reach the site of fertil-
ization [1, 2]. This filtering process not only reduces the
Figure 8.1 Behaviour of human sperm. (a, b) Activated and
probability of polyspermy, but favours progress of cells hyperactivated human sperm, respectively. (c–e) Tracks of sperm
that are functionally normal [1, 3]. As a sperm ascends (constructed by tracing the position of the sperm head in each
the female tract it must travel thousands of times its video frame) showing activated (c), transitional (d), and
hyperactivated (e) motility. The track in (f) is from a sperm showing
own length through fluids and viscous mucus, interact star-spin motility.
with the uterine and oviductal linings, find the oocyte,
and penetrate the surrounding cumulus and zona pel-
concentrating on internal fertilizers (mammals, par-
lucida. These different aspects of the sperm’s progress
ticularly man), but non-mammalian models, particu-
through the female tract vary in their mechanical and
larly the sea urchin, are also considered.
physiological requirements, but they are all essential;
any failure is probably sufficient to prevent fertiliza-
tion. Diverse motility patterns (behaviours) must be Sperm Behaviour
adopted by the sperm as they are needed. The ability of Freshly ejaculated mammalian sperm swim with a
sperm to detect and respond to cues from the tract and low-amplitude, symmetrical flagellar beat that gen-
cumulus–oocyte complex with appropriate behaviour erates progressive movement in low-viscosity fluid
is therefore a significant determinant of which, if any, (Figs. 8.1a, 8.1c). This pattern of movement, termed
reach the site of fertilization. In most species sperm activated motility, enables rapid progress of the sper-
achieve motility through the beating of a single flag- matozoa within the seminal plasma and penetra-
ellum [4], which drives the nucleated sperm head. tion into cervical mucus. Within the female tract,
Regulation of the flagellar beat provides the various sperm may adopt a number of behaviours, including
behaviours that the sperm requires. Here we consider activated motility and a strikingly different whiplash
what is known about regulation of sperm motility and type of movement termed ‘hyperactivation’, which is
the central role of [Ca2+ ]i signalling in this process, observed in cells retrieved from the oviduct [5]. This
University Press.
126
Chapter 8: Regulation of Sperm Behaviour: The Role(s) of [Ca2+ ]i Signalling
behaviour, which is normally associated with capaci- terns are loosely defined and almost certainly conflate
tation (functional maturation of the sperm within the mechanically and functionally different behaviours
female tract), is required for fertilization. Induction of [18]. For instance, subtle changes in activated motil-
capacitation in vitro is also accompanied by hyperacti- ity greatly enhance penetration into viscous substrates
vation, though it is possible to induce hyperactivation [19–22], and application of three-dimensional holo-
or capacitation (assessed by markers such as increased graphic tracking techniques to human sperm shows
tyrosine phosphorylation) independently [6]. the occurrence of diverse motility patterns that are not
Hyperactivation was first described by Yanagi- detectable by two-dimensional tracking [23].
machi in hamster sperm [7]. Compared with the Motility of free-swimming sperm, in semen or after
movements of activated sperm, it is characterized preparation, is typically assessed over a short period
by much greater bending of the flagellum, par- (⬍1 s). When more prolonged observation of indi-
ticularly in the anterior principal piece and mid- vidual cells is undertaken it becomes apparent not
piece (Figure 8.1b). In a low-viscosity medium this only that behaviour varies between cells but also that
results in a large increase in lateral movement of individual cells show marked variation or switching
the sperm head. Cells showing a partially hyperacti- of their behaviour (Figure 8.2) [24]. Clear alternation
vated motility pattern termed ‘transitional’ are pro-
gressive despite the exaggerated lateral movements of
the head (Figure 8.1d), but in strongly hyperactivated
cells, bending of the flagellum is markedly asymmet-
ric, so that the cell continuously turns or tumbles
(Figure 8.1e) and sometimes adopts a characteristic
pattern known as star-spin (Figure 8.1f). Computer-
assisted semen analysis (CASA) systems can be used a
to assess such movements by videomicroscopy (typi-
cally at 60 Hz). The track of each cell in the field of
view is constructed using the position of the sperm
head in each frame. This provides a range of kine-
matic parameters for each cell observed. Application of
standardized criteria can then be used to estimate the 50 μm
proportion of hyperactivated cells in a sample [8, 9].
Data of this type clearly show the simultaneous occur-
rence of different behaviours within a population of
b
sperm. In a higher-viscosity medium, such as mucus,
this extravagant bending is reduced and hyperacti-
hyper-
vated sperm may achieve progressive movement. In activated
fact, it has been reported that hyperactivated hamster
and mouse sperm progress more rapidly than activated
cells within viscous and viscoelastic media [10, 11].
Hyperactivation is also required for progression within transitional
the oviduct and for penetration through the vestments
that surround the egg [12–15]. Sperm are observed
repeatedly to bind to and then detach from the cells activated 0 100 200 300
seconds
lining the oviduct, so that ascent towards the ampulla
may occur by a series of hops [16, 17]. Mouse sperm Figure 8.2 Complex behaviour in human sperm. (a) A 45 s track
that cannot hyperactivate, once bound, cannot escape of a cell that switches repeatedly between activated and
hyperactivated motility (indicated by grey shading). Arrows show
from the oviductal epithelium, preventing ascent to the direction of movement. (b) Data from a different recording (lasting
fertilization site [12, 13, 16]. Hyperactivation also plays 300 s) in which a cell was videoed and its behaviour then classified
a key role in penetration of the oocyte vestments (see for each second as activated, transitional or hyperactivated. This cell
shows clear cycles of activity, with short bursts of hyperactivated
Regulation of Motility in Mammalian Sperm). Impor- swimming (duration 10–15 s) interspersed with longer periods of
tantly, both activated and hyperactivated motility pat- activated motility.
127
between activated and hyperactivated types has been tration [35]. This motility pattern is maintained in
described in rabbits [25], cattle [18], and humans [26– sperm that have traversed a cumulus-filled capillary
28], switching occurring at intervals of seconds to min- and emerged into culture medium, indicating that it
utes. In free-swimming sperm this produces appar- is a response to factors encountered in the cumu-
ently aimless or wandering behaviour, with straight or lus and not a behavioural change imposed by the
gently curving paths periodically interrupted by bursts viscoelastic properties of the cumulus matrix [36].
of hyperactivated motility. The functional significance In hamster sperm observed penetrating the cumu-
of this alternating behaviour has not been established, lus and zona pellucida, a number of different motil-
but there are a number of possibilities including facil- ities were observed, including high-amplitude, non-
itation of zona penetration [29] and periodic detach- propagating bends (described as ‘lever’ bending) of the
ment from the oviductal lining, allowing progress by proximal midpiece, resulting in a behaviour termed
hopping [16]. In sperm of many marine invertebrates ‘hatchet motility’ [18, 37, 38]. Sperm observed during
there is a stereotypic behaviour which has been called zona penetration clearly alternated between asymmet-
‘turn and run’ [30], which enables the sperm to fol- ric (lever) and symmetrical beating, changing mode
low a chemoattractant gradient to the egg (see Naviga- every 3–10 beats [38].
tion in Sperm of Marine Invertebrates). In mammalian
sperm there is no such stereotypic behaviour and the
occurrence of a chemotactic response is less clear. Nev-
Regulation of Sperm Motility – The
ertheless, there is considerable evidence from in vitro Role of [Ca2+ ]i
assays that a subset of cells (probably those that are Sperm motility is dependent on generation of waves of
capacitated) orient their movements towards the egg flagellar undulation driven by the axoneme, a complex
[31], the best-characterized response being to follic- structure composed of a central pair of microtubules
ular fluid and to very low concentrations of proges- surrounded by nine outer microtubule doublets, which
terone [32, 33]. It has been proposed that switching are connected to each other by interdoublet nexin links
between activated and hyperactivated motility gen- (Figure 8.3). Each of the nine microtubule doublets is
erates a random walk (periods of progression inter- composed of an A tubule and a B tubule. Two dynein
spersed with random direction changes) which can be arms (inner dynein arm and outer dynein arm) project
biased by extending the progressive period (between from each A tubule. Dynein is a molecular motor, and
bursts of hyperactivation) when the cell is travelling up force is generated when it contacts and ‘walks’ along
the chemoattractant gradient [34]. the B tubule of the neighbouring doublet. If struc-
In addition to hyperactivation, distinct behaviours tural elements of the axoneme are extracted, activity
have also been observed in sperm interacting with of dynein will cause the outer doublets to slide past
the oocyte vestments. Sperm moving within cumu- each other and the axoneme disintegrates (e.g. [39]).
lus matrix show ‘cumulus motility’, with a linear track However, in an intact flagellum, resistance to sliding is
and elevated beat frequency that may assist pene- present in the nexin links, at the base of the axoneme
outer doublet 1 Figure 8.3 Structure of the axoneme. The

axoneme is composed of a central microtubule pair
central surrounded by nine outer microtubule doublets.
B A microtubule Each doublet has an A and a B microtubule (labelled
9 2 pair on doublet 1). Dynein arms extend from the A
microtubule and can ‘walk’ along the B tubule of the
neighbouring doublet. This sliding motion is
converted to bending by the restriction of
8 movement at the base of the flagellum and by
outer dynein arm 3 axonemal structures. Selective activation of specific
radial spoke doublets at different positions along the axoneme
inner dynein arm generates flagellar waves.
7
4
nexin link
6 5
128
and in regions where dyneins are not active, so that turning [50]. The available evidence suggests that
sliding between a pair of doublets causes bending of this is the case. [Ca2+ ]i is higher in hyperactivated
the flagellum [40]. By regulating which dynein arms than in activated sperm [51], and hyperactivation is
are active and in which part of the flagellum, oscilla- induced by treatments that elevate [Ca2+ ]i [52]. In
tory waves are generated that propagate from the base sea urchin sperm, where chemotactic behaviour has
(sperm neck region) to the tip of the flagellum, driving been well characterized [30, 53, 54], the occurrence
the sperm forward. The ability of the sperm to vary the and amplitude of the asymmetric flagellar beat that
nature of the flagellar waveform may be dependent, at directs the sperm up-gradient is controlled by flagellar
least in part, on the presence of multiple dynein heavy- [Ca2+ ]i [55]. In some species, detergent-extracted
chain isoforms (encoded by different genes) and con- demembranated sperm can be reactivated to produce
sequent differences between the effects of activity of sperm models, and beat patterns can be observed in
the inner and outer dynein arms [41–43]. the presence of known [Ca2+ ]. Hyperactivation is
Since sperm are translationally (and largely induced by Ca2+ in triton-extracted bovine sperm
transcriptionally) silent, changes in flagellar beat- [56, 57], saturating at approximately 400 nM. In
ing, resulting in behavioural adaptation, must be demembranated macaque sperm, 10 ␮M Ca2+ causes
achieved by post-translational mechanisms [40]. strong asymmetry of flagellar beating. When [Ca2+ ] is
Cyclic nucleotides are of central importance in reg- raised to mM concentrations, demembranated sperm
ulation of sperm function, and there is no doubt from most species show arrest of the beat cycle, which
that they contribute to control of flagellar beating freezes in a characteristic shape. In mouse sperm this
[44–46]. This was elegantly demonstrated by Jansen is a tight coil in [39], whereas bovine sperm form a
et al. [46], who generated a transgenic mouse in U shape [56], and in demembranated sperm of the
which a photoactivated adenyl cyclase (bPAC) was sea urchin Tripneustes gratilla a tight (⬎300°) bend
expressed in sperm. These cells showed a marked occurs in the proximal flagellum immediately behind
increase in flagellar beat frequency upon illumination the sperm neck [58]. In contrast, demembranated
that was not associated with a change in [Ca2+ ]i . horse sperm failed to hyperactivate when [Ca2+ ] was
In cells lacking functional soluble adenyl cyclase, increased, showed arrest of beating at concentrations
which are infertile due to a failure of forward motility as low as 400 nM Ca2+ and maintained normal motil-
[45], bPAC restored both motility and fertility. In the ity when [Ca2+ ] was reduced to 27 pM, well below
dark these cells were immotile and attempts at IVF normal resting levels [59]. Regulation of flagellar beat
achieved no fertilization. However, when illuminated, may vary significantly even among mammals.
the sperm became motile and approximately 30%
fertilization occurred during IVF [46]. cAMP may
also contribute to controlling hyperactivated motility, Ca2+ Signalling in Sperm
particularly when this behaviour is prolonged [47], The use of Ca2+ as an intracellular messenger is ubiq-
but the prime regulator of sperm behaviour in most uitous. [Ca2+ ]i is maintained at submicromolar con-
species studied appears to be [Ca2+ ]i . In a wide range centrations in the cytoplasm of all cells, which is prob-
of organisms, elevation of [Ca2+ ]i has been observed ably a requirement for the use of a phosphate-based
to regulate asymmetry of ciliary and flagellar beating. energy currency (calcium phosphate is highly insol-
Typically an increase in [Ca2+ ]i causes an increase in uble). Active buffering of [Ca2+ ]i not only may have
beat asymmetry. This regulation of flagellar activity driven the evolution of Ca2+ -transport molecules, but
occurs through interaction of Ca2+ with response also may effectively have pre-adapted Ca2+ for use
elements that may be in the radial spokes or associated in coding of information, since movement of (rela-
with the dynein arms (Figure 8.3). These response tively) small numbers of ions, down large concen-
elements control kinases in the dynein arm regulatory tration gradients, will generate an instant and sig-
complex [48, 49]. Since [Ca2+ ]i regulates flagellar nificant change in cytoplasmic concentration that
beat, one would expect that [Ca2+ ]i signalling plays an can be sensed by Ca2+ -binding proteins [60, 61].
important role both in transitions in sperm behaviour A wide range of pumps, channels, receptors and
(such as adoption of hyperactivated motility in transducers are present in the plasma membrane,
capacitating sperm) and in more specific stimulus- organelle membranes and cytoplasm of somatic cells,
related responses such as chemoattractant-induced providing a ‘Ca2+ -signalling toolkit’ [62] for the
129
homeostatic regulation of [Ca2+ ]i and the generation shown to be localized to the principal piece of the
of spatiotemporal signals. Different cells with different flagellum in all species where this has been investi-
Ca2+ -signalling requirements express different com- gated. Though CatSper subunits are related to those
ponents of this toolkit. Since Ca2+ -signalling is cru- of voltage-gated Na+ and Ca2+ channels [72], the
cial for sperm function, not only controlling motility channel shows significant sensitivity to cytoplasmic
and behaviour but also playing a central role in acro- alkalinization (particularly in the mouse; [73]) and
some reaction and capacitation, one might expect that relatively weak voltage sensitivity. Immunostaining
sperm cells would have a sophisticated Ca2+ signalling of Ca2+ -permeable channels in sperm shows great
apparatus. In fact, the toolkit of sperm, though special- diversity in their distribution, suggesting that in
ized, appears to be simple. response to different types of stimuli, sperm might
have the ability to generate diverse [Ca2+ ]i signals,
with different spatio-temporal characteristics [50,
Plasma Membrane Channels and Pumps 74–78].
The plasma membranes of somatic cells include a The successful application of the whole cell patch
range of Ca2+ pumps, integral proteins which export clamp technique to mouse and human sperm has
Ca2+ from the cytoplasm to maintain low [Ca2+ ]i . enabled direct assessment of the presence of func-
These include four isoforms of the plasma mem- tional plasma membrane Ca2+ channels. Surprisingly,
brane Ca2+ ATPase (PMCA; [63]) and also Na+ –Ca2+ these studies have, to date, revealed the activity of
exchangers (NCXs) that are driven by the Na+ gra- only a small number of channels, with CatSper being
dient at the plasma membrane and thus indirectly by the only detectable Ca2+ -permeable conductance [73,
the Na+ –K+ ATPase [64, 65]. Contributions from both 79, 80]. In mouse sperm CatSper is weakly voltage-
of these mechanisms were detected in mouse sperm, sensitive and strongly activated by increased pHi [80]
with PMCAs being responsible for rapid extrusion (Figure 8.4). However, in human sperm, CatSper is
of excess Ca2+ after signalling events [66]. In mouse also activated by a wide range of ligands, including
sperm PMCA4 is the most common isoform present progesterone and some prostaglandins [81, 82]. It has
and is localized to the flagellar principal piece, show- been shown recently that this action of progesterone
ing the importance of tight Ca2+ regulation within the (but not stimulation of CatSper by prostaglandins) is
flagellum [67, 68]. mediated through the activation of alpha/beta hydro-
Entry of Ca2+ into the cell from the extracellular lase domain-containing protein 2 (ABHD2), which
fluid occurs through membrane Ca2+ channels. results in rapid breakdown of 2-arachidonoylglycerol
Somatic cell membranes possess a wide range of these (2AG) in the sperm membrane and consequent dis-
channels, including voltage-operated Ca2+ channels, inhibition of the channel [83]. Significantly, CatSper
ligand-activated Ca2+ -permeable channels, second- has been shown to be activated by treatments previ-
messenger-operated channels and store-operated ously considered to provide evidence for the presence
Ca2+ channels – channels that open upon depletion of of other Ca2+ -permeable channel types in sperm. For
Ca2+ -storage organelles [69]. Voltage-operated Ca2+ instance, cell-permeant cyclic nucleotide analogues
channels (VOCCs) alone are encoded by 10 different (used to probe for the presence of cyclic nucleotide-
genes, each with a number of splice variants, such gated channels) directly activate CatSper currents in
that activation of Ca2+ influx by depolarization of patch-clamped human sperm, whereas direct load-
membrane potential can vary in voltage sensitivity, ing of the cell with cAMP or cGMP from the patch
latency, kinetics and amplitude between and within pipette has no effect [84]. Whether CatSper provides
cells. Pharmacological investigations, combined with the only mechanism for influx of extracellular Ca2+
immunostaining and western blotting, provide evi- into mature sperm is questionable, since some obser-
dence for the presence of a range of Ca2+ -permeable vations are clearly not compatible with this conclu-
plasma membrane channels in mammalian sperm, sion. For instance, in human sperm, low doses of 2-
including VOCCs and cyclic-nucleotide-gated and aminoethoxydiphenyl borate (2-APB, a drug which
store-operated channels. Sperm-specific channels enhances activity of store-operated channels) strongly
have also been discovered, including the Ca2+ - potentiate progesterone-induced Ca2+ influx, yet par-
permeable channel CatSper [70, 71]. CatSper is tially inhibit progesterone-stimulated CatSper cur-
present in the majority of animal phyla and has been rents in patch-clamped sperm [85]. Sperm undergo
130
a
b
ligand (e.g. progesterone) ligand (e.g. progesterone)
alkalinity
proportion of CatSper channels open

O
CH3 CH3
CH3 Ca2+
O out
unstimulated
in
pHi
flagellar beating _
membrane potential +
Figure 8.4 Regulation of CatSper channels. (a) The stimulatory effect of increased pHi (observed primarily in mouse sperm) or ligand
binding (notably progesterone; human), leading to Ca2+ influx and modulation of motility. (b) The dependence of CatSper activity on the
membrane potential of the sperm. At the resting membrane potential (Vm; shown by dashed line) most channels are closed. CatSper can be
activated by depolarization of Vm or by a leftward shift of the relationship between voltage and channel activation, induced by a rise in pHi
(mouse) or binding of a range of ligands (human).
significant functional changes after ejaculation and much higher than [Ca2+ ]i so, as at the plasma mem-
ion channel activity, and availability may be regu- brane, Ca2+ channels allow rapid flux of Ca2+ into
lated by processes occurring during capacitation or in the cytoplasm and Ca2+ pumps remove Ca2+ from
response to stimuli encountered in the female tract the cytoplasm into the lumen of the store. The inosi-
(e.g. [86]). Whole cell patch clamping, which will effec- tol trisphosphate (IP3 ) receptor (IP3 R) and ryanodine
tively dialyze the sperm cytoplasm, may disrupt such receptor (RyR) are the best-characterized intracellu-
regulation and thus inhibit the functioning of some lar Ca2+ release channels. RyRs are activated by ele-
channels [87]. However, it seems likely that the vated [Ca2+ ]i (Ca2+ -induced Ca2+ release; CICR) and
rich diversity of Ca2+ -permeable channels reportedly are also sensitive to the second messenger cyclic-
present in mature sperm reflects, at least in part, the adenosine diphospho-ribose (cADPR) [62]. IP3 Rs are
promiscuous nature of CatSper and the presence of activated by the second messenger IP3 , but Ca2+ acts as
non-functional channel proteins inherited from the a co-agonist at these receptors, so a form of CICR may
immature germ cell. occur when IP3 is present. More recently, it has been
proposed that nicotinic acid adenine dinucleotide
phosphate (NAADP) is another Ca2+ -mobilizing sec-
Intracellular Ca2+ Stores ond messenger, acting to release Ca2+ from acidic
As well as having the ability to control entry and organelles such as lysosomes by activating Ca2+ -
removal of Ca2+ at the plasma membrane, somatic specific two-pore channels (TPCs) [88–91]. Uptake of
cells possess intracellular Ca2+ stores – organelles that cytoplasmic Ca2+ into intracellular stores is dependent
can accumulate Ca2+ and release it in response to stim- upon store-specific pumps. Two closely related Ca2+
ulation. The importance of the endoplasmic reticu- pumps, sarcoplasmic–endoplasmic reticulum Ca2+ -
lum as a Ca2+ store is well established and it is now ATPase (SERCA) and secretory pathway Ca2+ -ATPase
apparent that other organelles such as lysosomes and (SPCA), are present in most cells but their distribution
Golgi and secretory vesicles may also store Ca2+ . In differs, with SPCA being localized to Golgi and Golgi-
somatic cells and in oocytes these stores are central derived structures [92–94]. Since Ca2+ stored in intra-
players in the generation of complex spatio-temporal cellular organelles is finite (whereas the extracellular
patterns. Similarly to the plasma membrane, the mem- Ca2+ reservoir is effectively infinite), store-mediated
branes of Ca2+ storage organelles carry Ca2+ chan- Ca2+ signals are typically phasic, their duration reflect-
nels and pumps. [Ca2+ ] in the organelle lumen is ing the time taken for stored Ca2+ (or a portion of it)
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 28 May 2017 at 16:52:14, subject to the Cambridge Core terms of use, available at 131
to be mobilized and then removed from the cyto- where influx of extracellular Ca2+ should be abol-
plasm by cytoplasmic buffers and Ca2+ pumps. To pro- ished and also in mouse sperm which are null for
vide prolonged store-mediated Ca2+ signals (and to CatSper [102], but attempts to observe [Ca2+ ]i sig-
replenish stores after mobilization), capacitative Ca2+ nals induced by agonists (e.g. the effect of proges-
entry occurs, a process by which depletion of stored terone on human sperm (see Figs. 8.5 and 8.6a)) in
Ca2+ leads to activation of plasma membrane Ca2+ the absence of [Ca2+ ]o have produced variable results.
channels. This involves two components; (i) stromal Strunker and colleagues [82] observed that, in rapid-
interaction molecule (STIM) proteins, which moni- mixing experiments on human sperm, reduction of
tor luminal [Ca2+ ] of the store and thus detect deple- [Ca2+ ]o to 100 nM caused instant and complete inhi-
tion of stored Ca2+ , and (ii) plasma membrane chan- bition of the progesterone-induced [Ca2+ ]i signal. In
nels, which are regulated by STIM. Channels of the contrast, Espino and colleagues [103] observed small,
Orai family are the best characterized and probably the transient responses to progesterone in cells incubated
most widely distributed of the STIM-regulated plasma in ’Ca2+ -free’ medium supplemented with 100 ␮M
membrane channels, but other types have also been EGTA. In human sperm we and others have observed
proposed [69, 95–98]. the occurrence of [Ca2+ ]i oscillations which appear,
In comparison with somatic cells, sperm possess as in somatic cells, to be at least partly dependent on
few intracellular organelles. The endoplasmic reticu- cyclic mobilization of stored Ca2+ [104–107]. Appli-
lum and Golgi, the primary Ca2+ stores in somatic cation of EGTA-buffered saline (calculated [Ca2+ ] =
cells, are largely lost during spermiogenesis, but the 5 nM) abolishes these oscillations, but in some cells
nucleus, acrosome and mitochondria and some poorly a transient of reduced amplitude is generated, in the
defined membranous structures at the sperm neck absence of extracellular Ca2+ , before oscillations cease
remain. Loading of sperm with low-affinity Ca2+ dyes, completely (Figure 8.5). We consider it likely that
to reveal intracellular Ca2+ stores, shows localiza- the store responsible for [Ca2+ ]i oscillations is small
tion to the acrosome and to sperm neck/midpiece and/or labile, becoming rapidly depleted upon effec-
region [99, 100]. Staining of sperm for IP3 Rs, RyRs, tive buffering of [Ca2+ ]o.
SERCA and SPCA has shown the presence of pumps
and channels necessary for intracellular Ca2+ stor-
age at these locations, suggesting that at least two
separate Ca2+ stores are present, at the acrosome
Regulation of Sperm Behaviour by
and the sperm neck/midpiece [100, 101]. However, [Ca2+ ]i Signalling
demonstrating Ca2+ store function in intact sperm
has proved difficult and data are inconsistent. Func- Navigation in Sperm of Marine Invertebrates
tional effects induced by chemical mobilization of The best-studied and -characterized model for regula-
stored Ca2+ have been shown both under conditions tion of sperm motility is fertilization in echinoderms
progesterone [Ca2+]=10-6M EGTA Figure 8.5 [Ca2+ ]i oscillations in human sperm. Traces
[Ca2+]=5x10-9M show [Ca2+ ]i in five human sperm that responded to
stimulation with progesterone by generating a series of
[Ca2+ ]i oscillations. Saline with no added Ca2+ ([Ca2+ ]o
10–6 M) failed to inhibit oscillations, but when Ca2+
was buffered with the Ca2+ chelator EGTA (calculated
[Ca2+]i
[Ca2+ ] 5 nM), [Ca2+ ]i fell rapidly and oscillations

ceased, showing that they require Ca2+ influx at the
plasma membrane. However, in one cell a truncated
oscillation occurred ⬎2 min after application of EGTA,
consistent with involvement of stored Ca2+ in
generation of oscillations.
3 min
132
run’ [30]. When the sperm is in the part of its cir-

a cular path which is further from the chemoattractant
source, an increase in the asymmetry of flagellar beat-
ing causes it to turn further inward. This is imme-
b diately followed by a period of decreased asymmetry
and a straighter ‘run’ phase, which moves the sperm
up the chemoattractant gradient (Figure 8.6b). Sperm
of Arbacia punctulata and Lytechinus pictus respond
respectively to the peptides resact (14 amino acids)
and speract (10 amino acids) with this characteris-
Figure 8.6 Behaviour of echinoderm sperm. (a) Typical circular tic behaviour [30, 109]. Binding of speract activates
path seen when sperm swim next to the glass surface of a guanylate cyclase in the sperm (receptor and cyclase
microscopic observation chamber. (b) The looping track of a sperm are extracellular and intracellular regions of the same
migrating up a chemoattractant gradient (indicated by shading).
The sperm maintains its circling behaviour but intermittently makes molecule; [110–112]), and the consequent activation
a sharper turn (which occurs in the part of the loop distant from the of cGMP-gated K+ channels causes rapid hyperpo-
chemoattractant source) followed by a straighter run phase, which larization of Vm . This leads to a rise in pHi [113]
moves it up gradient.
due to activation of a Na+ –H+ exchanger and, upon
repolarization, activation of Ca2+ channels and conse-
quent generation of a [Ca2+ ]i spike [54]. It has been
[30, 53, 54, 108]. After spawning, sperm are guided by shown recently that, in A. punctulata, this channel is
a chemotactic peptide, which is released by the oocyte CatSper and that it responds to the speract-induced
and forms a gradient in the surrounding seawater. rise in pHi with an increase in voltage sensitivity, sim-
Sperm from different species are attracted by different ilarly to the CatSper channel of mice [114]. Due to the
peptides, but the behaviour and underlying signalling sperm’s circular swimming, it alternately experiences
events which enable navigation to the oocyte appear to rising and falling stimulation by the chemoattractant.
be conserved across species. When observed under the Ca2+ spikes, eliciting turn and run behaviour during
microscope, where movement is effectively restricted the falling phase of the stimulus function. Intriguingly,
to two dimensions at the interface of glass and water, it has been shown that it is not the absolute level of
the sperm adopt a characteristic circular motility pat- [Ca2+ ]i but its first derivative (rate of change) that
tern (Figure 8.7a). Upon detecting a chemoattractant determines flagellar beat asymmetry and thus turn-
gradient, the sperm show behaviour termed ‘turn and ing. During the rising phase of the [Ca2+ ]I , spike
a b
[Ca2+]i (normalised fluorescence)
3
progesterone (3 μM) 250
2.5 donors
200
current (pA/pF)
2 donors 150
1.5 100
50
1
patient
patient
0
0.5 -100 0 100
0 100 200 300 400 500 mV
-50
seconds
Figure 8.7 Failure of CatSper in a subfertile patient. (a) Mean [Ca2+ ]i response induced by progesterone (arrow) in donor samples (filled
symbols, n = 34) and in sperm from an IVF patient (open symbols, n = 2). (b) CatSper currents (recorded using Cs+ as charge carrier in a
divalent-free medium [81]) recorded from donors (n = 7; filled symbols) and from the cells provided by the patient (mean of three cells; open
symbols). Adapted from Williams et al. [22]. This patient fertilized 0/9 oocytes by IVF but subsequently fertilized 5/6 by ICSI [22].
asymmetry (and thus swimming path curvature) their ability to reach the site of fertilization. In vitro
increases, but as [Ca2+ ]i falls, asymmetry decreases, studies with trans-illuminated oviduct preparations
leading to the straighter run phase [55]. In sperm of show that sperm bind strongly to the epithelium of
Strongylocentrotus purpuratus, which also respond to the oviductal isthmus and that adoption of hyperac-
speract, the generation of Ca2+ spikes is not biased tivated motility facilitates their detachment [12, 16].
towards the falling stimulus function, so that the pep- Sperm null for CatSper, which cannot hyperactivate,
tide causes frequent changes of direction but no net fail to detach from the epithelium and cannot progress
movement up-gradient [109]. When sea urchin sperm beyond the isthmus [13]. Because of the asymmet-
are swimming freely and able to move in three dimen- ric (hooked) shape of the rodent sperm head, it is
sions, their tracks are helical rather than circular [115]. possible to identify the direction of the deep flag-
It has recently been shown that under these condi- ellar bend that occurs in hyperactivated cells (pro-
tions the sperm show two types of path correction. The hook or anti-hook). When mouse sperm are incu-
sperm’s trajectory is continuously corrected by ‘peri- bated with cumulus–oocyte complexes in vitro, ejac-
odic’ sampling generated by the helical path, but when ulated cells, which show a greater incidence of pro-
the sperm starts to move down gradient (falling stim- hook bending than epididymal sperm, are more effi-
ulus concentration), this evokes an ‘off’ response that cient in penetrating the cumulus. In contrast, sperm
causes a sharp change in direction [116]. inside the oviduct use anti-hook hyperactivation to
detach from the epithelium [17]. Intriguingly, phar-
macological induction of hyperactivation in vitro by
Regulation of Motility in Mammalian Sperm activation of CatSper results in pro-hook hyperactiva-
Motility of mouse sperm and its significance in fer- tion, whereas release of Ca2+ from storage organelle(s)
tilization have been studied extensively. When freshly at the sperm neck induces anti-hook bends [120].
isolated mouse sperm (normally obtained from the In human sperm the ability to adopt hyperactivated
epididymis) are dispersed in a ‘capacitating’ medium motility is believed to be similarly dependent upon
they swim with a characteristic activated pattern (see Ca2+ and CatSper [22, 122–124]. As in the mouse,
Sperm Behaviour), but within 60 min they adopt CatSper is expressed specifically in the principal piece
hyperactivated motility. This change in motility is of the flagellum, but regulation of the channel is
mediated by activation of the sperm-specific Ca2+ - markedly different in human cells. Similarly to mouse
permeable channel CatSper (see Plasma Membrane CatSper, the channel is weakly voltage-dependent, but
Channels and Pumps). Mouse CatSper is regulated pri- pHi sensitivity is limited and instead the channel is pri-
marily by pHi, which increases as the cells capacitate marily ligand-gated. It has been known for 25 years
in the female tract, probably due to activity of Na+ – that progesterone activates Ca2+ influx into human
Cl− /HCO3 − and Na+ /HCO3 − transporters and/or a sperm [125], but the mechanism of this non-genomic
Na+ /H+ exchanger [117, 118]. Experiments with mice steroid action defied characterization. Application of
null for CatSper have clearly shown the central role patch clamp to human sperm revealed that this Ca2+
of this channel in regulating motility and the impor- influx is mediated by CatSper, which is directly acti-
tance of this function for male fertility. Sperm from vated by progesterone binding [81, 82]. Further stud-
male mice null for CatSper resemble wild type sperm ies showed that in addition to progesterone and PGE1
in their abundance and morphology, but their motil- (another endogenous ligand for CatSper), a wide range
ity is clearly abnormal. The cells are progressively of small organic molecules activate human CatSper,
motile (though described as sluggish [70]), but they including a number of endocrine-disrupting chemi-
fail to hyperactivate [15, 119, 120]. Matings produce cals and pollutants [126, 127]. Macaque sperm have
no pups, and sperm from these animals fail to fertilize a progesterone sensitivity similar to that of human
at IVF. The IVF fertilization rate can, however, be fully cells [128], but in mice no such effect occurs, sug-
restored if the procedure is performed with zona-free gesting that ligand regulation of CatSper is char-
oocytes, leading to the conclusion that hyperactivation acteristic of primate sperm but does not occur in
is necessary for penetration of the zona and is depen- rodents.
dent on Ca2+ flux through CatSper channels [15]. Men with deletions or mutations in the CatSper
In addition to their failure to penetrate the zona, genes show reduced fertility and loss of CatSper cur-
mouse sperm null for CatSper are also impaired in rent [124]. However, in contrast to the phenotype of
134
the CatSper-null mouse, sperm from these men show sensor, which is activated by a wide range of ligands
multiple abnormalities [122, 129], so that it could not as well as membrane potential [84], enabling the con-
be concluded that loss of CatSper was responsible for vergence of diverse stimuli into a common signalling
or sufficient to explain the reduced fertility of these pathway (Figure 8.8). However, an important question
men. An alternative strategy has been to screen sub- is whether the flexibility of sperm behaviour (and the
fertile men (attending a fertility clinic) for CatSper discrete regulation of other Ca2+ -mediated processes
function. Progesterone reliably activates human sperm such as the acrosome reaction) can be achieved by such
CatSper, generating a [Ca2+ ]i transient followed by a an apparently simple signal pathway – is there a need
plateau (Figure 8.7a), which was observed in ⬎97% for spatio-temporal complexity of [Ca2+ ]i signals to
of donors and IVF patients but only 73% of ICSI generate different behaviour at different times? Since
patients [19, 20]. In IVF patients the amplitude of Ca2+ store mobilization appears to be far more effec-
this [Ca2+ ]i transient is correlated with the fertiliza- tive than CatSper activation as an inducer of hyperac-
tion rate [20, 130, 131]. By assessing the progesterone- tivation in human sperm, one possibility is that Ca2+
induced [Ca2+ ]i signal, Williams et al. [22] identi- signalling pathways effectively diverge downstream of
fied men with apparently normal semen but functional CatSper due to recruitment of (or failure to recruit)
loss of CatSper, which was confirmed by patch clamp Ca2+ stores. Alasmari et al. [19] suggested that in
(Figure 8.7). Only 3/135 donors and patients (includ- response to physiological activation of CatSper (such
ing some who had previously failed IVF) failed to as by progesterone), hyperactivation of human sperm
respond to progesterone, suggesting that this lesion is occurs only in cells where secondary mobilization of
rare. stored Ca2+ (Ca2+ -induced generation of IP3 [132]
Observations of human sperm exposed to acti- or CICR; [101]) occurs downstream of the CatSper-
vators of CatSper confirm that, as in mouse sperm, mediated flux of Ca2+ into the flagellum (Figure 8.8).
the channel plays a central role in motility regula- Uncaging of Ca2+ in the flagellum of human sperm has
tion. Treatment with progesterone potently stimu- been shown to evoke such secondary responses [101],
lates entry into viscous medium, and this effect is and studies on mouse sperm exposed to combined
inhibited by CatSper-blocking drugs [19, 21] and in depolarization/alkalinization, cAMP analogues, zona
sperm lacking functional CatSper [22]. The nature of proteins or BSA (all of which are believed to stimulate
the change in motility underlying this effect is not CatSper [84, 133–135]) suggest that active tail-to-head
clear. A burst of enhanced flagellar beating occurs in propagation of the [Ca2+ ]i signal also occurs in rodent
many cells during the period of initial progesterone- sperm. Mathematical modelling of CatSper-generated
induced [Ca2+ ]i transient (see videos in [126]), but [Ca2+ ]i signals in mouse sperm requires inclusion of
this effect is brief and only a small increase in the such secondary Ca2+ mobilization in order to fit these
proportion of hyperactivated cells is detected when observations [136]. In this way the nature of the Ca2+
motility is assessed by CASA [19, 20]. Attempts to signal (whether it is restricted primarily to the flagel-
activate CatSper by increasing pHi have a similarly lum or invades the sperm neck and head) and the con-
modest effect but, in contrast, treatments that mobi- sequent behaviour of the sperm may be determined
lize stored Ca2+ strongly potentiate hyperactivation by regulation of the ‘willingness’ of the Ca2+ store to
of human sperm [20]. Furthermore, hyperactivation mobilize Ca2+ . Channels that mediate release of stored
can be induced in sperm of men who are functionally Ca2+ are known to be regulated by signals encoun-
null for CatSper (and also in CatSper-null mice [102]), tered within the female tract and during capacitation,
apparently by mobilization of stored Ca2+ [22]. such as NO· [137, 138] and oxidative stress [139, 140]
(Figure 8.8).
In addition to diversity in the spatial distribution
Regulation of Mammalian Sperm of sperm Ca2+ signals, there is also marked varia-
Behaviour – Generation of Spatio-Temporal tion in their temporal pattern. In particular, a pro-
portion of human sperm stimulated with progesterone
Ca2+ Patterns or nitric oxide show [Ca2+ ]i oscillations (Figure 8.5),
The central role of CatSper in the generation of Ca2+ which modulate flagellar beating [105, 138, 141] and
signals in mammalian sperm is clearly established. also inhibit the occurrence of the acrosome reaction
Furthermore, in human sperm CatSper is a polymodal [142]. Extended tracking of human sperm behaviour
135
capacitation
•oxidative stress
Em, •cAMP/PKA
pH, CICR Ca2+ •???
progesterone CatSper store NO
prostaglandins agonists
small organic
molecules
penetration into mucus hyperactivation

Figure 8.8 Diagrammatic representation of model for interacting effects of CatSper and stored Ca2+ on sperm motility. CatSper (shown by
grey shading around flagellar principal piece) is sensitive to membrane potential (Em ), pHi (primarily observed in mouse sperm) and (in
human sperm) a range of small organic molecules including progesterone. CatSper-mediated Ca2+ entry into the flagellum regulates flagellar
beat, enhancing penetration into viscous medium. Ca2+ entering through CatSper diffuses forward to the sperm head, where it may induce
secondary release of stored Ca2+ at the sperm neck/midpiece and possibly from the acrosome. Release of stored Ca2+ , which induces
hyperactivated motility, is dependent on the strength of CatSper activation (and thus the [Ca2+ ]i signal in the flagellum) but also involves
sensitization of the Ca2+ store, which is regulated separately, potentially by aspects of capacitation and by effects of agonists encountered in
the female tract. Activation of CatSper can thus induce different behavioural responses in the sperm, depending on its capacitation status and
on cues from the female tract.
(seconds to minutes, rather than the short tracks behaviour. The central roles of CatSper and stored
acquired during CASA) shows alternation or switch- Ca2+ in this process are clear [22, 79, 80, 119, 124]
ing of behaviours between activated and periods of and the evidence suggests that in human (and probably
hyperactivated motility (Figure 8.2). It is therefore other mammalian) sperm, stored Ca2+ plays an impor-
probable (though not yet established) that this switch- tant role in shaping the [Ca2+ ]i signal to select appro-
ing is driven by the generation of [Ca2+ ]i oscilla- priate behaviour and potentially other responses such
tions. The occurrence of periodic bursts of hyperacti- as the acrosome reaction [107]. Studies on the motil-
vation has been proposed as a mechanism for human ity and behaviour of sperm of external fertilizers have
sperm chemotaxis (see Sperm Behaviour). The mul- now reached a stage where both the chemotactic sig-
tifunctional protein CRISP1, which is acquired by nalling cascade and the manner in which it regulates
sperm during epididymal transit and is implicated the characteristic behaviour seen as the sperm swim
in sperm–zona and sperm–oocyte interaction [143], at a glass–water interface have been characterized and
might mediate chemotaxis in this way. Ernesto et al. can be modelled. Progress is now being made in under-
[144] recently showed that this protein acts both as standing the motility and behaviour of these sperm in
a chemoattractant for mouse sperm and as a sup- three dimensions [115, 116].
pressor of hyperactivation (apparently by inhibiting The behaviour of sperm that fertilize internally is
CatSper channels). Sperm oriented up the CRISP1 gra- less well understood. We have some knowledge of the
dient showed lower levels of hyperactivation, consis- ability of sperm to progress within the tract (most fail
tent with this model for chemotaxis [144] (see Sperm early) and the complexity of their task [1, 3], but our
Behaviour). knowledge of the adaptive behaviour of sperm within
the tract – what sperm do, why they do it and how it
is regulated – is largely speculative. The vast majority
Conclusion of motility studies on both animals and humans have
Sperm function is dependent on regulation of the been done on cells in vitro without reference to the
activities of a set of proteins inherited from the differ- environment in which fertilization occurs. To under-
entiating germ cell. Ca2+ signalling is a major player stand human sperm motility and how its impairment
in this process and is particularly important in the causes failure of natural fertilization, we must address
regulation of flagellar beating and thus the sperm’s how the behaviour of the sperm is adapted to the
136
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tract. The ways in which cues from the oocyte and spermatozoa are unable to ascend beyond the
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[Ca2+ ]i signal are largely unknown. We need to under- 345–50.
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Chapter
Proteomics of Capacitation
9 Mark A. Baker
Introduction spermiogenesis and subsequent epididymal matura-

tion, the cell itself remains immature. Indeed, the
The development and indeed maturation of a sperma-
entire process of capacitation and the acrosome reac-
tozoon are so distinctive that it is difficult to com-
tion, beginning upon ejaculation into the female
pare its differentiation to that of any other cell type.
reproductive tract and ending with the fusion of the
Starting off as a round cell within the testis, after suc-
sperm and the oocyte, must take place in the absence
cessive rounds of mitosis and meiosis, a morpholog-
of de novo protein biosynthesis. Given this, post-
ically competent, but functionally incompetent sper-
translational modifications (PTM) of existing pro-
matozoon is formed during spermatogenesis. Apart
teins must be the dominant mechanism by which
from the transition from a diploid to a haploid cell,
an immature non-capacitated cell eventually obtains
spermatogenesis involves major chromatin structural
maturity.
changes including the replacement of histone pro-
teins with smaller, highly charged protamines [1].
This allows an initial compaction of the chromatin Post-Translational Modifications
during the shaping of the sperm head. Labelling of PTM affect the function of proteins in several dif-
spermatozoa with sulphhydryl compounds such as ferent ways simply by changing the chemistry of an
[N-3 H]ethylmaleimide [2], N-(4-carboxy-3-hydroxy- amino acid. For example, phosphorylation of a ser-
phenyl) maleimide (which forms fluorescent adducts) ine, threonine or tyrosine residue alters its proper-
[3] and [14 C]iodoacetamide has demonstrated that ties so that a normally polar site becomes hydrophilic.
chromatin compaction is complete following epididy- Alternatively, acetylation of a positively charged lysine
mal transit, in which protamines become extensively residue to bring about acetyllysine neutralizes the
cross-linked through cysteine bridges [4, 5]. Presum- charge and in doing so, changes the ionic strength of
ably chromatin compaction protects the integrity of the residue. The amount and diversity of PTM that
the DNA; however, it comes at a cost. Such packag- have been reported are large, with Uniprot1 now list-
ing means that spermatozoa are incapable of nuclear ing 307 different types of PTM, most of which can be
protein biosynthesis. Thus, even though incubation of studied using a proteomic approach. However, often
either mouse [6], human [7] or bovine [8] sperma- a post-translationally modified protein will not be as
tozoa with [3 H]-amino acid mixtures leads to pro- abundant as a non-modified counterpart. Thus, one
tein biosynthesis, this can be inhibited only by the major drawback when it comes to proteomic analy-
mitochondrial ribosomal protein synthesis inhibitor sis is that highly abundant proteins, which are often
chloramphenicol, but not the cytoplasmic protein syn- non-modified, make it troublesome to detect a post-
thesis inhibitor cyclohexamine, in all three cases. translationally modified peptide within the mixture.
Hence, while the mitochondrial DNA of spermato- Within the proteomics field, this is referred to as the
zoa is available for transcription, the nuclear DNA is dynamic range of a sample (the difference between
not. the most and least abundant protein). Considering
Importantly, despite the fact that nuclear tran- that most samples have a dynamic range beyond 1 ×
scription and translation are switched off during 104 (the detection diversity of a mass spectrometer),
University Press.
143
Chapter 9: Proteomics of Capacitation
enrichment techniques are slowly being developed knockout mice [14–16], it has been clearly estab-
which allow a more in-depth analysis of PTM taking lished that upon ejaculation, the influx of calcium
place during sperm capacitation. and bicarbonate (HCO3 − ) stimulate soluble adeny-
lyl cyclase (SACY) to produce cAMP from ATP. This
cyclic nucleotide then dissociates the sperm-specific
Capacitation – The Discovery and What protein kinase A regulatory subunits from the catalytic
It Means for Spermatozoa subunits, which is central to the signalling process of
In 1951 Colin Austin recognized that when sperm were capacitation. One of the major questions that arise
introduced into the fallopian tube hours before ovu- from this model is how, then, are sperm prevented
lation, ‘most of the eggs subsequently recovered were from undergoing premature capacitation while still
fertilized’. However, if sperm were introduced after residing inside the male reproductive tract? An indica-
ovulation, ‘the eggs rarely showed any sign of penetration of this came when a phosphoproteomic compari-
tion’ [9]. In that same year, Min Chueh Chang demon- son was performed of rat spermatozoa, from the caput,
strated that spermatozoa require roughly 6 h for a corpus and caudal regions of the epididymis. In this
physiological change to occur before they have ‘fer- analysis, 22 proteins were shown to undergo changes
tilizing capacity’ [10]. Based on these reports, Austin in their phosphorylation state, including the Na+ –
introduced the word ‘capacitation’ to define the phys- bicarbonate co-transporter SLc4a4 [17]. The presence
iological process sperm undergo in the female repro- of a Na+ –HCO3 − co-transporter had been predicted
ductive tract that allows the cell to penetrate the egg. in 2003 by Dalmarco et al. when they sequentially
However, the question still remained, what were sperm replaced the extracellular buffer and showed an active
doing over this period of time? uptake of HCO3 − -induced hyperpolarization – a first
In 1970, J. Michael Bedford used electron step in capacitation [18]. According to the phospho-
microscopy to look for structural (morphological) proteomic analysis, SLc4a4 was shown to be tyro-
changes during sperm capacitation. However, no sine phosphorylated once the spermatozoa were in
obvious changes were reported. Bedford reasoned the corpus region of the epididymis and was hypoth-
that while no structural changes were occurring, a esized to be involved very early in the capacitation
chemical change was likely to be under way [11]. This process [17]. The current model playing out is that
remarkable insight is what currently underpins the upon ejaculation, mammalian spermatozoa experi-
study of proteomics within capacitating spermatozoa. ence a significant elevation in the amount of extra-
cellular Na+ , from 30 mM in the cauda epididymis
to 100–150 mM in the seminal plasma [19]. At this
Sperm Capacitation – A Process moment SL4a4 may exploit the rise in extracellular
Defined by Post-translational Na+ as a signal to co-transport one Na+ ion together
with three HCO3 − ions across the plasma membrane
Modifications (Figure 9.1). Clearly HCO3 − is essential for the pro-
From the studies of Bedford, it stood to reason that cess of capacitation, as demonstrated by soluble adeny-
phosphorylation events may underpin signal trans- lyl cyclase (SACY) knockout mice [16]. SACY requires
duction pathways within ejaculated spermatozoa that both HCO3 − and Ca2+ to convert ATP in cAMP. The
are necessary before they are capable of fertilization. SACY knockout mouse demonstrated a complete lack
Indeed, in 1971, Lardy et al. demonstrated that the of male fertility, which could be overcome by addition
addition of either a phosphodiesterase inhibitor or a of cell-permeable cAMP analogues. Thus, it appears
cyclic nucleotide regulated sperm motility [12]. These that SACY is upstream of PKA and involved at the very
data clearly suggested that cAMP played a major role. initial stages of capacitation.
Only one year later, a highly active protein kinase A
(PRKACA) was defined to be central to sperm func-
tion [13]. As PRKACA is regulated by cAMP, this The Signalling Cascade of Capacitation
enzyme appeared be responsible for the sperm motility After the initial increase of cAMP and activation of
changes occurring upon addition of cyclic nucleotides. PKA, several studies have demonstrated that a hall-
Today we have a much more modern view of what mark of capacitation is the increased level of tyro-
is taking place during capacitation. Using transgenic sine phosphorylation [20–24]. Although PKA is a
144
serine/threonine kinase, it stands to reason that a tyro- phatase 2A (PP2A). The role of the latter is to stop PKA
sine kinase must be involved. Several kinase family from regulating the main tyrosine kinase pathway dur-
members have been proposed for this intermediate ing capacitation [33]. As such, c-SRC is not directly
species, including the src family members c-Yes [25] responsible, but allows the tyrosine kinase pathway to
and pp60cSRC (c-SRC) [26]. However, the location of proceed unhindered. A summary of the enzymes and
c-Yes within the sperm head suggested that this kinase pathways that we currently understand to be involved
does not play a dominant role, as the majority of tyro- in capacitation is shown in Figure 9.1.
sine phosphorylation events seen in capacitation occur So what are the tyrosine kinases regulating sperm
throughout the flagella [21, 23, 27–29]. The role of capacitation? Incubation of bovine [34] or mouse
C-SRC appeared to be important, since the knockout [35] spermatozoa with PF-431396 demonstrated a
animals were shown to be infertile [30], and further- dramatic inhibition of tyrosine phosphorylation dur-
more, c-SRC is acquired by the spermatozoa during ing capacitation. PF-431396 is an inhibitor of both
epididymal transit [31], which may also helpto pre- proline-rich tyrosine kinase 2 (PYK2) and the focal
vent capacitation from occurring within the male tract. adhesion kinase (FAK) family, suggesting one of
However, a further look at the spermatozoa from c- these as a likely candidate. However, further stud-
SRC knockout mice showed that they were still capa- ies demonstrated that only PYK2 was shown (i) to
ble of displaying increased tyrosine phosphorylation become (phospho)-activated during capacitation, (ii)
during sperm capacitation [31], suggesting that this to undergo activation dependent on the presence of
enzyme may not be directly involved [31]. So why, HCO3 − , and (iii) to be blockable through upstream
then, did a c-SRC inhibitor, namely SU6656, decrease inhibition of PKA with H89 [35]. Thus, the current
the phosphotryosine pathway associated with capac- model predicts that Ca2+ and HCO3 − regulate SACY,
itation [32]? Evidence suggests that a second, paral- which in turn regulates PKA. The latter phosphory-
lel pathway occurs during capacitation. Thus, kinases lates PYK2, which leads to a general increase in tyro-
sensitive to SU6656 down-regulate a protein, phos- sine phosphorylation of many proteins (Figure 9.1).
Phosphodiesterase
Figure 9.1 Our current understanding of the signalling cascade leading to tyrosine phosphorylation in spermatozoa. The influx of calcium
and bicarbonate activate SACY to increase the level of cAMP, which causes dissociation of PKA regulatory from the catalytic subunits. This in
turn directly activates the tyrosine kinase PYK2. In a parallel stream, the tyrosine kinase pp60c-SRC inhibits the phosphatase PP2A. (A black and
white version of this figure will appear in some formats. For the colour version, please refer to the plate section.)
145
PTMS of Proteins Involved in data clearly showed that A-kinase anchoring proteins
were tyrosine-phosphorylated, along with 56 serine
Capacitation and 2 threonine sites [37]. In addition, the Valosin-
Only a handful of studies actually exist in terms of containing protein P97 was also shown to undergo
proteomic analysis dedicated to capacitation. Two pro- tyrosine phosphorylation during capacitation, which
teomic approaches can be considered: the use of 2- leads to translocation of the protein from the neck
dimensional gels (2DE) and liquid chromatography region to the anterior head [37].
coupled to mass spectrometry. With regard to the for- In an alternative approach, our group used TiO2 ,
mer, by comparing normozoospermic human donors which is known to enrich for a different set of phos-
before and after capacitation, 25 protein spots were phopeptides than IMAC [38–40]. By comparing non-
shown to be more abundant in the non-capacitated capacitated and capacitated mouse spermatozoa, 288
sperm, while 23 spots were shown to be more abundant phosphosites were found from 120 different proteins.
in the capacitated sperm [36]. Many of these protein However, despite these data, very few phosphotyro-
changes included heat shock proteins and prostate- sine sites were identified [41]. This is mainly due to
derived proteins such as prolactin-inducible protein, the fact that phosphotyrosine is thought to represent
which may have the potential to be lost during capac- less than 10% of the overall phosphorylation events
itation. What is more difficult to understand is why that are occurring within a cell. A recent breakthrough
small fragments of A-kinase anchoring protein 4 or identifying tyrosine phosphorylation sites came about
outer dense fibre 2 (ODF2), both of which are embed- by using the CatSper knockout mice (CatSperKO);
ded within the tails of spermatozoa, were found to be these mice fail to undergo calcium influx, but they
more abundant within capacitated spermatozoa, yet do have what is described as a ‘rampant’ increase
tubulin – used as a loading control in many studies – in tyrosine phosphorylation – see Chung et al. [42,
is found to be less abundant [36]. Several possibilities Figure 4b]. Thus, CatSperKO and wild type sper-
arise. First, it is true that each of these proteins can be matozoa were compared using initial TiO2 enrich-
post-translationally modified, which may be why dif- ment followed by immunoprecipitation with an anti-
ferent charge train spots are seen. Nevertheless, this is phosphotyrosine antibody and MS analysis [42]. In
technically challenging, given that a non-linear immo- total, 62 phosphotyrosine residues could be detected,
bilized pH gradient strip of 3–10 was used in the first with 41 sites appearing with more than twofold in
dimension. Alternatively, it is possible that proteolytic CatSperKO spermatozoa, suggesting these are the
degradation is occurring on certain proteins during major proteins undergoing tyrosine phosphorylation.
capacitation and, as such, only fragments of the pro- Importantly, hexokinase, which in mice is constitu-
tein are reported as up- or “down-regulated. Finally, tively tyrosine-phosphorylated in non-capacitated and
however, it must be considered that 2DE is notoriously capacitated sperm populations, was reported to have
difficult to replicate. Furthermore, modern mass spec- a ratio of 0.8 (KO/WT), which suggests little to no
trometry can identify many (upwards of 80+) proteins change [42]. Furthermore, AKAP3 and AKAP4 were
within a ’single’ spot. So from this, it would be difficult also confirmed as phosphotyrosine targets. This is
to know exactly which of the proteins is the one that important, since many reports show that tyrosine
changes. Simply reporting the most abundant protein phosphorylation during capacitation occurs predom-
present in that spot may not necessarily reflect the true inantly along the midpiece and tail, which is precisely
situation. where PKA, PYK2, AKAP4 and AKAP3 reside [27, 43,
For the most part, liquid chromatography-mass 44]. Interestingly, the data confirmed the regulation of
spectrometry (LC-MS) has been used to investigate PKA during capacitation, showing that the regulatory
the proteomics of capacitation. Early analysis investi- subunit of PKA becomes tyrosine-phosphorylated.
gated which proteins become tyrosine-phosphorylated The largest fold change found among proteins that
using immobilized metal affinity column (IMAC) to become tyrosine-phosphorylated is reported to occur
enrich for phosphopeptide then compared changes in calmodulin (CaM) [42]. Calmodulin is a calcium-
during capacitation [37]. In this manner, not only binding protein that regulates a number of differ-
can proteins be definitively identified as being phos- ent targets, including PKA [45]. Calmodulin has four
phorylated, but also specific amino acids can often calcium-binding (EF-hand) domains and is known
be identified as the site of modifications. The IMAC to be phosphorylated by several different serine,
146
threonine and tyrosine kinases. One of these includes ity of ODF1 protein is found along the neck region
the Src-family kinases, which have been reported to of spermatozoa and appear to be important for the
tyrosine-phosphorylate CaM on residue 99 in humans, structural organization of the connecting piece [52].
or residue 100 in mice [46]. The latter is the same site In animal knockout mice lacking ODF1, several sper-
reported by Chung et al. [42] as the largest tyrosine- matozoa from the caudal region were found to be
phosphorylated fold change occurring between WT decapitated, suggesting that ODF1 may help form a
and CatSperKO mice. This site sits within the third rigid structure adjoining the head and neck region
Ca2+ binding domain of the protein. Phosphoryla- [52]. Importantly, during capacitation, ODF1 becomes
tion of tyrosine 99 within calmodulin appears to phosphorylated on tyrosine 154 [42]. In correlation
cause a reduction in the ability of antagonists such as with this, sperm transit from a forward progressive
W7 to inhibit the protein [47]. Phosphorylated CaM motile state (high beat frequency, low-amplitude beat-
has been shown to have different biological activity ing) to a more hyperactive one (low beat frequency,
compared with non-phosphorylated CaM. Interest- high-amplitude beating). Thus, there appears to be
ingly, although CAM can regulate phosphodiesterase a link between the phosphorylation of ODF1 during
(whose job it is to degrade cyclic nucleotides such as capacitation and the ability of spermatozoa to enter a
cAMP), the phospho(Try99) CaM is totally incapable hyperactivated state.
of activing calmodulin-dependent cyclic nucleotides
[48]. This suggests that the sperm cell does all it can to
increase the level of cAMP within the cell early on in Glycosylation
capacitation by not allowing phosphodiesterase to be In 1975, Austin published that the actual process of
switched on (see Figure 9.1). In agreement with this, capacitation consisted of removal of the glycoprotein
addition of high levels of the CaM antagonists triflu- coat on the spermatozoa, as well as the facilitation of
operazine and calmidazolium brings about a decrease hyperactivation [53]. However, despite this, there is
in the overall level of cAMP during capacitation in the a paucity of data when it comes to looking at pro-
mouse [49]. teomic characterization of glycosylation events dur-
A further role of CaM during capacitation has ing capacitation. Glycosylation can occur on either
been described. In sperm incubated with a higher asparagine (N-linked) or serine/threonine (O-linked)
level of extracellular Ca2+ , a lower level of tyro- residues and is a major post-translational modifica-
sine phosphorylation has been reported [50]. Inter- tion (PTM) event that occurs within developing sper-
estingly, addition of the CaM inhibitor deltamethrin matozoa. For example, during epididymal maturation,
under these conditions inhibited this loss of tyro- colloidal iron hydroxide labelling on rabbit, bull and
sine phosphorylation. The authors have suggested that hamster spermatozoa all demonstrate an increase in
calmodulin may regulate calcinuerin, a calmodulin- the cell surface net negative charge [54]. Hence, ejacu-
dependent phosphatase. In agreement with this, addi- lated spermatozoa are highly (negatively) charged. Yet
tion of cyclosporin A (an inhibitor of the calcineurin what functional role does this play? A recent model has
activator cyclophilin) also overcomes the loss of tyro- been proposed whereby during capacitation, sialic acid
sine phosphorylation seen when sperm are incubated appears to be actively removed from the spermatozoa
at high levels of calcium [45]. As such, calmodulin [55]. The manner in which this is achieved is through
likely down-regulates the activity of the protein phos- two neuraminidases (Neu1 and Neu3) which, although
phatase calcineurin during capacitation. initially present around the head of the sperm, become
Another major up-regulated tyrosine-phosphory- secreted into the surrounding fluid, where they act
lation change that occurs during capacitation is on the to cleave off terminal sialic acid residues [55]. Sup-
protein outer dense fibre 1 (ODF1). Four phosphoty- porting this model, inhibition of neuraminases during
rosine events have been reported to occur within this capacitation results in decreased amounts of tyrosine-
protein [42, 47]. In mice, three isoforms of ODF have phosphorylated proteins as well as ERK 1/2 phospho-
been detected, ODF1, ODF2 and ODF3(51). The role rylation [55]. Thus, it appears in mouse and human
of ODFs in general and ODF1 specifically in sperma- spermatozoa that proteolytic cleavage of sialic acid
tozoa is still unclear. Mice lacking the ODF1 gene pro- is necessary. However, this does not hold true for
duce normal numbers of spermatozoa with normal every species. Treatment of bovine spermatozoa with
motility but are completely infertile [52]. The major- a neurominadase inhibitor results in a higher level of
147
sperm–egg interaction [56], strongly suggesting that lation were found within AKAP3 and AKAP4. How-
terminal sialic acid groups within glycans are involved ever, due to the nature of the acetylation sites found
in bovine sperm–egg binding events. A similar pattern in the proteomic analysis (strong cation exchange, fol-
holds true in the chicken, whereby treatment of sper- lowed by LC-MS), it is unclear if any of these proteins
matozoa with neuraminidase results in complete dis- become acetylated during capacitation, since quantifi-
appearance of the sialic acid-binding lectin, Limulus cation was not performed [60]. Of interest, one pep-
polyphemus, and also severely impedes their ability to tide that was only found within capacitated, not within
populate the uterovaginal sperm storage tubule [57]. non-capacitated sperm, was the catalytic subunit of
Thus, in bovines and chickens, the presence of sialic PKA. Thus, it is possible that lysine 267 becomes acety-
acid appears to be necessary for sperm fertilization. lated during capacitation. (Note: In the paper, the
PRKACA 259 residue is acetylated. However, a close
Protein Acetylation during inspection reveals it is actually 267 [58].) These data
should be taken with caution nonetheless, since quan-
Capacitation tification based on the fact that it was only found can
As stated earlier, one major drawback of proteomics is be misleading in a proteomic analysis. This may also
the inability to detect less abundant proteins/peptides. mean that a tandem MS event did not occur on that
Enrichment of PTM peptides is necessary for a quanti- peptide in the non-capacitated sperm, despite that fact
tative analysis to become meaningful. Several methods that it is present. Although residue 267 is found within
exist for the enrichment of phosphorylation; however, a helix domain with PRKACA, it is unclear at this stage
with the exception of sialic acid glycopeptides, with what acetylation would confer on protein function.
which TiO2 can be used, the majority of PTM anal-
yses for proteomics use antibodies against the PTMs
of interest. Along these lines, anti-acetyl antibodies
Summary
have been used to immunoprecipitate acetylated pep- The use of proteomics is a valuable tool in trying to
tides in order to quantify the ‘acetylome’ and changes understand a cell such as the spermatozoon that lacks
therein during human sperm capacitation. Approxi- all capacity for nuclear protein synthesis. As such,
mately 973 acetylated lysine sites were found, corre- given the number of studies on sperm capacitation in
sponding to 456 proteins, suggesting that several pro- this area, it can still only be considered to be in its
teins are multi-acetylated [58]. Of interest, all of the infancy. Within somatic cells, over 3,000 phospho-sites
lysines shown to be acetylated were followed either by can be identified in a single LC-MS run. Yet within
a tyrosine, phenylalanine or histidine site. Thus, there spermatozoa, only several hundred phospho-sites have
appears to be a specific sequence necessary for lysine been identified. Despite the wealth of techniques that
acetylation and, interestingly, all three amino acids can be applied to looking at glycans, very few have been
are very polar in nature. Considering those proteins used to dissect what is really occurring during sperm
that were acetylated, many, like protein phosphoryla- capacitation. So it stands to reason that in the future
tion, were from both the glycolytic pathway and the decade, we are likely to see a large increase in the use
TCA cycle, suggesting that metabolism is highly reg- of proteomics to understand not only capacitation, but
ulated by acetylation within spermatozoa. Of interest, all aspects of sperm function.
alpha-tubulin was shown to be acetylated [58]. Oth-
ers have shown that a decrease in the acetylated alpha-
tubulin isoform is associated with reduced motility Note
within infertile sperm cells [59], suggesting that such 1. http://www.uniprot.org/docs/ptmlist.
an event is necessary for fertilization to proceed. Sev-
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151
Chapter
Current Concepts and Unresolved Questions
10 in Human Sperm Cumulus and Zona

Interaction
Christopher J. De Jonge and Christopher L. R. Barratt
Introduction Sperm Interaction with the

In several preceding chapters of this book, pre- Cumulus–Oocyte Complex
fertilization topics of pro-motility factors and require-
ments for motion changes, sperm-contributed egg During transit through the fallopian tube, spermato-
activation factors, and physiologic correlates of the zoa experience varied environmental stimuli that may
acquisition of fertilizing capacity, i.e. capacitation, serve in part to select [e.g. 1] and prime the sper-
have been presented. These cellular changes can, per- matozoa for subsequent encounter with the cumulus
haps, be considered as pre-fertilization maturational oocyte complex, such as (1) sperm motion alterations
changes. Without the ability or rather the competence as a consequence of physical binding to and release
of the spermatozoon to undergo change that is pred- from oviductal epithelial cells [e.g. 2, 3], (2) physiolog-
icated upon not only initial proper cellular assem- ical change resulting from exposure to oviductal-cell-
bly but also subsequent sensitivity and responsive- secreted proteins and peptides [e.g. 4], and (3) direc-
ness to extracellular signals while resident in both the tional responsiveness to tubal fluid movement [5],
male and female reproductive tract environments, the signals emanating from ovulatory factors, such as pro-
potential for conception rapidly deteriorates. gesterone and atrial natriuretic peptide [e.g. 6], and,
This chapter will focus on aspects of human fer- perhaps, proximal ovarian thermal effects [7]. In total,
tilization, and it is conspicuously brief because lit- then, spermatozoa en route to interaction with the
erature data regarding naturally occurring fertiliza- cumulus–oocyte complex are in a state of increasingly
tion in humans for the most part remain enigmatic heightened sensitivity, responsiveness and activity.
as compared with a plethora of data for nonhuman The cumulus–oocyte complex (COC) is composed
mammalian species, especially the mouse. Much of of an oocyte covered with layers of cumulus (granu-
what is known relies upon in vitro data, where culture losa) cells. The cumulus cells are held together by a vis-
conditions can influence sperm function, and from cous extracellular matrix of hyaluronic acid filaments
the mouse as a model, where significant differences (matrix) covered with proteinaceous granules [8].
between mouse and human are now featuring rather Upon ovulation the cumulus cell mass expands and
prominently. morphologically differentiates into several innermost
Fertilization is a complex of gametic events that layers in columnar organization, called the corona
culminate in the fusion of the male and female pronu- radiata (so named for the sunburst appearance of the
clei and the establishment of the next generation multicellular layers), and the overlying cumulus mass.
zygote’s genome. This chapter, expanding from evi- At the time and location where fertilization occurs in
dence provided in preceding chapters, will provide a the oviduct, the oocyte is surrounded mostly by the
description of component parts of fertilization from corona radiata. In order for fertilization to occur, sper-
the spermatozoon’s perspective, with a focus on cumu- matozoa must travel through any remaining cumulus
lus penetration and zona binding and penetration. mass cells and corona to contact the zona pellucida, the
University Press.
152
Chapter 10: Current Concepts and Unresolved Questions in Human Sperm Cumulus and Zona Interaction
outer shell that encases the oocyte. The question is how lar matrix facilitate COC penetration by the sperma-
sperm penetrate the corona. tozoa for subsequent zona interaction.
For obvious moral and ethical reasons, very little is While SPAM1 enzyme activity and sperm kine-
known about human sperm penetration of and passage matic changes have a role in facilitating sperm pas-
through the cumulus in vivo; thus, literature data relate sage through the cumulus, there is some evidence in
to more nonphysiological human in vitro and nonhu- humans [14] and cynomolgus macaques [15] to sug-
man animal data. gest that HA stimulates an increase in sperm intracel-
Tesarik and colleagues [9] first reported on the lular Ca2+ that may translate into a change in flagellar
motion characteristics of capacitated human sperma- motion. In the monkey, PH-20 is proposed to be the
tozoa interacting with cumulus oophorus dissected sperm receptor for HA that is responsible for aggre-
from cumulus oocyte complexes aspirated during rou- gating receptors that ultimately stimulate Ca2+ influx.
tine in vitro fertilization. They found that sperm To date, however, no similar HA-SPAM1 signal trans-
reacting with cumulus had a swimming pattern dis- duction mechanism has been demonstrated for the cal-
tinctly different from that of controls. While con- cium response in human spermatozoa.
trol sperm displayed a non-linear whiplash motion, A candidate factor responsible for stimulating
termed hyperactivation, sperm transiting the cumulus Ca2+ influx and subsequent motility change in human
had more linear motion. To restate, sperm within the sperm is progesterone (P4). The ovulatory follicle con-
cumulus were significantly less likely to display motion tains ultra-filtrate fluid, rich in P4, which bathes the
patterns characteristic of hyperactivation. They rea- COC during the final stage of maturation and at ovula-
soned that a more linear swimming motion was tion. Further, the ovulatory cumulus cells produce P4.
more likely to be facilitative for passage through the The P4 produced by the cumulus cells and the follicular
columnar-arranged cumulus extracellular matrix than fluid accompanying the COC act to stimulate intracel-
a nonlinear pattern. lular Ca2+ increases in human spermatozoa.
To more closely approximate sperm–cumulus Progesterone stimulates a biphasic intracellu-
interaction in the in vivo environment, an in vitro lar calcium increase in human sperm [16] via a
system of human cumulus cells drawn into a capil- unique nongenomic cell surface receptor [17, 18].
lary with the end submerged in a droplet of washed P4-stimulated Ca2+ influx results in human sperm
human sperm was developed. Various functional motion change [19] and the acrosome reaction [20].
parameters of sperm that passed through the cumu- Apropos of the former, Lishko et al. [21] demonstrated
lus and into an overlying medium were compared that P4 stimulates the primary calcium channel in
with those of sperm that did not pass through [10]. human sperm, termed CatSper.
Sperm that traversed the cumulus had more normal CatSper activation causes an increase in flagel-
morphology, changes in sperm kinematic parame- lar intracellular calcium that translates into motion
ters similar to what was described by Tesarik et al. requirements for sperm penetration through a vis-
[9], higher percentages of capacitated and acrosome- cous environment, such as the cumulus extracellu-
reacted sperm, and better zona-binding capacity [10, lar matrix. CatSper has been theorized to be a ‘poly-
11]. Thus, it can be concluded that one or more fac- modal stimulus integrator’ [22], meaning that the
tors associated with the cumulus mass regulate sperm channel senses the chemical and physical environmen-
motion. tal cues during and throughout sperm transit through
One factor purported to be, in part, responsible for the female reproductive tract and responds by selec-
inducing the sperm kinematic changes is hyaluronic tive channel activation required and necessary for the
acid (HA). As mentioned earlier, HA is a principal locale, which will further support sperm movement
component of the COC extracellular matrix. A GPI- ultimately to the oocyte and its interior.
anchored sperm plasma membrane protein (SPAM1),
formerly PH-20, has hyaluronidase activity that acts on Sperm Interaction with the Zona
the HA substrate [12]. SPAM1 is active at both neutral
and acidic pH and, in addition to an HA binding site, Pellucida (ZP)
contains a zona-binding domain [13]. It is believed In contrast to the relatively minimal work on human
that changes in sperm swimming motion in conjunc- sperm–cumulus interaction, there are a significant
tion with proximate degradation of the HA extracellu- number of studies examining human sperm–zona
153
interaction. The initial experiments were focused on thought to contribute to the structural integrity of the
the ZP as the site of initiation of the acrosome reac- ZP matrix, acting as a linker molecule between ZP
tion (AR) [23]. When the acrosomal status of sperm in filaments, ZP2 was involved in the secondary bind-
the ZP and in the surrounding material (usually cul- ing for AR sperm [37], and ZP3 was accepted to be
ture dishes) was examined, many of the data indicated the primary sperm receptor responsible for binding
that sperm initially bound to the zona were acrosome- to acrosome-intact capacitated sperm and induction
intact and then were induced to undergo an AR [24]. of the AR [38]. The human zona was presumed to
Some data, notably from Morales and colleagues [25], have a similar structure and thus, with progress in
indicated that acrosome-reacted human spermatozoa recombinant technology, a plethora of experiments
could bind to the ZP, although the nature of this bind- were performed using recombinant human zona pro-
ing (tight vs. loose) was difficult to determine. Limi- teins to study sperm–zona interaction in more detail.
tations in technology such as an accurate method for Additionally, recombinant ZP3 was placed on beads
assessment of the dynamics of the AR in live spermato- to mimic a sperm function test [39]. Production of
zoa (it was the 1980s, after all) meant it was difficult to biologically active recombinant human zona proteins
resolve the issue of the ZP as the primary site of AR proved to be very challenging (solubility, purification,
induction [26]. Although not in humans, live imag- folding, consistent biological activity, etc.) and the lack
ing experiments in rhesus macaques show that sperm of consistent data made understanding the biological
binding to the ZP are acrosome-intact and that AR picture difficult. Breakthroughs in genetics revealed
occurs upon binding to the ZP [27]. It will be interest- that the human and mouse ZP were very different –
ing to do comparable experiments using human sper- humans had four zona genes (not three as in mice) and
matozoa. Nevertheless, in vitro experiments such as subsequent experiments demonstrated that all four
this are relatively artificial. For example, almost all proteins were expressed in the human ZP [40]. Per-
human sperm–zona experiments are performed with- haps, with hindsight, the difficulty with the recombi-
out a cumulus, which is of course an artificial system. nant system was that we were using the wrong (mouse)
If the cumulus (see above) plays a significant role in model – concentrating on a three-protein, not a four-
sperm priming/activation, then the state of functional protein system [41, 42].
readiness of spermatozoa that interact with a cumulus- Jurrien Dean developed a ‘difference system’ to
free zona will be different than when there is initial study the sperm–ZP interaction. Based on expression
exposure to cumulus prior to contact with the ZP. This of a mixture of human and mouse zona genes in the
may or may not change the interpretation of the data. same structure, he elucidated that it was in fact human
With the development of IVF as a treatment for ZP2 (which does not bind to mouse sperm) that was
infertility, human zonae from unfertilized oocytes the primary ZP-binding protein for human sperma-
became available for research and a human ZP sperm tozoa [43]. Subsequent experiments utilizing recom-
function test was developed [28, 29]. Human sperm binant production of ZP2 peptides have identified a
bound to the zona were proportionately more mor- region on ZP2 which is the primary binding site for
phologically normal [30] and had less DNA dam- human spermatozoa [44]. Very recently, Avella and
age [31] and a lower percentage incidence of ane- colleagues produced human ZP2 peptide beads that
uploidy [32]. Several studies demonstrated that the bind human spermatozoa and provide preliminary
number/proportion of sperm bound to the zona was a evidence that sperm bound to these beads are better
good sperm function test [33] and, in particular, could able to penetrate the human ZP [45].
identify men with what appeared to be normal semen Where can we go from here? The potential of
parameters but an abnormality in sperm–zona inter- recombinant peptides enables (1) the potential for
action [34]. Subsequent experiments identified a sub- selection of higher-quality sperm for use in ART, (2)
group of men where the sperm bound to the ZP but development of a potential sperm function test (20
could not undergo a physiological AR [35]. Addition- years after originally proposed!), and (3) study of the
ally, selection of zona-bound human sperm for ICSI molecular interaction between human sperm and the
resulted in higher-quality embryos and higher implan- human zona. Historically it has been very difficult to
tation rates than with scientist-selected sperm [36]. determine what the candidate receptor(s) on sperm are
Experiments in mice had shown that the ZP was when we cannot identify what the sperm binds to on
composed of three glycoproteins, where ZP1 was the zona. In ART we have a number of clear phenotypic
154
abnormalities in men, for example, those whose sperm 11. Hong SJ, Chiu PCN, Lee KF, Tse JYM, Ho PC, Yeung
do not bind to the zona, and these can then be used as WSB. Cumulus cells and their extracellular matrix
tools to assist the dissection of the molecular details of affect the quality of the spermatozoa penetrating the
the sperm–zona interaction process [see 46]. Bearing cumulus mass. Fertil Steril 2009; 92: 971–8.
in mind the clear differences between mice and human 12. Lin Y, Mahan K, Lathrop WF, Myles DG, Primakoff P.
(e.g. ZP structure, difference in sperm physiology) it is A hyaluronidase activity of the sperm plasma
membrane protein PH-20 enables sperm to penetrate
very unlikely, despite our hopes, that the mouse rep- the cumulus cell layer surrounding the egg. J Cell Biol
resents a good model for the human ZP interaction 1994; 125: 1,157–63.
[42]. To understand human ZP interactions better, we
13. Sade A, Banerjee S. SPAM1 (sperm adhesion molecule
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156
Chapter
Sperm-Specific WW-Domain-Binding
11 Proteins
Richard Oko, Mahmoud Aarabi, Jiude Mao, Hanna Balakier
and Peter Sutovsky
Introduction types of PAWP correlate with fertility in livestock ani-

mals and with treatment outcomes in couples under-
The sperm perinuclear theca (PT) is a cytoskeletal cap-
going assisted reproductive therapy (ART). In addition
sule that shapes the mammalian sperm nucleus during
to PAWP, its somatic orthologue, WBP2 is present in
spermiogenesis and protects it from mechanical, phys-
mouse but not human or ungulate sperm PT, which
ical and chemical injury during sperm maturation in
could explain the lack of male-infertile phenotypes
the epididymis and sperm transport and fertilization
in recently created PAWP-null mice. The discovery
within the female reproductive system. Besides amal-
of PAWP expression in breast cancer cells and the
gamating cytosolic components and male-germ-line-
presence of anti-PAWP antibodies in cancer patients’
specific proteins, the PT contains unique signalling
serum add a new level of significance to the study
molecules that are released into oocyte cytoplasm
of PAWP protein and its somatic orthologue. These
through PT solubilization at fertilization. Among
findings are discussed here in the context of evolu-
them, the postacrosomal sheath WW-domain bind-
tionarily conserved fertilization mechanisms, current
ing protein (PAWP or WW-binding protein 2 N-
paradigms of oocyte activation, male fertility evalua-
terminal-like/WBP2NL, in HUGO nomenclature) is
tion and infertility treatment, cancer diagnostics and
a major, evolutionarily conserved, male-germ-line-
transgenic animal models of human disease.
specific protein component of the postacrosomal PT.
Studies in various mammalian species and frogs indi-
cate that the release of PAWP from the sperm head at
the time of sperm–oolemma fusion coincides with the
Structure and Biogenesis of Sperm
hallmark events of oocyte activation, such as induc- Perinuclear Theca
tion of repetitive calcium release from oocyte endo- The sperm PT is a multi-layered cytoskeletal capsule
plasmic reticulum, cortical granule exocytosis and the covering all parts of the sperm head nucleus [1]. The
onset of zygotic development, marked by the for- PT is inserted between the inner acrosomal mem-
mation of maternal and paternal pronuclei. Conse- brane (IAM) and the nuclear envelope in the ante-
quently, oocyte activation can be blocked by sperm rior sperm head and sandwiched between the sperm
co-injection with anti-PAWP antibodies or competi- plasma membrane and the nuclear envelope in the
tive peptides that interfere with PAWP’s ability to inter- posterior part [2]. Tracing the major segments of the
act with its substrate WW-domain containing pro- sperm head, the PT can be divided into the sub-
teins. Recently, we demonstrated that the expression acrosomal layer (SAL), including the outer periacro-
of PAWP protein through oocyte microinjection with somal layer (OPL) residing over the equatorial seg-
PAWP complementary RNA (cRNA) elicits ooplas- ment (ES) of the acrosome, and the postacrosomal
mic calcium release, a hallmark of oocyte activation sheath (PAS)[3] (Figure 11.1). It should be emphasized
in both vertebrates and invertebrates. Flow cytomet- that antibodies raised against SAL proteins immuno-
ric studies revealed that the sperm levels and pheno- label both the SAL and OPL, suggesting an identical
University Press.
157
Chapter 11: Sperm-Specific WW-Domain-Binding Proteins
Perinuclear Theca
1. Subacrosomal Layer
2. Outer Periacrosomal
Layer
ES
N
3. Postacrosomal Sheath
PM
SG
Centriole Mid-sagittal section of sperm
head as seen by EM
T
Figure 11.1 Diagrammatic representation of a midsagittal section through the head of a spatulate-shaped eutherian spermatozoon, in
relation to its three-dimensional configuration. The perinuclear theca is a layer of condensed cytosolic proteins found between the membrane
systems of the sperm head. It is divided into two compositionally different regions, the subacrosomal layer and the post-acrosomal sheath.
The subacrosomal layer is found between the inner acrosomal membrane and the nuclear envelope and is continuous with another layer of
material, referred to as the outer periacrosomal layer, which is sandwiched between the plasma membrane (PM) and the outer acrosomal
membrane overlying the equatorial segment (ES). The post-acrosomal sheath begins caudally where the acrosome ends and is sandwiched
between the plasma membrane and the nuclear envelope. The surface groove (SG), a circumferential indentation of the plasmalemma at
the base of the sperm head, often referred to as the post-nuclear ring, marks the caudal extent of the perinuclear theca. It is here that the
plasmalemma comes into close apposition to the outer nuclear membrane. A = acrosome; N = nucleus; T = sperm tail.
composition (see Table 11.1). Importantly, the OPL tion of PAS during spermatid differentiation. Develop-
could serve to stabilize the plasmalemma overlying the mentally, the deposition of PAS-PT coincides with the
outer acrosomal membrane of the ES, preventing it acquisition of oocyte-activating factors, endowing the
from undergoing the acrosome reaction and preserv- spermatid with the capability to activate oocytes when
ing it for sperm–oolemma fusion. The deposition of microinjected into the ooplasm [9, 12].
PT starts at the round spermatid stage of spermio-
genesis and occurs from two distinct foci. The SAL
is primarily derived in association with Golgi secre- Molecular Composition of
tory vesicles that give rise sequentially to the acrosomic
vesicle and the acrosomal cap [4, 5]. The nascent SAL,
Perinuclear Theca
sometimes referred to as acroplaxome [6], is probably
instrumental in the anchoring of the acrosomic vesi-
More than Just Amalgamated Germ
cle to the anterior nuclear envelope, which is a pre- Cell Cytosol
lude to distal extension of the acrosomal cap [7, 8]. The Perinuclear theca could be considered a product of the
PAS of PT primarily arises from proteins that are pro- highly condensed cytoplasm of a spermatid. However,
duced in the cytoplasmic lobe of the elongating sper- proteins are inserted into the PT in a compartmental-
matids and travel to both PAS and the ES region along ized fashion and the process of PT assembly is not a
the microtubule-based caudal manchette [9–11]. It fol- merely random amalgamation of the cytosol [1, 34].
lows that the deposition of SAL predates the forma- Thus, the proteome of perinuclear theca arises from
158
Table 11.1 Identified perinuclear theca (PT) proteins: PAS postacrosomal sheath, SAL subacrosomal layer, OPL outer periacrosomal layer
residing over the equatorial segment of the acrosome
Location
Molecular spermatozoa
Identity mass in kDa Type of protein in PT References
Identified by Dr. Oko’s group
PERF 15 15 Fatty-acid-binding family SAL Oko and Morales 1994 [13]
(only in murids)
SubH2Bv (PT15) 15 Histone variant SAL, OPL Aul and Oko 2002 [14]
RAB2A (PT24) 24 Vesicular transport SAL, OPL Mountjoy et al. 2008 [15]
KPNA6 60 Importin alpha SAL, OPL Tran et al. 2012 [5]
KPNB1 75 Importin beta SAL, OPL Tran et al. 2012 [5]
Calicin (PT60) 60 Basic and structural PT Oko et al. 2001 [16]
GSTO2 28, 31 Glutathione S-transferase PAS Hamilton and Oko
Omega2 (in preparation) [17]
PAWP (PT32) 32 WW domain-binding protein PAS Wu et al. 2007a [18]
(signal transduction)
H3 19 Somatic core histones PAS Tovich and Oko 2003 [19]
H2B 18 Somatic core histones PAS Tovich and Oko 2003 [19]
H2A 17 Somatic core histones PAS Tovich and Oko 2003 [19]
H4 14 Somatic core histones PAS Tovich and Oko 2003 [19]
Identified by Dr. Franke’s group
Cylicin I 74 Basic and structural PAS Hess et al. 1993 [20]
Cylicin II 58 Basic and structural PAS Hess et al. 1995 [21]
Calicin (PT60) 60 Basic and structural PAS von Bülow et al. 1995 [22]
Capping protein ␣3 (CP ␣3) 31 Actin capping PAS von Bülow et al. 1997 [23]
Capping protein ␤3 (CP ␤3) 37 Actin capping PAS von Bülow et al. 1997 [23]
Actin-related protein T1 40 Structural PAS Heid et al. 2002 [24]
(ArpT1)
Actin-related protein T2 40 Structural PAS Heid et al. 2002 [24]
(ArpT2)
Identified by other researchers
C-YES 60 SRC tyrosine kinase SAL Leclerc and Goupil 2002 [25]
TR-KIT (truncated -KIT) 30, 50 Tyrosine kinase receptor AL, OPLa Muciaccia et al. 2010 [26]
MN13 (detected by ? Candidate egg-activating PAS Manandhar and Toshimori
monoclonal antibody) factor (properties unknown) 2003 [27]; Ito et al. 2010 [9]
STAT14 85 Transcription activator PAS Herrada and Wolgemuth
1997 [28]; Lachance and
Leclerc 2011 [29]
CYPT1 19 Basic and structural PAS Kitamura et al. 2004 [30]
ZNF645 49 E3 ubiquitin-protein ligase PAS Liu et al. 2010 [31]
Glutamine synthetase 45 Enzyme PASb Francou et al. 2012 [2]
Dp71f-like, Dp71dc 70 Short dystrophin family PAS Hernandez-Gonzalez et al.
products 2001 [33]
a Location in equatorial region (OPL) of human sperm identified by immunofluorescence. Without the resolution of EM it is difficult to
distinguish if immunolabelling is over the equatorial segment of the acrosome or is in the proximal region of the PAS. Narrow
immunobanding pattern is reminiscent of PAWP labelling in primate sperm (see [18]).
b Location to the PAS is questionable. By EM looks like glutamate synthetase is located in the region of the redundant nuclear envelope of
the sperm head just below the PAS.
c Plasmalemma associated overlying PAS.
selective inclusion and differs from the proteome of segments as well as varied resistance to extraction by
residual spermatid cytoplasm/residual body or that ionic detergents and protein solubilizing agents [34,
of the cytoplasmic droplet [35]. The major proteins of 36].
PT include cytoskeletal proteins, signalling molecules, The structural continuity of the PT is retained after
transcription factors and histones (see Table 11.1). non-ionic detergent extraction (e.g. NP40, Triton-
These proteins have distinct localization within PT X100 and Lysolecithin) of sonicated and isolated
159
SSpH PT proteins Nucleus
Sonication 0.2% TX-100 0.1 M NaOH

+
centrifugation centrifugation centrifugation
STEP 1 STEP 2 STEP 3

pellet pellet pellet
supernatant
Figure 11.2 Approach used to selectively extract perinuclear theca (PT) proteins from sonicated and isolated sperm heads (SSpH). After
sonication of spermatozoa and separation of heads from tails in a sucrose gradient (Step 1), the SSpH are incubated with non-ionic detergents
(e.g. 0.2% Triton X-100, 1% NP-40 or 0.4% Lysolecithin) (Step 2), which solubilize the nuclear and acrosomal membranes and release
membrane-associated proteins, of which the inner acrosomal-membrane-associated proteins (stars and dots) are the most abundant [36]. On
centrifugation, the released membrane-associated proteins end up in the supernatant while the sperm nucleus, surrounded by the intact PT,
is pelleted. The PT proteins can then be selectively extracted from this membrane-free head pellet by incubation with an alkaline solution,
such as 100 mM NaOH (Step 3). Following centrifugation, the PT proteins end up in the supernatant while the barren nucleus is pelleted.
PT-associated core somatic histones that are ionically bound to the PT can be removed prior to alkaline extraction by high salt
extraction [19].
sperm heads (Figure 11.2). Essentially, all that remains proteins [36, 37] and can be used to improve oocyte
is a shell of PT material surrounding the condensed activation and embryonic development after intracy-
nucleus (Figure 11.3). This sperm head fraction serves toplasmic sperm injection [38, 39]. Surprisingly, this
as an ideal starting point for extraction of the PT head fraction has been found to be devoid of the
candidate spermborne oocyte activating factor PLC␨
[40], yet still able to induce calcium oscillations [39].
Because of this, we tested whether we could inhibit cal-
cium oscillations induced by Triton-X100-extracted
human spermatozoa utilizing a peptide competitive
inhibitor to candidate oocyte activating PAWP and
found that we were successful (Figure 11.4).
Structural/Cytoskeletal Proteins
The rigidity of the PT is most likely due to the presence
of a structural protein framework made up of basic
and cysteine-rich proteins (Table 11.1; [42]) such as
calicin, cylicin I and II and CYPT1 [30, 43, 44], which
Figure 11.3 Sonicated and isolated mouse sperm heads (SSpH)
that were extracted with nonionic detergent, leaving behind only may serve to anchor the other categories of PT pro-
the condensed nucleus (N) and PT. The subacrosomal layer of the teins by ionic and disulphide bonds. Calicin appears
PT (SAL-PT) can be clearly distinguished from the postacrosomal to be found throughout the PT and therefore may be
sheath of the PT (PAS-PT). The dashed white line divides the two
regions of the PT from each other. Compare the ventral spur (VS) of the major structural link between the SAL and PAS
the PAS to the dorsal apex (arrow) of the SAL. Bars 0.2 µm. [45]. The actin-related proteins T1 and T2 (ARPT 1
160
Figure 11.4 The PAWP-derived PPGY competitive peptide inhibits calcium oscillations induced by Triton X-100 (TX)-treated sperm in
humans. Human spermatozoa were treated with TX as described in [39] and injected into the human metaphase II oocytes (n = 6 oocytes
from four different oocyte donors). Dynamics of intracellular calcium was monitored in injected oocytes for 2 h after injection by confocal
microscopy as described in [41]. The resulting calcium oscillations (A) resembled sperm-induced oscillations were observed post-ICSI (B) and
were inhibited when TX-100 spermatozoa (C; n = 7 oocytes from three different oocyte donors) or sperm (D) were coinjected with the
synthetic PPGY competitive peptide derived from the human PAWP sequence.
and 2), which have many epitopes in common with Histones

actin, may also form part of this structural frame-
Histones include somatic-cell-type histones [19] as
work [24]. Whether they form a filamentous network
well as testis-specific histone variants [45]. Somatic-
remains to be explored. Previous localization studies,
cell histones are transported by the caudal manchette
with anti-actin antibodies and phalloidin, have not dis-
mainly to PAS during spermatid elongation [10], while
criminated between actin and ARPT proteins within
the testis-specific histone H2B variant, SubH2Bv, is
the sperm head. PERF 15, a fatty-acid-binding pro-
inserted into the SAL during acrosomal biogenesis
tein and the major protein constituent of the perforato-
[45]. In the early phases of acrosomal formation,
rium, the complex of subacrosomal PT and inner acro-
SubH2Bv, by the nature of its bipartite nuclear local-
somal membrane, forms a triangular rodlike structure
ization signal, appears to be involved in the transport
in the apical part of SAL. It was one of the first SAL PT
of the acrosomal vesicle to the nucleus for attachment
proteins characterized and is by far the most abundant
[5]. Although both types of histones could be released
PT protein in the mouse and rat [13]. However, PERF
from PT in the ooplasm and incorporated into the
15 was not found in spatulate mammalian sperm heads
nascent paternal pronucleus, such a paternal contribu-
[45], casting doubt on the old concept that a vestige of
tion to the zygote has not been confirmed by fertiliza-
the perforatorium is present in them. This data coin-
tion studies (unpublished observations).
cided with a study correlating the presence of PERF 15
with apical and ventral processes of falciform sperm
heads ([46]. Even though PERF 15 mRNA is expressed
in the human testis [47], it does not appear to be a con-
Perinuclear Theca Proteins Associated with
stituent of the human sperm (unpublished data, RO). the Acrosome
The idea that PERF 15 is involved in providing shape So far the SAL-PT proteins that have been character-
to the falciform head was borne out in the fertile sperm ized in our lab (see Table 11.1) appear to be involved
phenotype of mice lacking FABP9/PERF15 [48]. in acrosome vesicle growth and transport during early
161
spermiogenesis. In common, all of these proteins coat the PAS-PT [33]; DFP71D is associated with the plas-
the peripheries of both proacrosomic and acrosomic malemma overlying the PAS-PT, whereas DP71F-like
vesicles during acrosomal biogenesis. It has been pro- is not readily extractable by non-ionic detergent from
posed that SubH2Bv, along with two karyopherins, the PAS-PT.
KPNA and KPNB, whose remnants are also found In our review of the articles on the localization of
in the SAL-PT, form a trimeric complex that targets PT proteins we noticed that the terms equatorial seg-
and docks the acrosomic vesicle onto the nucleus of ment, equatorial segment region and equatorial region
the round spermatid [5, 36]. Reflecting its origin in are often used interchangeably or incorrectly to des-
the Golgi-derived acrosomal precursor structures, the ignate the location of immunofluorescence in spatu-
vesicular transport protein RAB2A is also enriched late sperm heads. Strictly speaking, localization to the
in SAL ([15]. Two other proteins, C-YES [25] and equatorial segment should be used only if the protein
TR-KIT [26], have been localized to the SAL–PT of localizes to the content of this most caudal segment of
human sperm (see Table 11.1). The residence of TR- the acrosome or to the inner or outer acrosomal mem-
KIT in the SAL-PT of spermatozoa corresponds to brane associated with it. If it is uncertain where the
its origins in round spermatids and association with localization is in relation to the depth of the equatorial
acrosome formation, while the origin of C-YES dur- segment, which usually is the case, then the labelling
ing spermiogenesis has not been analyzed. C-YES, a should be designated to the equatorial segment region.
SRC tyrosine kinase, may be responsible for some of Often, fluorescent immunolabelling forms a rectangu-
the capacitation-induced increases in protein tyrosine lar banding pattern in the ‘equatorial region’, which
phosphorylation and also has a more soluble form could depict labelling either in the proximal part of the
which may be associated with the outer acrosomal PAS or in the equatorial segment region. The only way
membrane and plasmalemma [25]. TR-KIT, a trun- to resolve this issue is by the higher resolution achieved
cated form of tyrosine kinase receptor, has been shown by immunogold electron microscopy. It is also impor-
to have oocyte-activating activity in the mouse involv- tant to note that the equatorial segment in most spat-
ing the activation of FYN kinase in the oocyte cor- ulate sperm heads is cup-shaped with its apical end
tex and phosphorylation of phospholipase C Gamma concave and its caudal end flat. Therefore, if this pat-
1 (PLC␥ 1) in the ooplasm, resulting in calcium release tern is achieved by immunofluorescence, it most likely
from oocyte stores [49, 50]. However, previous local- depicts labelling in the equatorial segment region.
izations of TR-KIT in mouse sperm [51, 52] do not
appear to correspond with its localization in human Post-fertilization Fate of Sperm
sperm [26].
Perinuclear Theca
Sperm–oolemma fusion starts at the ES, during which
Transcription Factors and Other Proteins time PT appears to come off in layers with the help of
The activator of the transcription protein STAT4 is actin-rich oocyte microvilli and solubilization in the
present in the PT of human [29] and mouse [28] ooplasm [53]. Such sperm head incorporation, assisted
spermatozoa, the former study localizing STAT4 by by microfilaments, can be disrupted by cytochalasin
immunofluorescence to the PAS-PT. The zinc fin- treatment, which, however, does not prevent PT solu-
ger domain containing RING finger ubiquitin ligase bilization in the area of initial sperm–oolemma fusion
ZNF645 is present in PAS PT in addition to its local- or subsequent oocyte activation [54]. The PT solubi-
ization in the sperm flagellum. Glutamine synthetase lization requires reduction of disulphide bonds (S–S)
has been localized by immunofluorescence in the most and proteolysis, and thus can be hindered by the deple-
caudal region of the sperm head and implied to be tion of oocytes’ intrinsic S–S reducing peptide glu-
part of the PAS-PT [32]; however, the EM immuno- tathione [55] and by protease inhibitors [56], respec-
gold micrograph shown by these authors clearly tively. Solubilization of ES PT or OPL is followed by the
indicates that glutamate synthetase is just distal to the dispersion of PAS PT. In accordance with this chronol-
caudal end of the PAS, most likely localized in the ogy of ES PT and PAS PT solubilization, the sperm-
redundant nuclear envelope region. The dystrophin borne oocyte activating factors (SOAF) may be com-
family proteins DFP71D and DP71F-like are local- partmentalized as two distinct fractions in the mouse
ized by immunofluorescence to the sperm tail and to sperm head: the first, highly soluble, freeze–thaw- and
162
Figure 11.5 Localization of PAWP in normal and defective mammalian spermatozoa and during the early stages of pronuclear
development post-fertilization. (A) Localization of PAWP (red; bottom) and its intensity profile generated by MetaMorph imaging (top)
of ejaculated boar spermatozoa. (B–F) Solubilization and nuclear translocation of PAWP in the porcine ICSI zygotes starts early after injection
(B, C – detail of PPN from B) and is still traceable during sperm nuclear decondensation (D), paralleled by the excision of the sperm flagellum
(arrow in E). Note the persistence of subacrosomal PT (arrowheads, inset E), which does not contain PAWP and is uniquely observed after ICSI,
as opposed to natural fertilization. PPN = paternal pronucleus; MPN = maternal pronucleus. (G) Deposition of PAWP (red) along the caudal
manchette (green) in a murine elongating spermatid. (H, I) Individual bull spermatozoa show varied intensities of PAWP labelling (H; red),
which is completely missing from defective spermatozoa coated with ubiquitin (I; green). DNA in all panels was counterstained with DAPI
(blue). (A black and white version of this figure will appear in some formats. For the colour version, please refer to the plate section.)
detergent-extractable fraction could be present in the and protecting it from proteolysis, it is possible that the
ES PT; the second fraction is less soluble, detergent- epitopes used to detect the histones become hidden in
resistant and localized mainly in the PAS [57]. Further the PPN.
studies suggested that at least two distinct protein com-
ponents contribute to mouse SOAF, which have differ-
ential sensitivity to heat treatment [56].
Sperm-Borne WW-Domain Proteins
While the SAL PT appears to dissolve relatively and Their Candidate Substrates
early during natural fertilization, the complex of SAL
PT, ES and IAM persists on the nuclei of spermato- WW-Domain-Binding Proteins
zoa after intracytoplasmic sperm injection (ICSI) and The WW-domain-binding proteins (WBP) are a group
occasionally interferes with the formation of the pater- of proline-rich proteins involved in signalling and
nal pronucleus (PPN) [58, 59]. Against expectation, transcriptional regulation that bind specifically to
the histones of PAS PT do not appear to diffuse into the the WW-domain-containing proteins. The repetitive
PPN (R. Oko and P. Sutovsky, unpublished), but PAWP, PPxY motifs of WBP mediate WBP interactions with
discussed next, does (Figure 11.5) [18]. However, since their substrates in cell signalling. Of significance, short
the expectation is that core histones would aid in the PPxY peptides with appropriate flanking sequences
development of the PPN by stabilizing the chromatin can be used for affinity purification of substrate
163
Figure 11.6 Localization of the candidate PAWP-interacting ooplasmic WW-domain proteins in porcine oocytes and embryos.
(A–D) The YES-associated protein YAP (red) is present in the meiotic spindle of fertilization-competent metaphase-II (MII) oocytes (A) and in
pronuclei of IVF zygotes (B) and embryos reconstructed by SCNT (C). Note the high ooplasmic YAP signal at all stages. Negative control (D)
was performed by replacement of anti-YAP antibody with a nonimmune rabbit serum followed by anti-rabbit Ig conjugate. (E–H) The
WW-domain ubiquitin ligase NEDL2 (syn. HECW2; green) is prominent in the germinal vesicle (GV) and its resident nucleolus of the immature
GV-stage oocytes (E) in the zygotic pronuclei (F; 2PN), in all stages of embryonic development up to and including the blastocyst (G; BL), and
in oocytes activated parthenogenetically by microinjection of Pawp cRNA (H). In all immunofluorescence panels, DNA was counterstained
with DAPI (blue). Corresponding intensity profiles generated by MetaMorph are shown below each panel. (A black and white version of this
figure will appear in some formats. For the colour version, please refer to the plate section.)
WW-domain proteins as well as competitive inhibitors approximately 40 amino acid (AA) residues that
of signalling involving WBP interactions with WW- features conserved, evenly spaced tryptophan residues
domain proteins. and forms a triple-stranded beta sheet [60]. Of sig-
The WW-domain-containing proteins, the sub- nificance for the proposed role of WBP2NL/PAWP
strates of WBPs, have high affinity for proline-rich in oocyte activation is the ability of WW-domain
proteins, particularly those containing repetitive PPxY proteins to compete with the SH3 domain of protein
motifs, such as sperm WBP2 and WBP2NL/PAWP. tyrosine kinases [61, 62].
This group contains a large number of related and
unrelated proteins, both structural and signalling
molecules involved in cytoskeletal architecture, Substrates of WBP Relevant to Fertilization
nuclear function, protein phosphorylation and Among the potential substrates of the sperm-
protein turnover, among others. The WW domain released PAWP in the ooplasm are the YES-kinase
is a small protein–protein interaction module of associated protein (YAP) and the WW-domain-
164
containing ubiquitin ligases HECW1 (NEDL1) and PAWP and Its Somatic Orthologue
HECW2 (NEDL2). The YAP protein binds to YES
tyrosine kinase to activate tyrosine-phosphorylation- WBP2
dependent signalling [63]. Significantly for oocyte
activation research, PAWP has been shown to bind PAWP/WBP2NL
YAP by Far Western blotting [18], and YAP is bounti- The postacrosomal sheath WW-domain-binding
fully present in mammalian oocytes (Figs. 11.6A–6D). protein (WW-binding protein 2 N-terminal-
Ubiquitin ligases HECW1 and 2 are the members like/WBP2NL in HUGO nomenclature) is a major
of NEDD4 subfamily of E3 ubiquitin ligases, which protein component of PAS-PT in mammals [18]. It is a
share a similar domain composition: an N-terminal C2 product of an evolutionarily conserved gene expressed
domain that mediates anchoring of E3s to intracellular exclusively in the male germline in vertebrates.
membranes, WW domains (2-4) that mediate protein– The expression of the Pawp gene is first observed
protein interaction, and a catalytic HECT domain at in secondary spermatocytes in the rat, and PAWP
the C-terminus. It was demonstrated that HECW1 protein synthesis peaks during spermatid elongation
associated with the C-terminus of tumour protein p53 [18]. Significantly, this transcription and translation
and increased its pro-apoptotic functions independent pattern coincides with the timing of the acquisition
of HECW1’s E3 ligase activity [64]. The HECW2 lig- of the oocyte-activating capability by the elongating
ase, whose mRNA is preferentially expressed in neu- mammalian spermatids [9]. The PAWP protein
ronal tissue, interacts with two isoforms of p73, ␣ and appears to be synthesized in the spermatid cyto-
␤ (p73 is another tumour protein related to the p53); plasmic lobe and deposited in the PAS by transport
it binds to p73, but not to p53, and ubiquitinates p73. along the caudal manchette in the bull, boar and rat
This ubiquitination leads to stabilization of p73␣/␤ spermatids [11].
and increases p73␣ transcriptional activities [65]. The
HECW2 ligase is also involved in regulation of cell
cycles [66]. WBP2
Both Hecw1 and Hecw2 genes are expressed and Wbp2nl arose from gene duplication of Wbp2
translated in the testis and the ovary. In particular, we (Ensembl: GeneTree ENSGT530000063718). As
detected HECW2 protein in the pro-acrosomic gran- early as the existence of fish, both genes were found
ules of round spermatids and in the caudal manchettes together in the same genome; however, unlike the
of elongating spermatids, as well as in the oocyte Wbp2nl gene, which appears to have a testis-specific
germinal vesicle, zygotic pronuclei and embryonic expression in mammals, Wbp2 has a ubiquitous tissue
blastomere nuclei up to and including the blastocyst expression including expression in the testis. The
(Figs. 11.6E–6H). Association of HECW2 with nascent N-terminal halves of their protein products, WBP2
acrosomal structures indicates involvement in acro- and WBP2NL, share high sequence homology and
somal biogenesis, while its presence in the caudal contain a Pleckstrin homology domain shown in
manchette at later steps of spermiogenesis is consistent other proteins to be involved in membrane coupling
with the proposed role of the ubiquitin–proteasome via phosphoinositides (Figure 11.7). In the C-terminal
system in protein transport from the cytoplasmic half, there is little sequence homology between these
lobe to the equatorial and postacrosomal regions, and two proteins except for a high proline content and
in the shaping of sperm nuclei [67]. Association of the common occurrence of PPxY motifs. Importantly,
NEDL2 with the caudal manchette mimics that of our BLAST search uncovered a mouse WBP2 isoform
PAWP [11], which makes it possible that the two pro- (unnamed, GenBank: BAB23594.1) that has an amino
teins interact during spermatid elongation. High lev- acid sequence length similar to that of WBP2NL
els of HECW2 were detected in the single pronu- (Figure 11.7), and based on its molecular mass of
clei of parthenogenetic porcine zygotes resulting from approximately 40 kDa and immunoblotting, appears
the microinjection of Pawp cRNA (Figure 11.6H). to prevail as the dominant isoform in the lung and
It remains to be assessed whether this NEDL2 pro- testis but not in the brain, where the shorter WBP2
tein pattern is different from that of partheno- isoform prevails. It is this unnamed longer WBP2
genetic oocytes activated chemically or by an electrical isoform which we believe we are co-localizing with
pulse. WBP2NL/PAWP in the PAS of the sperm head (see
165
Figure 11.7 Multiple mouse protein sequence comparison between two isoforms of WBP2 (NCBI reference sequence: NP_058548.1 and
GenBank: BAB23594.1) and WBP2NL/PAWP (NCBI access code: Q9D529.1). The N-terminal halves of these proteins share high sequence
homology and contain a Pleckstrin homology domain (boxed in). There is little sequence conservation in the C-terminal end of these proteins
except for high proline content and the presence of PPxY motifs (bold font).
WW-Binding Proteins as Proposed Oocyte Activation first found to interact with the SH3 domain of C-YES, a
Factors). nonreceptor kinase of the SRC family [63]. Since these
WBP1 and 2 were the first proteins identified to initial studies, many WW1-domain-containing pro-
interact through their PPxY (PY) motifs with the WW teins and their cognate PY ligands have been identified
domain of Yes-associated protein (YAP) [68, 69]. YAP and demonstrated to be involved in a wide spectrum
is a proline-rich phosphoprotein of 65 kDa that was of cellular events ranging from cell cycle control to
166
ubiquitin ligation, transcriptional activation and reg-

ulation of cell proliferation and apoptosis. WBP2‘s
interaction with YAP was shown to augment the
transcriptional activity of oestrogen and progesterone
receptors [70]. More recently, the YAP-WBP2 interac-
tion was shown to play a key role in the Hippo tumour
suppressor pathway [71, 72]. For a detailed biophysi-
cal analysis of the binding of WW domains of YAP to
PPxY peptides derived from WBP1 and WBP2m, refer
to [73].
WW-Binding Proteins as Proposed

Oocyte Activation Factors
Single-peak or repetitive, oscillatory calcium release is Figure 11.8 Non-ionic-detergent-extracted human spermatozoa
a hallmark of both vertebrate and invertebrate sperm- are devoid of the functional isoform of PLCZ but retain another
induced oocyte activation. Currently, the prevalent candidate SOAF, PAWP. Immunoblots showing pellet (P) and
supernatant (S) fractions of whole human sperm (WS) obtained after
hypothesis is that a SOAF induces calcium oscilla- incubation with non-ionic detergents (e.g. 0.2% Triton X-100, 1%
tions via direct or indirect activation of an ooplas- NP-40 or 0.4% Lysolecithin). The left panel of the Western blot
mic phospholipase, which cleaves the phosphatidyl- labeled with anti-hPLCZ antibody (Covalab, Villeurbanne, France)
clearly shows that the functional 72 kDa isoform of PLCZ (arrow) is
inositol-diphosphate (PIP2 ) to produce a second mes- extracted by the detergent and consequently accumulates in the
senger molecule, inositol-3-phosphate (IP3 ); IP3 binds supernatant, while the 50 kDa tail isoform remains in the pellet. In
to its receptor on the endoplasmic reticulum (ER), contrast, the right panel labeled with an anti-PAWP antibody shows
that PAWP (arrow) is resistant to detergent extraction and is
from which calcium is released [74]. Alternatively, retained in the pellet fraction.
a sperm-borne phospholipase has been proposed to
directly cleave ooplasmic PIP2 , and the resultant initial
spike in free Ca2+ then activates ooplasmic phospho- amounts of human Pawp cRNA, though the second
lipases that propagate oscillatory Ca2+ release from study recorded a reduced frequency of calcium oscilla-
ER and a coordinated repetitive refilling of Ca stores tions in mouse oocytes co-injected with WW-domain-
by extracellular calcium influx through ion chan- blocking peptides at ICSI [79]. It should be noted that
nels on the oolemma [75]. While claims have been the peptides were diluted in 120 mM KCl-containing
made in the past that the SOAF is a single protein, Hepes solution, which could have affected the out-
evidence suggests that several sperm-borne cytosolic come. Similarly to Plcz1 cRNA injection experiments,
factors from different sperm cytosol/PT fractions con- the amount of PAWP produced by this method greatly
tribute to the induction of calcium entry/release in ver- exceeded a single sperm equivalent delivered by the
tebrate and invertebrate oocytes (e.g. [56, 57, 76, 77]). fertilizing spermatozoon, as seen by a large PAWP
This scenario is supported by the differential solubil- band detected in the cRNA-injected oocytes by West-
ity of two major SOAF candidates, PLCZ1 and PAWP ern blotting [79]. An important omission in this study
(Figure 11.8). was the detection of activation events other than cal-
Competitive PPxY peptides and anti-PAWP anti- cium oscillations, such as meiotic resumption (sec-
bodies block oocyte activation when co-injected with ond polar body extrusion), pronuclear formation and
spermatozoon by ICSI [18, 41, 78]. Although this is oocyte cleavage. Most of our studies, using bovine,
most likely due to competition of injected peptides porcine and Xenopus models, have used these indi-
with sperm-released PAWP, there could be an oocyte- cators of oocyte activation rather than calcium mea-
derived WW-binding protein that is also involved in surements. Furthermore, in our more recent article,
the activation-signalling cascade. A preliminary trial indicating that human PAWP elicits ooplasmic cal-
conducted by a research group promoting PLCZ1 cium release in human oocytes [41], we initially estab-
as the sole SOAF component failed to detect Ca lished the dose and efficacy of Pawp cRNA by mea-
oscillations in mouse oocytes injected with excessive suring the rate of pronuclear formation/cleavage it
167
part of PAS, as is PAWP (Figs. 11.9B, 11.9C). Their

localizations, as well as their differential solubility,
fit in with the thesis that SOAF is released in two
stages during sperm–oolemma fusion: first the more
soluble non-ionic-detergent-extractable component is
released, followed by solubilization of the detergent-
resistant component from PAS [57].
PAWP as a Biomarker of Sperm Quality

and Fertility in Men and Livestock
Animals
The amount and subcellular localization of PAWP
reflect the size of the sperm head and the structure and
Figure 11.9 Immunoblotting and immunofluorescence show the integrity of PAS (Figure 11.5). Consequently, sperma-
presence of WPB2 in mature mouse spermatozoa and elongating
mouse spermatids. (A) Immunoblotting indicates that WBP2 is
tozoa with abnormal phenotypes show altered PAWP
found in the mouse, human and bovine testis but that only mouse content and localization. Image-based flow cytometry
spermatozoa retain WBP2. (B) Immunofluorescence shows that demonstrated that macrocephalic bull spermatozoa,
WBP2 antigenicity is retained in the mouse sperm head’s
postacrosomal sheath (arrow) after sperm sonication. (C) WBP2
which could arise from aneuploidy and/or incomplete
appears to be expressed in the cytosol of elongating spermatids packaging of sperm chromatin, tend to have abnor-
(asterisk) and assembled as part of the postacrosomal sheath of mally large PAS with elevated PAWP content. On the
elongated spermatids (arrow). Blue = DAPI, Green = anti-WBP2
(Santa Cruz, sc-160905). (A black and white version of this figure will
opposite end of the spectrum, microcephalic sperma-
appear in some formats. For the colour version, please refer to the tozoa often lack PAWP completely and spermatozoa
plate section.) with flagellar defects show ectopic PAWP in addition
to or instead of anticipated PAS localization [81]. A
large field trial of 300 bulls yielded a significant
could elicit. Only when we were sure that the rate correlation between PAWP and other biomarkers of
of pronuclear formation/cleavage approximated the sperm quality (ubiqitin, lectins LCA and PNA, aggre-
rate obtained with spermatozoa (see Figure 3F in somes) as well as with sire conception rates in artificial
[41]) did we proceed with the calcium analysis. Based insemination (AI) [81]. Independent of the aforemen-
on our experience, we recommend first establish- tioned study, recent proteomic analysis identified the
ing whether PAWP can induce pronuclear formation association of PAWP with field AI fertility in Holstein
and/or cleavage before proceeding to calcium mea- sires with varied but acceptable fertility [82].
surements examining whether PAWP can elicit cal- Significantly for the diagnostics and management
cium oscillations in metaphase II oocytes. of male-contributed human infertility, sperm PAWP
The reported Pawp null male mouse has no dis- levels correlated significantly with ART outcomes and
tinct sperm phenotype, proposed to be a result of com- particularly with embryo development after ICSI [83].
pensation by the somatic orthologue Wbp2 or other While it remains to be determined if such PAWP
SOAF-like molecules [80]. Uniquely, mice but no other insufficiency is due to lack of oocyte-activating capac-
mammals examined thus far carry both PAWP and ity in spermatozoa, a recent study found that nor-
WBP2 in their sperm, even though WBP2 is expressed mal levels of enzymatically active PLCZ1, the most
in the testes of all these species (Figure 11.9A). Inter- thoroughly studied SOAF candidate, were present in
estingly, WBP2 remains on mouse spermatozoa after spermatozoa of men with infertility due to oocyte
plasmalemma and acrosomal disruption by sonication activation failure [84]. Sperm extracts of a patient
and is extractable in non-ionic detergents, whereas whose spermatozoa contained what was described
PAWP is resistant to both. Surprisingly, WBP2 local- as ‘disrupted’ PLCZ1 patterns and failed to activate
izes to the PAS of mouse spermatozoa in the same human oocytes had normal oocyte-activating activ-
region as PAWP and appears to be expressed in the ity towards mouse oocytes, suggesting that the SOAF
cytosol of elongating spermatids and assembled as components may differ between species [85]. High
168
variance of PLCZ immunofluorescence signal inten- Conceptual Considerations

sity and localization was reported in the sperm of fer-
tile men, which were not different from sperm of men Oocyte Activation Concepts
with oocyte activation failure [86]. A possible correla-
The currently favoured SOAF candidate is phospho-
tion between PLCZ immunofluorescence signals and
lipase C ␨ (PLCZ1) [100], originally thought to be a
ART fertilization rate has been suggested recently [87],
male germline-specific enzyme but later shown to be
although the protein expression was again detected in
expressed in the epididymis and pancreas [40, 101,
various sperm compartments in this study. The lack
102]. The PLCZ hypothesis is attractive yet somewhat
of PLCZ in globozoospermia patients with round-
counterintuitive, as the oocytes themselves have an
headed, perinuclear theca- and acrosome-less sperma-
abundant cytosolic phospholipase that can induce Ca
tozoa that are unable to activate oocytes by ICSI [88,
release [103]. Contrary to the well-documented sol-
89] does not necessarily support PLCZ in this role,
ubilization of PAWP in the ooplasm at fertilization
because many other proteins, including PAWP, are also
[18], the release of PLCZ1 from the fertilizing sperma-
absent or in low supply in globozoospermia [83]. Fur-
tozoon to the ooplasm has never been demonstrated
ther investigation is required to measure both PAWP
convincingly. Most attempts at localizing PLCZ in the
and PLCZ levels in sperm samples of infertile patients
sperm head point to the acrosome [40, 104, 105],
to determine the possible diagnostic roles of these pro-
which is lost before the sperm and oocyte cytosols co-
teins. To this end, we highly recommend establishing
mingle [40]. Overexpression of the Plcz1 gene through
flow cytometric techniques, as established for PAWP
intraooplasmic cRNA injection induces calcium oscil-
[83], which provide a reliable analysis of protein con-
lations reliably, but so does oocyte treatment with Ca-
tent in a larger number of sperm in each sample as a
ionophores or electrical pulses. Furthermore, recent
reliable alternative to subjective immunofluorescence
studies suggest the involvement of acrosomal PLCZ1
analysis.
in sperm capacitation rather than in oocyte activation
[106].
Supporters of the PLCZ hypothesis relentlessly
WW-Binding Proteins as Cancer dispute the proposed role of PAWP. Their prelimi-
Biomarkers nary attempts at replicating Pawp cRNA-induced cal-
cium oscillations in mouse oocytes failed. As pointed
While PAWP is a male-germ-cell-specific gene not
out by a recent review, this could have been due to
expressed at all in females, two recent studies reported
interlaboratory differences in reagents, protocols and
the expression of the Pawp gene in breast cancer tis-
instrumentation [107]. Neither the Plcz-null [108] nor
sues ([90, 91]. Independent of those findings, cir-
the Pawp-null mice show a normozoospermic phe-
culating anti-PAWP antibodies have been detected
notype with disabled oocyte-activating ability, which
in breast cancer patients’ blood [92]. These studies
supports the idea that the two factors may be cross-
strongly recommended PAWP as a potential cancer-
compensating [80, 108]. Alternatively, is it possible
testis-specific (CT) antigen. The CT antigens, such
that we have misread the oocyte activation mechanism
as MAGE-A, SYCP3 and TSGA10, are normally
and prematurely discounted the importance of extra-
expressed during spermatogenesis in the immuno-
cellular calcium entry in favour of intracellular release
privileged testis but absent from normal (i.e. non-
from ER as the initial impulse for oocyte activation and
malignant) somatic cells [93–95]. The immunogenic
the perpetuation of calcium oscillations? Consistent
effects of a subset of CT antigens qualify them as
with studies suggesting the crucial role of the extracel-
potential targets for cancer immunotherapy and the
lular calcium influx for complete mouse oocyte activa-
vigorously pursued therapeutic cancer vaccines [96,
tion [109], the calcium wave could be propagated from
97]. The somatic orthologue of PAWP, WBP2, has
the sperm entry site along the oolemma in addition to
been implicated in the regulation of tissue growth
percolating across the ooplasm.
[72] and tumorigenesis [98] through the Hippo path-
way. Thus, the sperm-borne WW-binding proteins and
their substrate WW-domain proteins could serve as Possible Compensatory Effects
diagnostic markers and potential targets for cancer Genetic ablation of Pawp produced fertile male
therapy [99]. mice and normal litter sizes, suggesting that in the
169
domestic mouse, the lack of PAWP protein does recognize PAWP protein by immunofluorescence in
not reduce male fertility [80]. Routine phenotyping the wild type spermatozoa.
of Pawp-null males did not reveal significant sperm
abnormalities, while a thorough histological and ultra- Biomedical Considerations and
structural analysis, as well as investigation of the fer-
tile lifespan of Pawp-null males, is pending. However, Concluding Remarks
caution should be exercised in interpreting the phe-
notypes of single-gene disruption in mice and gener- Human Infertility Contributed by
alizing the conclusions to include humans and other SOAF Deficiency
nonrodent mammals. A good example of why over-
Characterization of the SOAF components and their
reaching conclusions should be avoided is the abla-
possible interactions during oocyte activation may
tion of the gene for the cystic fibrosis transmem-
lead to infertility treatments that mimic the signalling
brane receptor (CFTR). This gene, associated with
cascade of natural fertilization more closely. Such
cystic fibrosis in humans, has no phenotype in Cftr
approaches should consider both the composition and
mutant mice yet replicates all typical human patholo-
dosage of SOAF (i.e. the equivalent of the SOAF
gies including lung disease, newborn intestinal meco-
amount delivered to oocyte by a single spermatozoon).
nium ileus and male congenital absence of the vas def-
Normozoospermic unexplained infertility cases with
erens in Cftr mutant pigs [110].
oocyte activation failure appear to be rare, yet cases
Compensatory overexpression of both Wbp2 and
with heritable defects causing the absence of SOAF-
Plcz1 have been suggested as possible reasons for the
bearing PT from individuals’ spermatozoa, such as
lack of infertile phenotype in Pawp null males [80].
globozoospermia, could also benefit from an artifi-
While the double mutant for Wbp2 and Pawp is pend-
cial oocyte activation method that mimicked natural
ing and could be potentially embryo-lethal or infer-
fertilization [114, 115]. Caution should be exercised
tile, phenotypic data are available for the single-Wbp2
with regard to potential toxic and epigenetic effects of
mutant from the International Mouse Phenotyping
ionophores and inhibitors used to treat oocyte activa-
Consortium1 . The web site indicates that the reproduc-
tion failure after human ICSI [116].
tive system was tested but not found to be significantly
Focus on the individual SOAF components
abnormal in the Wbp2-null mice; litter size data are not
should not distract from considering the interplay
shown [111].
between sperm-contributed factors and oocyte sig-
Contradicting the notion that PAWP protein is
nalling pathways involved in the conductance of the
not critical for spermiogenesis, Pawp gene expres-
sperm-delivered activation signal [117]. Fertilization-
sion is significantly up-regulated in a fertile Plin1
mimicking induction of oocyte activation by Pawp
-/- mutant mouse with testicular phenotype with a
cRNA has recently been shown in the mouse and
markedly increased number of haploid germ cells
human oocytes [41], as well as in the pig (see
[112]. Pawp was one of the two most prominently
Figure 11.6H), but the technique is far from routine.
up-regulated genes among a total of 538 signifi-
Improvements could be made by using truncated
cantly up-regulated spermatogenesis-related genes
Pawp cRNA versions, optimizing the injection dose to
identified. Such an increase could merely reflect an
deliver the exact amount of cRNA producing a PAWP
increase of the total number of spermatids, but it
protein equivalent of a single spermatozoon. Efforts
was disproportionate to other testis-expressed genes,
will also be made to produce purified PAWP protein
suggesting that overexpression of Pawp had a specific
as well as recombinant PAWP, for which the major
role in accelerating spermiogenesis in this mutant.
obstacles have been solubility and proper folding.
Furthermore, a recent genomic study in pigs asso-
ciated higher fertility of Chinese breeds (compared
with Western-type pigs) with genomic duplica- Safeguarding and Improvement of ART
tion/increased copy number of Pawp [113]. If such As mentioned, the disassembly of the IAM-SAL com-
duplication is revealed in mice, the existing Pawp- plex lags only slightly behind the solubilization of
‘null‘ phenotype will have to be re-evaluated, more PAS and ES during natural fertilization. However, after
so since the antibodies used to characterize the ICSI, and particularly in the absence of treatments
purported Pawp-null spermatozoa did not seem to that disrupt the acrosome and PT, the intact SAL still
170
covered by IAM and acrosomal components can Competitive Grants no. 2013–67015–20961 and 2015–
hinder the decondensation of the sperm nucleus 67015–23231 from the USDA National Institute of
(Figure 11.5). This delay can occur to the point of Food and Agriculture, by Grant 1R24OD12221 from
preventing the development of the paternal pronu- National Institutes of Health and by seed funding from
cleus, causing asynchrony of development with the the Food for the Twenty-First Century program of the
maternal pronucleus and triggering early blocking of University of Missouri. RO is supported by Canadian
embryo cleavage due to unsatisfied cell cycle check- Institute of Health Research Grant no. MOP-84440
points [59, 118]. Due to preferential topology of the sex and Natural Science and Engineer Council Grant no.
chromosome in the SAL-covered region of the sperm RGPIN/192093.
nucleus, such post-ICSI delay of PPN development has
been mentioned as a possible cause of sex chromo-
some abnormalities found in some ICSI babies [119].
Sperm treatments have been devised to disrupt sperm Note
PT before/during ICSI to facilitate both the disruption 1. http://www.mousephenotype.org/.
of the acrosome and SAL, and the release of SOAF and
oocyte activation. These include piezo-driven microin- References
jection used in rodents, pigs and humans ([58, 120, 1. Oko R, Maravei D. Distribution and possible role
121], mechanical or piezo-driven disruption of human of perinuclear theca proteins during bovine
sperm PT by tapping with the injection needle [123] spermiogenesis. Microsc Res Technol 1995; 32: 520–32.
and cysteine supplementation of post-ICSI culture 2. Yu Y, Xu W, Yi YJ, Sutovsky P, Oko R. The
media to promote S–S reduction in the injected sperm extracellular protein coat of the inner acrosomal
head PT [122]. membrane is involved in zona pellucida binding and
penetration during fertilization: Characterization of its
most prominent polypeptide (IAM38). Dev Biol 2006;
Concluding Remarks 290: 32–43.
Further research will focus on identifying the down- 3. Sutovsky P, Manandhar G, Wu A, Oko R. Interactions
stream interactors/substrates of PAWP during sper- of sperm perinuclear theca with the oocyte:
matogenesis and following fertilization, as well as sim- Implications for oocyte activation, anti-
plifying/optimizing the use of synthetic PAWP for the polyspermy defense, and assisted reproduction.
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Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Sutovsky P, Day BN. Improved fertilization and
Hanfland RA, Smith TL, Samuel M, Wax D, Murphy embryo development resulting in birth of live piglets
CN, Rieke A, Whitworth K, Uc A, Starner TD, after intracytoplasmic sperm injection and in vitro
Brogden KA, Shilyansky J, McCray PB Jr., Zabner J, culture in a cysteine-supplemented medium.
Prather RS, Welsh MJ. Disruption of the CFTR gene Theriogenology 2007; 67: 835–47.
produces a model of cystic fibrosis in newborn pigs.
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Palermo GD. Does ICSI require acrosomal disruption?
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(2015). Available at http://www.mousephenotype.org/.
176
Chapter
Fundamental Role for Sperm Phospholipase
12 C ␨ in Mammalian Fertilization
Michail Nomikos, Karl Swann and F. Anthony Lai
Introduction a single large transient [1, 5]. In contrast, in mam-

Fertilization involves the fusion of gametes to initi- mals, the sperm-stimulated Ca2+ signal is delivered as
ate the development of a new individual organism. In a long-lasting train of periodic Ca2+ transients, known
mammals, the process involves the fusion of an egg as Ca2+ oscillations (for example, see Figure 12.1). The
(oocyte) with a sperm, which first creates a zygote and frequency and duration of Ca2+ oscillations appear to
then leads to the development of an embryo. Fertil- be species-dependent, with some mammalian eggs dis-
ization can be described as a chain of events rather playing a Ca2+ transient every 10 minutes and other
than a single, isolated phenomenon. Interruption of species’ eggs once every hour [1, 6, 7].
any step in this chain will almost certainly cause fer- Numerous studies have indicated that the
tilization failure. The chain begins with a group of generation of fertilization-associated Ca2+ oscilla-
changes affecting the sperm, which prepares it for the tions specifically requires stimulation of the 1,4,5-
next fertilization steps. Within this chain, the most trisphosphate (IP3 ) signalling pathway [8–11].
important function of the sperm after its fusion with This process involves the increased production of
the fertilized egg is the stimulation of egg activation, IP3 via the hydrolysis of phosphatidylinositol 4,5-
which ‘kick-starts’ the events that produce successful bisphosphate (PIP2 ), leading to IP3 -mediated Ca2+
embryonic development. The term ‘egg activation’ is release from internal stores, primarily the endoplas-
used to describe a series of pre-programmed early bio- mic reticulum (ER). Intracellularly, IP3 is generated by
chemical events within the mammalian egg that are the stimulation of phospholipase C (PLC) enzymes,
necessary to prepare the egg for development into an a ubiquitous family of cytoplasmic proteins that
embryo [1]. It is now well established that a spermato- play a key role in hydrolyzing specific membrane
zoon stimulates egg activation by triggering an acute lipids that activate a number of intracellular signal
rise in cytosolic free Ca2+ concentration [Ca2+ ] [1–3]. transduction pathways. The involvement of the IP3
This major cytosolic [Ca2+ ] increase is both necessary signalling pathway in fertilization has been illustrated
and sufficient for completion of the series of egg acti- by studies which demonstrated that Ca2+ oscillations
vation events to occur, including cortical granule exo- at fertilization can be inhibited by injection of a
cytosis, which acts to block polyspermy, followed by function-blocking IP3 receptor (IP3 R) monoclonal
resumption and completion of meiosis (where neces- antibody or by down-regulation of IP3 R protein
sary) and subsequent pronuclei formation [1, 4, 5]. The expression [8, 10, 11]. Moreover, the microinjection
importance of this intracellular Ca2+ signalling phe- of IP3 , or of adenophostin A, which is an IP3 ana-
nomenon extends beyond mammals, since egg activa- logue, can lead to a series of Ca2+ oscillations in all
tion is accompanied by an increase in the level of intra- mammalian eggs examined [12–14]. Hence, in mam-
cellular [Ca2+ ] in all nonmammalian species studied malian eggs, the gamete-fusion-initiated production
to date, and the critical role of Ca2+ was previously dis- of IP3 appears to be both necessary and sufficient to
covered in such species as fish, frogs and sea urchins, explain the generation of Ca2+ oscillations observed
where the observed Ca2+ rise in the egg composes at fertilization.
University Press.
177
Chapter 12: Fundamental Role for Sperm Phospholipase C ␨ at Mammalian Fertilization
Sperm induced Ca2+ oscillations in a mouse egg

6
4
Rhod dex (F/F0)
0
1 hour
Figure 12.1 Sperm-induced Ca2+ oscillations in mouse eggs. Representative traces of mouse sperm-induced cytoplasmic Ca2+ oscillations
recorded using the calcium indicator Rhod dextran following in vitro fertilization of a mouse egg.
From the ‘Sperm Factor’ Hypothesis to factor from the sperm head into the egg cytoplasm,
which activates the 1,4,5-inositol trisphosphate (IP3)
the Discovery of Sperm PLC␨ signalling pathway [15]. The sperm factor is intrinsi-
Multiple hypotheses have previously been proposed cally capable of initiating the Ca2+ release within the
to explain the generation of the Ca2+ signal in fer- egg cytosol that leads to the appropriate pattern of
tilized eggs [1]. The hypothesis that currently has waves or oscillations of Ca2+ , depending on species.
gained the most experimental support in mammals is The initial experimental evidence for the sperm fac-
known as the ‘sperm factor’ hypothesis (Figure 12.2). tor hypothesis came from studies in sea urchins, where
This hypothesis proposes that fusion of sperm and it was shown that microinjection of cytosolic sperm
egg membranes leads to the introduction of a soluble extracts from sea urchin sperm could trigger fertil-
ization envelope elevation in unfertilized sea urchin
eggs [16, 17]. The first clear evidence for this sperm
Figure 12.2
Schematic illustration factor came from the finding that the microinjec-
Sperm of the sperm factor tion of cytosolic sperm extracts could cause sustained
hypothesis. The Ca2+ oscillations in hamster eggs [15]. Further sup-
hypothesis proposes
that the sperm delivers port came from experiments where injection of sperm
SF
a sperm factor to the cytosolic extracts or intact sperm by intracytoplasmic
ooplasm (egg sperm injection (ICSI) into mammalian eggs were able
cytoplasm) that
IP3 generates IP3 , which in to initiate Ca2+ oscillations that mimicked the pattern
turn causes the of Ca2+ changes observed during IVF [15, 18–20].
Ca2+ intracellular Ca2+ The evidence that sperm-mediated Ca2+ oscilla-
release that leads to
IP3R egg activation and tions are caused by activation of the IP3 signalling
embryo development. pathway suggested that the sperm factor might itself be
Ca2+ a PLC enzyme. In addition, biochemical experiments
on mammalian sperm extracts using an in vitro PIP2
ER hydrolysis assay suggested that these extracts possess
178
significant PLC enzymatic activity, even at the resting of PLC␨ protein that has been measured within
cytosolic free Ca2+ levels (100nM) typical of mam- a single mouse sperm. Moreover, transgenic mice
malian eggs at the time of fertilization [21]. Microin- exhibiting significantly reduced expression of PLC␨
jection of mouse eggs with recombinant proteins cor- through RNA interference (RNAi) yielded sperm that
responding to the most characterized somatic cell PLC induced premature termination of Ca2+ oscillations,
isoforms at the time either failed to trigger any Ca2+ and while not infertile, these mice with low PLC␨ in
oscillations, or was able to initiate some disparate Ca2+ their sperm exhibited a reduced litter size [30]. Finally,
release events at nonphysiological concentrations the importance of PLC␨ in mammalian fertilization
[1, 22, 23]. Moreover, chromatographic fractionation has been emphasized by a number of recent clinical
of sperm extracts revealed that none of the known, pre- reports that have directly linked defects of deficiencies
viously identified PLC isoforms was present in the pro- in human sperm PLC␨ (reduced expression levels or
tein fraction displaying the Ca2+ oscillation-inducing mutated forms of PLC␨ ) with documented cases of
activity [1, 23]. All this evidence pointed to the pos- male infertility [27, 31–35]. This cumulative evidence
sibility of a novel PLC isoform as the trigger of Ca2+ strongly supports the assertion that sperm-specific
oscillation at mammalian fertilization, which eluded PLC␨ is the long-sought mammalian sperm factor that
investigators for some time. initiates the Ca2+ oscillations leading to egg activation
A set of novel, overlapping PLC sequences that and early embryonic development at mammalian
were all derived specifically from the testis was fertilization.
revealed following the homology search of a mouse
database of expressed sequence tags using the
sequences of known somatic PLCs. A two-step RACE
Other Proposed ‘Sperm Factor’
(rapid amplification of cDNA ends)–PCR successfully Candidates
amplified a 2.2 kb product from a mouse spermatid A number of other sperm factor candidates have been
cDNA library, which contained a 1,941 bp open proposed over the years, including a 33 kDa pro-
reading frame encoding a novel 647-residue protein tein, termed ‘oscillin’ [36], and a truncated form of
(24). This spermatid-derived protein sequence shared the kit receptor, tr-kit [37]. However, neither of these
significant homology to other mammalian phos- two proteins is able to elicit the characteristic pat-
pholipases, especially those of the ␦ type. However, tern of Ca2+ oscillations observed during mammalian
Northern and Western blot analysis indicated that fertilization [38, 39]. Interestingly, a recent study by
expression of this protein was strictly confined to Aarabi et al. [40] reported that post-acrosomal sheath
the testis [24, 25]. This novel PLC isoform, termed WW domain-binding protein (PAWP), a sperm head
PLC␨ , was subsequently demonstrated to play a protein that exclusively resides in the post-acrosomal
fundamental role in egg activation [24, 25]. Since its sheath region of the perinuclear theca, is able to trigger
discovery, PLC␨ has been identified in many different Ca 2+ oscillations and pronuclear formation in human
mammalian species, suggesting that it could play a and mouse eggs, similar to what is observed dur-
fundamental role in fertilization in all mammals [1, 5, ing ICSI [40]. The authors also reported that sperm-
24, 25]. Immunodepletion of PLC␨ from native sperm induced Ca2+ oscillations could be blocked by co-
extracts using an anti-PLC␨ specific antibody abol- injection of a peptide derived from the WWI domain-
ished their ability to trigger Ca2+ oscillations in mouse binding motif of PAWP, which acts as a competitive
eggs [24]. Microinjection of recombinant PLC␨ pro- inhibitor of PAWP [40]. This report followed the ini-
tein or complementary RNA into mouse eggs not only tial proposal from the same group, previously suggest-
causes Ca2+ oscillations indistinguishable from those ing that PAWP promotes meiotic resumption as well
observed at fertilization but also triggers embryonic as pronuclear development during fertilization [41].
development to the blastocyst stage [24–28]. Quan- Despite these recent observations consistent with a
tification of the amount of PLC␨ protein expressed potential role of PAWP in mammalian egg activation,
in mouse eggs following cRNA injection experiments these data could not be replicated in other studies that
indicated that PLC␨ is effective at triggering the independently demonstrated that PAWP was unable to
physiological pattern of Ca2+ oscillations at 40 fg cause Ca2+ release in eggs [42, 43]. Significantly, fur-
per egg [24, 29]. This estimate of the amount of PLC␨ ther experiments [43] revealed that the PAWP-derived
required for egg activation correlates with the amount inhibitory peptide was unable to block sperm-induced
179
Ca2+ oscillations following IVF or ICSI. Interestingly, 39]. However, the X and Y catalytic domains of PLC␨
a further independent study has recently been pub- are separated by an unstructured region referred to as
lished that describes the generation and analysis of the XY-linker. In PLC␨ , a part of this linker region
the fertilizing ability of male PAWP-null mice [44]. that is proximal to the Y catalytic domain contains
This important PAWP ‘knockout’ study has reported a distinctive cluster of positively charged amino acid
results from ICSI with a single spermatozoon from the residues that is not found in the homologous regions of
PAWP-null mouse, which confirmed that the absence any of the other PLC isoforms [5]. PLC␨ is most simi-
of PAWP did not cause any quantitative differences in lar in domain structure to the PLC␦ isoforms, show-
Ca2+ oscillations or in subsequent development of the ing the greatest sequence identity with PLC␦1 (33%
embryos compared to control, leading the authors to identity) and the least with PLCε (9% identity) [24].
conclude that PAWP does not play an essential role in The major structural difference distinguishing PLC␨
mouse fertilization [44]. These observations indicat- from PLC␦1 and all other PLC isoforms is the lack
ing that PAWP is not the sperm factor protein have of a typical Pleckstrin homology (PH) domain at its
resulted in PLC␨ currently remaining as the sole can- N-terminus [24]. PH domains are well-defined struc-
didate for the sperm factor that provides the physi- tural modules of 120 amino acid residues that have
ological Ca2+ signalling phenomenon to successfully been identified in more than 100 different proteins
trigger egg activation and initiate early embryo devel- and are believed to mediate the membrane binding of
opment at mammalian fertilization. somatic PLC isoforms [45]. The PH domain of PLC␦1
is essential for the specific and high-affinity binding of
this enzyme to its phospholipid substrate PIP2 in the
Structure and Distinctive Molecular plasma membrane [46, 47]. The notable absence of a
Properties of PLC␨ PH domain for sperm PLC␨ suggests that it employs
Sperm PLC␨ is the smallest known mammalian PLC a novel mechanism to target biological membranes
isozyme and although it has the most elementary [5, 39].
domain organization, its Ca2+ oscillation-inducing
activity and fertilization potency are distinctively The Highly Conserved X and Y Catalytic
superior to those of somatic PLCs [1, 39]. The reason
that PLC␨ is so effective in generating Ca2+ oscil- Domains
lations that subsequently orchestrate the egg activa- The X and Y catalytic domains represent the catalytic
tion process is the unique molecular and physiologi- sites of PLC enzymes and are essential for PLCs to
cal properties of PLC␨ compared with somatic PLCs, hydrolyze the phosphoinositol lipid substrate, PIP2
which may be attributable to its discrete structural [48]. These domains are the most highly conserved
domains [39]. PLC␨ exhibits a typical PLC domain regions of PLCs compared with the other regulatory
structure consisting of four tandem Ca2+ -binding EF- domains [45, 48]. The X and Y domain sequence sim-
hand domains at the N-terminus of the catalytic X and ilarity among all PLC isoforms is 60%, and even
Y domains, which form the active site responsible for higher among those of the same class [45, 48]. The
the PIP2 hydrolysis, which is followed by a single C2 PLC␨ catalytic domain displays 64% similarity with
domain (Figure 12.3) (24). All these domains are com- that of PLC␦1 [24]. However, replacement of the XY
mon to the other PLC isoforms (␤, ␥ , ␦, ε and ␩) [5, 24, catalytic domain, including the XY linker region, of
PLC␨ with that of PLC␦1 results in the complete aboli-
tion of Ca2+ oscillation-inducing activity in the resul-
tant PLC␨ /PLC␦1 protein chimera in mouse eggs [49].
E F E F X Y C2 By homology with the catalytic XY domain of PLC␦1,
the PLC␨ XY domain is predicted to be organized in
XY-linker repetitive beta sheet/alpha helix sequences, forming a
Figure 12.3 Schematic linear representation of the protein distorted barrel. Mutagenesis of conserved active site
domain organization of sperm PLC␨ . PLC␨ exhibits a typical residues within the catalytic domain of PLC␨ leads to
mammalian PLC domain structure consisting of two pairs of complete loss of its enzymatic activity and thus its Ca2+
N-terminal EF-hand domains, followed by the catalytic core
encompassing the X and Y domains, which are separated by the oscillation-inducing ability in mammalian eggs. The
XY-linker region, and finally the C2 domain at the C-terminus. active site residues that are required for PIP2 hydrolysis
180
are also present and conserved in the PLC␨ sequences cleavage within the XY-linker region, suggesting that
determined for a wide variety of different species [1, 5]. an intact polypeptide is not essential for PIP2 sub-
Notably, point mutations within the catalytic domain strate hydrolysis [60]. The authors proposed that
of PLC␨ have been associated with loss of function in the proteolytic cleavage of PLC␨ within the XY-
human sperm and consequently with male infertility linker region may have important regulatory functions
[27, 32, 33, 35, 50]. during mammalian fertilization [60]. However, fur-
ther investigation is required to fully understand the
importance of proteolytic cleavage for PLC␨ function
The Multifunctional XY-Linker and mode of regulation.
The sequence segment that joins the X and Y catalytic In contrast to the other domains, the XY-linker
domains, termed the XY-linker, plays an important sequence of PLC␨ is the most poorly conserved
multifunctional role, with evidence of specific involve- region between the mammalian PLC␨ species thus
ment in regulating the enzymatic activity, PIP2 lipid far sequenced. Interestingly, the PLC␨ XY-linker
substrate targeting and the nuclear translocation of sequences of all species retain an overall net positive
PLC␨ [1, 39]. In contrast to that for PLC␦1, PLC␨ charge [1, 61]. Although the significance of this XY-
contains a more extended XY-linker that is notably linker diversity remains unclear, it might explain the
rich in basic residues [1, 24]. Structural and biochem- different rates of PIP2 hydrolysis and the relative Ca2+
ical evidence suggests that the XY-linker region of oscillation-inducing ability between PLC␨ isoforms of
the somatic PLCs, PLC␤, ␥ , ␦ and ε, mediates potent different species [61]. A recent study demonstrated
auto-inhibition of their enzymatic activity, prevent- that replacement of the human PLC␨ XY-linker with
ing PIP2 access to the active site, by a combination the corresponding region of mouse PLC␨ caused sig-
of steric exclusion and electrostatic repulsion of neg- nificant reduction in protein stability [62].
atively charged membranes [51, 52]. In contrast, the
XY-linker region of PLC␨ does not mediate auto-
inhibition, but it is required for maximal enzymatic
EF-Hands Confer High Ca2+ -Sensitivity and
activity, as deletion of the PLC␨ XY-linker significantly Mediate Binding of PLC␨ to PIP2
diminishes both its in vitro PIP2 hydrolysis and its PLC␨ contains four EF-hand motifs at the N-terminal
in vivo Ca2+ -oscillation-inducing activity [53]. This is end of the protein. The EF-hand motifs are arranged
consistent with our previous findings suggesting that in two pairwise lobes similar to those involved in
the positively charged residues within the XY-linker Ca2+ binding in calmodulin [1, 5]. It has been pro-
region of PLC␨ are directly involved in the targeting of posed that the EF-hand domains of PLC␨ confer its
this enzyme to biological membranes via electrostatic high Ca2+ sensitivity relative to the other somatic
interactions with the negatively charged substrate PIP2 PLC isoforms [29, 63]. PLC␨ is 100-fold more sen-
[54, 55]. sitive to Ca2+ stimulation than PLC␦1, with an EC50
It has been reported that the XY-linker region of of 80nM [29]. This suggests that PLC␨ is already
mouse PLC␨ contains a nuclear localization signal half-maximally active at resting cytosolic Ca2+ lev-
(NLS) sequence located close to the start of the Y els (100 nM) in eggs, and increasing basal Ca2+
domain [56, 57]. The NLS might play a significant would further increase its PIP2 hydrolytic activity [29,
role in the mode of regulation of PLC␨ by localiz- 39]. It has been demonstrated that deletion of both
ing this enzyme at the pronucleus upon the cessa- EF-hands dramatically increases EC50 of PLC␨ from
tion of Ca2+ oscillations [56–58]. The mouse PLC␨ 80 nM to 30 ␮M, resulting in complete loss of its
NLS corresponds to an octapeptide (KKRKRKMK). Ca2+ oscillation-inducing activity in mouse eggs [29].
Although a putative NLS, involving similar clusters of Furthermore, replacement of PLC␨ EF-hand domains
basic residues, has been predicted in PLC␨ sequences with that from PLC␦1 results in an 10-fold decrease
of various mammalian species, it has been demon- in PLC␨ Ca2+ sensitivity [49]. A mathematical model
strated that rat, human and medaka fish PLC␨ do not based on the empirical data of a study investigating the
translocate to the pronuclei when expressed in mouse molecular properties of PLC␨ /PLC␦1 chimaeras sug-
eggs [59]. gested that the exquisite Ca2+ sensitivity of PLC␨ is
Another study has reported that porcine PLC␨ largely mediated by its EF-hand domain, and this is a
remains functionally active after proteolytic protein major determinant of the superior potency of PLC␨ at
181
triggering high-frequency Ca2+ oscillations compared get, and this biochemical association may be respon-
with other PLC isoforms [49]. Moreover, a recent study sible for targeting PLC␨ to discrete cytoplasmic vesi-
proposed an additional role for the EF-hand domains cles, as discussed in more detail below [38]. Despite
of PLC␨ . It was demonstrated that the N-terminal lobe these interesting speculations on potential physiologi-
of the EF-hand domain of PLC␨ , a region that is rich cal properties of the C2 domain, further investigation
in basic residues, has an essential role in the interac- is required to delineate the empirically observed vital
tion of PLC␨ with its substrate, PIP2 [64]. It is plausible role of the C2 domain in PLC␨ cellular function.
that PLC␨ is attracted to the anionic PIP2 -containing
component of the intracellular vesicular membranes
through electrostatic interactions with both the first Localization of PLC␨ in Sperm
EF-hand domain and the XY-linker polybasic regions The distribution of PLC␨ within the sperm is consis-
[64]. tent with the idea that PLC␨ is the moiety that trig-
gers Ca2+ signalling in eggs, as localization to par-
The Essential but Currently Unknown Role of ticular regions would enable rapid diffusion of the
enzyme into the ooplasm to initiate Ca2+ oscillations
the C2 Domain within 10 minutes following sperm–egg fusion [5].
The C2 domain is an 120 residue structural motif Figure 12.4 presents the distribution of PLC␨ pro-
that has been identified in numerous proteins, includ- tein in human sperm as detected using an antibody to
ing all isoforms of protein kinase C, phospholipase PLC␨ , indicating that the predominant immunolocal-
A, synaptotagmin and PLC [1, 65]. In many of these ization of PLC␨ is in the post-acrosomal and equato-
proteins it was found that C2 domains can bind rial region of the head, with minor localization in the
to phospholipids either in a Ca2+ -dependent or a neck as well as the midpiece. In immunocytochemi-
Ca2+ -independent manner with different affinities cal studies of sperm from mice, hamsters and boar,
and specificities [65]. In PLC␦1, the C2 domain has two distinctive populations of PLC␨ localization have
been shown to interact with phosphatidylserine (PS), been reported, acrosomal and post-acrosomal [67–
forming a C2-Ca2+ -PS quaternary structural complex, 69]. Notably, PLC␨ in equine sperm has been proposed
which enhances its enzymatic activity [66]. Although to be present in the acrosome, equatorial segment and
the exact role of the C2 domain in PLC␨ function head midpiece, as well as the principal piece of the flag-
is still unresolved, it appears to play a vital role in ellum [70]. This apparently widespread localization in
its function. It has been reported that deletion or equine sperm may be significant, as it would be con-
replacement of the C2 domain of PLC␨ with the cor- sistent with the demonstration that specific microin-
responding domain of PLC␦1 completely abolishes the jection of isolated equine sperm tails into mouse eggs
Ca2+ oscillation-inducing activity of PLC␨ in intact triggered Ca2+ oscillations [50, 70]. It should be fur-
eggs, although the ability of the enzyme to hydrolyze ther noted that a study by Aarabi et al. [71] disagrees
PIP2 and its Ca2+ sensitivity is unaffected [29, 49]. with the majority of the published literature regard-
Although the C2 domain of PLC␨ does not play a ing the localization of PLC␨ . The observations by these
direct role in the interaction of PLC␨ with its sub- authors suggest that PLC␨ is localized to the acro-
strate PIP2 , the current biochemical data indicate that some of mouse and human sperm, with further pop-
it can bind with low affinity to the membrane inos- ulations on the surface of the sperm head, following
itol phospholipids phosphatidylinositol-3-phosphate PLC␨ secretion during sperm maturation within the
(PI3P) and phosphatidylinositol-5-phosphate (PI5P) epididymis [71]. However, this study used PLC␨ poly-
[55, 63]. It has been proposed that the interaction of clonal antibodies of disparate specificity, recognizing
the C2 domain with PI3P may play a role in PLC␨ a variety of sperm proteins in immunoblots. More-
localization, or even perhaps in regulation of enzy- over, the Aarabi et al. observations are clearly inconsis-
matic activity, as the presence of PI3P has been shown tent with previous findings that PLC␨ is biochemically
to reduce the in vitro PIP2 hydrolysis activity of PLC␨ detectable in extracts from the perinuclear theca of
[63]. mouse sperm that have also been functionally demon-
There is also potential for an additional or alterna- strated to cause oocyte activation [50, 67].
tive role that the C2 domain of PLC␨ could have by An important question that still has not been
functionally interacting with a specific egg protein tar- addressed is how PLC␨ remains enzymatically
182
Figure 12.4 Immunocytochemical localization of human sperm PLC␨ . The distribution of PLC␨ protein in normal human sperm
(photomicrograph, left panel), as determined with an antibody that recognizes human PLC␨ (immunofluorescence, right panel), indicates a
predominant localization within the post-acrosomal and equatorial region of the head, and minor localization in the neck as well as the
midpiece.
inactive within the sperm, although it is present at an els has been observed [73]. In addition, diacylglyc-
order of magnitude higher concentration than in the erol (DAG) probes developed to report cellular DAG
egg. It is therefore plausible either that PLC␨ could changes following Ca2+ ionophore treatment have
be discretely packaged into intracellular compart- revealed that there is no detectable DAG increase at the
ments to ensure it is kept away from its substrate, or plasma membrane at fertilization, or after microinjec-
alternatively that the sperm might contain inhibitory tion of PLC␨ [74]. Both these data suggest that sperm
factor(s) that specifically bind and retain PLC␨ in an and PLC␨ are not hydrolyzing PIP2 in the plasma
inactive state. Another possibility may be that there membrane, which is surprising, since PLC␨ mediates
is a sperm-dependent post-translational modification its effects via InsP3 production. Interestingly, immuno-
of PLC␨ that results in inhibition of activity, which cytochemistry experiments examining the distribu-
would therefore need to be relieved once PLC␨ was tion of PLC␨ expressed in mouse eggs indicated that
delivered to the egg cytosol. The silence of PLC␨ in the recombinant PLC␨ is localized to small (⬍1 ␮m)
the sperm, despite the presence of significant Ca2+ cytoplasmic vesicles [72]. By using a PIP2 -specific anti-
rises during induction of motility, capacitation and body, it was also demonstrated that the population of
the acrosome reaction, might be necessary to prevent PIP2 in mouse eggs similarly appears to be localized
an early and uncontrolled acrosome reaction. in small vesicles. This suggests that IP3 can not only
be generated by hydrolysis of plasma membrane PIP2 ,
but in eggs this PIP2 can be derived from intracel-
Targeting of PLC␨ within Eggs lular sources [72]. Further support for this hypoth-
Due to the similarity of sperm PLC␨ to somatic PLCs, esis has come from experiments where a phospho-
it should be reasonable to assume that PLC␨ would inositol lipid phosphatase, which catalyzes removal
likewise target the plasma membrane of the fertil- of phosphates from PIP2 , was used to deplete cellu-
ized egg, as the somatic cell plasma membrane nor- lar PIP2 levels. Overexpression of this inositol phos-
mally contains the bulk of the PIP2 . However, the evi- phatase in the plasma membrane of mouse eggs in
dence currently indicates that PLC␨ targets specifi- order to deplete this pool of PIP2 was unable to inhibit
cally to intracellular vesicles that exhibit a uniform or block sperm- or PLC␨ -mediated Ca2+ oscillations.
distribution within the egg cytoplasm [57, 72]. This This indicates that plasma membrane PIP2 may not
localization pattern of PLC␨ within mammalian eggs be essential for the Ca2+ oscillations at fertilization.
further supports its role as the mammalian sperm fac- In contrast, targeting of this inositol phosphatase to
tor, because upon fertilization, there is no detectable cytosolic small vesicles by tagging it with an inac-
decrease in the amount of PIP2 at the plasma mem- tive PLC␨ mutant significantly inhibited sperm- or
brane in mouse eggs. Instead, an increase in PIP2 lev- PLC␨ -mediated Ca2+ oscillations [72]. Interestingly,
183
experiments where PLC␨ was expressed in CHO cells, the plasma membrane. We speculate that following
at 1,000 times higher levels than that which is active sperm–egg fusion, PLC␨ may enter the ooplasm and
in eggs, showed no significant Ca2+ changes follow- specifically interact with a distinct egg protein (‘egg
ing ATP-induced Ca2+ release, despite extensive bio- factor’) that may be present either in the egg cytosol
chemical analysis indicating that the PLC␨ -transfected or on these cytosolic vesicles [38]. We propose that
CHO cell extracts exhibited significant in vitro PLC this putative egg-derived protein partner of PLC␨
enzymatic activity (75). In contrast, injection into enables the specific targeting of PLC␨ to a discrete
mouse eggs of PLC␨ -transfected CHO cells, or cytoso- cytosolic vesicle population. The consequent asso-
lic extracts made from these cells, triggered the dis- ciation of PLC␨ with the negatively charged PIP2
tinct pattern of fertilization-like Ca2+ oscillations [75]. enriched within these vesicles is mediated via elec-
This observation would be consistent with the absence trostatic interactions with the positively charged first
of PIP2 -containing vesicles in the cytoplasm of CHO EF-hand domain and the XY-linker region of PLC␨
cells, as determined by immunocytochemistry exper- (Figure 12.5). This molecular interaction provides a
iments using PIP2 antibody [75]. Despite recent evi- stabilizing tether that facilitates effective PIP2 sub-
dence for the ability of PLC␨ to hydrolyze an intra- strate access and binding at the PLC␨ active site,
cellular PIP2 population, the precise targeting mech- enabling the catalytic XY domain to proceed with effi-
anism of PLC␨ requires further investigation to define cient hydrolytic cleavage of PIP2 on these cytoplas-
the nature of the PIP2 -containing cytoplasmic vesicles mic vesicles. The putative egg factor may possibly be
that PLC␨ appears to target specifically within mam- an egg-specific protein, which would explain the supe-
malian eggs. rior potency of sperm PLC␨ in generating Ca2+ oscil-
lations in eggs but not in other cell types. Moreover,
the egg factor may also confer the high specificity of
PLC␨ and the Search for a Putative eggs for the PLC␨ isoform, consistent with the obser-
‘Egg Factor’ vation that other somatic PLC isoforms are much less
The intracellular targeting of sperm PLC␨ to dis- effective in triggering Ca2+ oscillations in eggs. The
crete cytosolic vesicles/organelles within mammalian existence of an egg factor would also explain some
eggs cannot be explained solely by the phospholipid species-specific differences in the potency of PLC␨
specificity of PLC␨ for PIP2 , first because the PLC␨ when introduced into eggs of different species. For
sequence lacks the phospholipid-binding PH domain example, mouse PLC␨ is more potent than bovine
present in other PLCs, and second because PLC␨ PLC␨ when injected into mouse eggs, while bovine
does not bind to the PIP2 -rich regions present within PLC␨ is more effective in bovine eggs than its mouse
Figure 12.5 Schematic illustration of the

Egg proposed regulation of PLC␨ function mediated
Vesicular PIP2 PI(3)P factor by PLC␨ domains. PLC␨ targets either a distinct
membrane
intracellular vesicular PIP2 -containing membrane
or a PIP2 -containing microdomain within the
? plasma membrane. The high Ca2+ sensitivity
PLCζ ? conferred by the EF-hands enables PLC␨ to be

active at resting Ca2+ levels (nM) within the egg.
PLC␨ associates with PIP2 , involving electrostatic
interactions via the positively charged first
Ca2+ sensitivity EF-hand domain and XY-linker region, followed
X Y by enzymatic cleavage of PIP2 by the catalytic X
and Y domains. Association of PLC␨ with a
specific vesicular membrane may be mediated by
NLS
interaction of the C2 domain with PI(3)P or an as
yet unidentified membrane or cytosolic protein.
In mouse PLC␨ , the XY-linker region contains a
nuclear localization signal (NLS) which targets the
protein to pronuclei in a cell cycle-dependent
manner. Source: Modified from [1].
Nucleus
184
counterpart [76]. As similar doses of functional PLC␨ sensitivity of eggs to various species’ PLC␨ . This is
are delivered for each species, such observations are illustrated by data showing that although mouse PLC␨
not readily explained or interpreted by the differences appears to be more active than rat PLC␨ in mouse eggs,
in intrinsic PLC␨ enzymatic activity. However, they mouse and rat eggs are very similar in size [59].
would be in accord with the presence of a specific Comparison of the primary structure of mon-
PLC␨ -binding egg protein (‘PLC␨ receptor’) in mam- key and human PLC␨ revealed that their amino acid
malian eggs with different species-dependent affinities sequences are nearly identical except for the XY-
for PLC␨ from the different species’ sperm. Another linker region, which in the human PLC␨ is notably
important parameter is the expression level of this shorter than for monkeys, due to the absence of a
putative egg factor/PLC␨ receptor, as this may also single exon [25]. Analysis of several primate PLC␨
be variable in eggs from different species and could sequences (Figure 12.6) indicates that the single exon
potentially account for the disparity in the frequency exclusion occurs in the XY-linker region of many
of Ca2+ oscillations observed upon fertilization in dif- higher primates and results in their clustering together
ferent mammalian eggs. in the phylogenetic tree. In contrast, this exon is
retained in monkey and marmoset XY-linker regions
Species-Specific Differences in the (Figure 12.6), as is also the case for other mammalian
PLC␨ sequences [24, 70]. The single exon excluded
Activity of PLC␨ from the XY-linker region results in a reduction in
Although the presence of PLC␨ is not a species-specific the relative molecular mass of the PLC␨ protein from
characteristic of mammalian sperm, a number of stud- 74 kDa, found in most mammals, to 70 kDa in
ies have suggested that there are significant species- human sperm [25]. The 33-amino acid sequence cor-
dependent differences in the relative potency of PLC␨ responding to the single exon that is present in the
[61, 62, 70, 77, 78]. Quantitative and qualitative data
from a recent study directly compared the relative
potencies of human and mouse PLC␨ in inducing Marmoset
Ca2+ oscillations in unfertilized mouse eggs [62]. It Rhesus_monkey
was demonstrated that human PLC␨ exhibits superior Gibbon
0.958
0.987 Human
potency, being 5 times more effective in generating 0.806 Chimpanzee
Ca2+ oscillations in mouse eggs than its mouse coun- Orangutan
terpart [62]. This distinctive difference was in line with A 0.03
the disparate in vitro enzymatic properties of these
proteins in hydrolysis of PIP2 , as recombinant purified Figure 12.6 Sequence analysis of primate PLC␨ s. (A) Phylogram
human PLC␨ protein exhibited a 76% higher spe- depicting the phylogenetic relationship between primate PLC␨
sequences, demonstrating the close similarity of the higher primate
cific activity than mouse PLC␨ [62]. Moreover, anal- sequences (human, chimpanzee, gibbon, orangutan). Evolutionary
ysis of human/mouse PLC␨ chimaeric proteins has divergence occurs between the higher primates and the
suggested a novel role of the EF-hand domain in the marmoset/rhesus monkey. The most recent evolutionary
divergence is between the human and chimpanzee PLC␨ . Higher
species-specific differences in PLC␨ activity [62]. A primate PLC␨ s are shorter in sequence length than the macaque,
more complete explanation of the physiological role rhesus monkey and other mammalian PLC␨ sequences. The scale
of the apparent species-specific differences in PLC␨ bar at the bottom represents 0.5 substitutions per amino acid
residue. Branch support values are numbers (0–1.0) representing
activity requires further investigation. However, we the statistical probability that the sequences to the right of the
can speculate that the species-specific intrinsic activity node cluster together to the exclusion of any other. (B) The Clustal
of PLC␨ might be related to the relative size of the eggs sequence alignment of the various primate PLC␨ sequences shown
in the phylogram (A) identifies the specific absence in the higher
being fertilized. Considering that the range of various primates of a 33-amino acid sequence at the centre of the protein,
species’ sperm size is not as significant as that of the within the XY-linker region. The XY-linker is positioned between the
egg, it is plausible to propose that a human sperm may catalytic X and Y domains that are responsible for the PIP2 hydrolytic
mechanism (Figure 12.3). This XY-linker sequence corresponds to a
need to deliver a more potent package of PLC␨ to gen- single exon that is retained in the marmoset and rhesus monkey
erate the requisite Ca2+ oscillations in human eggs ver- PLC␨ sequences. The marmoset also has an additional 11-amino
sus, for example, mouse eggs, which are smaller [62]. acid sequence insertion near to the amino terminus. Conserved
amino acids are denoted by an asterisk (∗ ) below the sequence
However, it should be noted and not excluded that alignment, while conservative substitutions are denoted by a colon
there may also be some differences in the functional (:) and nonconservative substitutions by a period (.).
185
Figure 12.6 (cont.)
186
monkey and marmoset XY-linker contains a large and subsequent oocyte activation exhibited abnormal
number of negatively charged amino acids (e.g. an PLC␨ levels]31]. The first genetic link between male
octa-glutamic acid sequence) that would significantly infertility and a defective PLC␨ gene was made after
alter the ratio of negative versus positive residues identification of a PLC␨ substitution mutation in an
within the XY-linker region, a region of PLC␨ which infertile male with failed fertilization after ICSI treat-
notably has been shown to play an important role in ment [32]. This PLC␨ catalytic domain mutation of
electrostatic interaction with the negatively charged a conserved histidine residue to a proline (H398P)
PIP2 substrate [54, 55; see above on The Multifunc- correlated with absence of Ca2+ oscillation-inducing
tional XY-Linker]. However, further understanding of activity when it was present in human and mouse
the precise functional role of residues encoded by PLC␨ , resulting in complete abolition of its PIP2
this XY-linker exon in the enzymatic and/or targeting hydrolytic activity, likely due to deleterious changes
activity of PLC␨ requires further study. in protein secondary/tertiary structure [27, 34]. A
Early studies have also suggested disparities in the subsequent study identified a second PLC␨ muta-
relative solubility of PLC␨ in sperm from different tion, also in the catalytic domain, in the same H398P
species [61, 79]. The different PLC␨ solubility of var- patient, involving a replacement of a histidine with
ious species may be related to the observed timing of a leucine residue (H233L), although this particu-
the initiation of Ca2+ oscillations. For example, at fer- lar histidine residue is not conserved [33]. Interest-
tilization of a hamster egg, the hamster sperm, which ingly, this study also showed that the heterozygous
contains highly soluble PLC␨ , initiates Ca2+ oscilla- PLC␨ H398P and PLC␨ H233L mutations had different
tions within 10 s following sperm–egg fusion. In parental origins, as PLC␨ H398P was paternal in origin,
contrast, at mouse fertilization, due to the relatively while PLC␨ H233L was maternal [33]. These findings
low solubility of mouse PLC␨ , there is a several-minute represent the first description of an autosomal point
delay between sperm–egg fusion and the first Ca2+ mutation resulting in male infertility via the maternal
spike [61, 80]. lineage [33, 82].
More recently, the first homozygous PLC␨ muta-
tion was identified in two infertile brothers with
PLC␨ , Oocyte Activation Failure and isoleucine substituted for proline at residue 489
Male Infertility (I489P). This PLC␨ mutation was found to produce
Human infertility is a condition affecting 1 in 7 cou- abnormal Ca2+ signalling and defective egg activation,
ples [35, 81, 82]. Despite recent developments and which resulted in the arrest of early embryo devel-
new IVF methods, several conditions such as severe opment [86]. Importantly, a third brother who was
male infertility, which accounts for 19–57% of cases of proven fertile was heterozygous for the I489P muta-
infertility [81, 82], often remain untreatable. Even with tion, indicating that the specific disruption of nor-
ICSI, which is a very powerful modified IVF technique mal PLC␨ function results in infertility [86]. Inter-
where the sperm is directly injected into the eggs, 1– estingly, the same study further demonstrated that
5% of treatment cycles still fail, affecting at least 1,000 the sequence of PAWP protein, another recently pro-
couples per year in the United Kingdom alone [5]. posed sperm factor candidate (see section above on
In such incidents of ICSI failure, the main cause has Other Proposed ‘Sperm Factor’ Candidates), was unal-
been shown to be the lack of oocyte activation [83–85]. tered in the infertile homozygous PLC␨ I489P patient
Although the aetiology of ICSI failure in such cases and hence does not contribute to the observed
is likely to be multifactorial in nature and may be infertility (86).
attributable to a number of factors in the oocyte, sperm
defects are considered the leading cause of activation PLC␨ as an Effective Therapeutic
failure. A steadily increasing number of clinical reports
have linked defective or abnormal forms of PLC␨ with Option for Egg Activation Failure
oocyte activation failure and subsequently with male At present, in cases of failed or poor rates of fertiliza-
infertility. tion after ICSI due to egg activation failure, the only
Yoon et al. [31] first demonstrated that sperm from available treatment option is the use of artificial oocyte
patients who displayed repeated failure to fertilize activation agents. Ca2+ ionophores, such as A23187
after ICSI due to inability to trigger Ca2+ oscillations or ionomycin, have been used successfully by some
187
IVF clinics to overcome fertilization failure, although with clinical semen parameters in males proven fer-
there are only a few studies on the efficacy and safety of tile, indicating that the accurate routine analysis of
these agents with regard to embryo viability and future PLC␨ status of sperm specimens may benefit the
health [87]. In addition, Ca2+ ionophores cause a sin- wider male population and not just cases of ICSI
gle large Ca2+ increase that does not mimic the phys- failure [39].
iological series of Ca2+ oscillations triggered at fertil-
ization. Although Sr2+ medium has the ability to trig-
ger repetitive Ca2+ oscillations in mouse oocytes, it Concluding Remarks
has not been shown to cause Ca2+ release in human Since the discovery of sperm PLC␨ in 2002, mounting
oocytes [87]. Thus, at present, PLC␨ remains the only experimental and clinical evidence has strongly sup-
physiological agent and presumably the safest option ported the assertion that this molecule is likely to be
that could potentially replace the current synthetic the sole physiological trigger of egg activation during
methodology utilized by IVF clinics. Microinjection mammalian fertilization. Sperm-specific PLC␨ oper-
of recombinant human PLC␨ protein was able to trig- ates in a way quite distinct from the other somatic
ger Ca2+ oscillations in mouse and human eggs within PLC isoforms. Despite all the recent advances and
the physiological range [27]. This study also demon- understanding of how the discrete molecular and bio-
strated that recombinant PLC␨ could be used to over- chemical properties of PLC␨ contribute to its supreme
come the deleterious effects of mutant forms of PLC␨ effectiveness in mammalian eggs, the complete molec-
in a prototype model of male infertility [27]. Moreover, ular mechanism that sperm PLC␨ uses to trigger Ca2+
a recent study reported that fertilization failure and oscillations remains to be fully elucidated. Some of the
poor embryo development after ICSI with heat-treated major remaining scientific issues to be resolved include
sperm can be counteracted by subsequent microinjec- the determination of the intracellular vesicle/organelle
tion of recombinant human PLC␨ protein, which is that sperm PLC␨ specifically targets within the egg
more effective than the use of Sr2+ medium or Ca2+ as well as the exact mechanism that PLC␨ utilizes to
ionophores [87]. It was also found that PLC␨ can be target the intracellular source of its substrate, PIP2 .
used successfully even if the sperm already contains Another important unresolved question is whether
native PLC␨ protein and thus has endogenous Ca2+ there is a requirement for a PLC␨ -specific egg fac-
releasing activity [87]. However, to further translate tor that plays an important role in the regulation
the recent advances using recombinant human PLC␨ of PLC␨ function following sperm–egg fusion. If so,
protein, more work is needed for the current progress could this egg factor be involved in the unresolved
to be extrapolated from laboratory models to an IVF cases of female infertility? What is the precise molec-
clinical setting. ular and physiological function of the essential PLC␨
C2 domain? Answering these and other open ques-
PLC␨ as a Prognostic Indicator of Male tions about PLC␨ should help to provide a complete
molecular explanation of the way in which mam-
Infertility malian sperm triggers egg activation at fertilization.
Besides its role as a potential therapeutic agent, Furthermore, the potential therapeutic application of
PLC␨ can potentially serve as a powerful diagnostic recombinant human PLC␨ in IVF clinics would also
biomarker of sperm functional competency [39]. Pre- represent a major clinical advance, as would the possi-
vious immunofluorescence studies of human sperm bility of its utility in regenerative medicine approaches,
have shown a particular PLC␨ localization pattern in via the generation of parthenogenetic embryos and
the sperm head that is consistent with fertile sperm, blastocysts that may enable stem cell derivation and
while abnormal patterns were observed in ICSI-failed differentiation.
sperm [32, 88]. A more recent clinical report demon-
strated that evaluation of the total levels, localiza-
tion patterns, and proportions of sperm exhibiting
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192
Chapter
Male Infertility and Assisted Reproduction
13 Nigel Pereira, Queenie V. Neri, Tyler Cozzubbo, Stephanie Cheung,

Zev Rosenwaks and Gianpiero D. Palermo
Introduction Epidemiology
In most medical conditions, the diagnosis and treat- Infertility is commonly defined as the failure to con-
ment are directly linked, and successful treatment ceive after one year of unprotected intercourse. It
hinges on the functional relationship between a patient is thought to affect approximately 15% of repro-
and medical provider. Infertility, in contrast, involves ductive age couples worldwide [1], with up to 50%
a couple whose general and reproductive health is of cases having some degree of male factor infer-
evaluated by multiple medical providers in paral- tility [3]. Population-based estimates in the United
lel, with treatment focused on the two partners [1]. States suggest that more than 1.1 million men sought
Successful treatment not only entails an orchestrated fertility care in 2002 and that there were 131–172
collaboration between the couple and providers, but infertility-related physician visits per 100,000 insured
also requires congruity of the two partners. Thus, men between 1994 and 2006 [3]. Recent reports have
apart from being a medical condition, infertility often revealed temporal and geospatial variation in the
evolves into a social condition, which encompasses prevalence of male factor infertility [3]. Specifically,
several psychosocial stressors [2]. While the diagno- its prevalence in the United States is highest in New
sis, medical treatment and psychosocial management Mexico (56.4%) and lowest in Mississippi (24.2%). The
of infertility have evolved rapidly over the past four aforementioned distribution of male factor infertility
decades, some difficulties still persist. These difficul- is generally multifactorial [3].
ties are especially apparent in the field of male infer-
tility, where we perennially strive to discover novel
mechanisms underlying the etiology of male infertility, The Spermatozoon [4]
as well as propose accurate diagnoses and treatments The antiquated perception of the spermatozoon as a
of male reproductive dysfunction [1]. In this chapter, delivery device for the male genome has been replaced
we review the epidemiology and diagnostic workup of by more recent findings on the cell’s complex role in
male infertility based on various facets of sperm pro- oocyte fertilization. The general structure of the sper-
duction, genetics and environmental factors. We high- matozoon includes the head and flagellum, which are
light various therapeutic strategies, including sperm both enclosed by a regionally differentiated plasma
retrieval and assisted reproductive techniques, which membrane. The head is mostly occupied by the nucleus
are frequently utilized to help couples conceive. We and is covered by the caplike acrosome, which is
also present the clinical outcomes associated with the derived from the Golgi complex. The acrosome con-
aforementioned approaches and appraise their safety. tains several hydrolytic enzymes that are involved in
Finally, we describe the most recent attempts pertain- the acrosomal reaction, a physiological event essential
ing to and future directions for the treatment of male for oocyte fertilization and subsequent embryo devel-
infertility. opment. The flagellum or tail of the spermatozoon
University Press.
193
Chapter 13: Male Infertility and Assisted Reproduction
consists of three regions – the midpiece, which is cen- Table 13.1 Reference values for semen analysis per WHO
standards
trally located and generally defined by an aggregated
sheath of mitochondria surrounding the centrosome, Reference value (fifth
the principal piece and the end piece. percentile with 95%
Parameter confidence intervals)
Screening the Male Patient and Volume (mL) 1.5 (1.4–1.7)

Concentration (106 per mL) 15 (12–16)
Semen Analysis Total motility (%) 40 (38–42)
Screening the Male Patient Progressive motility (%) 32 (31–34)

Morphology (normal forms %) 4 (3.0–4.0)
The initial screening of an infertile man [5] should
involve a thorough developmental, medical, surgical,
family, social and sexual history, as well as a metic-
try. Presence of varicoceles should be noted by exam-
ulous physical examination. The developmental his-
ining the patient in both the supine and standing
tory should include a review of hypospadias, cryp-
position. A rectal examination can reveal large cysts,
torchidism, midline defects, hypogonadism and con-
prostate masses or infection, or dilated seminal vesi-
genital infections. Any maternal exposure to diethyl-
cles, and should be performed routinely.
stilbesterol should also be noted. Medical problems
such as unexplained fevers, diabetes, hypertension,
cystic fibrosis or malignancy must be reviewed and Semen Analysis
noted. Given the potential adverse effects that many Evaluation of the ejaculate as a whole has been utilized
medications can have on male fertility, all medica- for over 60 years now, and it continues to be a useful
tions used by a patient, including dosage and route clinical and research tool to investigate the male part-
of administration, should be noted in detail. Surgical ner’s fertility status. In 2010, the World Health Organi-
procedures such as herniorrhaphy, orchidopexy and zation (WHO) published lower reference limits for tra-
retroperitoneal, bladder, pelvic or prostate surgery can ditional semen parameters [6]. Of note, raw data from
impair fertility and should therefore be reviewed. Any about 400–1,900 semen samples, from recent fathers
family history, particularly paternal history of mid- in eight countries spanning three different continents,
line defects, hypogonadism or infertility, should be were used to generate the following reference values in
elicited. The social history should comprise a thor- Table 13.1.
ough review of alcohol consumption, as well as use As evident in Table 13.1, a semen analysis provides
of anabolic steroids, recreational drugs and tobacco useful information regarding viability, production and
products. Furthermore, any occupational exposure to motility of spermatozoa, as well as the patency of the
chronic heat, ionizing radiation, pesticides, herbicides male genital tract. In general, spermatozoa account for
and industrial solvents should be investigated. Elicit- ⬍10% of the total semen, while the remainder of the
ing history of any pregnancies with the current or pre- ejaculate consists of products secreted by the semi-
vious partners, ejaculatory or erectile dysfunction, the nal vesicles (55%), prostate (25%) and bulbouretheral
use of spermicidal lubricants and incorrect patterns of gland (10%) [4]. Assessment of the volume and con-
timing intercourse generally comprises the sexual his- sistency of the ejaculate can offer insight into the con-
tory. dition of the accessory glands. While quantifying the
The physical examination includes a detailed number and motility of spermatozoa is perhaps intu-
assessment of body habitus, specifically obesity or itive, assessment of sperm morphology is more com-
gynecomastia. Genital examination generally involves plex, owing to the variability in criteria utilized to eval-
evaluation of the phallus and testes. For the former, uate their shape and size [4].
any evidence of chordee, plaques, venereal lesions or Human spermatogenesis is completed in 60–80
hypospadias should be noted. For the testes, the size, days, and therefore an individual’s semen analy-
volume, consistency and contours should be assessed. sis reflects biological activity occurring 2–3 months
The epididymides, vas deferens and spermatic cords before [4]. Given the inherent biological fluctuations
should be palpated for nodularity, fullness or asymme- between semen samples, a minimum of two samples
194
should be examined. Ideally, semen samples should be sperm becomes highly condensed and its histones
produced after 2–3 days of sexual abstinence, prefer- are replaced by protamines [12, 13]. This complex
ably without lubricants, and kept at body tempera- organization of DNA and protein into a structure
ture if transport is required [6]. If an individual’s his- called chromatin is highly regulated and different from
tory suggests recent insults to spermatogenesis such as that in somatic cells [12, 13]. During the later stages
medical illness, testicular injury or chemical or toxin of spermiogenesis, breakage of a sizable amount of
exposure, then semen analysis should be expanded single- or double-stranded DNA occurs to allow tight
over several months. Although there can be consid- chromatin compaction and, under ideal conditions,
erable biological variation in semen analyses, men only those spermatozoa with fully repaired chromatin
whose semen contains ⬎48 × 106 sperm per mL are would reach the ejaculate [14]. The integrity of the
deemed fertile [7], while those with ⬍10 × 106 sperm sperm genome is important for embryo development,
per mL are considered subfertile, especially when the specifically blastocyst development and early implan-
specimen contains many immotile sperm and the few tation, and it is thought that DNA breakage may con-
motile sperm have abnormal morphology [4, 7]. It tribute to infertility in a way that is not revealed by
must be noted that a normal semen analysis does simple morphological evaluation of spermatozoa [14].
not guarantee fertility [6] and does not provide func- Thus, tests (biomarkers) of sperm DNA integrity may
tional information about the sperm; that is, a semen be incorporated into the clinical assessment of male
analysis does not predict whether spermatozoa can infertility patients [15].
undergo capacitation or acrosome reaction or fertilize While several studies use the terms DNA integrity
an oocyte [8]. A semen analysis, therefore, correlates and chromatin integrity interchangeably, most tests
with fertility, but does not prove an individual’s fertil- (biomarkers) measure only specific parameters of
ity potential [8]. chromatin [9, 15]. It is postulated that sperm DNA
integrity is closely associated with sperm quality,
male fertility potential and pregnancy outcomes [12].
Other Markers of Male Infertility Specifically, an abnormal DNA fragmentation index
The need for new male infertility biomarkers largely (DFI, %) is thought to have an inverse relationship
arises from the challenges in translating in vivo sper- with male fertility success [16], and if pregnancy
matogenic function into fertility success using semen does occur, then such pregnancies are thought be
analyses [9]. For a long time, a testicular biopsy was at risk for miscarriage [17]. Some of the available
considered the cornerstone in the evaluation of vari- methods for detecting sperm DNA integrity include
ous forms of male infertility [10]. However, its inva- sperm chromatin structure assay (SCSA), terminal
siveness can pose undue risk for the health of the testes deoxynucleotidyl transferase (TdT) dUTP Nick-End
[9]. Furthermore, it provides only a small sample of tis- Labelling assay, TUNEL assay and comet assay, which
sue, and its histology is often unable to reveal the actual will be discussed below [9]. However, it is important
cause of infertility [10]. Thus, evaluation of sperm to note that these assays are consumptive; in other
and sperm-derived biomarkers has been proposed as words, they require permanent fixation of the sperm,
an alternative for evaluating reproductive success [9]. which renders them unsuitable for clinical practice
These biomarkers aim to highlight spermatic function, [18]. Thus, in current clinical practice, there is limited
specifically the fertilization capacity of sperm. Ideally, ability to select sperm with varying degrees of DNA
these biomarkers would aid in the diagnosis of sperm damage for immediate use, assisted reproduction or
dysfunction, would predict fertilization or pregnancy cryopreservation [9].
rates and would indicate suitable therapies for sperm
dysfunction [9].
The packaging of DNA in sperm and its integrity SCSA
have important fertility-related implications. In gen- Variants of SCSA have been commercially avail-
eral, the sperm’s DNA is bundled very densely, owing able for close to 30 years now. It relies on the
to the action of testis-specific serine kinase 6 (TSSK6) metachromatic properties of acridine orange, which
prior to sperm’s transit to the oocyte [11]. During changes colour from green to red when associ-
spermatogenesis and spermiogenesis, the DNA in the ated with single-stranded DNA or RNA [18]. The
195
results of the assay are generally expressed as (␣t) tions [9]. The assay derives its name from the gel elec-
in three ways: red fluorescence/(red + green) fluo- trophoresis pattern of DNA fragments, which splay out
rescence, red fluorescence/total green florescence, and in the shape of a comet and associated tail [9]. Specif-
red fluorescence/COMP␣t. COMP␣t is the number ically, intact DNA remains in place as the comet head,
of sperm outside of the normal population and com- while damaged and fragmented DNA is smaller and
monly represents the DFI [9]. As acridine orange asso- forms the comet tail. Similarly to the TUNEL assay,
ciates poorly with condensed DNA, most SCSA proto- the comet assay lacks a reference assay and relies on
cols require denaturing of DNA with low pH or high the microscopic observation of a few hundred sperm
temperature to promote thorough penetration of DNA [9, 22]. Thus, its cutoffs vary by institution. At least
by acridine orange [18]. The assay also requires flow one study has suggested that higher DNA damage as
cytometry and the use of a reference for successful cal- measured by the comet assay is predictive of failure
culation of COMP␣t [18]. While cutoff values vary by of embryo development after intracytoplasmic sperm
institution, most data indicate that a DFI ⬎27% may injection [22].
be associated with a reduced probability of pregnancy
with assisted reproductive techniques [19].
Chromosomal Markers and FISH
TUNEL Assay In general, autosomal trisomies (93% of trisomy 18,
95% of trisomy 21 and 100% of trisomy 16) originate
The TUNEL assay was first used in somatic cells
in the maternal line, whereas sex chromosomal aneu-
and then subsequently applied to spermatozoa [20].
ploidies are more frequently of paternal origin (50% of
The assay involves transfer of a fluorescence-labeled
47,XXY, 100% of 47,XYY and 70–80% of 45,X) [4,24].
nucleotide to the 3 -hydroxyl group of damaged DNA
While meiotic errors that lead to foetal aneuploidy
strands using the activity of deoxynucleotidyl trans-
occur in both the male and the female gametes, the fre-
ferase [9, 20]. The fluorescence intensity of each sper-
quency of these errors is lower in spermatozoa (9%)
matozoon is then evaluated and a designation of ‘dam-
than in oocytes (20%) [4, 24]. Although fluorescent
aged’ (fluorescence) or ‘undamaged’ (no fluorescence)
in situ hybridization (FISH) has increased the ability
is given [9]. A laboratory technician uses a fluorescent-
to detect chromosomal abnormalities, indications for
light microscope or flow cytometer to report the num-
FISH on sperm are not well established currently [9].
ber of TUNEL positive sperm. In the absence of a refer-
Furthermore, with FISH, only selected regions of inter-
ence assay, most laboratories develop their own proto-
est can be visualized and any estimate of aneuploidy
cols for the TUNEL assay. In addition, a single techni-
from this procedure refers only to the chromosomes
cian is frequently required to perform fluorescent-light
analyzed [4].
microscopy or flow cytometry to ensure consistent
interpretation of the TUNEL assay results [9]. Thus,
given differences in protocols between institutions, Sperm RNA
TUNEL result cutoffs are often institution-specific. In
The recent discovery of RNA in spermatozoa has
general, higher fractions of TUNEL-positive sperm
raised several interesting questions regarding its role
are seen with increasing male age [20]. However, sev-
in male fertility [25, 26]. Analysis of sperm RNA tran-
eral other individual and environmental factors can
scripts reflects prior events in spermatogenesis as well
increase the percentage of TUNEL-positive sperm
as highlighting potential factors that may be critical
[20]. Within the realm of assisted reproduction, a
to fertilization and embryo development [25, 26]. In
higher percentage of TUNEL-positive sperm has been
addition to mRNA, human sperm has been found to
associated with reduced pregnancy rates [21].
carry small noncoding RNAs (sncRNAs). The distri-
bution of sncRNAs in ejaculated specimens is as fol-
Comet Assay lows: 65% repeat-associated small RNAs, 17% Piwi-
The comet assay is used frequently in somatic cells interacting piRNAs, 11% quiescent RNAs, and 7%
to measure single- and double-strand breaks [22, 23]. micro RNAs [26]. Such a complex population of sncR-
When used to assess sperm, the assay involves mix- NAs suggests a role in post-fertilization development,
ing sperm with liquefied agarose gel, followed by elec- making them an emerging biomarker of male infertil-
trophoresis under either alkaline or neutral pH condi- ity [26].
196
Genetic and Epigenetic Basis of have shown that methylation patterns in the testes are
diminished eightfold relative to somatic tissue [31].
Male Infertility Whether this hypomethylation is present in mature
Male germ cell development begins in early embryo- spermatozoa, or whether it represents an epigenetic
genesis, but mature spermatozoa first appear only at process to prepare spermatozoa for increased tran-
puberty [1]. Genetic disorders can disrupt this male- scription following pronuclear development, is still not
specific cell differentiation and maturation at the chro- known [1].
mosomal or molecular DNA level. Genes involved in It is important to note that our comprehen-
spermatogenesis may be expressed functionally in the sion of genetic and epigenetic aspects of human
germ line, during the development of male gonads or spermatogenesis is still poor, and mostly deduced
in testicular somatic cells, but those expressed specifi- from animal studies [1]. Such extrapolation should
cally in the germ line are assumed to be the most rel- be treated with caution due to the myriad altered
evant to regulation of germ cell maturation [1]. For spermatogenesis/spermiogenesis states seen in
example, the RBM, SPGY and DAZ genes are known humans.
to regulate male fertility, and disruption of these genes
may lead to infertility or sterility [27]. There are other
recessive and dominant mutations in somatic cells Scope of Assisted Reproduction
that may indirectly induce infertility as a consequence Following the first successful birth in 1978 using
of other problems, as seen for instance in men with assisted reproductive technologies (ART), their use to
Kartagener’s syndrome, cystic fibrosis or myotonic overcome infertility has increased steadily [32]. An
dystrophy [1]. estimated 456 ART clinics in the United States per-
Chromosomal abnormalities account for approx- formed 157,635 ART procedures in 2012, and these
imately 5% of all male infertility cases and 15% of procedures resulted in 51,261 live deliveries and 65,151
infertility in azoospermic males [9]. Aneuploidy lead- infants [32]. In 2012, ART contributed to 1.5% of all
ing to male infertility may involve the sex chromo- infants born in the United States [32]. ART procedures
somes, for example an additional X-chromosome in consist of several steps over a two-week period, begin-
Klinefelter’s syndrome, or the autosome, for example ning with drug-induced ovarian stimulation, progress-
trisomy 21 [1]. Structural chromosome abnormalities ing to oocyte retrieval and fertilization with sperm in
such as small deletions, inversions, or translocations the laboratory and ultimately leading to embryo trans-
can lead to male infertility and may involve both sex fer [32]. In general, ART includes treatments such as
and autosomal chromosomes [1]. In fact, deletions in IVF, gamete intrafallopian transfer (GIFT) and zygote
the Yq region can be associated with azoospermia. intrafallopian transfer (ZIFT), with IVF accounting for
Specifically, in the region designated AZF (azoosper- approximately 99% of all ART procedures. ART, how-
mia factor), three loci (AZFa, AZFb, AZFc) associated ever, does not include treatments such as intrauterine
with nonobstructive azoospermia have been identified insemination, in which only sperm is handled, or ovu-
[1, 28]. Chromosomal rearrangements like reciprocal lation induction, which involves stimulating oocyte
translocations can also give rise to abnormal meiotic production [32].
chromosome pairing, thus disrupting spermatogene- After the establishment of IVF, it soon became clear
sis [1, 28]. that as many as 40% of the inseminated in vitro cycles
During spermatogenesis, sperm chromatin under- were affected by fertilization failure or by extremely
goes dramatic reorganization, including protamine low fertilization [33]. This was particularly problem-
replacement of histones, histone modifications and atic in patients with marginal semen characteristics
DNA methylation [1, 11]. Modifications of the N- and poor spermatozoa [33]. Specifically, diminished
terminal region of histones confer an epigenetic reg- sperm motility and/or poor morphology presented a
ulatory mechanism of gene expression [29]. Generally, complex obstacle for spermatozoa to penetrate the
methylation of the histones is associated with silenc- zona pellucida (ZP), a thick glycoprotein layer sur-
ing of the gene, while acetylation is associated with rounding the oocyte [34]. Traditional means of over-
transcription [30]. Methylation is carried out by DNA coming such hurdles were limited and dealt primarily
methyltransferases, which transfer a methyl group to with increasing sperm concentration and at enhanc-
deoxycytosines found in CpG islands [30]. Studies ing their selection [1]. Embryologists often increased
197
sperm concentration within the inseminating suspen- Intracytoplasmic Sperm Injection

sion, or if extremely few spermatozoa with impaired
Intracytoplasmic sperm injection (ICSI) is a proce-
motility were available, then a lesser volume of the
dure that involves the injection of a single spermato-
insemination medium was used to facilitate com-
zoon directly into the cytoplasm of an oocyte. ICSI
mingling of the two gametes [1]. Simple procedures
bypasses both the zona pellucida barrier and sperm
included the swim-up method, which selected only
defects in the male gamete that compromise its abil-
highly motile cells in the upper fraction [1]. Other
ity to fertilize [1]. Documented ICSI trials on mam-
methods utilized multilayer density gradients to select
malian gametes date as far back as 1966 [44]. How-
highly motile and, in general, morphologically normal
ever, initial efforts with human gametes yielded a
spermatozoa that also exhibited higher penetration
high incidence of oocyte damage and an unsatisfac-
capacity [35]. While these selection procedures opti-
tory level of embryo implantation [45]. The early use
mized the ability to treat IVF patients with moderately
of ICSI required some adjustments to identify the
compromised semen characteristics, often described
best method for piercing the membrane and identi-
by moderate oligoasthenospermia, they fell short in
fying the best location within the ooplasm in which
dealing with more severe issues of sperm dysfunction
to release the spermatozoon [37]. However, following
such as severe oligoasthenospermia or teratospermia
these adjustments and success in conceiving pregnan-
[1, 36, 37].
cies [43], it soon became apparent that ICSI was capa-
The focus then shifted to efforts to assist the sper-
ble of fertilizing nearly every mature oocyte injected
matozoon in penetrating the ZP. Early attempts involv-
[36, 37]. Moreover, the ability to pinpoint the differ-
ing complete removal of the ZP resulted in polyspermy
ent steps of pronuclei appearance and to monitor the
or impaired embryonic development [1, 38]. These
observation of the first embryonic cleavage without
were followed by attempts to overcome the zona as
the obstructive layer of cumulus cells would facili-
a barrier either by softening it enzymatically with
tate tracking these critical steps in early embryonic
trypsin or pronase [39] or by penetrating it chemically
development [37].
via localized or pinpoint exposure to acidified Tyrode’s
solution prior to sperm exposure [40]. The latter tech-
nique became known as zona-drilling (ZD), but the Popularity of ICSI
low-pH solution involved proved to possibly damage The implementation of micromanipulation techniques
the oocyte [40]. A variant of ZD opened a fissure in in the past 20 years has made it possible to over-
the ZP via mechanical means, called partial zona dis- come male gamete production deficiencies and fer-
section (PZD), and utilized a smaller opening than tilization defects to allow infertile male partners to
did ZD, thus mitigating the rate of polyspermy [41]. reproduce at rates that previously would have been
Still, a large obstacle persisted in the form of sperm deemed unachievable [37]. In a cross-sectional sur-
cells that required further assistance to properly inter- vey of ART procedures performed in 55 countries
act with oolemma [1]. This procedure was replaced by during 2007, the International Committee for Mon-
a more refined technique in which spermatozoa were itoring Assisted Reproductive Technologies reported
brought through the ZP with a pipette and deposited that 65.2% (400,617 of 614,540) of all cycles utilized
beneath the zona into the perivitelline space – sub- ICSI [46]. However, there was considerable variation
zonal insemination (SUZI) [1, 42]. This provided some in ICSI rates, ranging from 49.1% in Asia to 97.8%
success in patients with more impaired sperm motility in the Middle East [46]. In another recent publica-
while controlling the incidence of polyspermy, but still tion analyzing trends in ICSI use between 1996 and
yielded relatively low fertilization rates. This was pri- 2012 in the United States, ICSI use increased from
marily because spermatozoa still needed to undergo 36.4% in 1996 to 76.2% in 2012 [47]. At our centre
a complete acrosome reaction, a necessary precursor (Figure 13.1), there has been a steady and progres-
of fusion with the oolemma [1, 42]. These preliminary sive increase in ICSI prevalence, starting at 32.2% in
efforts to artificially assist sperm penetration soon 1993, rising to 48.8% in 1995 and reaching 73.6% by
became obsolete with the introduction of a microsur- 2002 [37]. Since 1993, ICSI has been used in 29,998
gical method for insertion of spermatozoa directly into cycles compared with 13,454 cycles with conventional
the oocyte [42, 43]. insemination. ICSI has yielded reproductive outcome
198
IVF extremely low sperm count, impaired motility and

100 ICSI poor morphology cannot be helped with IVF alone
90 26.4
[37]. In such cases, ICSI appears as a superior modality
Proportion of ART (%)
80 51.2 in achieving pregnancy. One of the crowning achieve-

70 67.8
60
ments of ICSI is perhaps its role in helping many
50
azoospermic men achieve pregnancy. Until advanced
40 73.6 micromanipulation techniques were available, such
30 48.8
couples relied on donor sperm or adoption [1]. A com-
20 32.2 plete absence of spermatozoa in the ejaculate occurs in
10 1% of all men and approximately 10–15% of all infer-
0 tile men [1]. The diagnosis is further subcategorized
1993 1995 2002
into obstructive azoospermia (OA) and nonobstruc-
Figure 13.1 Prevalence of ICSI cases at our centre in 1993, 1995 tive azoospermia (NOA) [51]. OA can result from con-
and 2002. genital absence of the vas deferens (CBAVD), trauma,
infection, vasectomy or failed vasectomy reversal [1,
comparable to those of conventional IVF, but is also 51]. In general, any form of male infertility due to
capable of consistently overcoming unforeseen sperm obstruction can be treated by ICSI with spermato-
cell dysfunction [37]. The overall fertilization rates zoa microsurgically recovered from either epididymal
(1993–2015) after ICSI and conventional IVF were aspirations, including microscopic (MESA), percuta-
74.4% (161,842/217,449) and 60.6% (74,741/123,316), neous (PESA) and fine-needle aspirations (FNA) [52,
respectively. However, with standard IVF, the two- 53] or testicular aspiration (TESE) [54, 55]. NOA usu-
pronucleated (2PN) formation rate is calculated over ally occurs due to a failure of spermatogenesis and
the total number of oocytes retrieved, so once cor- requires surgical intervention to obtain spermatozoa
rected for ICSI, the fertilization rate is comparable for subsequent injection [1]. TESE and the now refined
between the two insemination methods (58.5% ICSI micro-TESE retrieve seminiferous tubules for search,
vs. 60.6% IVF). The clinical pregnancy rate as defined with the latter achieving a higher probability of sperm
by the presence of a foetal heartbeat on ultrasound was retrieval and maintaining greater anatomical integrity
45.7% (12,066/26,429) for ICSI compared with 40.0% of the testicle [56–58]. It is also important to note that
(4,473/11,189) for IVF. Thus far 16,511 babies have when no motile spermatozoa can be retrieved from
been born from the two ART procedures, of which the epididymis due to epididymal fibrosis or for other
10,199 were conceived with ICSI. reasons, the use of testicular spermatozoa is indicated
There has been an increase in ICSI rates over the [36].
past two decades; however, the use of ICSI for patients Although ICSI requires only a spermatozoon with
with borderline or even normal semen characteristics a functional genome, oocyte activating factor and cen-
has also increased, without clear evidence of a bene- trosome for the fertilization of the oocyte, indications
fit over conventional insemination [48, 49] from using for ICSI are not restricted to low sperm count; they
ICSI. In fact, the American Society for Reproductive also include morphologically impaired spermatozoa
Medicine and the Society for Assisted Reproductive and impaired sperm kinetics [37]. In these scenarios,
Technology confirm that there is insufficient evidence high fertilization and pregnancy rates can be achieved
to support the routine use of ICSI in patients without when a viable spermatozoon is injected, independent
male factor infertility [50]. Thus, it is important to dis- of its characteristics [59]. In addition to these pri-
cuss the indications for ICSI. mary indicators, ICSI is the preferred option for male
patients experiencing various sperm-related problems
that include ejaculatory dysfunction and retrograde
Indications for ICSI ejaculation, as well as complications stemming from
While IVF has become a well-established treatment paraplegia [1]. Conditions linked to ultralow ejaculate
for most types of infertility, including tubal disease, volumes include retrograde ejaculation, lack of emis-
endometriosis, unexplained infertility and even some sion, ejaculatory duct obstruction, hypogonadism and
mild forms of male factor, some couples with an CBAVD. In such patients, high-speed centrifugation of
199
the semen sample and examination of the pellet under Table 13.2 Indications for ICSI
oil at 400× magnification with an inverted microscope Male factor Non-male-factor
is carried out to identify sperm [37].
Ejaculated spermatozoa Prior failed fertilization with IVF
ICSI has also been used in other non-male-factor Oligozoospermia Oocyte dysmorphism
settings. For example, ICSI can be used successfully Asthenozoospermia Low number of oocytes
in patients with complete fertilization failure (CFF) Teratozoospermia Low oocyte maturity
Antisperm antibodies Cryopreserved oocytes
after conventional IVF [39]. It has been hypothesized Fertility preservation In vitro maturation (IVM)
that ICSI might be a better method of fertilization Ejaculatory disorders Preimplantation genetic
than conventional IVF for patients with poor-quality screening
Acrosomeless spermatozoa HIV and hepatitis C discordant
oocytes, as determined by morphologic assessment couples
[60]. The advantage of using ICSI may be twofold in Cryptozoospermia Restrictive legislation
these situations: ICSI confers the ability to confirm Surgically retrieved
that the retrieved oocytes are indeed mature following Epididymal
Obstructive azoospermia
cumulus removal and specifically enhances chances Congenital bilateral
of fertilization by direct sperm injection [37]. ICSI is absence of the vas
commonly used in poor responders with the idea of deferens
Young syndrome
improving fertilization rates in the few oocytes that Failed vasoepididymostomy
are available for fertilization [37, 61]. Another possi- Failed vasovasostomy
ble benefit for ICSI is the prevention of polyspermia. Testicular spermatozoa
In fact, the reported incidence of triploidy in human Necrozoospermia
embryos after conventional IVF ranges from 2 to 10%, All indications for
epididymal sperm
with dispermy being the most common cause [62]. A Nonobstructive
retrospective analysis of 95 couples with ⬎20% inci- azoospermia
dence of 3PN zygotes in their initial conventional IVF
cycles followed by the use of ICSI in a subsequent cycle niques for men with obstructive and nonobstructive
showed that after ICSI, the rate of normally fertilized azoospermia, respectively. The optimal sperm retrieval
zygotes (2PN) was enhanced (65 vs. 34.1%), with a neg- technique depends upon the etiology of azoospermia,
ligible occurrence of 3PN (5.0 vs. 33.9%) [63]. There the technical capabilities of the embryology laboratory,
was no difference in cleavage and quality of embryos and the skill set and preferences of the clinician per-
derived from normal zygotes by the two insemina- forming the sperm retrieval procedure.
tion methods [63]. Thus, ICSI generated a higher num-
ber of diploid zygotes without compromising embryo
development. Table 13.2 summarizes the indications
Clinical Results with ICSI
for ICSI. Between September 1993 and June 2015, we per-
formed 29,998 ICSI cycles. Of these, approximately
91% (n = 27,284) of all ICSI cycles were per-
Sperm Retrieval Methods formed using ejaculated spermatozoa and the remain-
Technical refinements in sperm retrieval methods in der involved specimens that were surgically retrieved
conjunction with ICSI have enabled biological pater- from the epididymis or testis at our centre. In cycles
nity in azoospermic men who were previously con- utilizing ejaculated spermatozoa, a total of 224,247
sidered untreatable [64]. In general, a sperm retrieval MII were oocytes injected, resulting in a survival rate
technique that minimizes trauma to the reproductive of 97.3%. Of those that survived, 75.1% oocytes were
tract and yields the highest-quality sperm in sufficient fertilized normally, with 1PN and 3PN in only 2.4%
quantity for immediate and later use is desirable [64]. and 3.5% oocytes, respectively. No fertilization was
Surgical techniques for sperm retrieval can vary by noted in 16.3% oocytes (Figure 13.2).
anatomical target (epididymis vs. testis) and whether Table 13.5 summarizes the fertilization and
or not the procedure is assisted by intraoperative clinical pregnancy rates in ICSI cycles using ejac-
optical magnification (conventional vs. microsurgical) ulated, epididymal and testicular spermatozoa.
[64]. Tables 13.3 and 13.4 summarize the technical Figure 13.3 compares the fertilization rates between
aspects and spermatozoa yield of sperm retrieval tech- fresh and frozen ejaculated, epididymal and testicular
200
Table 13.3 Sperm retrieval techniques and corresponding yield in men with obstructive azoospermia [65–68]
Technique Yield Sperm retrieval rate Anaesthesia

Percutaneous epididymal sperm Thousands to millions of sperm 80–100% Local with or without sedation
aspiration (PESA) Cryopreservation for later use
possible in most cases
Testicular fine-needle aspiration Hundreds of thousands to millions 52–100% Local with or without sedation
of sperm
Cryopreservation for later use
possible in some cases
Testicular percutaneous biopsy Hundreds of thousands to millions 82–100% Local with or without sedation
of sperm
Microsurgical epididymal sperm Average of 15–95 × 106 total sperm 95–100% General, regional plus sedation,
aspiration (MESA) Cryopreservation for later use or local plus sedation
possible
Testicular sperm extraction Hundreds of thousands to millions 100% General, regional plus sedation,
(TESE) of sperm or local plus sedation
Microsurgical testicular sperm Hundreds of thousands to millions 100% General, regional plus sedation,
extraction (micro-TESE) of sperm or local plus sedation
spermatozoa. When the three different sperm sources Our centre also treats severely oligozoospermic
were examined, encompassing all maternal ages, the men with a concentration of spermatozoa of ⬍1 ×
ejaculated cohort displayed the highest fertilization 106 /mL [36]. Outcomes of ICSI cycles in these men
rates despite having older women (P ⬍ 0.001). Epi- are highlighted in Table 13.6. If the initial semen speci-
didymal spermatozoa achieved a somewhat lower men examination showed no spermatozoa, then high-
fertilization rate but attained the highest clinical speed centrifugation is used. In 311 cycles, after high-
pregnancies, as defined by the presence of at least one speed centrifugation, a mean density of 0.60 ± 1.1 ×
foetal heartbeat. Cycles using testicular spermatozoa 106 /mL and a motility of 39.2 ± 34% were reached. In
had the lowest fertilization rates in spite of having this cohort, a fertilization rate of 59.7% (1,881/3,150)
the youngest women (P ⬍ 0.001). The pregnancy and a clinical pregnancy rate of 37.6% (117/311) were
rates were somewhat lower compared with those in achieved [37].
the other groups. It must be noted that this analysis In cases of NOA, the degree of spermatogenic fail-
is purely academic, because the surgically retrieved ure often varies, and consequently, sufficient sperma-
spermatozoa address different clinical indications. tozoa for ICSI can be identified in only 40–60% of
Table 13.4 Sperm retrieval techniques and corresponding yield in men with nonobstructive azoospermia [65–68]
Technique Yield Sperm retrieval rate Anaesthesia

Testicular fine-needle aspiration ⬍10 to thousands of sperm 17–59% Local with or without sedation
Cryopreservation for later
use possible in some cases
Testicular sperm extraction (TESE) ⬍10 to thousands of sperm 17–70% General, regional plus sedation,
Cryopreservation for later or local plus sedation
Microsurgical testicular sperm ⬍10 to thousands of sperm 33–77% General (preferred), regional plus
extraction (micro-TESE) Cryopreservation for later sedation, or local plus sedation
201
3PN
MII Injected MII Injected 1PN 3.5%
n = 224,247 n = 224,247 2.4%
Lysed
2.7%
No fert
16.3
2PN
Surviving oocytes 75.1%
97.3%
Figure 13.2 Fertilization characteristics of ICSI cycles performed between September 1993 and June 2015 at our centre.
all patients. It is often necessary to search the biopsy profiles and numbers of oocytes retrieved. The fertil-
for an extended period of time in cases where only ization rate was 44.0% in the search group and 57.1%
a few sperm cells are present. In one study [36], we in the control group (P = 0.002), and live birth rates
investigated whether an extended search for sperma- were 34.3% and 46.8%, respectively (P ⬍ 0.001). Fer-
tozoa in NOA patients has an effect on ICSI outcome. tilization and pregnancy rates were plotted according
The average search time for routine TESE cases was to the length of time spent on each search (30 min–1
no more than 30 min (control), mostly in relation to h, 1–2 h, 2–3 h, ⬎3 h). The fertilization rates for these
the oocyte cohort. The extensive searches, often car- four groups were 54.2, 46.3, 28.0, and 25.4%, respec-
ried out by several embryologists, were divided into tively (R2 = 0.9315; P ⬍ 0.001). A progressive decrease
four groups based on time – 30 min to 1 h, 1–2 h, 2– in pregnancy rate with lengthening search times was
3 h, and ⬎3 h – and compared with clinical outcome. also observed. Specifically, the clinical pregnancy rates
A total of 739 NOA men who underwent 1,087 ICSI were 44.1, 37.8, 31.8 and 23.8%, respectively. Simi-
cycles were included in this study. The mean ages of the larly, the live birth rates were 32.4, 23.5, 18.2 and
female and male patients were 37.2 and 35.4, respec- 9.5%, respectively. Pregnancy loss rates were compa-
tively. Of the 1,087 cycles included in this study, 225 rable between all the extended search groups and con-
(26.1%) required an extended search. The length of trol. Thus, it appears that the length of time required to
sperm search ranged from 30 min to as long as 10 h extensively search a testicular tissue sample and to per-
with a mean of 82 min. The average number of embry- form ICSI on all the oocyte cohorts is inversely related
ologists involved in the searches was 4 ± 2. Pentoxi- to fertilization and pregnancy outcomes. In spite
fylline was used in almost all of the extended search of the time-dependent clinical performance, search-
cycles and in about 57% of the control cycles. The con- ing for precious spermatozoa is still warranted even
trol and the extended search groups had similar patient after several hours. Although labour-intensive and
Table 13.5 ICSI outcomes using ejaculated, epididymal and testicular spermatozoa
Parameter Ejaculated Epididymal Testicular

Maternal age (years) 37.9 ± 5 35.2 ± 5 33.3 ± 6
Cycles 27,284 1,083 1,631
Fertilization rate 168,411/224,247 (75.1%) 7,326/10,314 (71.0%) 8,466/16,188(52.3%)
Clinical pregnancy 12,469 (45.7%) 550 (50.8%) 661 (40.5%)
202
Fresh Figure 13.3 Fertilization rates using

fresh and frozen ejaculated, epididymal
Frozen and testicular spermatozoa between
75.0% September 1993 and June 2015 at our
148,019/197,342 centre.
Ejaculated 75.8%
20,392/26,905
2,602/3,610 72.1%
MESA 4,724/6,704 70.5%
6,314/12,008 52.6%
TESE 2,152/4,180 51.5%
0 10 20 30 40 50 60 70 80 90
Fertilization rate (%)
time-consuming, this procedure still grants many cou- ICSI has also been proposed as a method for rein-
ples the opportunity to conceive [36]. seminating oocytes that failed to fertilize following
conventional IVF [68–71]. Although reinsemination
When Not to Use ICSI of unfertilized oocytes has been performed with ICSI
15 to 18 h after initial insemination, normal fertil-
ICSI requires technical skills that conventional insem-
ization rates after rescue ICSI remain relatively low,
ination does not. In general, it needs to be performed
and the generated embryos achieve low pregnancy
in a highly regulated laboratory environment. It is per-
rates [72]. It is thought that oocyte quality diminishes
formed out of the laminar flow hood, on a heated
during the 24 h after retrieval, and although some
stage outside of the incubator, and requires enzy-
oocytes may still be fertilized, embryos derived from
matic/mechanical removal of the cumulus oophorus
rescue ICSI procedures often arrest at early stages of
[37]. In fact, early ICSI adopters indirectly improved
development [68–72]. Furthermore, high rates of poly-
their pregnancy rates because of the required adjust-
ploidy are reported in embryos fertilized by rescue
ments to more stringent laboratory conditions [37].
ICSI [72, 73], and the proportion of abnormally fer-
Most importantly, ICSI must be performed in an expe-
tilized oocytes seems to be a function of the length of
dited fashion [37]. Thus, ICSI should not be performed
time the oocyte is in culture following the initial failed
when a regulated laboratory environment is not
fertilization assessment [71].
available.
Table 13.6 ICSI outcomes in men with severe oligospermia

(⬍1 × 106/mL of spermatozoa)
Safety of ICSI
ICSI’s safety has often been criticized because the fer-
Parameter Value tilizing spermatozoon neither binds to the zona pel-
Cycles 1,820 lucida nor fuses with the oolemma [74, 75]. Bypass-
Mean concentration (106 per mL ± SD) 0.3 ± 0.3 ing these physiological steps, together with the arbi-
Mean motility (% ± SD) 19.1 ± 24.0 trary selection of the spermatozoon, has been reason
Mean morphology (% ± SD) 0.9 ± 1
for concern [74, 75]. Thus far, ICSI offspring undergo-
ing adolescence and beyond have provided sufficient
Fertilization 11,082/17,360 (63.8%)
information to reassure these qualms. Follow-up stud-
Clinical pregnancy 748 (41.1%) ies of ICSI children, beginning in the mid-1990s, have
203
revealed an incidence of malformations within the found in the IQ assessment between ICSI and natu-
expected range for the general population of New York rally conceived children. No differences were found
state [76]. Another series investigating the outcomes between ICSI and control children in regard to general
of neonates generated by different assisted conception health, such as chronic illnesses or physical develop-
procedures, ICSI versus IVF with conventional fertil- ment. Thus, ICSI and IVF appeared to exert a negative
ization, provided further confirmation of the expected effect on the wellbeing of offspring mainly because of
rate of malformation [77]. In one study evaluating the the association with multiple gestations [74].
medical and developmental state of 1-year-old chil-
dren born after ICSI or IVF as well as after natural con-
ceptions, the authors found that most 1-year-old ICSI Conclusions
children were healthy and were developing normally, Infertility is a common condition, and problems in
as measured by the Bayley Scales of Infant Develop- the male partner are one of the common causes. The
ment [78]. However, about 17% displayed an increase information generated by conventional semen analysis
in learning difficulties compared with those conceived has historically classified patients into categories lack-
by IVF or naturally. A later report dismissed this con- ing knowledge of causality and leaving conventional
cern in 2-year-old ICSI toddlers [79]. In a different therapy somewhat empirical. However, a better under-
follow-up of 10-year-old children, it was found that standing of spermatogenesis and its genetic control
ICSI children and their naturally conceived counter- has in recent years quite rapidly improved our knowl-
parts had similar motor skills and IQ [80]. edge regarding the epidemiology of male reproduc-
Our centre has compared the pregnancy outcomes tion. ICSI remains the most effective means of treat-
and the developmental wellbeing of children con- ing couples with male factor infertility and previous
ceived from 12,866 ICSI cycles with those of chil- fertilization failures. However, assisted reproduction
dren from naturally conceived singleton pregnancies techniques such as IVF and ICSI do not address the
[74]. From a total of 3,277 couples delivering 5,891 underlying cause for infertility, potentially increasing
neonates, the incidences of low birth weight and ges- the risk of transmitting both identified and concealed
tational length were comparable with those for the genetic anomalies [37]. Thus, basic research is needed
naturally conceived counterparts, after controlling for to elucidate the biological mechanisms underlying the
maternal age. Rates of malformation in ICSI offspring genetic and epigenetic effects of ART. Although there is
ranged from 3.5 to 6.2%, compared with 6.5% in still very little known about the long-term health con-
the natural conception group. In the ICSI group the ditions of both infertile men and their offspring, recent
major malformations included two neonates with a data suggest that infertility may serve as a proxy for
cardiac disease (ventricular septal defect and severe general medical ill health [81], with infertile or sub-
tricuspid regurgitation), one with talipes, and one fertile men possibly having increased mortality rates
with trisomy 7 mosaicism. Among the naturally con- [82]. The adverse outcomes in offspring conceived by
ceived pregnancies, there were two neonates with car- IVF or ICSI are generally due to the occurrence of
diac defects (ventricular septal defect and patent fora- high-order pregnancies. Thus, single embryo transfers
men ovale/atrial septal defect), one with encephalopa- are paramount in reducing such adverse outcomes.
thy, one with polydactyly, and one severe midshaft Although perinatal outcomes such as prematurity,
hypospadias with penile angulation. At 3 years of age low birth weight, perinatal mortality and increased
(n = 811), the proportion of children at risk for devel- incidence of malformations have been linked to the
opmental delays was 10.4% in ICSI and 10.7% in IVF techniques of IVF and ICSI, infertility itself seems
singletons. However, high-order gestations were char- to be the larger issue that leads to negative clinical
acterized by 19.4% of the children having compro- outcomes.
mised development. To study the long-term effect of
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207
Chapter
The Genetic Basis of Male Infertility
14 Amin S. Herati, Peter R. Butler and Dolores J. Lamb
Introduction nial stem cells (SSCs) undergo mitosis to maintain

the spermatogonial population and differentiate to
Infertility is defined as a couple’s inability to con- enter the meiotic pathway to produce daughter game-
ceive after one year of unprotected intercourse and tocytes. Sertoli cells mediate the actions of andro-
affects 10% of couples, for approximately 50% of which gen and FSH on spermatogenesis and function to
it is attributable to a male factor [1]. The standard maintain a favourable environment for the develop-
clinical evaluation of an infertile male includes a ing spermatogenic cells. The proper function of this
complete medical history, physical examination, hor- milieu of cells depends on the stimulation of Leydig
mone level measurement, and semen analysis (SA). cells by luteinizing hormone (LH) to produce testos-
The routine SA parameters include sperm density, terone and the stimulation of Sertoli cells by follicle-
motility and morphology. Suboptimal SA parame- stimulating hormone (FSH) to promote spermatoge-
ters include absent sperm in the ejaculate (azoosper- nesis and testosterone to complete the meiotic divi-
mia), low sperm density (oligozoospermia), abnormal sions and spermiogenesis/spermiation. Genetic and
sperm morphology (teratozoospermia) and impaired genomic abnormalities, such as aneuploidy, chromo-
motility (asthenozoospermia). Although the SA allows somal structural defects, genetic mutations and epige-
the classification of infertile men into various cate- netic dysregulation, can cause infertility or subfertility
gories based on their kind and degree of spermato- through disruption of spermatogenesis or sperm func-
genic impairment, this is a crude indicator of the tion. The resulting phenotypes range from structurally
underlying etiology. Indeed, a SA result cannot distin- normal but nonfunctional spermatozoa to the com-
guish the fertile from the infertile population except in plete absence of spermatogonia, meiotic cells and/or
the case of azoospermia [2]. This diagnostic approach haploid cells to mature spermatozoa.
identifies a spermatogenic defect in half of infertile Over the last three decades, our understanding
males, whereas in the other half an etiology is not iden- of the regulation of spermatogenesis, genetic and
tified and the patient is considered to have idiopathic genomic anomalies and their associated pathophys-
infertility [3]. It is estimated that a genetic or genomic iology has increased tremendously. Elucidating the
defect contributes to nearly 50% of male factor infer- genetic basis of male infertility has significant impli-
tility [4]. Not surprisingly, with the advent of advanced cations for the diagnosis and management of the sub-
genetic testing, such as next generation sequenc- fertile male and associated family counselling of risks
ing (NGS), the medical community is identifying a of genetic disease transmission to the offspring. We
genetic basis with ever-increasing frequency in these present a systematic review of known genetic and
individuals. genomic causes of male infertility.
In humans, steroidogenesis begins during foetal
testicular development, but is subsequently suspended
at birth or shortly after birth, and resumes at puberty Chromosome Anomalies
with the resurgence of gonadotropins. Within the The identification of the correct number of human
testicle and its seminiferous tubules, spermatogo- chromosomes in 1956 by Tjio and Levan [5] marked
University Press.
208
Chapter 14: The Genetic Basis of Male Infertility
a turning point in the field of medical genetics. Chromosome Aneuploidy and Male
Their discovery dispelled a three-decade-long belief
that individual human cells possessed 48 chromo- Infertility
somes and allowed future dichotomization of chro- Compared with fertile males, infertile males with
mosomal complement number as normal and abnor- impaired SA parameters have 3- to 10-fold increased
mal. Since the 1950s, significant progress has been levels of sperm aneuploidy (reviewed in [17]). Oocyte
made in linking chromosomal anomalies to infer- fertilization with an aneuploid sperm will create an
tility. There have also been substantial gains in aneuploid conceptus, the majority of which sponta-
our understanding of the molecular mechanisms by neously abort [18]. Chromosomal aneuploidies that
which these abnormalities may contribute to impaired can produce a viable offspring include abnormalities
spermatogenesis. of chromosomes 13, 18, 21, X and Y. Patients with tri-
Cytogenetic anomalies can be categorized as disor- somy of either chromosome 13 (Patau syndrome) or
ders of chromosome number or structure. Numerical 18 (Edwards syndrome) succumb to early deaths and
chromosomal anomalies are referred to as aneuploidy do not reach reproductive age; however, their fertility
if there is a loss or gain of a chromosome, or poly- potential is likely impaired, as evidenced by the associ-
ploidy if the entire chromosome complement has been ation between isolated trisomy of 18p with NOA [19].
amplified. Structural chromosomal anomalies include While case reports of men with trisomy 21 with proven
reciprocal and Robertsonian translocations, small fertility exist, they are generally sterile [20–23]. The
supernumerary marker chromosomes, deletions, and theorized mechanism by which this occurs is through
inversions. impaired proliferation and increased apoptosis of the
Studies that compared the chromosomal abnor- primordial germ cell (PGC) line during their migra-
mality with the degree of impairment in spermatogen- tion to the gonadal ridge [24]. In contrast, aneuploidy
esis identified a higher likelihood of detecting a gono- of the X chromosome is less detrimental, due to the
somal abnormality with nonobstructive azoospermia cell’s ability to inactivate supranumerical X chromo-
(NOA) and autosomal abnormality among men with a somes, and is more commonly implicated in the devel-
less severe phenotype, such as severe oligozoospermia opment of male infertility.
(sOS), oligoasthenoteratozoospermia (OAT), oligoter-
atozoospermia (OT), and isolated oligozoospermia
(OS) [6, 7]. Reciprocal and Robertsonian transloca- Aneuploidy of the X Chromosome
tions occur at a higher frequency in OS men than in The most common chromosomal disorder of males
NOA men (1.7% vs. 0.6% and 0.9% vs. 0.3%, respec- and similarly the most common sex chromosome
tively) [8]. Importantly, even infertile men with nor- disorder of infertile males is Klinefelter’s syndrome,
mozoospermia display a higher incidence of kary- 47XXY (KS) [25, 26]. Cytogenetic studies investigat-
otypic anomalies [6]. ing the causes of NOA and OS detected KS among 10–
It is well established that as cytogenetic abnormali- 26.7% and 1.4–5% of cases, respectively [8, 27, 28].
ties increase, the degree of spermatogenic impairment KS is clinically characterized by the presence
increases. In a retrospective review by Yatsenko et al. of hypergonadotropic hypogonadism, gynecomastia,
[6] of 668 subfertile males who underwent karyotype small testes and tall stature. It is diagnosed in the
analysis, the rate of cytogenetic abnormality was found late teen to early adult years secondary to evaluations
to be highest among azoospermic men (13.3%), fol- of subfertility and/or hypogonadism. Eighty percent
lowed by men with sOS (10.9%), OS (4.2%) and mild of KS cases possess a 47XXY karyotype, whereas the
OS (1.2%). The overall incidence of cytogenic abnor- remaining 20% either are mosaic (46XY/47XXY) or
mality in this study was 8.2%, which is more than 10 have higher-grade aneuploidy (48XXXY, 49XXXXY);
times the incidence in the general population (0.6%) however, the rate of mosaicism may be underestimated
and 20 times the incidence among healthy fertile men due to false negative interpretation of conventional
(0.4%) [6, 9, 10]. Several other studies have found sim- karyotypes (as high as 21.2% for low-level mosaicism)
ilar incidences ranging from 4.3 to 10.3% among sub- [7, 29]. The extra X chromosome is inherited due to
fertile men and up to 15.4% among azoospermic men nondisjunction, which can occur during either meio-
[7, 11–16]. sis I, meiosis II, or postzygotic mitosis [29]. Paternal
209
X chromosome aneupoloidy accounts for almost half mosome 14, 15 and 22 and none from chromosomes
of KS cases, in contrast to other trisomies, which are 10, 19 and X. Of the 149 male patients identified, the
typically maternal in origin [30–32]. sSMCs were associated with OAT in 40 cases (26.8%),
Two mechanisms were described to explain the recurrent pregnancy loss in 32 cases (21.5%) and idio-
severity of the KS phenotype, including the number pathic infertility in 77 cases (51.7%). Early meiosis
of short-stature homeobox (SHOX) gene copies, which studies demonstrated spermatogenic impairment and
are located in the pseudoautosomal region (PAR) 1 meiotic arrest at the spermatocyte stage [13]. Although
of Xp, and the presence of CAG repeats within the the mechanism by which sSMCs cause infertility has
androgen receptor [25]. The number of SHOX copies not been fully characterized, it stands to reason that
determines the severity of the stature phenotype in if the sSMCs contains genes associated with spermato-
a nonlinear, direct correlation [33]. A recent analy- genesis, the microduplication of those genes could per-
sis of copy number variation (CNV) using a single turb sperm development. Alternatively, the presence of
nucleotide polymorphism (SNP) array demonstrated sSMCs might disrupt meiosis.
a higher frequency of CNVs on the X chromosome
in nonmosaic KS subjects than in control males and
females [34]. Half of the CNVs mapped to 34 genes, Chromosome Structural Anomalies and
including 8 in the regions of PAR1 and Xq21.31 that Male Infertility
escape X-inactivation. An alternative theory involves Structural anomalies associated with male infertility
triple nucleotide repeat polymorphisms (CAG and can involve deletions, reciprocal balanced transloca-
GGC) of the androgen receptor gene on the X chromo- tions (X–Y, X–autosome, Y–autosome), unbalanced
some, which influences the sensitivity of the receptor translocations, Robertsonian translocations, disomies
to androgens [35]. Larger numbers of CAG repeats are or inversions. Central to many of these structural
predictive of increased height, the presence of gyneco- anomalies is the tendency to disrupt the formation
mastia, smaller testis size and an increased risk of male of normal bivalents due to errors in synaptonemal
infertility among KS patients [36–38]. complex formation and/or recombination, both in
Unassisted fertility among Klinefelter patients is chromosomes directly affected by the mutation and
uncommon, as 74.7% of mosaic KS patients and 90% in unrelated chromosomes. Synaptonemal complex
of nonmosaic KS patients are azoospermic. Testis length and location depend on the genetic density of
biopsy in these individuals often reveals progressively individual chromosomes; therefore gene-rich chromo-
degenerating seminiferous tubules with hyalinizing somes are more likely to be affected by recombina-
fibrosis and gradual depletion of germ cells hastened torial events [17]. Additionally, structural anomalies
at puberty [39–41]. Despite this, Ramasamy et al. can invoke abnormal synaptic and recombinational
[42] showed that two-thirds (45/68) of nonmosaic KS behaviour of one or more bivalents unrelated to the
patients had successful sperm retrieval by microscopic original anomaly causing aneuploidy, a process called
testicular sperm extraction and 45% of the sperm interchromosomal effect (ICE) [45, 46]. Fluorescent in
retrieved resulted in live birth after IVF. situ hybridization (FISH) studies detected ICE among
38.5% of Robertsonian translocations and 34.5% of
reciprocal translocations.
Small Supernumerary Marker Chromosomes These structural anomalies are of clinical interest,
The improved sensitivity of FISH and aCGH have as they result in reproductive problems such as con-
increased the detection of small supernumerary genital anomalies, recurrent pregnancy loss and pri-
marker chromosomes (sSMCs) among infertile males. mary infertility. The type and severity of these prob-
It is estimated that infertile males carry sSMC at a five lems depends on the anomaly’s location, size and
times higher frequency than newborns [16]. In a recent effects on other chromosomes during meiotic recom-
systematic review of 234 infertile males and females bination [47].
with sSMC, a higher frequency was found relative to
the general population (0.125% vs. 0.043%) and at a
higher rate in males than in females (7.5:1) [43, 44]. Chromosomal Translocations
In this study, 72% of the sSMCs were derived from Chromosomal translocations, including reciprocal
acrocentric chromosomes, predominantly from chro- and Robertsonian translocations, account for up to
210
3.6% of autosomal abnormalities among infertile men The impact of A–A translocations on fertility
[6]. Translocations can be either balanced or unbal- depends on the location, size and characteristics
anced depending on the conservation of genetic mate- of the translocations. Synaptic pairing abnormalities
rial, and occur between gonosomes, between gono- and recombination frequencies can significantly vary
somes and autosomes and between autosomes. The between two balanced translocation carriers and result
balance of genetic material is critically important for in variable presence of sperm in the ejaculate. FISH
the development of structurally and functionally nor- and multicentromere fluorescent in situ hybridization
mal spermatozoa and embryos inheriting the genetic (cenM-FISH) studies comparing the chromosomal
material from the spermatozoa. constitution of a normozoospermic carrier of t(10;14)
with that of an azoospermic carrier of t(13;20) revealed
synaptic pairing abnormalities more frequently (71%)
Reciprocal Translocations in the spreads of the t(13;20) carrier than in the
In contrast to unbalanced translocation carriers, who t(10;14) carrier (30%) (50). One mechanism by which
manifest neuropsychiatric and/or morphologic dis- autosomal translocations are thought to cause steril-
orders, carriers of balanced reciprocal translocations ity is through the interference of unpaired autoso-
possess normal karyotypes and rarely manifest a phe- mal fragments, produced as a byproduct of non-
notype, such as infertility. Deleterious phenotypes homologous pairing during prophase, with the sex
manifest if the translocation breakpoint occurs within chromosomes and impairing proper XY metabolism
a gene or its regulatory regions, disrupting that copy (51).
of the gene and thus causing haploinsufficiency. Bal-
anced translocations can, however, have a significant Robertsonian Translocations
impact on the genetic constitution of the carrier’s sper- Robertsonian translocations occur among the acro-
matozoa, which are subject to a higher rate of meiotic centric chromosomes (chromosomes 13, 14, 15, 21,
errors (such as impaired synaptic complex pairing or and 22) and are detected in 0.7% of infertile males
recombination), resulting in either disomy or unbal- (8.5 times higher than in newborn children) [16].
anced translocations in 29–81% of the spermatozoa Acrocentric chromosomes usually possess only one
[17, 45]. These unbalanced gametes in turn produce recombination focus in the q-arm of the chromosome.
chromosomally abnormal or mosaic embryos. Therefore, dysfunction of that single focus can result
Reciprocal translocations can occur between gono- in achiasmate bivalents (bivalents lacking recombina-
somes (X–Y), between gonosomes and autosome (X– tion foci), which can subsequently lead to aneuploid
A, Y–A) or between autosomes (A–A). Rearrangement sperm and impaired spermatogenesis [17]. Robertso-
of chromosomal material between the X chromosome nian translocations produce an unbalanced gamete in
and autosome produces sterility via disruption in sper- 3 to 36% of spermatozoa [17]. Similarly to reciprocal
matogenesis regardless of the position of the X chro- translocations between A–A chromosomes, Robertso-
mosome break [48]. Sterility is thought to occur due nian translocations may also produce autosomal frag-
to genetic imbalance created by the escape and activa- ments that interfere with the XY bivalent and impair
tion of the mis-segregated portion of the X chromo- fertility [52]. Despite this, studies found that these
some from an inactivated state [16]. Rearrangements translocations result more commonly in OS than NOA
between the Y chromosome and autosomes result in (1.6% vs 0.09%) [16]. The most common Robertso-
sterility if the breakpoint occurs in a PAR of the Y chro- nian translocation associated with male infertility is
mosome, which possesses genes integral to meiosis t(13q;14q), which is identified in 71.4% of cases involv-
and fertility, or involves Yq11 or its boundaries. PARs ing a Robertsonian translocation [16]. Other notable
of the X and Y chromosomes, which are located on the translocations include t(14q;21q) and t(21q;22q).
short arms of both sex chromosomes, possess homol-
ogous sequences capable of recombining. Within the
PAR are 29 genes, including the SHOX and other genes Inversions
critical for XY pairing, synapsis and recombination Inversions can be either paracentric or pericentric.
[49]. Translocations can also result in disorders of sex- They result in reproductive problems similar to those
ual differentiation (46XX, +SRY) and male infertility for other structural anomalies but with lower fre-
due to meiotic breakdown. quency. Inversions have been identified eight times
211
more frequently in infertile males than in the gen- 10% of infertile YCMD males with normal karyotypes
eral population (0.16% vs. 0.02%) [16]. Similarly to and in 100% of men with abnormal karyotypes.
other structural anomalies, unbalanced gametes occur The association between Yq microdeletion and
at higher frequencies (range 0.7–54.3%) depending on NOA was first reported by Tiepolo and Zuffardi [59]
the size and location of the inversion [45]. Inversions in 1976 in six men who possessed breaks in the Y chro-
of chromosomes 1–3, 5–7, and 9 have been impli- mosome at the distal portion of Yq11. In 1996, Vogt et
cated in male infertility; however, chromosome 1 car- al. [60] performed mapping studies of 360 idiopathic
ries the highest risk [16]. Meiotic and cytogenetic stud- azoospermic males using sequence-tagged site (STS)
ies performed on testicular tissue of an sOS carrier of and polymerase chain reaction (PCR) technology. That
a pericentric chromosome 1 inversion showed a wide study revealed three predictable histologic patterns of
range of synaptic disturbances (including reduced or impaired spermatogenesis depending on the subre-
absent chiasma for inverted and non-inverted biva- gion affected: ranging from Sertoli cell only (SCO)
lents, heterosynapsis and rare formation of chromoso- among proximal subregion deletions, spermatogenic
mal loops), a 45% apoptosis rate, and a small number arrest at the spermatocyte stage among mid-subregion
of cells (4%) possessing an intact sex chromosome pair deletions and intermittently present, mature sperma-
beyond the early pachytene stage of meiosis I [53]. tozoa in the seminiferous tubules of distal subregion
deletions. These three subregions were collectively
characterized as azoospermia factors (AZF) and indi-
Y Chromosome Microdeletion vidually termed AZFa, AZFb, and AZFc from proximal
The Y chromosome q11 band is of particular impor- to distal, respectively [60].
tance in evaluating the genetic causes of male infer- Although these subregions were initially thought to
tility, as it possesses genes essential for spermato- be nonoverlapping, a subsequent study by Repping et
genesis and is error-prone due to the abundance al. [61] in 2002 used finer molecular characterization
of palindromic sequences, which are mirror-image to reveal a 1.5 Mb overlap between the AZFb and AZFc
genomic sequences of similar polarity composing subregions and novel deletion patterns spanning both
almost a quarter of the q11 band [54, 55]. Gene bal- intervals (AZFb+c). Despite this, complete deletions
ance is maintained by the homologous palindromic of AZFb (P5/ proximal P1) and AZFc (b2/b4) express
sequences not crossing over following double-strand distinct phenotypes consistent with those presented by
break (DSB) creation. Intrachromosomal crossover (or Vogt et al. [60], and thus the original nomenclature has
nonallelic homologous recombination [NAHR]) cre- persisted. Several other deletion patterns exist, includ-
ates intrachromosomal recombinational errors, such ing combined AZF mutations (AZFa+b, AZFb+c, and
as inversions and gene deletions, with potentially dev- AZFa+b+c) and partial deletions of AZFc (such as
astating perturbation of spermatogenesis [56]. These gr/gr, b1/b3, and b2/b3).
microdeletions can in turn result in loss of large frag- A literature review of 6,620 OS and NOA men
ments of Y chromosome DNA, creating structural by Massart et al. [62] showed microdeletions of Yq11
changes that disrupt proper X–Y bivalent formation present in 7.4% of the total cohort and 9.7% of the
[57]. Analogously, sister chromatid crossover follow- subset of NOA men. Another large systematic review
ing DSB formation creates an acentric Y fragment of 4,800 infertile males by Foresta et al. [63] found
and an isodicentric Y chromosome (idicY), which can an overall prevalence rate of YCMD of 8.2% and sub-
occasionally be visualized with light microscopy but is set prevalence rates of 4% in OS, 14% in sOS, and up
detected with more sensitivity using FISH and array to 18% in NOA men. However, demographic differ-
comparative genomic hybridization (aCGH) [56]. Sev- ences are present, with AZF deletion frequencies as
eral mechanisms can explain spermatogenic failure high as 24.2% in the Middle East [64]. The most preva-
associated with idicY, including deletion of the sper- lent type of AZF deletion detected by Massart et al.
matogenic genes in the acentric fragment and disrup- [62] was complete deletion of AZFc (69%), followed
tion of X–Y pairing [56]. Jorgez et al. [58] evaluated by AZFb (14%) and AZFa (6%) deletions. Significant
for structural defects of the PAR of the Y chromo- clinical differences exist between AZFa, b and c, which
some in 87 infertile males with known Y-chromosome are likely due to loss of genes thought to be required
microdeletions (YCMD) using aCGH or qPCR of for spermatogenesis within the respective subregions.
select PAR genes. PAR abnormalities were present in Moreover, combined deletions of two to three AZF
212
subregions accounted for almost one-fourth of dele- drome 5 and extending to the proximal arm of palin-
tions in a review of 187 men with YCMD from 20 stud- drome 1. Deletion of AZFb (P5/Proximal P1) removes
ies by Foresta et al. [65]. Men with combined muta- 32 gene copies, many of which are critical for pro-
tions, such as AZFb+c and AZFa+b+c, had a higher gression through meiosis. It should not come as a sur-
rate of azoospermia than men with single AZF subre- prise that the histologic phenotype associated with
gion deletion. AZFb (P5/Proximal P1) deletions is maturation arrest
at the spermatocyte stage. Other overlapping loss-of-
AZFa function (LoF) variants of clinical relevance have been
Complete deletions of the AZFa subregion origi- described, including P5/distal P1 and P4/distal P1 (see
nate from intrachromosomal recombination involv- Figure 14.1) [61, 64].
ing two homologous palindromic sequences within Controversy remains as to which AZFb gene is
AZFa, human endogenous retroviral sequences 1 and responsible for this phenotype. Genes within the DNA
2 (HERV15yq1 and HERV15yq2), and clinically result sequence of AZFb but not overlapping with AZFc
in hypogonadism with small testes and SCO on testis include lysine demethylase 5D (KDM5D), eukaryotic
biopsy [55]. The AZFa subregion is approximately translation initiation factor 1A, Y-linked (EIF1AY),
1,100 kb long and possesses single copies of two ribosomal protein S4, Y-linked 2 (RPS4Y2), chro-
candidate fertility genes, ubiquitin specific peptidase mosome Y open reading frame 15 (CYORF15), X
9 (USP9Y) and DEAD-box polypeptide 3 (Ddx3y) Kell blood group precursor-related, Y-linked (XKRY)
[64, 66]. USP9Y regulates protein turnover by de- and heat shock transcription factor, Y-linked (HSFY).
ubiquitinating target proteins. Its role in spermatoge- The KDM5D gene codes for the H3 lysine 4 (H3K4)
nesis, however, appears to be marginal, as complete demethylase enzyme, which interacts with the DNA
deletion of USP9Y has been identified in an NS fer- repair factor MutS protein homolog 5 (MSH5) at the
tile male [67]. In contrast, deletion of the Ddx3y gene prophase stage of meiosis [66, 68]. KDM5D and MSH5
results in complete absence of germ cells and is likely proteins complex together in the leptotene/zygotene
responsible for the phenotype characteristic of AZFa stage of meiosis and are thought to be responsible
[60]. for chromatin remodelling [57]. Mutations of KDM5D
are therefore posited to cause maturation arrest via
AZFb, AZFb+c and AZFc impaired chromatin remodelling and chromosome
The partially overlapping AZFb and AZFc subregions condensation during meiosis.
span a stretch of 8.2Mb and contain multiple copies of The HSFY gene codes for transcriptional activators
24 genes [61]. AZFb is 6.2Mb long, beginning at palin- of the heat shock proteins (HSP), which localize to the
PAR1 Yp Yq PAR2 Deletion Phenotype
AZFa Sertoli Cell Only
AZFb Spermatogenic Arrest
AZFc Azoospermia to Severe Oligozoospermia
AZFabc Sertoli Cell Only
AZFbc Sertoli Cell Only to Spermatogenic Arrest
Partial AZFc Azoospermia to Normal Spermatogenesis
Figure 14.1 AZF microdeletions with associated phenotype. Image modified from [151]. Creative Commons License http://
creativecommons.org/licenses/by/3.0/legalcode.
213
Inter-relations for epigenetic marking acquisition during spermatogenesis
Histone Histone Histone Protamines RNA

DNA methylation
Acetylation Methylation Variants substitution production
Fetal Life PGC
Demethylation
Demethylation of H3K4
Gonad Differentiation PGC Demethylation
Deacetylation
of H3 & H4 of H3K9
and H3K27
Progressive Action of
Proliferation
methylation DNMT3A, 3B
Spermatogonia & 3L
(2n)
Birth Methylation
Spermatocytes I of H3K9
Puberty
(2n)
Meiosis I
Maintenance Methylation
Spermatocytes II of H3K4
(n) of DNA methylation
Incorporation
Meiosis II Acetylation of histone
Spermatids of H3 & H4
(n) Demethylation variants Replacement
of H3K9 of histones
by protamines
Spermatozoa
(n) Histones
retention
Fertilization
Histone
Retention
Specific
Histone marking
Specific Bivalent marking
Contribution of the paternal genome to the embryo
demethylated H3K4Me-H3K27Me
development
genes
Sperm
RNAs
Figure 14.2 A temporal schematic of epigenetic regulators of spermatogenesis, spermiogenesis, fertilization, and foetal life. Adapted from
[136].
nuclei of spermatogenic cells ranging from spermato- and modulation of STAR and T-STAR protein func-
gonia to round spermatids and in the cytoplasm of tion, which are members of signal transduction path-
Sertoli cells (69). Although isolated deletions of HSFY ways necessary for completion of the cell cycle [57].
have been associated with maturation arrest and NOA, Members of the DAZ and CDY gene families also
its contribution to fertility has been questioned by the localize to the overlapping region of AZFb+c and
finding of HSFY deletion transmission over several have established roles in the regulation of spermato-
generations in the family trees of four males with HSFY genesis. The DAZ gene family includes three gene
deletion [70]. members, Boule-like RNA-binding protein (BOLL),
Among the genes within the AZFb+c overlap Deleted In Azoospermia-like (DAZL), and DAZ. The
regions are RNA binding motif protein, Y-linked, fam- DAZ gene, which is expressed exclusively in the testis
ily 1 (RBMY1), PTPN13-like, Y-linked (PRY), Chro- and is present in four copies (DAZ1–4) on the Y chro-
modomain Y, Y-linked (CDY), Basic protein Y2, Y- mosome, is highly homologous with DAZL, which is
linked (BPY2), and Deleted In Azoospermia (DAZ) autosomal [72]. This is of evolutionary significance, as
[66, 71]. The RBMY1 gene is a member of the RBM DAZ is thought to have originated from the transposi-
gene family and, unlike other ubiquitously expressed tion of DAZL to the Y chromosome and can compen-
members of the RBM family, is solely expressed in the sate for DAZ mutations, allowing a less severe pheno-
testis. Functional studies investigating RBMY1 have type [57]. The DAZ genes express proteins that possess
shown its role in mRNA splice regulation, transport an RNA binding motif that allows the activation and
of mRNA from the nucleus during spermatogenesis transport of mRNA transcripts from the nucleus to the
214
cytoplasm and the repression of microRNA (specifi- tors for testicular cell adhesion molecule 1, pseudo-
cally miR430), which if unchecked leads to depletion gene (TCAM1P), testis expressed gene 101 (TEX101),
of the spermatogonial germline [72]. Of the four DAZ ADAM metallopeptidase domain 2 (ADAM2) and t-
genes, proper function of DAZ1 is essential for sper- complex 10 (TCP10) genes are necessary for com-
matogenesis, as its deletion more consistently results pletion of meiosis in mice but not in humans [76].
in infertility, unlike the other three DAZ genes [73]. Moreover, a multihit phenomenon is likely nec-
The CDY gene family is composed of the CDY1, essary to destabilize cellular processes and path-
CDY2, CDYL1 and CDYL2 genes, which localize to ways, given the evolutionary conservation of the
AZFb, AZFc, chromosomes 6 and 16, respectively reproductive system. Nevertheless, advances in high-
[57]. Similarly to the DAZ family, significant redun- throughput genomic sequencing technology, such as
dancy exists between the CDY isoforms, with 98% aCGH and NGS, have expanded our candidate gene
amino acid homology between CDY1 and CDY2 [57]. pool through more efficient audits and comparisons
The CDY protein family exhibits histone acetyltrans- of the infertile male’s whole-genome or whole-exome
ferase activity to promote chromatin remodelling dur- sequence.
ing spermiogenesis, and deletion of either isoform can A recent transcriptome analysis of human germ
be partially compensated for by up-regulation and cells at different developmental stages of spermato-
increased acetylation activity of the other homologous genesis identified 6,622 differentially expressed
isoform [74]. genes involved in spermatogenesis (Table 14.1)
The AZFc sub-region is 3.5Mb in length and is [76]. Although the roles of many of these genes in
the most commonly affected AZF subregion, as it spermatogenesis still need to be confirmed, several
is highly susceptible to NAHR events [61]. Putative have been confirmed through prior associations of
genes in this subregion include BPY2, CDY, DAZ, infertility with a genetic syndrome or as a single-/
Golgi autoantigen, golgin subfamily A, 2-like, Y-linked multiple-gene cause of infertility. In addition to the
(GOLGA2LY1) and chondroitin sulphate proteoglycan genes described in prior sections of this chapter, we
4 pseudogene 1, Y-linked (CSPG4LY). The important will highlight select infertility genes with syndromic
candidate genes in this subregion overlap with AZFb association, gene mutations grouped by their impact
and have been discussed above. on spermatogenesis and sperm function, and char-
acterize other notable, recently described deleterious
mutations. A comprehensive review of all the genes
Gene Polymorphisms and Infertility associated with male infertility is beyond the scope of
Spermatogenesis is a highly conserved process. Suc- this chapter.
cessful differentiation of primordial germ cells to
mature spermatozoa and maintenance of the germ
cell line require intact expression of several gene Syndromic Genetic Causes of Male Infertility
families, such as those that control the cell cycle,
signal transduction pathways, cellular metabolism Congenital Bilateral Absence of the Vas Deferens
and cell-structure-related proteins. Extrinsic regula- In many cases, infertility may be part of a syndromic
tion is similarly controlled through the intact expres- condition, with many symptoms in unrelated physi-
sion of genes necessary for endocrine signalling and ologic systems, arising from a common genetic etiol-
somatic–germ cell interactions. Linkage studies con- ogy. One of the most widely recognized of these syn-
necting these genes and gene families to infertility have dromic infertility conditions is due to mutations in the
historically been difficult to validate due to the het- cystic fibrosis transmembrane conductance regulator
erogeneity of clinical presentation, histologic phe- (CFTR) gene. The protein encoded by the gene is a
notype of the testis on testis biopsy and the con- chloride channel expressed in epithelial cells, through
founding effects of epigenetic regulation even in the which chloride ions (and subsequently water, by osmo-
context of identical genomic sequences [75]. Addi- sis) pass out of the cell into secreted mucus. Loss
tionally, validation of putative infertility genes with in function of CFTR leads to production of abnor-
in vivo experiments is challenging due to species- mally viscous mucus, which has deleterious effects
specific gene expression patterns. For example, per- on multiple organ systems, including the reproduc-
sistently high expression levels of transcription fac- tive tract. There is some clinical evidence of impaired
215
Table 14.1 Pathways and Genes Up-Regulated in Spermatogenesis
Expression
Pattern Pathway Gene Name
Spermatogonia Purine metabolism ATP1B1, TOR3A, ARL8A, RAB38, GNB5, RAB31, ARL4C, GNL3, FGF2, RHOBTB3,
up-regulated TAP2, TAP1, TAPBP, RAB23, AGAP3, TOR1A, GBP3, GBP1, RRAD, MRAS,
DCTPP1, ADK, AK3, PRPS2, PNP, RUNX1
Cytokine-cytokine receptor LIF, ACVR1, CCR7, CCL2, CXCL3, IL13RA1, CXCL2, VEGFA, PDGFRA, NGFR,
interaction TGFBR2, LTBR, BMP2, TNFRSF1B, IL7R, IL6, IL24, IL8, TNFRSF1A, KITLG,
TNFRSF12A, TNFRSF10D, INHBA
Pathways in cancer MMP9, TPM3, COL4A2, FGF13, FGF2, MMP1, CDKN1A, VEGFA, PDGFRA, GSTP1,
BID, TGFBR2, FGFR1, BMP2, PTGS2, MYC, IL6, IL8, NFKBIA, KITLG, RUNX1, ITGAV
MAPK signalling pathway PLA2G4A, DUSP2, GNG12, DUSP6, FGF13, FGF2, MYC, MAP3K8, TNFRSF1A,
MRAS, PDGFRA, NGF, TGFBR2, FGFR1
Lysosome SCARB2, CD68, CTSS, CTSO, CD63, CTSB, HEXB, LAPTM5, LAPTM4B, HGSNAT,
ARSB, NEU1
Focal adhesion ITGB8, COL4A2, VAV2, CAV1, PARVA, ITGAV, VEGFA, PDGFRA, SRC, MYL12A,
CAV2, THBS1
Chemokine signalling pathway GNG12, GNB5, CCR7, CCL2, CXCL3, VAV2, NFKBIA, IL8, CXCL2, LYN, FOXO3
Regulation of actin cytoskeleton GNG12, ITGB8, FGF13, FGF2, VAV2, MRAS, ITGAV, PDGFRA, MSN, MYL12A, FGFR1
TGF-beta signalling pathway DCN, BMP2, ACVR1, MYC, ID2, FST, TGFBR2, INHBA, THBS1
Phagosome CTSS, COLEC12, SEC61A1, TAP1, STX7, TAP2, C1R, ITGAV, THBS1
Tuberculosis LTBR, PLK3, CTSS, IL6, TNFRSF1A, SPHK1, BID, SRC, CD74
Jak-STAT signalling pathway LIF, IL7R, IL6, MYC, IL24, IL13RA1, SPRY4, SPRY2
Spermatocyte Purine metabolism MSH4, MYO1A, DNM1P46, SRCAP, RNF112, TOP2A, DDX17, DMC1, KIF20A,
up-regulated PMS2P5, ATP8B5P, PPP2R4, SETX, POLR2A, POLE
Progesterone-mediated oocyte SPDYA, BUB1, CCNB3
maturation
Oocyte meiosis SMC1B, SPDYA, BUB1
Cell cycle SMC1B, BUB1, CCNB3
Spermatid Purine metabolism ALLC, LINC00282, ATP10A, ATP6V1E2, CHD5, ATP1A2, ATP1A4, ATP7B, DNAH9,
up-regulated KIF2B, DHX57, ABCB11, ABCA12, LOC100131047, DNAH11, PDE4A, PDE11A,
PDE1A, PDE6A, PDE1C, AK8, ADCY8, NT5C1B, EHD1, GNG2, TUBA4A, KIF17,
DNALI1, MYO7A, MYH7, STARD9, DYNLRB2, MYH1, RAB27B, KIF5C, MYH7B,
TUBA8, MYO18B, KIF15
Neuroactive ligand-receptor VIPR1, PTH1R, P2RX3, GRID1, CHRM4, OPRM1, GRM3, GLRA3, GRIK1, GRM5,
interaction GRIN2B, GRM7, GRID2, OPRL1, CHRNB4, GPR156, GABRG3
Calcium signalling pathway PDE1A, CACNA1C, ADCY8, P2RX3, PLCD4, PLCG2, CACNA1A, PLCE1, PDE1C,
CAMK4, GRM5, NOS3
Glutamatergic synapse GNG2, CACNA1C, ADCY8, GRM7, SHANK2, CACNA1A, GRM3, GRIK1, GRM5,
PLA2G4E, GRIN2B
MAPK signalling pathway MAPKAPK2, HSPA1L, MKNK1, FGF14, CACNA1C, CACNA1A, MAP3K14, IL1A,
MAPK10, MAPK8IP1, PLA2G4E
Pathways in cancer CDKN2B, FGF14, DCC, PLCG2, FZD9, WNT7A, TRAF4, MAPK10, CDKN2A, CTNNA2,
LAMB4
Axon guidance EPHA6, EFNB3, ROBO2, DCC, SLIT3, EPHA1, SLIT1, NTN1, SLIT2, UNC5D
Tight junction EPB41L3, MYH7, PARD6A, IGSF5, PPP2R2C, MAGI1, SYMPK, MYH1, MYH7B,
CTNNA2
Regulation of actin cytoskeleton FGF14, FGD3, CHRM4, GIT1, DIAPH3, PFN3, TIAM2, ARHGAP35, IQGAP2, PIP5K1B
Pancreatic secretion SLC26A3, CA2, SLC4A4, ADCY8, KCNMA1, ATP1A4, RAB27B, ATP1A2, PLA2G4E
Insulin signalling pathway ACACB, MKNK1, SLC2A4, INPP5D, LIPE, HK1, MAPK10, G6PC2, HK3
Bile secretion CA2, SLC4A4, ADCY8, ATP1A4, ABCB11, SLC51B, SLC27A5, ATP1A2
Note: Adapted from [76].
spermatogenesis in patients with cystic fibrosis (CF), increased acidity, as well as smaller testes and epi-
but the most common presentation of infertility in didymides, and absence of the vasa deferentia on phys-
patients with CFTR mutations is congenital bilateral ical and surgical examination [77]. The mechanism
absence of the vas deferens (CBAVD) [77]. by which CFTR mutation leads to CBAVD is not
The clinical presentation of CBAVD involves completely understood. CBAVD can entail not only
reduced semen volume with low fructose content and absence of the vasa deferentia but also absence or
216
malformation of the epididymides. CFTR has been absence prevents this migration, leading to the Kall-
localized to the reproductive tract during human foetal mann phenotype [80]. The semaphorin 3A (SEMA3A)
development, suggesting that it is involved in normal gene is similarly involved in neuronal migration and
development of Wolffian duct structures such as the implicated in Kallmann syndrome [81]. Fibroblast
vasa deferentia [78]. growth factor signalling may activate this migration,
There is substantial variability in CFTR mutations and mutations in fibroblast growth factor receptor 1
and their associated phenotypes. Not all male CF (FGFR1) have been associated with Kallmann syn-
patients have CBAVD, and not all CBAVD patients drome as well [82]. Alternatively, mutations in either
have CF. Frequently, patients possess different muta- kisspeptin 1 (KISS1) or kisspeptin receptor 1 (KISS1R)
tions in both copies of CFTR. These mutations vary in appear to lead to Kallmann syndrome of altering
severity, with the most severe mutation determining GnRH secretion. In animal models in which Kiss1r
the phenotype. One of the early explorations of the is knocked out, the hypothalamus contains a normal
connection between CFTR and CBAVD examined concentration of GnRH, but the HPG axis is nonethe-
exonic mutations known to cause classical CF and an less disrupted, resulting in a Kallmann-like phenotype.
intronic mutation, 5T, whose prevalence was enriched This suggests the KISS1/R pair is involved in secretion
in CBAVD patients [79]. The 5T mutation leads to or processing for secretion of GnRH [83].
omission of exon 9 during mRNA splicing, producing
a shortened but still largely functional protein. Most Deafness-Infertility Syndrome
patients with CF symptoms have mutations in the
Kallmann syndrome is not the only association of
coding region of each allele, while most CBAVD
male infertility with sensory dysfunction. Deafness-
patients without CF symptoms have a mutation in
infertility syndrome (DIS) involves sensorineural
the coding region of one allele and the intronic 5T
hearing loss in both men and women, as well as
mutation in the other allele. The inheritance pattern
asthenoteratozoospermia in men. DIS has been asso-
of CBAVD arising from the 5T allele appears to be
ciated with microdeletion of chromosome 15q15.5,
incomplete penetrance, as many but not all CBAVD
encompassing the final two exons of cation channel
patients have this variant [79].
sperm associated 2 (CATSPER2) and the entirety of
Kallmann Syndrome stereocilin (STRC) (84). The CATSPER family encodes
calcium ion voltage-gated channels expressed in the
Kallmann syndrome is another disease affecting
flagella of sperm and necessary for capacitation. This
male fertility as well as other physiological func-
particular family member may also be necessary
tions. Specifically, this syndrome entails congenital
for normal development of the spermatozoa, as
gonadotropin-releasing hormone (GnRH) deficiency
the DIS phenotype specifically involves abnormal
(idiopathic hypogonadotrophic hypogonadism, IHH)
morphology.
as well as anosmia. Loss of GnRH activity disrupts
the hypothalamic–pituitary—gonadal (HPG) axis at
its origin, leading to failure of the pituitary to release HOX Genes
FSH and LH and subsequent failure of the gonads to The homeobox (HOX) genes are well-studied master
produce sex hormones and gametes (GnRH deficiency regulators involved in body planning and patterning
can occur in both sexes, but it is approximately five in the developing embryo, resulting in major anatom-
times as common in men). The male patient may expe- ical defects in an organism when deleted. They are
rience a failure to complete, or even enter, puberty and characterized by the homeobox DNA-binding domain,
may also present with micropenis and cryptorchidism. and in many organisms they are grouped in clusters
This set of symptoms may arise from any of a number with other HOX genes in the genome. These genes
of genetic mutations. are relevant to male infertility, as their disruption
Most of the genes associated with Kallmann syn- can lead to improper development of the urogenital
drome affect the migration of GnRH neurons or the system. Homeobox A10 (Hoxa10) is associated with
processing and secretion of the hormone itself. Dur- male infertility in mice, as homozygous Hoxa10 muta-
ing foetal development, GnRH neurons migrate from tion results in impaired spermatogenesis secondary to
the olfactory bulb to the hypothalamus. Anosmin- bilateral cryptorchidism, in addition to defects of the
1 (ANOS1, aka KAL1) encodes a protein whose lumbar vertebrae (85).
217
Functional Failures Arising from Genetic remain to be determined. Nevertheless, more than 130
polypeptides have been localized to the axoneme, and
Mutations loss of function (LoF) of the genes that either express
Primary Ciliary Dyskinesia or regulate these polypeptides can contribute to ciliary
Primary ciliary dyskinesia (PCD) refers to a condi- dyskinesia and fertility [87].
tion characterized by defective ciliary function rang- Globozoospermia
ing from completely immotile cilia to abnormal move-
ment of cilia. Amongst the earliest discovered and Sperm fertilization potential requires proper forma-
most common forms of PCD is Kartagener syndrome tion and function of the acrosome. Defective sperm
(PCD/KS), which was originally recognized in 1975 head elongation and acrosome formation results in
in two asthenozoospermic brothers who possessed globozoospermia, which can portend devastating out-
ultrastructural changes of the axoneme with absence comes despite the use of IVF/ICSI. Koscinski et al. [93]
of dynein arms and abnormally arranged longitu- recently used aCGH to detect microdeletions of the
dinal columns of the fibrous sheath [86]. PCD/KS dpy-19-like 2 (DPY19L2) gene in up to 19% of globo-
has subsequently been associated with the triad of zoospermic human males who otherwise had normal
bronchiectasis, situs inversus and sinusitis. Autoso- sperm density [93]. Although mutations of DPY19L2
mal recessive mutations of the genes dynein axone- have been associated with poor conventional ICSI out-
mal intermediate chain 1 (DNAI1) and dynein axone- comes, application of assisted oocyte activation (AOA)
mal heavy chain 5 (DNAH5) have most commonly has improved pregnancy and live birth rates of cou-
been identified in PCD/KS [87]. Moore et al. [88] ples with DPY19L2 mutations in the male partner [94].
recently identified biallelic mutations of zinc finger Similarly to DPY19L2 but with more devastating ART
MYND-type containing the 10 (ZMYND10) gene in outcomes, mutations of the genes spermatogenesis-
a subset of PCD families with dual loss of inner and associated 16 (SPATA16), and protein interacting with
outer dynein arms, using whole-exome and Sanger C kinase 1 (PICK1) result in globozoospermic sperm,
sequencing. Although infertility was not described in but neither has been associated with a live birth.
these families, Drosophila with ZYMYD10 mutations
were infertile. Similarly, whole-exome and Sanger Single-Gene Mutations
sequencing identified autosomal recessive mutations Although 1,200 genes are associated with male fertil-
of the HYDIN axonemal central pair apparatus pro- ity, very few have both been identified among human
tein (HYDIN) gene among three consanguineous Ger- patients and validated in the murine model. Vali-
man siblings, including two brothers who have not dation in the murine model is challenged by vari-
fathered children [89]. Videomicroscopy of the ejacu- ability in molecular mechanisms of gene expression
late obtained from one of the index brothers showed and the absence of orthologous genes across species
markedly reduced sperm motility, with only 8% of [95]. A small number of genes, however, have been
sperm with progressive movement. validated in both organisms, including spermatoge-
Axonemal defects can have more ominous con- nesis and oogenesis-specific basic helix-loop-helix 1
sequences due to interactions of cilia/flagella in cell (SOHLH1) [96], synaptonemal complex protein 3
signal pathways, affecting embryologic development, (SYCP3) [97], nuclear receptor subfamily 5 group A
tissue morphogenesis and homeostasis [90]. This is member 1 (NR5A1) [98], heat shock transcription
particularly true for polycystin 1 (PKD1) mutations, factor 2 (HSF2) [99], protamine 1 (PRM1) [100],
which cause autosomal dominant polycystic kidney protamine 2 (PRM2) [100], testis expressed gene 11
disease (ADPKD) and sterility in men likely, through (TEX11) [101] and E2F transcription factor 1 (E2F1)
interactions with the TGF-␤/BMP signalling pathway [102].
[91]. Copy number variation (CNV) analysis and/or
WES performed on 20 families, who did not pos- SOHLH1
sess known PCD mutations, has recently uncovered SOHLH1 is a spermatogonium-specific transcription
11 clinically significant variations, including 4 CNVs factor and modulates the expression of KIT, which is
and 7 novel biallelic mutations [92]. The consequences a tyrosine kinase necessary for spermatogonial pro-
of these CNVs and gene mutations for male fertility liferation and differentiation into spermatocytes [96].
218
Viral transfection studies and Sohlh1 knockout mice trance despite heterozygosity in five separate men [99].
both showed reduced KIT levels. In a comparison of 96 As predicted by the murine model, these men pos-
Korean NOA men with 156 control males, Choi et al. sessed NOA and had maturation arrest at the sperma-
[96] detected three novel SNPs of SOHLH1 exclusively tocyte level.
in the NOA cohort, including one intronic variant and
two nonsynonymous extronic variants. PRM1 and PRM2
SYCP3 PRM1 and PRM2 code for nuclear proteins, called
protamines, which are found only in the chromatin
The synaptonemal complex is a protein structure
of mature spermatozoa. These highly basic proteins
that mediates meiotic homologous recombination.
are protonated at physiological pH and carry a pos-
SYCP3, a component of the synaptonemal complex,
itive ionic charge [107]. This neutralizes the nega-
is necessary to bind the chromosomal DNA in order
tive charge of DNA and allows tighter DNA packag-
for recombination to occur. As one would expect,
ing than is possible in any other cell type. In the ter-
homozygous mutations of SYCP3 were shown to result
minal stages of spermatogenesis, protamine binds to
in murine sterility, with arrest at prophase stage of
nuclear DNA to supercondense the gamete’s nuclear
meiosis, by Yuan et al. [103] in 2000 and subse-
volume approximately tenfold from its uncondensed
quently identified by Miyamoto et al. in 2003 in two
size [108]. This process, referred to as protamination,
human patients [97], who possessed a heterozygous
allows further chromatin maturation as the sperma-
single-nucleotide deletion resulting in a premature
tozoa pass through the epididymis and improves the
stop codon and a truncated, nonfunctional protein.
hydrodynamics of the sperm head [108]. Imken et
NR5A1 al. [100] identified a nonsynonymous SNP in PRM1
and a promoter sequence SNP of PRM2 in a cohort
NR5A1 encodes the transcriptional activator protein
of 125 infertile males screened for PRM gene fam-
steroidogenic factor 1 (SF1), which plays a key role in
ily mutations. Although these SNPs were validated
gonadal development and steroidogenesis. LoF muta-
in the murine model, a recent meta-analysis com-
tions of SF1 are a known endocrinological cause of
paring 7,350 infertile cases with 6,167 controls from
disorders of sexual differentiation characterized by
data culled from 13 published case-control studies val-
microphallus, hypospadias, bilateral anorchia and/or
idated the association of only one SNP (rs2301365)
primary adrenal insufficiency in 46XY boys [98]. In
with infertility and failed to associate the three novel
a mutational screen, the DNA sequences of 270 NOA
SNPs identified by Imken et al. with infertility [109].
and 218 sOS infertile males were compared by Ropke
et al. [98] with those of 237 NS males. Three heterozy-
gous missense mutations were identified unique to the TEX11
infertile cohort, with a comparable frequency between Similarly to SCPY3, TEX11 is involved with synap-
NOA (1.5%) and sOS (1.8%). tonemal complex formation in prophase I but also
functions in double-stranded DNA break repair [101].
HSF2 Moreover, Tex11 knockout mice have recapitulated
Heat shock proteins are transcription factors cyto- the infertile phenotype with AZO and meiotic arrest
protective against noxious stimuli (such as heat). The on testis biopsy. Yatsenko et al. [101] recently iden-
HSF2 gene is exclusively expressed in the human testis tified a 90-kb hemizygous loss of TEX11 on Xq13.1
and codes for the HSF2 protein [104]. The protein sub- in an aCGH screen of 15 unrelated NOA men. High-
sequently activates the HSPA2 gene, which encodes resolution microarray mapping of this area identified
a 70kDa HSP2 chaperone protein that prevents inap- a 91,042-bp segment deletion of the long arm of the
propriate protein aggregation and similarly recognizes X chromosome, resulting in a 79-amino acid deletion
non-native protein aggregation1 (HPSA2) [105]. Hsf2- of the SPO22 protein [101]. Sanger sequencing of the
knockout mice have reduced testis size and disorga- TEX11 coding regions was then performed on 289
nized synaptonemal complexes in majority of sperma- infertile males, which identified 7 (2.4%) individuals
tocytes [106]. Mou et al. performed Sanger sequenc- with TEX11 mutations. Testis biopsy of these individ-
ing on 766 idiopathic infertile males and identified five uals corroborated the murine model, with complete
missense mutations of HSF2 with phenotypic pene- meiotic arrest in five of seven individuals harbouring
219
the TEX11 mutation, while two individuals possessed spermatogenesis is dependent on a distinct wave of
mixed testicular atrophy. gene expression regulated by phase-specific miRNAs
that can either inhibit or promote translation [111,
E2F1 112]. As spermatogenesis transitions to spermiogen-
E2F transcription factor 1 (E2F1) is a transcription esis, spermatozoal RNA becomes less transcription-
factor associated with the p53 cancer pathway and ally active and is inactive in the mature spermato-
expressed in spermatogonial stem cells and sperma- zoon. Using RNA sequencing and in situ hybridiza-
tocytes. In addition to being associated with several tion (ISH), a complex population of RNA has been
malignancies, it has been identified as an infertility identified in the periphery of the nucleus, close to the
gene in mice, as both knockout and overexpression nuclear envelope and in the mid and principal pieces
models have spermatogenic defects. Genetic muta- of the tail [113–115]. It has been demonstrated that
tions and CNVs in human E2F1 have been associ- during spermatogenesis, RNA synthesis peaks dur-
ated with NOA. In a cohort of 110 NOA men, 4 ing the early pachytene stages of meiosis and at the
(3.6%) exhibited microdeletion and 4 (3.6%) exhib- round spermatid phase [116, 117]. Dysregulation of
ited microduplication of E2F1, while 3 (2.7%) exhib- miRNA has the potential to perturb spermatogene-
ited nonsynonymous SNPs in coding regions of E2F1 sis and has been linked to spermatogenetic failure
[102]. in patients with various spermatogenic impairments,
such as NOA [118, 119], asthenozoospermia [120] and
Epigenetic Regulation of Male Fertility OS (120).
Dysregulation of miR-449a has been implicated
Gene expression during spermatogenesis is a highly
in impaired spermatogenesis. Using Northern blot
regulated, complex process that enables the creation
analysis, Commazzetto et al. [121] demonstrated that
of mature, functional spermatozoa capable of trans-
both miR-449a and miR-34b/c have ubiquitous lev-
mitting genetic material across generations. Epige-
els of expression in postmitotic germ cells with
netics refers to the extragenomic regulation of gene
increased expression at the pachytene spermatocyte
expression through heritable factors. Epigenetics con-
stage. Although the miR-449 cluster of miRNAs have
trols cell-specific gene expression, allowing a single
high postmitotic germ cell expression levels, miR-449-
genomic sequence to give rise to a highly special-
null mice exhibit normal spermatogenesis with com-
ized, multicellular organism. The three best-studied
pensatory increased levels of miR-34b/c levels, imply-
mechanisms of epigenetic regulation include post-
ing functional redundancy [122].
transcriptional modulation with small, non-coding
The causal role of miR-34b/c in impaired sper-
RNAs (sncRNA), particularly microRNA (miRNA),
matogenesis and male factor infertility has been
and transcriptional modulation via DNA methylation
demonstrated [123–125]. Commazzetto et al. [121]
and modification of chromatin structure. These mech-
showed that miR-34b/c knockout mice not only were
anisms can be influenced by environmental exposure,
infertile when mated with wild type mice, but also
allowing cells to have a memory of exposure to stim-
had a 60-fold drop in the quantity of mature sperm
uli such as endocrine disruptors and dietary factors.
and a significant reduction in the quality of sperm
In mature spermatozoa, these epigenetic factors play
with poor motility secondary to separation of sperm
important roles in post-fertilization embryogenesis,
heads and tails. Compared with wild type (WT) mice,
such as programmed gene activation and induction of
miR-34b/c knockout mice have up-regulation of the
pluripotency in embryonic cells [110].
miR-34b/c target genes SH3KBP1 binding protein 1
(SHKBP1), forkhead box J2 (FOXJ2) and dystrobrevin
microRNA alpha (DTNA). Notably, FOXJ2 is a member of the
miRNAs are highly conserved, short (18–22 nucleo- Fork Head transcription factor family and regulates
tides long), single-stranded noncoding segments of expression of the gap junction protein Connexin-43
RNA that bind to the 3’ or 5’ untranslated regions of and the cell–cell contact protein E-Cadherin. In a
messenger RNA and interfere with translation of the separate study, its overexpression has been associated
tagged messenger RNA. Each miRNA is capable of reg- with male factor infertility [126]. This was seen in
ulating the post-transcriptional activity of several mes- a study by Martı́n-de-Lara et al. [126], who showed
senger RNAs (mRNAs). In this context, each phase of that male transgenic mice overexpressing FOXJ2 were
220
sterile, with no mature spermatozoa in their seminif- as primordial germ cells (PGCs), are largely unmethy-
erous tubules. Beyond spermatogenesis, sperm-borne lated and thus capable of expressing a wide range of
miR-34c plays a role in post-fertilization epigenetic genes that are silenced in more differentiated tissues
control in a mouse zygote. Inhibition of miR-34c in with methylation (134).
the zygote disrupted DNA synthesis and prevented the DNA methylation is particularly interesting in the
first cleavage of the zygote [127]. context of infertility, as both male and female germ
Herati et al. [128] recently identified 35 miRNA cells exhibit unique methylation patterns relative to
that were aberrantly expressed in teratozoospermic somatic cells. Although most epigenetic markers are
sperm. Of the differentially reduced miRNAs, miR- not sex-specific, some CpG sites are differentially
449a has the largest reduction in expression relative methylated by sex in germ cells. As a result, genes
to NS. Similarly, miR-34b-3p demonstrated reduced associated with these loci are expressed in a monoal-
expression in teratozoospermic sperm relative to NS lelic, parent-of-origin-specific manner, a phenomenon
controls. In contrast, the most overexpressed miRNA referred to as genomic imprinting [135]. This spe-
in this study was hsa-let-7b-5p. The hsa-let-7 family cific methylation pattern is introduced, and erased,
(let-a to let-f) represents the most abundant miRNA at two points in foetal development at which the
in the testis, constituting 11% of the total miRNA genome undergoes two waves of demethylation [136].
in the adult testis [129]. ISH has demonstrated high The first wave of demethylation occurs in the post-
levels of the hsa-let-7 family in premeiotic and mei- fertilization zygote, in which the methylation patterns
otic germ cells. This family of miRNA is thought to of the ovum and sperm are erased and a new somatic
silence the self-renewing program of spermatogonia pattern is gradually established as the embryo grows
through the suppression of Lin28, V-myc avian mye- [137]. The second wave of demethylation occurs in the
locytomatosis viral oncogene neuroblastoma derived PGCs as they migrate into the primordial gonad dur-
homologue (Mycn), Cyclin D1 (Ccnd1) and Collagen I ing subsequent embryologic development. The PGCs
␣ 2 (Col1a2) genes [130]. Given the abundance of hsa- will proceed through sex-specific differentiation and
let-7 and its putative role in spermatogenesis, Wu et al. enter either mitotic arrest in the male or meiotic
[131] investigated the role of hsa-let-7a as a stable and arrest in the female before the process of remethylation
reproducible seminal plasma biomarker for diagnos- begins. At this point, the oocytes and the spermatogo-
ing NOA. This study, however, found no statistically nia establish their separate, sexually distinct patterns
significant difference between the hsa-let-7a levels of of methylation [137]. In mammals only a few genes
OS patients and those of fertile controls. are paternally imprinted. They include the insulin-like
growth factor 2/H19 imprinted maternally expressed
(IGF2/H19, aka H19) locus, Ras protein specific gua-
DNA Methylation nine nucleotide releasing factor 1 (RASGRF1), mater-
DNA methylation is the modification of cytosine nally expressed 3 (MEG3 aka GTL2, and zinc finger
nucleotides with a methyl group at the carbon 5 DBF-type containing 2 (ZDBF2) [136].
position. In vertebrates, methylation predominantly There are several reported associations between
occurs in CpG (5’-cytosine-phosphate-guanine-3’) male-factor infertility and aberrant methylation of the
dinucleotides. DNA methylation is mediated by DNA paternally imprinted genes. Kobayashi et al. [138] have
methyltransferases (DNMTs), but CpG sites may demonstrated that aberrant methylation of H19 and
experience highly variable rates of methylation, as GTL2 is associated with OS, with greater percentages
the process depends on multiple factors, includ- of abnormal methylation associated with sOS. Mar-
ing methylation-determining regions (MDRs) in the ques et al. [139] similarly reported hypomethylation
genomic sequence [132]. CpG sites are frequently clus- of H19 as well as hypermethylation of mesoderm spe-
tered in what are referred to as CpG islands, which are cific transcript (MEST), a maternally imprinted gene,
located in or near the promoter regions of genes. These in their cohort of men with OS. Additionally, Minor
CpG islands inhibit transcription when methylated. et al. [140] reported in 2011 that men with NOA and
Consequently, hypermethylation is usually associated OA were also more likely than NS men to have H19
with silencing of gene expression, while hypomethyla- hypomethylation in sperm.
tion is associated with increased expression [133]. The Given the important role of methylation in
zygote and blastomeres of the early embryo, as well PGC development, it is not surprising that global
221
dysfunctions of methylation have been associated or less compact in others (euchromatin). Euchromatin
with impaired spermatogenesis. Broad, genome-wide is more easily accessible than heterochromatin and
hypermethylation has been reported in sperm DNA is thus more readily transcribed and expressed [135].
from OAT men [141]. Furthermore, methylenete- Methylation of lysine residues is generally associated
trahydrofolate reductase (MTHFR) hypermethylation with compaction of DNA and gene silencing, although
has been specifically associated with NOA. MTHFR is some methylation sites, such as H3K4 (lysine 4 on
an enzyme involved in processing methyl groups for histone 3), are associated with increased gene expres-
various biological functions, including establishment sion [135]. Acetylation is generally associated with
of new DNA methylation[142]. There is also some increased gene expression, as it relaxes the affinity of
evidence in the literature that oxidative stress due to DNA for histones, creating more accessibility for poly-
reactive oxygen species (ROS) may globally impair merases. Ubiquitylation can either activate or inacti-
DNA methylation, leading to spermatogenic defects vate expression, and SUMOylation is generally associ-
[143]. ated with decreased gene expression and blocking of
Aberrant methylation is associated with several acetylation [147].
spermatogenesis-specific genes in males with infertil- The mature spermatozoon is unique in that the
ity. DAZL is a germ line regulator that encodes an major structural protein associated with DNA is the
RNA-binding protein found in spermatozoa, where it protamine, not the histone. As described above, tight
is involved in the stabilization and storage of sperm DNA compaction is facilitated by protamines in the
RNAs, as well as translational initiation. DAZL hyper- sperm nucleus. There is some evidence that in addition
methylation was reported in sperm from OAT men to compressing the DNA, this compaction also confers
in 2010 [144]. cAMP-responsive element modulator protection against DNA fragmentation [136].
(CREM) is a DNA-binding protein which is known to The modification of histones and protamination of
be involved in signal transduction during spermatoge- DNA during spermatogenesis occur in several phases.
nesis, and hypermethylation of its promoter has been Several markers of histone methylation have been
similarly reported in men with OS [145]. Discoidin specifically associated with spermatogenesis. H3K4,
domain receptor 1 (DDR1) is a tyrosine kinase whose H3K9 and H3K27 are all essential to spermatogene-
ligands include collagen and E-cadherin, which are sis in mouse models [136]. H3K4 methylation is high
associated with spermatogenesis and germ cell pro- in spermatogonial stem cells and then decreases dur-
liferation, respectively. DDR1 is hypermethylated in a ing meiosis. In contrast, H3K9 and H3K27 methy-
subset of men with NOA [146]. These findings suggest lation increase during spermatogenesis, and subse-
that a wide array of genes involved in spermatogenesis quently H3K9me but not H3K27me must be removed
may be affected by aberrant methylation. in order to initiate spermiogenesis [148]. Similarly,
existing acetylation is erased prior to the initiation of
meiosis, which is followed by partial reacetylation dur-
Histone Modification and Protamination ing the spermatocyte stage [136]. This is necessary to
Epigenetic regulation also includes modification of initiate the multistep process of protamination dur-
chromatin structure. Chromatin compaction affects ing spermiogenesis. In this process, histones are first
the accessibility of a particular genomic locus to replaced by four transition proteins, which are sub-
RNA polymerases, and thus modification of chro- sequently replaced by protamines [135]. This process
matin compaction may result in alterations to gene never results in complete protamination, however. In
expression. sperm from NS men, approximately 10–15% of chro-
Histone modification is the primary means by matin structural proteins are still histones, and most of
which this form of epigenetic regulation is accom- those histones are associated with loci known to play
plished. In most cells, DNA is organized around his- important roles in embryonic development [149]. This
tone proteins. The N-terminal ends of histones are rich suggests that the incomplete protamination of sperm
in lysine residues, which can be covalently modified DNA may be a functional modulation of gene expres-
in a number of ways, including methylation, acety- sion in the developing offspring.
lation, ubiquitylation and SUMOylation [135]. As a Though little is known about the specific interac-
result of these covalent modifications, chromatin may tions between DNA and protamines, it is well estab-
be more compact in certain areas (heterochromatin) lished that a relatively precise ratio of PRM1 to PRM2
222
(P1/P2) is required for properly functioning sperm. 4. Hwang K, Yatsenko AN, Jorgez CJ, Mukherjee S,
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7. Elghezal H, Hidar S, Braham R, Denguezli W, Ajina
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229
Chapter
The Sperm Epigenome
15 Timothy G. Jenkins and Douglas T. Carrell
Introduction ciation with infertility, but also in consideration of

offspring health and disease susceptibility. This chap-
Classically, epigenetic marks have been defined by two
ter will focus on the sperm epigenome (DNA methy-
major requirements. First, that a chemical modifica-
lation, noncoding RNAs and nuclear protein con-
tion is capable of perturbing gene expression with-
tent) and the evidence supporting the importance of
out altering the coding sequence and, second, that
these unique epigenetic marks in transgenerational
these perturbations are heritable. Today, we often think
inheritance.
about epigenetics in a slightly different fashion, where
the only consideration is that the mark in question is
capable of gene expression alteration in the absence of Sperm Epigenetics
clear genetic mutations [1]. In effect, this change in Sperm contain one of the most distinctive epigenetic
definition is a result of important studies investigat- landscapes known in biology [3]. These unique epi-
ing epigenetic alterations to elucidate the mechanisms genetic marks work in concert and are necessary to
of disease progression in somatic cells that, by their facilitate the specialized function of the mature sperm
very nature, are incapable of meeting the latter classi- in delivering the paternal DNA blueprint safely to the
cal requirement, namely that altered epigenetic marks oocyte. DNA methylation patterns, histone tail mod-
are heritable. However, in the germ cell, the classical ifications and noncoding RNAs all play a role in this
epigenetic definition remains relevant. process. Specific patterns of these important epige-
Mammalian sperm have among the most highly netic marks contribute to gene activation and silenc-
specialized epigenetic landscapes yet known in biol- ing (Figure 15.1). In addition to these traditional epi-
ogy, contributing to the cell’s extremely unique func- genetic marks, there are other nuclear proteins found
tion [2, 3]. In addition, sperm are highly susceptible only in sperm that play an important role in germ cell
to epigenetic change, likely due to the high degree quiescence and extreme chromatin compaction. Each
of adult germ line stem cell proliferation that occurs of these individual epigenetic marks is important in
over time and/or as a result of various exposures [4]. many ways to the mature sperm and its unique func-
The key difference between the paternal germ line tion. Perturbation of these marks has the capacity to
and somatic tissues is that these nongenetic alterations affect the phenotype (Figure 15.2).
have the potential to affect fertilization, embryogenesis
and even offspring health.
It should be noted that there are multiple steps in DNA Methylation
epigenetic reprograming that occur following fertiliza- DNA methylation is capable of transcription inhibi-
tion and in the primordial germ cells, which make the tion through multiple mechanisms and is thus con-
heritability of altered epigenetic marks over multiple sidered to be among the most prominent epigenetic
generations difficult to explain. Despite this, there is marks. This chemical modification occurs on the 5 car-
accumulating evidence to suggest that such patterns bons of cytosine residues and is most frequently seen
of inheritance do occur and should be accounted for in cytosine phosphate guanine dinucleotides (CpGs),
in the study of germ line disease, not only in asso- though non-CG methylation has been reported [5].
University Press.
230
Chapter 15: The Sperm Epigenome
Activation Marks Activation Histone Tail Modifications

GENETICS EPIGENETICS
ub
ac
Target DNA
K4me2/3 Gene Methylation
SNP H
Mutation CH
H
Hypomethylated Promoter
Histone
ac Modification
CNV
Inactivation Marks Inactivation Histone Tail Modifications
ub
K9me ncRNA
Alteration
Target
K27me3 Gene
5-mC
PHENOTYPE / DISEASE
Hypermethylated Promoter
Figure 15.2 Complex diseases can be derived from multiple

Totipotent Marks Bivalent Histone Tail Modifications sources and are often multifactorial. This figure describes both the
epigenetic and genetic aspect that can lead to disease or altered
K4me3
phenotypes and demonstrates the need to be cognizant of all
potential causes of phenotypic alterations.
Target
K27me3 Gene
a high degree of enrichment of methylation at CpG
islands, thought to contribute to or even drive the
silencing events on the X chromosome [6]. Similar pat-
terns can be found at imprinted loci and during cellu-
5-hmc
lar senescence [7, 8]. While much of this work has been
5-hmc Enriched Promoter performed in somatic cells, the role of CpG methyla-
Figure 15.1 Epigenetic modifications and their influence on gene tion and its proposed mechanism of action appear to
activation/silencing. Additionally, the profile of a poised gene is hold true in the paternal germ line as well.
described.
Evidence suggests that mature sperm harbour
poised genomic regions that are marked, in part,
Functionally, increased methylation (hypermethyla- by hypomethylated promoters. These regions consist
tion) at gene promoters is believed to be a potent largely of genes important in embryonic development.
inhibitor of transcriptional machinery (though the Thus, upon fertilization, the mature sperm can deliver
exact details of this mechanism are poorly understood) epigenetic marks that allow swift activation of genes
and is thus considered to be a strong inhibitor of gene important in developmental processes [2]. Despite the
transcription. Conversely, the absence of DNA methy- fact that the mature sperm have no transcriptional
lation at promoters is indicative of an activated gene. activity, it appears that these cells can prepare genes for
The role of promoter DNA methylation enrich- activation in an epigenetic fashion similar to what has
ment is supported by the methylation patterns of CpG been shown in somatic cells.
dense regions (termed CpG islands), commonly asso- Multiple important genes play a role in DNA
ciated with gene promoters, on the X chromosome methylation and demethylation. Foremost among
during X chromosome inactivation. In brief, there is these are genes in the DNA methyltransferase
231
(DNMT) family, which supports methylation estab- lation signatures and dramatically decreased sper-
lishment and maintenance [9]. DNMT3a and matogenesis and even retarded gestational growth and
DNMT3b both contain catalytic domains and are embryo lethality [16–20]. General sperm defects and
responsible for de novo methylation patterns [10]. various forms of infertility have also been associated
DNMT3l is also essential for the establishment with perturbed methylation signatures at DAZL and
of methylation marks by working in concert with MTHFR promoters [21–23]. Aberrant DNA methyla-
DNMT3a/b but plays a unique role by ensuring tion at the CREM gene promoter has also been seen in
proper placement of de novo marks [10]. DNMT1 patients with male factor infertility [24]. Time to preg-
plays a key role in the maintenance of methylation nancy also appears to be associated with methylation
marks by targeting hemimethylated DNA during patterns at specific genomic loci [25].
DNA replication/cell division [11, 12]. Importantly, it Among IVF patients, there is also evidence to sug-
is this maintenance activity that is the molecular basis gest that global levels of sperm DNA methylation may
for the heritability of methylation marks [13]. be predictive of poor pregnancy outcomes, though
Multiple recent studies have analyzed the nature the utility of global DNA methylation in screening a
of DNA methylation across the entire population of patient for risk can be low due to assay variability [26].
sperm. Specifically, these studies have assessed the Even the likelihood that a couple may need to utilize
variability of methylation marks within the same ejac- IVF to achieve a pregnancy may be predictable via
ulate. In brief, it appears that the sperm DNA methy- sperm DNA methylation analysis. Specifically, a recent
lation patterns between different fractions of individ- study has shown that algorithms considering all of the
ual ejaculates are largely identical. Krausz et al. com- CpGs tiled on Illumina’s 450k methylation array col-
pared global methylation and a few targeted regions lectively may be more predictive of an infertile couple’s
of methylation between two fractions of sperm sep- need to utilize advanced reproductive therapies than
arated via a swim-up experiment. This study found a semen analysis alone [27]. The accumulating data
no significant difference in average methylation sig- strongly suggest a link between sperm DNA methy-
natures, either globally or at the selected regions of lation patterns and male factor infertility in general,
interest, between the sperm that are in the swim-up though the true nature of this association is unclear.
fraction versus those in the lower fraction [14]. Our The associations identified in these studies, as well
laboratory performed a similar experiment utilizing as many more that have established similar findings,
density gradient centrifugation to isolate two distinct may be the result of a direct causative relationship
sperm populations within a single ejaculate (90% and where methylation signatures impact gene expression
35% fractions). Our study correlated with the limited either in the developing sperm or in the embryo,
regions studied by Krausz et al.; however, our study ultimately leading to the phenotype identified. Con-
identified a number of moderately significant regions versely, these signatures may be reflective of a more
of the genome with subtly altered DNA methylation global issue in the sperm that is also causative of
signatures in regions which Krausz et al. did not study anomalies that actually drive the alterations in fertil-
[15]. Interestingly, these subtle methylation changes ity status. In some ways, the latter explanation may be
appeared to be more associated with increases in ran- more logical, as the alterations seen in sperm DNA
dom variability rather than programmatic changes. methylation are typically very subtle. However, the
This was also suggested by the finding of increased areas where these alterations occur often make a great
coefficient of variation in the low-quality fraction of degree of biological sense (for example, altered methy-
sperm analyzed when compared with the high-quality lation patterns at spermatogenesis genes in patients
fraction [15]. Taken together these findings suggest with poor sperm parameters [28]) and thus could very
that the average DNA methylation patterns in sperm clearly be directly causing the phenotypes identified.
from a single ejaculate are tightly conserved but may The uncertainty in this field is a result of the major
have slight increases in variability in the fraction of difficulty associated with DNA methylation studies in
lower-quality sperm. sperm. To detect the real impact of any given methyla-
From the available data, it appears that sperm DNA tion signature, it must be coupled with an alteration
methylation may play a role in fertility. Most notable to transcription at an associated gene. However, in
among these studies are knockout experiments of the the quiescent sperm, such transcriptional alterations
DNMT family that resulted in globally altered methy- cannot be identified. In effect, alterations seen in the
232
mature sperm may reflect transcriptional alterations in a nuclear structure that is approximately 20 times
the adult germline stem cells or the spermatocyte or denser than nucleosome-bound chromatin [3, 30].
spermatid precursors, or these alterations may cause Such a tightly bound nucleus facilitates quiescence and
transcriptional deregulation in the early embryo or effectively protects the paternal DNA blueprint from
potentially even the offspring. Very little work has damage in a cell that, at maturity, lacks repair mecha-
been performed on this specific issue, and it must be nisms [31]. This compaction is the result of the step-
performed to help us understand the true impact of wise removal of histones and replacement with tran-
altered sperm DNA methylation signatures. sition proteins and subsequently protamine proteins.
While DNA methylation in sperm is difficult to Importantly, this replacement occurs with two specific
study, it does have some very intriguing attributes that variants in the protamine family, P1 and P2, which are
make it an ideal target in many ways for important expressed in approximately a 1:1 ratio [32–35]. This
work in the field of infertility and beyond. Most of very unusual chromatin structure helps to facilitate the
the epigenetic landscape seen in the mature sperm is highly specialized sperm function.
far different from what is found in the adult germline Histone variants in general are potent regulators
stem cell. For example, there are massive rearrange- of gene activation or silencing. In sperm there are
ments of sperm nuclear proteins that occur during a number of influential forms of histone tail chemi-
spermiogenesis alone and, when the dynamics of the cal modifications including acetylation, methylation,
earlier stages of spermatogenesis is considered, RNA ubiquitination and phosphorylation. These modifica-
profiles are strikingly different, as these are helping to tions typically occur at lysine (K) or Serine (S) residues.
drive the process of meiosis and the commitment to In general, these variants provide some degree of
mature sperm development [29]. In consideration of regulatory control over nearby genes. Gene activa-
these data, the analysis of the mature sperm epigenome tion can be driven, independently or in concert with
is a poor reflection of events in sperm development. other marks, by Histone3K4 (H3K4) methylation, H3
While this is not problematic in all studies, many of acetylation, H4 acetylation, and H2B ubiquitination
the correlative analyses performed are investigating [36–39]. Conversely, gene silencing is promoted by
sperm function, and the majority of important events the absence of acetylation at H3 and H4, as well
contributing to development of sperm function occur as H3K9 methylation, H3K27 methylation and H2A
well before sperm maturation. Thus, we are not able ubiquitination [38, 39] (Figure 15.1). These marks
to identify the real alterations, only remnants of these are catalyzed by various enzymes, including histone
changes. However, in the case of sperm DNA methy- methyltransferases/demethylases and histone acetyl-
lation, it was recently identified that the vast major- transferase/deactylases [36, 37].
ity of sperm DNA methylation patterns remained One of the most intriguing aspects of the sperm
unchanged throughout the process of spermatogen- epigenome is the high degree of specialization, not
esis and even between the committed and prolifer- because of what this adds to the sperm, but what it
ative adult germline stem cells, effectively indicating potentially removes. It is well established that histones
that sperm DNA methylation marks are established and their modifications play a very important role in
prior to spermatogenesis and are maintained through- gene regulation and have the capacity to either turn on
out that process [29]. The resultant DNA methyla- or suppress gene expression [36–39]. Further, it is also
tion signature may actually provide some insight into known that histones, including testes-specific histone
gene activation in the stem cells and can also offer variants, play a crucial role in spermatogenesis [38,
more insight into potential etiologies of some forms of 40–42]. However, due to the widespread replacement
sperm functional abnormalities. Taken together, while of histones with protamine proteins, large portions
difficult to study and interpret, these marks offer a of these potentially informative and regulatory marks
unique opportunity for study that should be taken are simply removed from the genome to facilitate the
advantage of. dramatic condensation described earlier. While this
generates a mature sperm cell that is well suited to per-
form its key function of safely delivering the pater-
Nuclear Proteins nal genome to the oocyte, it may subsequently limit
The complex and dynamic chromatin remodelling in the male gamete’s potential regulatory impact on the
human spermatozoa during spermiogenesis results in embryo and beyond.
233
Importantly, studies have shown that the histone- on fertility status. Mice exposed to trichostatin-A had
to-protamine transition is incomplete, and thus some dose-dependent decreases in spermatogenesis with
histones (approximately 5–15% of the chromatin) associated alterations in histone acetylation [41, 51].
are retained [43, 44]. This is particularly interest- These and many other studies demonstrate the regu-
ing because nucleosome retention is enriched at sites latory role of histone modifications in the developing
known to be important in development and in partic- sperm.
ular in the early embryo [2, 45]. Interestingly, these Our knowledge is constantly growing in regard to
retention findings also identified histone modifica- the impact of histone modifications on the process of
tion patterns that resembled the bivalency at promot- spermatogenesis. A recent study from Hammoud et al.
ers seen in embryonic stem cells, believed to be a [29] has actually traced these marks throughout sper-
distinctive mark of totipotency. Specifically, repres- matogenesis. In general, histone modifications are rel-
sive H3K27 methylation modifications and the acti- atively consistent from the adult germline stem cell
vating mark H3K4 methylation co-localize at the same stage to the beginning of spermiogenesis. However, a
developmental promoters in mature sperm. This find- very unusual and intriguing process occurs in the com-
ing suggests that these marks contribute to a poised mitment of adult germline stem cells when they tran-
genomic state at these developmental genes and that sition from the proliferative state (the hallmark of pro-
these marks may actively contribute in the develop- liferative cells is the expression of THY1) to becoming
ment of the embryonic stem cell epigenetic program. committed to gametogenesis (identified by the expres-
Clearly these data suggest that while many of the reg- sion of KIT). It was shown that during the transition
ulatory histone marks are removed, the portion that from THY1+ to KIT+ the repressive H3K27me3 is
remains have a potential to impact events beyond that removed from the promoters of both aldehyde dehy-
associated with mature sperm function alone. drogenase (Aldh2) and Stimulated by Retinoic Acid 8
Data suggesting that nuclear protein composi- (Stra8), and thus the increased transcription of these
tion is important to sperm function, including fer- genes is facilitated. It is also known that both Stra8
tilization and early embryo development, comport and Aldh2 are important in the retinoic acid pathway,
well with studies that have assessed the negative which is essential to drive commitment to meiosis [29].
impact of perturbed sperm nuclear protein compo- This transition highlights the functionality of histone
sition on fertility. Hammoud et al. have described tail modifications, particularly in the dynamic process
altered histone retention patterns in infertile men of spermatogenesis. While we do know a great deal
[46]. Loss-of-function mutations at JmjC-domain- about the role of histone modifications in the devel-
containing-histone demethylase 2A (JHDM2A) have oping sperm, it is still unclear how these marks con-
shown altered histone-to-protamine transition during tribute to the embryo directly. The data regarding the
the process of spermiogenesis [42]. This is particularly localization of the marks are intriguing, but require
of interest in the discussion of the role of histone tail further study to elucidate just how essential sperm-
modifications in spermatogenesis, as this enzyme has derived histone tail modifications actually are.
known H3K9 demethylase activity.
A recent study utilizing a knockout model of
Ash1-like (ASH1l), a known histone methyltrans- RNA
ferase, demonstrated that ASH1l is required for nor- RNA plays many important roles in all cell types,
mal hox gene expression in the developing sperm and mammalian sperm are no exception. However,
[47]. As might be expected, ASH1l -/- mutants that likely due to the quiescent nature of the mature male
survived suffered from multiple ailments, including gamete, levels of this important biomolecule (partic-
infertility. Other recent studies have shown that poly- ularly mRNA) are very low. This has driven some
morphisms in members of the H2B family are likely to consider the RNA contribution from sperm to be
associated with azoospermic patients [48]. Further limited in its ability to contribute important func-
studies have suggested that varying degrees of infertil- tional processes in the mature sperm and in the early
ity and even sterility can be associated with alterations embryo. Such a conclusion is logically reached con-
in histone methylation alone [49, 50]. Other direct sidering that the RNA content in sperm may very
studies using the deacetylase inhibitor trichostatin-A well be residual in nature and not directly designed to
have demonstrated the effect of histone acetylation contribute to downstream processes (fertilization and
234
embryogenesis). However, a growing body of evidence interest among these transcripts is mir-34C. Interest-
suggests otherwise. These data suggest that this limited ingly, it appears that the enrichment of mir-34C in
view of sperm RNAs is incorrect and that the RNA load sperm is not merely representative of a remnant of
in the mature sperm may not only contribute to but highly expressed transcripts earlier in spermatogene-
also be essential for proper embryogenesis. sis, but that mir-34C also likely plays a role in early
As was described earlier, the nuclear reprogram- embryogenesis. In fact, a recent study showed that this
ming events that occur during the process of sper- transcript was essential for the first cell division in the
matogenesis are dynamic and remarkable in terms embryo [56], though these data are somewhat con-
of scope. Among the most highly altered epigenetic troversial [57]. Despite some conflicting data, there
marks during this process is the nuclear protein con- is strong accumulating evidence that this transcript
tent, with massive chromatin remodelling from a (mir34C) in particular is associated with fertility in
nucleosome-bound DNA structure to a largely pro- general in multiple mammalian species and even with
taminated landscape [3, 30]. While this process results outcomes of in vitro fertilization (IVF) and intracyto-
in a drastically altered chromatin structure, not all his- plasmic sperm injection (ICSI), suggesting that it likely
tones are removed in the process and it appears that, to plays an important role in the embryo [58–61]. Fur-
a large extent, the retention of histones through this ther study suggests that mir-34C and four other tran-
process is programmatic [2, 45]. Interestingly, avail- scripts have such a high degree of association with
able data suggest that RNAs in the developing sperm fertility that this panel of transcripts has been sug-
may play a role in this process [52, 53]. It has been gested to be ideal for diagnostic screening purposes
suggested that RNA transcripts may be capable of [62]. These are not the only transcripts that may have
inhibiting the protamination process directly, effec- potential diagnostic value. A recent study has identi-
tively maintaining histone-bound chromatin region- fied a broader cohort of RNAs (‘RNA elements’) that,
ally [54]. In fact, RNA transcripts co-localize with in concert, may be very informative to clinicians in
regions where histones are retained, and this occurs the diagnosis and guided treatment of unexplained
near the nuclear envelope in mature sperm, as has infertility [63]. This study demonstrated that patients
been shown with insulinlike growth factor 2 (IGF2) who possessed normal levels of all sperm RNA ele-
[44, 54]. ments defined by the author (648 in total) had sim-
Further, while the chromatin transitions are often ilar pregnancy rates with either timed intercourse or
discussed and studied in the context of sperm devel- IUI when compared with IVF (73.3% and 75%, respec-
opment, a similarly complex and dynamic process tively). These rates declined sharply (27%) in couples
can be seen in the transition between spermatogenic attempting timed intercourse or IUI where at least one
stages and transitional RNA profiles [29]. Multiple of these sperm RNA elements was absent, though IVF
RNA species including piRNAs, mRNAs, long non- results remained similar. The utilization of this infor-
coding RNAs (lncRNA) and even not yet annotated mation for diagnostic testing has a great potential to
RNA species were shown to fluctuate greatly between provide results that can inform clinical decision mak-
different stages of spermatogenesis [29]. This suggests ing and begin to realize the goals of precision medicine
that the role of RNA in the developing sperm is quite in the context of male factor infertility. This is an area
significant and that the dynamic process of spermato- of research that clearly warrants a great degree of fur-
genesis is driven, at least in part, by changes to RNA ther scientific exploration to determine its true clinical
transcript content at each stage of the development of utility.
the male gamete. It is clear from accumulating data that sperm RNAs
As described above, recent studies have directly play a large role in the generation of mature sperm, as
demonstrated the importance of mammalian sperm well as in the process of fertilization and embryoge-
RNA in the development of mature sperm. It is also nesis. Moving forward, it will be important to assess
important to note the growing data that support the these marks for their utility in clinical diagnostic test-
idea that sperm RNA is important in the process of ing, which may provide data upon which physicians
embryogenesis. One family of transcripts that is highly can offer patients insight with a high degree of pre-
expressed in the sperm is microRNA 34 (mir-34), dictive power (or at least a well-defined degree of pre-
which is found at higher levels in the mature sperm dictive power) in an effort to guide the process of
than any other set of transcripts [55]. Of particular care.
235
Difficulty in Studying Sperm particularly true in the study of sperm epigenomics,

as their patterns are so disparate from other cell types
Epigenetics and Potential Downfalls that any contamination can be difficult to overcome in
In many ways, mammalian sperm provide one of downstream analyses. In addition, the epigenetic alter-
the most exceptional and promising opportunities for ations found in sperm can be very difficult to interpret
study, due largely to the fact that these cells are eas- for one main reason, that the function of mature sperm
ily attained and have a number of phenotypic mea- is typically unaffected by these alterations unless the
sures that can be quickly and thoroughly assessed. alteration in question results in important perturba-
However, sperm offer very unique challenges as well. tions in development.
These challenges are particularly evident in the study Because the function of the mature sperm does
of sperm epigenetics. It is well established that the not rely on active transcription and gene regulation
sperm epigenome is far different than that of any other and because epigenetic changes are meaningful only
somatic cell in the body. This includes drastic differ- if they affect phenotype through transcriptional alter-
ences in nuclear protein content, DNA methylation ation or otherwise, hypotheses about the importance
and RNA expression patterns [3, 29, 64]. This becomes of a particular mark can only be tested through obser-
particularly problematic considering that typical ejac- vation and not with direct approaches. As a result, the
ulate is far from homogenous and often contains mul- majority of papers reporting alterations to the sperm
tiple cell types, including white blood cells, which are epigenome must make an effort to describe the poten-
highly differentially methylated at imprinted genes and tial impact of alterations, but are typically unable to
other loci and have distinct RNA expression patterns. test these. There are, however, some areas that will
You can then imagine that any degree of consistent allow direct testing, such as the impact of sperm-
somatic cell contamination in either a study group or a derived RNAs on embryonic development. With the
test group could easily sway results of a study of sperm use of conditional knockouts or siRNA degradation,
epigenetic patterns. This is further compounded by specific transcripts believed to be important in the pro-
the fact that, for DNA methylation analysis, the sperm cess of embryogenesis can be assessed directly as a con-
are haploid cells and thus have one-half the DNA of sequence of their removal. Importantly, intact tran-
diploid somatic cells contained in the ejaculate. Thus scripts may also be injected to preserve the process,
a single white blood cell is twice as influential in DNA thus offering direct evidence of the role of sperm RNAs
methylation analyses as a single sperm cell. in early embryogenesis. In the case of DNA methy-
The scenario is even more difficult in the case of lation alterations, it is not unreasonable to assume
RNAs, where the mature sperm are quiescent and do that gene editing techniques will develop to allow
not contain large amounts of RNA content, whereas the direct testing of methylation signatures and their
the somatic cells contain large amounts of transcripts, downstream impact on embryogenesis as well.
thus making even a small number of contaminat- It is clear that a great deal of research is still needed
ing white blood cells problematic. It is essential that to help us better understand the actual impact of sperm
robust somatic cell lysis steps be employed to ensure epigenetic alterations. While we have learned much
the absence of potentially contaminating cells. Utiliz- about these alterations, what causes them and altered
ing techniques to purify the cells before the somatic associated phenotypes, we have little direct evidence
cell lysis steps, such as swim-up protocols or density to suggest that these marks are directly causative. This
gradient centrifugation, is also advisable. While there will be required as we continue to learn of the impor-
may be some selection of sperm subpopulations when tance of these marks in the mature sperm.
these protocols are utilized, the available data suggest
that, at least in methylation studies, the differences
between these populations exist more in variability Conclusions and Future Directions
than in clear programmatic alterations to the methy- A great number of data have accumulated in regard
lome that could easily affect the findings of a study. In to the impact of epigenetic alterations associated with
fact, the decreased variability may ultimately lead to the complex disease of infertility. Still, we are left
more clear data sets for analysis. with no real conclusions in terms of what impact
It is clearly important in the study of epigenetics these perturbations actually have on the developing
to isolate your cell population of interest, but this is sperm or in the process of embryogenesis. It is clear
236
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virtually every aspect of human male infertility, but regulation of transcription. Curr Opin Genet Dev 1994;
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Future targeted work designed to directly assess methyltransferases Dnmt3a and Dnmt3b are
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development. Cell 1999; 99: 247–57.
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Chapter
Environmental Factors and Male Fertility
16 Tina Kold Jensen, Hanne Frederiksen, Katrine Bay and Niels E. Skakkebaek
Introduction life stage from conception to adulthood and old age.

However, as delineated below, the reproductive system
Environmental effects on male fertility will be defined
seems particularly vulnerable during foetal develop-
in their broadest sense in this chapter as the influ-
ment, and exposures occurring here may have lifelong
ence of lifestyle, including diet, exercise, psychological
consequences. For example, phthalates administered
stress, cosmetics, recreational drugs, stress, indoor and
perinatally to rats can cause dysgenesis of the testis and
outdoor environment and exposure to endocrine dis-
result in poor semen quality in adulthood, even when
rupting chemicals (EDCs). We will mainly review liter-
the exposure ceases after the neonatal period [5]. This
ature studying the effects on semen quality as a marker
substantial lag time between exposure and outcome
of fertility but will also report on other reproductive
and the complex exposure patterns and lifestyle sce-
health outcomes such as testicular cancer.
narios described above are factors that complicate the
A clear proof of principle that environment plays
research linkage between environmental factors and
a role in male fertility comes from studies of work-
reproductive outcomes.
ers who were exposed to the pesticide dibromochloro-
propane (DBCP) and consequently became infertile
due to azoospermia or oligozoospermia [1, 2]. Envi- Testicular Dysgenesis Syndrome and
ronmental effects on male reproductive function are,
however, often subtle and difficult to evaluate. An the Role of the Environment
individual’s environment changes all the time, not In 1972, precursor cells of testicular germ cell carci-
only from day to day and season to season, but also noma (TGCC) were identified in two infertile men,
from one life stage to another. In addition, indus- who later developed TGCC [6]. After that, numerous
trialization has introduced numerous new products research projects confirmed that the link between tes-
and food items and changed our health behaviour. ticular cancer and infertility was not a coincidence [7–
Thus the lives of people today are fundamentally dif- 9]. Therefore, physicians who manage male infertil-
ferent from the lives of previous generations. We are ity must be aware that they are dealing with a group
all exposed to unique combinations of chemical mix- of patients with increased risk of TGCC. The testic-
tures, although recent analyses of human breast milk ular cancer precursor cells, called germ cell neopla-
have shown country-specific chemical signatures [3]. sia in situ (GCNIS) are very similar to gonocytes in
In other words, although we modify our exposure that they express a number of embryonal pluripo-
when we select a certain diet, smoke cigarettes, or use tency markers [10–12]. These and other findings sug-
personal care products, we cannot escape a general gested that germ cell cancer is of foetal origin and
exposure to chemicals. Examples of such omnipresent supported the hypothesis of the testicular dysgenesis
bulk chemicals are phthalates and bisphenol A (BPA), syndrome (TDS) [12–13]. TDS links testicular cancer
which are commonly used in everyday products of all with poor spermatogenesis, infertility, cryptorchidism
modern societies [4]. and hypospadias (Figure 16.1). All these symptoms
Importantly, environmental exposure and result- share the same risk factors and are risk factors for each
ing effects on reproductive health can occur at any other, essentially because they are all manifestations of
University Press.
240
Chapter 16: Environmental Factors and Male Fertility
Fetal germ cells Fetal Leydig cells
Environmental Lifestyle
exposure factors
Sertoli
cells
Genetic defects and Epigenetic
polymorphisms factors
Testicular dysgenesis
Decreased Leydig cell function Disturbed Sertoli cell function
Decreased INSL3 Decreased Impaired germ cell

production testosterone production differentiation
Hypospadias Short AGD
Decreased Impaired GCNIS

Cryptorchidism
testosterone production spermatogenesis Testicular cancer
Reduced male fecundity influencing pregnancy rates
Figure 16.1 The testicular dysgenesis syndrome (TDS). Normally, not all symptoms are present in individual patients. In its mildest form only
decreased spermatogenesis and low fecundity may be found. AGD: anogenital distance; INSL3: insulin-like peptide 3, a Leydig cell hormone
important for testicular descent. Reproduced from Skakkebæk et al. [12] with permission.
a disrupted testicular development. Thus, infertile men one or more symptoms of TDS, for example, poor
have an increased risk of developing testicular can- semen quality in a man with a history of cryp-
cer and vice versa. Also, men with a history of cryptorchidism, GCNIS cells in an infertile man, or even
torchidism have an increased risk of being infertile, just infertility due to poor semen quality in a man
and the same is the case for men born with hypospa- who does not have any other symptoms (Figure 16.1)
dias [13]. [12].
Various genetic disorders that result in poor devel- As a rodent counterpart to the TDS syndrome,
opment of the testicular Sertoli cells (which support experimental studies have described a so-called phtha-
the development of sperm cells) and/or the Leydig late syndrome, where rats exposed to dibutyl phtha-
cells (which produce testosterone) can cause all the late in utero present with cryptorchidism, hypospa-
symptoms of TDS, sometimes in one single indi- dias, testis abnormalities and infertility in adult life
vidual. In the most severe genetic cases, the male [15]. In addition to being a useful tool for investigat-
gonad does not develop at all. However, such genetic ing the pathogenesis of TDS, this rodent model also
cases are quite rare. Much more common is the pres- demonstrates how exposure in utero can indeed lead
ence of one or a few TDS symptoms in one individ- to TDS.
ual, notably in the absence of any identified genetic A factor complicating the distinction between
causes [14]. genetic and environmental factors is the finding of epi-
We have hypothesized that environmental factors genetic alterations induced by environmental factors.
in their broadest sense may disrupt early gonadal Thus, influence from the environment, for example in
development and interfere with the endocrine func- the form of EDC exposure, may persist through gen-
tions of the Sertoli and Leydig cells. This may cause erations [16, 17].
241
Testicular Dysgenesis Syndrome and mon among young men from the general popula-
tions in Europe, Japan, the United States and Aus-
Anogenital Distance tralia [25–28]. Remarkably, only a minor part of the
A new biomarker of testicular dysgenesis came with younger generation of men in these industrialized
the advent of anogenital distance (AGD) measure- countries have semen quality within the World Health
ment as a novel epidemiological tool in male repro- Organization (WHO) reference range for fertile men
ductive health research. AGD provides a link between [12, 26, 29]. Although some cases of poor semen
androgen-dependent events occurring in foetal life quality are clearly genetic, an underlying genetic
and reproductive health outcomes in postnatal life, disorder cannot be demonstrated in most infertile
including adulthood [18]. The AGD is, as the name men [12].
suggests, the distance between the genitals and the As mentioned, male infertility is associated with
anus, a distance which has for many years been used testicular cancer. TGCC is particularly common
to determine the sex of newborn rodent pups. The dis- among Caucasians, and registry studies from the
tance is determined by the degree of testosterone activ- WHO (IARC, Lyon) [30] have demonstrated a remark-
ity in foetal life and is normally about twice as large in able worldwide increase in this disease (Figure 16.2).
males as in females. Experimental animal studies have Even though the rapid increase in testicular cancer
shown shorter AGD in animals after chemical expo- incidence can only be explained by (largely unknown)
sure and resulting androgen deprivation during foetal environmental factors, genetic factors are also at play
development [19–21]. In humans, associations have [31]. Thus, African Americans have much lower rates
been found between a shorter AGD and infertility, low of TGCC than Caucasians living in the same areas of
testosterone levels, cryptorchidism and hypospadias the United States (Figure 16.2), and sons and broth-
[22–24]. Although a shortening of AGD in a boy or ers of patients with testicular cancer have increased
a man may have little impact at the individual level, risk of the disease [12]. However, generation stud-
the usage of AGD as a non-invasive epidemiologi- ies have shown that men emigrating from a coun-
cal marker linking male reproductive health problems try with low incidence to a country with high inci-
such as infertility to events occurring in prenatal life dence as adults carry the same low incidence as the
has greatly improved our possibilities of studying and country from which they emigrated, whereas second-
understanding the linkage between early events and generation immigrants carry the same risk as the men
later reproductive health problems. in the country they immigrated to, again suggesting a
Bearing in mind that TDS, disruption of the testis strong environmental influence.
in foetal life, may give rise to infertility in adulthood, Direct and unequivocal links between environ-
it is important to emphasize that current exposure mental factors and male reproductive problems are
can also interfere with testicular function, such as extremely limited, one example being the DBCP-
azoospermia in DBCP-exposed workers. Other exam- exposed workers mentioned in the chapter introduc-
ples come from the use of anabolic steroids or other tion. However, as the trends with more men devel-
lifestyle habits, as described below. Thus, men may be oping testicular cancer and poor semen quality have
infertile without having TDS and without having a occurred over only a few generations, nongenetic, that
shortened AGD. is, environmental factors most likely play a major role
in their etiology.
Evidence for Trends in Male
Reproductive Health Endocrine-Disrupting Chemicals
Manifestations of TDS are widespread and increas- During the past century the number and volume of
ingly common. Several retrospective studies have indi- manmade industrial chemicals have increased enor-
cated that semen quality has decreased, although mously. About 82,000 substances are regulated under
the topic is still controversial. A recent review of the Toxic Substances Control Act and 8,600 food addi-
numerous trend studies is contained in Skakkebaek et tives, 3,400 cosmetic ingredients, 1,800 pharmaceuti-
al. [12]. In addition, prospective investigations have cals and 1,000 pesticide active ingredients are regu-
shown that poor semen quality is currently very com- lated under federal statutes [32]. About 800 of these
242
Northern Europe The Americas Asia

12 Norway 12 12
10 Denmark 10 10
UK New
Age-standardized (World) incidence rate per 100000

Scotland Zealand
7 Ireland 7 USA 7
Iceland White*
Australia
5 5 5
Finland
Ecuador* Israel
Costa
Canada* Rica
3 Estonia 3 3
Latvia
Columbia*
2 Lithuania 2 2
Japan*
1.5 1.5 1.5
USA Singapore
Black* China*
Philippines*
1 1 1
0.7 0.7 0.7 India*
0.5 0.5 0.5
1960 1970 1980 1990 2000 2010 1960 1970 1980 1990 2000 2010 1960 1970 1980 1990 2000 2010
Year Year Year
* Regional registries
Eastern, Southern and Western Europe

12 12 12
10 10 10 Switzerland*
Slovenia Germany*

Czech
7 Republic 7 Croatia 7 UK
Slovakia Italy* The England*
5 5 5 Netherlands
Bulgaria
France
3 Poland* 3 3
Spain*
2 2 2
Belarus
1.5 1.5 1.5
Russian
Federation
1 1 1
0.7 0.7 0.7
0.5 0.5 0.5
1960 1970 1980 1990 2000 2010 1960 1970 1980 1990 2000 2010 1960 1970 1980 1990 2000 2010
Year Year Year
* Regional registries
Figure 16.2 Trends in testicular cancer; age-standardized (world) incidence (regional or national), all ages. Modified from Znaor et al. [30].
Courtesy of Ariana Znaor and Mathieu Laversanne, WHO, International Agency for Research in Cancer (IARC), Lyon, France. Reproduced from
Skakkebæk et al. [12] with permission. (A black and white version of this figure will appear in some formats. For the colour version, please refer
to the plate section.)
chemicals are known or suspected to have endocrine- its progeny, or (sub) populations’, while a suspected
disrupting effects [33]. endocrine disruptor is defined as ‘an exogenous sub-
WHO and the United Nations Environment Pro- stance or mixture that possesses properties that might
gramme (UNEP) define an endocrine disruptor as be expected to lead to endocrine disruption in an intact
‘an exogenous substance or mixture that alters func- organism, or its progeny, or (sub) populations’ [34].
tion(s) of the endocrine system and consequently Thus endocrine-disrupting chemicals (EDCs)
causes adverse health effects in an intact organism, or can act at several biological levels, such as at the
OH OH
CH3 CH3
CH3
O Testosterone HO 17β- Estradiol
H 3C OH
NH CH3
O
Cl1 - Cl10 HO CH3 HO
Polychlorinated biphenyls Diethylstilbestrol Paracetamol
(PCBs) (DES)
CCl3 Cl OH
H3C CH3
O
Cl Cl Cl Cl HO OH
Dichlorodiphenyltrichloroethane Triclosan Bisphenol A
(DOT) (TCS) (BPA)
CH3 HO O
O
O CH3
O CH3 OH O
OH
Di-ethylhexyl phthalate Genistein
O
CH3 (DEHP)
Figure 16.3 Chemical structures of the natural hormones testosterone, 17␤–estradiol, the natural plant phytoestrogen, and genistein and
examples of some of the most commonly used and well-known EDCs, including two pharmaceuticals diethylstilbestrol (DES) and
paracetamol. Note the structural similarity between the synthetic hormone DES and the normal steroid hormones.
hormone receptor level as agonists or antagonists Figure 16.3 illustrates the similarities between the
or at the level of enzymatic processes necessary chemical structures of natural reproductive hormones
for normal hormone production. Many EDCs can and some of the well-known EDCs. It is easy to
mimic or interfere with the action or synthesis of envisage how these structures can replace or interfere
natural reproductive hormones, such as testosterone, with the natural reproductive hormones in the body.
oestrogen and insulin-like peptide 3 (INSL3). This As described below, many EDCs have shown
may lead to hormonal imbalance and thus play a adverse effects on the male reproductive system
central role in the pathogenesis of disorders associated in animal experiments, including reduced AGD,
with declining male reproductive health (Figure 16.1). decreased testicular weight, reduced semen quality
244
and decreased testosterone levels as well as changes in The few studies looking into prenatal PCB expo-
other reproductive hormones [33]. Also, very direct sure and male reproductive health are inconsistent,
effects on mature sperm function have been suggested presumably due to differences in study designs and
[35]. exposure levels. One Swedish study found that moth-
ers of sons with testicular cancer had higher current
Exposure Routes and Sources serum levels of PCBs than to controls [37]. Another
study among a small group of Taiwanese men reported
Humans are exposed to EDCs via ingestion, inhala-
that exposure to high levels of PCBs in prenatal life was
tion and direct skin contact or dermal uptake directly
associated with impaired sperm parameters in adult-
from air. In general, EDCs can be classified into three
hood [38]. Conversely, PCB levels in biobank sam-
different groups: persistent organic pollutants (POPs),
ples from pregnant Danish women did not associate
nonpersistent environmental chemicals and natural
with semen quality among their sons 20 years later
compounds such as reproductive hormones and plant
[39]. More concordant data come from a number of
phytoestrogens (Table 16.1).
studies on current PCB exposure and male repro-
The sources of human exposure to EDCs are
duction. These are reviewed by Meeker and Hauser
contaminated food, water, soil, air and dust. The
[40] and report associations between various levels
chemicals derive, for instance, from pesticides
of PCB exposure and altered circulating reproductive
(dichlorodiphenyltrichloroethane, DDT), electronics
hormone levels and poorer semen quality, especially
and building materials (e.g. polychlorinated biphenyls,
impaired sperm motility [40]. Also, another POP, the
PCBs), electronic equipment (e.g. brominated flame
environmental pollutant dioxin, has been associated
retardants), contaminants from industrial production
with male reproduction health problems. For exam-
and waste incineration (PCBs and dioxins), all kinds
ple, the population prenatally exposed to high levels of
of plastic products (phthalates and BPA), coatings of
dioxin as a consequence of the Seveso accident (indus-
food packing materials (PFAS) and ordinary consumer
trial accident in Italy, 1976) suffered from reproduc-
products including food, beverages, drinking water,
tive problems, including decreased semen quality and
pharmaceuticals and personal care products. Most of
changed sex ratio of offspring [33, 41].
the EDCs in Table 16.1 are high-production-volume
Another important group of persistent pollutants
(HPV) chemicals; for example, BPA is produced in
is per- and polyfluorinated alkyl substances (PFASs)
millions of [millions of] tons every year.
such as perfluorooctane sulphonic acid (PFOS) and
perfluorooctanoic acid (PFOA), which are widely
Persistent Endocrine-Disrupting Compounds used, for example, as surface coating agents and fire-
Persistent organic pollutants (POPs) are highly fighting foam. They are persistent and accumulate by
lipophilic organic compounds that are resistant to binding to proteins instead of lipids. PFOS is of spe-
chemical, biological and photolytic environmental cial concern and has been banned for some purposes;
degradation. They can bind to lipoproteins in the however, these compounds have been replaced by oth-
organism where they bioaccumulate. For biomoni- ers whose adverse health effects have not been stud-
toring, POPs are most often measured in biopsies of ied. One study has shown associations between PFOS
adipose tissue, in lipid fractions in blood or breast levels in serum and decreased sperm parameters and
milk and in hair samples. Polychlorinated biphenyls altered testosterone levels among healthy young men
(PCBs) are a group of POPs that consist of 209 differ- [42]. Also indicating a prenatal effect, PFOA levels
ent congeners. They were produced as HPV industrial in serum samples from pregnant women stored for
products from 1929 to the mid-1980s and used 20 years are associated with semen quality in their
mainly as insulating agents in electronic equipment sons [43]. Lack of association has also been reported.
and as sealants in building construction. As PCBs Specifically, a European study found no clear asso-
are highly persistent and distributed globally via air ciations between current PFOA and PFOS exposure
and water, both wildlife and humans are exposed to and semen quality among men from four different
these bioaccumulating compounds. Concentrations countries [44]. In conclusion, the role of PFAS in
of individual PBCs in serum lipids increase with age male reproductive health, especially the more recently
(Figure 16.4) [36]. introduced PFAS, remains to be delineated.
245
Table 16.1 Some EDCs, their classifications and possible sources
Class Group Compound Common use or source

Bioaccumulative and persistent organic pollutants (POPs)
Persistent halogenated Polychlorinated biphenyls (PBCs) Insulation and sealants
chemicals
Polybrominated diphenyl ethers Flame retardants
(PBDEs)
Dioxins Waste incineration
Dichlorodiphenyldichloroethylene Metabolite of the pesticide
(DDE) DDT
Per- and polyfluorinated Perfluorooctane sulphonic acid Surfactants and surface coating
alkyl substances (PFAS) (PFOS), Perfluorooctanoic acid agents (e.g. Teflon) and
(PFOA) fire-fighting foam
Non-persistent environmental chemicals

Phthalate diesters Di-n-butyl phthalate (DnBP),
Butylbenzyl phthalate (BBzP),
Di-ethylhexyl phthalate (DEHP),
Di-iso-nonyl phthalate (DiNP) Plasticizers and dissolving
agents
Halogenated phenolic Triclosan (TCS) Antibacterial and antifungal
chemicals agent
Vinclozolin Fungicide
Nonhalogenated phenolic Bisphenol A (BPA) Polycarbonate and epoxy
chemicals resins
Benzophenone-3 (BP-3),
4-Methylbenzylidene camphor UV-filters and absorbers
(4-MBC), 3-Benzylidene
camphor (3-BC)
Natural compounds
Phytoestrogens Genistein, Daizein Isoflavones, natural compound
in several plants, e.g.
soybeans
70
ng PCB / g of lipid in serum
60
50
40
12 - 19 y
30
20 - 39 y
20
10 40 - 59 y
0 60+ y
58
0
15
17
18
15
15
17
18
1
8,
8,
B
PC
PC
PC
PC
PC
PC
13
13
B
B
PC
PC
Male Female
Figure 16.4 Polychlorinated biphenyls (PCBs) in serum lipids from the American NHANES studies divided into gender and age groups by
years (y) and expressed as weighted arithmetic means (ng/g of lipid). Samples were collected in 2007–8 and were pooled. All data shown are
from the white non-Hispanic part of the NHANES studies. More information on other ethnic groups is available at www.cdc.gov/
exposurereport/ [36]. The higher levels observed in the elderly reflect both the bioaccumulating nature of the PCBs and the fact that older
generations were exposed to higher levels of these compounds before the compounds were banned.
246
Table 16.2 Urinary excretion (ng/mL) of some nonpersistent EDCs from American and Danish population studies, expressed
as median (95th percentile).
NHANESa Denmarkb
Men Women Men Pregnant women
Sample size 1,399 1,350 901 565
Collection years 2011–12 2007–9 2011–12
Age (years) 20+ 18–28 18–42
Phthalate metabolitesc
Mono-iso-butyl phthalate, (MiBP) 6.6 (39.3) 6.1 (32.7) 57.7 (176) 29.3 (110)
Mono-n-butyl phthalate (MnBP) 9.2 (56.9) 9 (51.8) 28.0 (90.5) 12.5 (50.8)
Mono-benzyl phthalate (MBzP) 4.8 (37) 4,2 (33.9) 34.0 (166) 2.45 (16.6)
Mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) 14.6 (71.5) 12 (64.9) 15.4 (54.4) 5.19 (16.8)
Mono-iso-(carboxyoctyl) phthalate (MCiOP) 20.2 (255) 16 (180) 7.70 (40.6) 3.79 (22.1)
Environmental phenols
Triclosan (TCS) 6.4 (566) 7.6 (545) 2.64 (377) 0.82 (411)
Bisphenol A (BPA) 1.5 (9.5) 1.3 (8.5) 3.23 (14.9) 1.52 (7.52)
Benzophenone-3 (BP-3) 16 (594) 22.2 (1720) 2.98 (80.4) 3.22 (446)
a Center for Disease Control [36].
b Frederiksen et al. [45].
c Major metabolites of phthalate diesters: DiBP, DnBP, BBzP, DEHP and DiNP.
Nonpersistent Endocrine-Disrupting ucts and other consumer products. Generally, expo-

sure to these nonpersistent chemicals varies a lot from
Compounds one individual to another. In addition, longitudinal
We are exposed to numerous nonpersistent EDCs studies point to a substantial day-to-day variation
daily. They can be excreted directly in urine, but are within the same person. Still, several studies suggest
most often very rapidly metabolized in the body to that some people tend to remain highly exposed over
hydroxylated, oxidized and/or conjugated metabolites time while others have a persistently lower exposure
before they are excreted. Metabolites are most often [47, 48].
measured in urine. Many of the nonpersistent chem- Several nonpersistent EDCs, including some of the
icals can also be measured at lower concentrations in phthalates and pesticides, have been associated in ani-
serum, amniotic fluid and breast milk, as well as in mal studies with a range of effects on the male repro-
adipose tissues. Table 16.2 shows examples of urinary ductive system [33]. Newer studies have reported asso-
excretion of some phthalate metabolites and environ- ciations between shorter AGD in newborn boys and
mental phenols measured in the United States and in higher prenatal phthalate exposure [22, 49]. Higher
European countries. We found that 95–100% of the concentrations of phthalates in adult men have been
Danish population was exposed to virtually all the associated with poorer sperm quality. Data from a
listed EDCs. While it is possible to avoid some of meta-analysis of associations between semen quality
the nonpersistent EDCs, such as the parabens used as and urinary concentrations of phthalate metabolites
antibacterial additives in cosmetic products, nobody showed that urinary mono-n-butyl phthalate (MnBP)
living in a modern society can avoid the EDCs listed and monobenzyl phthalate (MBzP) were especially
in Table 16.2. For instance, benzophenone-3 (BP-3), inversely associated with sperm concentration, given
which is a UV filter used in sunscreens and cos- the right-side weight in Figure 16.5 [50]. For the other
metic products, has been detected in almost all indi- semen quality parameters, associations were not or
viduals – children, adolescents and adults – and not were only borderline significant (Figure 16.5) [50].
only during the summer season [45, 46], suggest- During the past decade, bisphenol A (BPA) has
ing that the sources for BP-3 exposure are not lim- been one of the most studied EDCs. Many contro-
ited to sunscreens, but include personal care prod- versial studies have shown harmful effects of BPA,
247
A B
Study Urinary MBP level : 26.0–14459.0 μg/L Odds ratio %
Study Urinary MBP level : 7.4–25.3 μg/L
Odds %
(95% CI) Weight
ratio (95% CI) Weight Sperm concentration
Sperm concentration Duty (2003a) 3.30 (0.90, 12.60) 17.87
Duty (2003a) 1.40 (0.30, 6.00) 20.71 Hauser (2006) 3.30 (1.20, 8.50) 27.17
Wirth (2008) 0.50 (0.10, 3.60) 10.88
Hauser (2006) 2.80 (1.25, 6.28) 71.33
Liu (2011) 12.00 (1.01, 143.00) 6.12
Liu (2011) 6.80 (0.60, 75.30) 7.96 Han (2014) 1.97 (0.95, 4.08) 37.95
Total 2.60 (1.32, 5.15) 100.00 Total 2.39 (1.26, 4.53) 100.00
Sperm motility
Sperm motility Duty (2003a) 3.00 (1.20, 7.60) 15.93
Duty (2003a) 1.80 (0.70, 4.60) 29.13 Hauser (2006) 1.80 (1.10, 3.20) 28.80
Hauser (2006) 1.50 (0.83, 2.71) 42.75 Wirth (2008) 0.80 (0.20, 3.90) 7.70
Liu (2011) Liu (2011) 0.70 (0.30, 2.10) 14.82
0.50 (0.20, 1.40) 28.12
Han (2014) 1.08 (0.69, 1.69) 32.74
Total 1.16 (0.58, 2.34) 100.00 Total 1.35 (0.86, 2.11) 100.00
Sperm morphology Sperm morphology

Duty (2003a) 2.20 (0.80, 6.10) 21.06
Duty (2003a) 1.50 (0.50, 4.30) 24.40
Hauser (2006) 0.80 (0.40, 1.60) 34.47
Hauser (2006) 0.88 (0.48, 1.63) 75.60 Wirth (2008) 3.30 (0.70, 16.20) 10.46
Total 1.00 (0.59, 1.71) 100.00 Han (2014) 1.53 (0.76, 3.09) 34.02
Total 1.43 (0.83, 2.47) 100.00
Sperm volume
Liu (2011) 0.40 (0.10, 2.10) 39.99
0.02 0.08 0.30 1.00 4.00 15.00 75.30 Han (2014) 1.26 (0.40, 3.93) 60.01
Total 0.80 (0.26, 2.40) 100.00
0.01 0.05 0.23 1.00 5.00 28.00 143.00

C Study Urinary MBzP level : 14.0–540.2 μg/L Odds %
ratio (95% CI) Weight
Sperm concentration
Duty (2003a) 5.50 (1.30, 23.90) 20.36
Hauser (2006) 1.90 (0.80, 4.30) 61.02
Wirth (2008) 1.40 (0.30, 6.30) 18.62
Total 2.23 (1.16, 4.30) 100.00
Sperm motility
Duty (2003a) 2.10 (0.80, 5.30) 25.32
Hauser (2006) 1.30 (0.70, 2.30) 63.98
Wirth (2008) 1.30 (0.30, 5.50) 10.70
Total 1.47 (0.91, 2.36) 100.00
Sperm morphology
Duty (2003a) 2.20 (0.80, 6.00) 24.30
Hauser (2006) 1.10 (0.60, 2.10) 62.86
Wirth (2008) 0.90 (0.20, 3.20) 12.83
Total 1.27 (0.77, 2.08) 100.00
0.04 0.12 0.36 1.00 3.00 8.00 23.90
Figure 16.5 Pooled odds ratio (ORs) of low sperm parameters associated with medium (a) and high (b) levels of urinary monobutyl
phthalate (MnBP) and pooled ORs for low sperm parameters associated with high urinary monobenzyl phthalate level (MBzP) (c). Squares
represent study-specific ORs (size of each square reflects the study-specific statistical weight, that is, the inverse of the variance). Horizontal
lines represent 95% CIs for individual studies. Diamonds represent summary OR (width of each diamond represents 95% CIs for the summary
estimates). A random-effects model was used. Meta-analysis is reproduced from Cai et al. [50] with permission. References to the individual
studies included in the meta-analysis can be found in Cai et al. [50].
while other studies could not confirm these findings triclosan (TCS), an antimicrobial agent, and UV filters
[51]. Recent studies suggest that BPA might work in used in sunscreens.
a complicated manner and exert both anti-androgenic
and anti-estrogenic effects on the male reproductive
hormone system. In adult men, higher levels of BPA Trends in Exposure Over Time
have been associated with decreased semen quality Human POP exposure levels have decreased during
[51, 52]. Also, several other nonpersistent EDCs are the past decades due to regulation initiatives from
under investigation for reproductive effects, including national authorities. For instance, PCBs and dioxins
248
have shown decreasing trends in breast milk [53]. studies have shown negative effects similar to those of
Newer chemicals introduced onto the market in the DEHP and DnBP [54].
early 1970s such as PBDEs, PFOS and PFOA increased
from the midseventies and peaked at the end of the
nineties.
Lifestyle Effects
Also, for several of the nonpersistent EDCs,
changes in production volume and concomitant
Current Smoking and Exposure to
changes in human exposure level have been observed Smoking in utero
during the past years. For instance, the production Many studies have examined the association between
of the phthalate diethylhexyl phthalate (DEHP) has current smoking and semen quality (reviewed by
decreased from 250,000 tons/year to less than 100,000 Harlev et al. [55]), but the results have been conflict-
tons/year in the period from 1988 to 2003. In the same ing. Two meta-analyses both found reduction in sperm
period, corresponding declining trends were observed concentration, total sperm count and semen volume
for the estimated daily intake of DEHP from 4.0 to among smokers [56, 57] (Figure 16.6) and concluded
2.5 µg/kg bodyweight/day based on biomonitoring of that large studies are necessary to have enough power
urinary DEHP metabolites [33]. Some of the phased- to detect an association.
out EDCs have been replaced with other similar prod- More studies [58] have found an association
ucts. Reflecting this, a declining trend in urinary con- between maternal smoking and semen quality in the
centration of DEHP and di-n-butyl phthalate (DnBP) offspring, but some only among sons whose moth-
metabolites was followed by increasing urinary lev- ers smoked more than 10 cigarettes daily during
els of di-iso-nonyl phthalate (DiNP) and di-iso-butyl pregnancy [59, 60]. In a study among 1,770 young
phthalate (DiBP) metabolites, respectively. Replace- men from six Northern European countries, maternal
ment of DEHP and DnBP with DiNP and DiBP, smoking was associated not only with reduced semen
respectively, is particularly worrying, as both of the quality but also with smaller testis size [61]. Smok-
latter chemicals are categorized as EDCs and animal ing men are more often sons of smoking mothers.
Semen volume Sperm density Total sperm count

Kumosani 2008 Colagar 2009 Chen 2007
Kumosani 2008 Chang 2006 Colagar 2007
Chang 2006 Chen 2007 Colagar 2009
Chen 2007 Colagar 2007 Colagar 2007
Chia 1998 Colagar 2007 Colagar 2009
Colagar 2009 Elshal 2009 Elshal 2009
Colagar 2009 Eskenazi 2003 Eskenazi 2003
Colagar 2007 He 2008 He 2008
Colagar 2007 Härkönen 1999 Kumosani 2008
Elshal 2009 Künzle 2003 Kumosani 2008
Eskenazi 2003 Künzle 2004 Künzle 2003
He 2008 MAK 2000 Künzle 2004
Künzle 2003 Martini 2004 Ramlau-Hansen 2007
Künzle 2004 Nan 1992 Richthoff 2007
Omu 1998 Omua 1998 Rubes 1998
Ozgur 2005 Ozgur 2005 Wallock 2001
Pasqualotto 2004 Pasqualotto 2006 Zavos 1998
Ramlau-Hansen 2007 Ramlau-Hansen 2007 Total (95%CI)
Richthoff 2007 Richthoff 2008
Rubes 1998 Sobreiro 2005
Shen 1997 Sofikitis 1995 –100 –50 0 50 100
Sobreiro 2005 SOFIKITIS 1995
Sofikitis 1996 Tai 2008
Tai 2008 Trummer 2002
Trummer 2002 Viloria 2005
Wallock 2001 Wallock 2001
Wang 2001 Wang 2006
Wang 2006 Zhang 2000
Zavos 1998 Zhang 2003
Zhang 2000 Zhang 2002
Zhang 2002 Total (95%CI)
Zhang 2003
Total (95%CI)
–50 –25 0 25 50
–1 –0.5 0 0.5 1
Figure 16.6 Mean differences (MDs) in the effects of smoking on semen quality. The figure shows the forest plots for the meta-analysis on
the association between smoking and semen quality. Reproduced from Li et al. [56] with permission.
249
Therefore, the association between current smoking semen quality among men for whom the last week
and semen quality may be mediated through prenatal was not a typical week. To our knowledge, no studies
exposure to smoking. This was disentangled in a study have assessed the impact of binge drinking on semen
among 3,486 young Danish men reporting that prena- quality.
tal exposure to smoking was associated with impair- Given that young men in the Western world have a
ment of testicular function, which was even more pro- high alcohol intake, men should be advised that high
nounced if the man smoked himself [62]. habitual alcohol intake may affect not only their gen-
Both men at fertile ages and pregnant women eral but also their reproductive health.
should therefore be advised to avoid cigarette smok-
ing in order to improve not only their general health
but also their fertility and the fertility of future Marijuana Use
generations. Marijuana is the most widely used illicit recreational
drug in the Western world, with reported use among
Caffeine Intake 13.7% in the United States and users being predomi-
nantly males. Few studies have investigated the asso-
Previous studies of caffeine intake and semen quality
ciation between marijuana and male reproduction
have shown contradictory results [63–72]. Some stud-
and most have been conducted among men attending
ies have found increased numbers of neck abnormali-
infertility clinics, or in small populations of chronic
ties and sperm nuclear morphometry [64, 65, 68–70,
users, and among men suffering from malnutrition
72]. Two Danish studies among healthy young men
and using other recreational drugs. In our study
found no adverse effect of caffeine intake on semen
among more than 1,200 healthy young men [77], of
quality [63, 67], but one found an inverse association
whom 45% had smoked marijuana during the past
between cola intake and semen quality. This was con-
three months, we found associations between regu-
firmed in a recent study among 796 healthy Chinese
lar use of marijuana more than once per week dur-
men [70]. Therefore, more studies are urgently needed
ing the past three months and reduced semen qual-
before advice can be given to men at fertile ages.
ity. No adverse association was found for irregular
use. The combined use of marijuana and other recre-
Alcohol Intake ational drugs decreased semen quality further [77].
Studies among infertile men have assessed alcohol Men should therefore be informed that habitual mar-
intake and found an association with semen qual- ijuana use may be detrimental to their semen quality,
ity; however, it is difficult to assess whether the men but further studies are urgently needed.
have changed their alcohol intake due to infertility.
To date not many studies assessing the association
between alcohol intake and semen quality have been Body Mass Index
conducted among healthy men, and the results have Two meta-analyses of the effects of BMI on semen
been contradictory [73–76], probably because of dif- quality have been conducted. The first was published
ferences in intake and assessment of alcohol con- in 2010 and concluded that there was no evidence
sumption between studied populations. Some stud- of an association between BMI and sperm concen-
ies reported alcohol intake for the month [76], week tration or total sperm count [78]. However, data
[73, 74], or five days [75] prior to semen sampling from most studies could not be aggregated for this
and did not find any convincing associations. How- meta-analysis, and more than 30 original studies have
ever, misclassification is likely, as alcohol intake may been published since then. The second meta-analysis,
vary, and therefore one study assessed habitual alco- from 2014, which was based on more than 13,000
hol intake by including the men who reported that men showed a J-shaped association between BMI and
their intake the previous week represented a typical abnormal sperm count: underweight was associated
week. Among these men semen quality was reduced with an increased but nonsignificant risk of abnormal
from an intake of more than 5 units/week, although sperm count, whereas overweight and obese men had
the decreasing trend was most apparent for men with a significantly elevated risk of abnormal sperm count
a typical weekly intake above 25 units. Interestingly, compared with normal weight men [79] (Figure 16.7).
the last week’s alcohol intake was not associated with Men should therefore be advised that being overweight
250
2.75
2.50
2.25
Odds ratios (95% Cls)
2.00
1.75
1.50
1.25
1.00
0.75
<18.5 18.5–24.9 25.0–29.9 30.0–39.9 >40.0
Cases: Events /N 65/288 1285/4649 476/1480 148/244

Controls: Events /N 1363/5687 1623/6416 1623/6416 1345/4852
Number of studies 13 21 21 14
Body mass index (kg/m2)
Figure 16.7 Association between BMI and oligozoospermia or azoospermia according to categories of BMI. Reproduced from Sermondade
et al. [79] with permission.
may negatively affect not only their general health but and vegetables, low intake of meat/fat/processed food)
also their reproductive health and semen quality. was associated with increased sperm motility [82],
and four studies [83–86] reported that fat-rich foods
(e.g. processed food or red meat), soy isoflavones, and
Antioxidant Supplementation sweets decreased semen quality. Two studies reported
A Cochrane review suggested that antioxidant supple- the positive effects of fish and low-fat dairy intake
mentation in infertile men may improve the chance (particularly low-fat milk) [82, 86] on semen quality.
of a live birth among infertile couples undergoing Among U.S. men attending an infertility clinic and
assisted reproductive technology [80]. However, pub- among young healthy Danish men, a high intake of sat-
lication bias cannot be excluded, and most of the urated fats was negatively associated with semen qual-
included studies were not designed to investigate the ity at the same order of magnitude [87, 88]. A signif-
effects of antioxidant treatment on semen quality. In icant dose–response association was found in one of
addition, the men were treated with many different the studies [88] (Figure 16.8). The role of diet in male
combinations and doses of antioxidants, and it is not infertility still needs further research. However, avail-
possible to recommend infertile men a type and dose able studies support the suggestion that a healthy diet
of antioxidant. A recent study reviewed 16 published is a safe way to improve at least one measure of semen
papers on dietary supplementation and semen quality quality.
[81] and came to the same conclusion. It is therefore
not possible to advise men regarding supplementation
and semen quality. Future studies should be large, ran- Exercise
domized placebo-controlled trials. Previous research on the effect of physical activity on
semen quality has been inconsistent, with some stud-
ies finding a positive association [72, 89, 90], others
Diet finding no association [91–94] and some finding a neg-
A recent review of seven papers on diet and semen ative association [95–98]. This is most likely due to
quality [81] concluded that diet plays a key role in the differences in the type, range and intensity of phys-
improvement of sperm parameters. One study found ical activity across studies. Several studies, particu-
that prudent dietary patterns (high intake of fruits larly among long-distance runners and cyclists, have
251
Percentage change in total sperm count

80
60
40
20
8
0
0 –8
–20 –14
–19 –23
–38
–40 –42
–42
–60
–65
–80
–100
Percentage intake of saturated fat in centiles
Figure 16.8 Percent reduction in total sperm count (and 95% confidence intervals) with increasing percentage caloric intake of saturated fat
(divided into deciles). Adjusted for period of abstinence (transformed by the natural logarithm), BMI, alcohol consumption, smoking,
cryptorchidism, total energy intake, protein intake, and remaining fatty acids in multiple linear regression. Reproduced from Jensen et al. [88]
with permission.
found reduced semen quality among very active men, phone use had no adverse effects on semen param-
which is probably because these sports are strongly eters in human studies but indicated that radiofre-
associated with negative energy balance. In the largest quency radiation had a detrimental effect on sperm
human study to date, Wise et al. [91] found no asso- motility and viability in vitro and had a harmful effect
ciation between overall self-reported physical activity on sperm concentration and motility in animal studies
and semen quality parameters in 2,261 men attending [104]. Radiation from phones has declined since first-
a fertility clinic. This is in contrast to a Danish [98] and generation cell phones, and further studies are needed
a U.S. study [99] reporting beneficial effects of exer- to determine possible effects on semen quality.
cise among young, healthy men with a higher level of
exercise.
Television watching and semen quality have been Psychological Stress
investigated in two studies. Both found a negative asso- Several studies have investigated associations between
ciation, which was further modified if the men were semen quality and stress due to different types of stres-
inactive. This is supported by a study reporting asso- sors: occupational stress, stressful life events, stress
ciation between sedentary position at work and scro- due to infertility, etc. (summarized in Nordkap et al.
tal temperature [100]. However, three studies found no [105] and Li et al. [56]). Overall, they provide evi-
clear negative association between sedentary work and dence that semen quality is impaired by psychological
semen quality [101, 102]. Further research is needed stress. Several studies included men undergoing infer-
into the impact of physical activity and sedentary tility treatment. The majority of such studies found
work, including television watching, on semen quality a link between stress and reduced semen quality in
to quantify the levels and types that are beneficial. men undergoing infertility treatment (summarized in
Nordkap et al. [105]). It is, however, difficult to differ-
entiate between stress as a cause or a consequence of
Cell Phones decreased semen quality in such studies.
Radiofrequency electromagnetic radiation (RF-EMR) Results from studies using ‘‘stressful life events’’
from mobile phones could potentially affect sperm or environmental disasters as indicators of stress have
development and function. Two meta-analyses have been equivocal (summarized in Nordkap et al. [105]).
been conducted to determine whether exposure to The recording of life events does not address how
RF-EMR emitted from mobile phones affects human stressful a certain exposure is perceived as being,
sperm quality. One found reduced sperm motility which might explain the mixed results. Three stud-
and viability, but the effects on concentration were ies investigated self-reported ‘‘daily life stress’’ in men
more equivocal [103]. The other reported that mobile from the general population while controlling for
252
30
20
Difference in sperm concentration (%)
10
0
0 –5
–7
–10
–11 –12
–20 –17
–23
–30
–38
–40
–50
–60
–70
0 1–10 11–20 21–30 31–40 41–50 51–60 >60
Stress score (points)
Figure 16.9 Adjusted change in sperm concentration according to stress score (reference stress score 21–30 points). Reproduced from
Nordkap el al. [105] with permission.
relevant confounders [105–107]. One found an inverse may also affect their semen quality. Therefore, and
U-shaped form (Figure 16.9), so that both high and despite taking confounders into account in the data
low stress reduced semen quality [105], and one a analysis, it is difficult to disentangle the adverse effect
linear negative association between perceived stress of one single lifestyle factor.
and semen quality [107]. However, a Danish study Mixed exposure to various chemicals is also a con-
among pregnancy planners did not find associations siderable challenge in terms of delineating the possi-
between stress and any semen parameters, but found ble health effects of chemical exposure. Humans are
that fecundability decreased with increasing stress exposed to a complex mixture of EDCs, and when the
scores in men with low semen quality [106]. Explana- effect of one chemical is considered, it may be another
tions for these mixed results might include differences correlating co-exposure that actually does the harm, or
in age and stress assessments used. a mixture of a number of chemicals, the so-called cock-
Even though different markers of stress have been tail effect.
used, it seems that stress may adversely affect semen Male fertility studies are often based on couples
quality and therefore men of fertile age should take undergoing infertility treatment. Men from infertile
that into consideration. However, more studies with couples constitute a very heterogeneous population,
biological markers are urgently needed. consisting both of men with impaired semen qual-
ity and men with normal semen quality but with an
infertile female partner. Other studies included young
Research Challenges healthy men or semen donors. It is difficult to obtain
Most studies of the association between environmen- a participation rate above 30% in such studies, and
tal exposure (including lifestyle) and semen quality the participants may either be healthier or more con-
have been purely observational, without any kind of cerned about their fertility than nonparticipants.
intervention or randomization. Thus, only associa- Older studies especially are relatively small. As
tions have been studied and conclusions regarding semen quality varies considerably both among indi-
causation cannot be drawn. Also, for cross-sectional viduals and within the same individual at different
studies (which constitute the majority), reverse cau- time points, the power to find statistically significant
sation is a possibility, as men with poor semen qual- results is limited. In addition, it is difficult to perform
ity may have an unhealthier lifestyle. In general, men adequate confounder control in small studies.
exposed to one unhealthy lifestyle factor often have Spermatogenesis is a time-consuming process,
other inappropriate lifestyle behaviours as well, which lasting 70 days from early spermatogonia to late
spermatid stage. Often questionnaires focus on and avoidance of obesity. In addition, specific work-
behaviour and lifestyle in the recent four to six weeks, place exposure to EDCs such as pesticides should be
and thus do not cover the entire timespan of the avoided.
spermatogenesis process. In addition, due to the lack
of intervention, the question of whether a change References
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Chapter
Susceptibility of the Testis to Lifestyle
17 and Environmental Factors During

the Life Course
Richard M. Sharpe
Introduction ity in adult men are expensive, difficult to manage

and insensitive, which are significant deterrents to
As chapters elsewhere in this book demonstrate, funders and researchers alike. It is also an impor-
semen quality, sperm number and fertility rates in nor- tant determinant of our continuing ignorance about
mal human males are remarkably poor in comparison the causes of male infertility and other reproductive
with those in the majority of the animal kingdom. This disorders.
appears to be largely an inherent property of the pro- The other major function of the testis, the produc-
cess of spermatogenesis in man, the organization of tion and secretion of testosterone, also shows consider-
which is far less efficient than in domestic and labo- able variation between individuals, but not as dramatic
ratory animals, as well as in those nonhuman primates as that for sperm count, perhaps due in part to the fact
for which robust data are available [1, 2]. Adding to that its regulation is largely homeostatic; thus, if blood
this sorry situation is the fact that the actual number testosterone levels fall, increased secretion of pituitary
of sperm produced per day is hugely variable between LH should be triggered to correct the fall. However,
individual apparently normal men and is probably the other important factors impinge on testosterone levels,
primary determinant of sperm concentration and total such as time of day, age (especially older age) and body
sperm count in an individual [3]. The reasons for this composition (discussed later), and emerging evidence
are discussed later, but from the perspective of assess- suggest that birth weight and year of birth may be other
ing the susceptibility of the human testis to lifestyle and influential factors, as discussed below. Therefore, there
environmental factors, it has fundamentally important is sufficient noise in the system to ensure that what is
implications. Most obviously, it means that those indi- considered a normal testosterone level covers a wide
viduals with constitutionally low sperm production are range. This has opened the door in recent years to the
inherently more vulnerable to any exogenous factor(s) increasing diagnosis of age-related testosterone defi-
that might adversely affect sperm production. How- ciency in older men, even though testosterone levels
ever, there are also other consequences. For example, for the majority of such men remain within the (wide)
the poor quality and high variability of sperm pro- normal range and do not lead to any clinically identi-
duction/sperm counts in men means that any study fiable signs of androgen deficiency [4]. As with sperm
seeking to evaluate whether a particular lifestyle, diet counts, our rather poor understanding of the cause of
or exposure is detrimental or not faces an uphill task the variation in testosterone levels between individu-
in overcoming the high ‘normal’ level of background als and how and when these may be impacted by age
noise. This means that each study has to recruit rel- and environmental/lifestyle factors helps create a vac-
atively large numbers and to make allowance for the uum within which speculative treatments can be justi-
high normal variability, which increases costs. In turn, fied in the absence of good evidence [5]. As Benjamin
this means that studies to answer even simple ques- Franklin pointed out, ‘the only thing more expensive
tions about impacts on spermatogenesis/semen qual- than education is ignorance’.
University Press.
260
Chapter 17: Susceptibility of the Testis to Lifestyle and Environmental Factors During the Life Course
As detailed below, and in chapter 16, there is grow- developmental effect, there is likely to be high inac-
ing evidence that the perinatal development and func- curacy in recall – the exceptions may be factors such
tion of the testis are critically important in determin- as mothers smoking or drinking in pregnancy, which
ing how well it will function in adulthood. This implies appear to be recalled accurately, perhaps because of
that this period may also be one of vulnerability to feelings of guilt.
exogenous factors. The difficulty that this poses is that
studying what lifestyle or environmental factors are
important during this period means that the outcome
Susceptibility of the Human Testis to
is some 18–25 years down the road! This creates even Lifestyle/Environmental Factors
bigger issues for researchers and funders than the stud- The aim of this chapter is not to consider what
ies in adult men referred to above, as it effectively rules lifestyle/environmental factors might negatively affect
out prospective studies that are ‘designed for purpose’. testis function in men per se, as this is covered to
Instead, researchers have to use ingenuity to tap into various extents in other chapters, but rather to con-
existing birth cohorts that were set up for other pur- sider this from a susceptibility perspective. Put another
poses, which inevitably means that the studies will way, what is it about the development and func-
have limitations, such as unavailability of data, low- tion of the human testis that might make it espe-
quality data relevant to reproductive development or cially vulnerable to specific lifestyle/environmental
absence of relevant (e.g. blood) samples. The alterna- factors? In addressing this, another important aspect
tive approach is to work backwards, recruiting adult to consider is that there are important human-specific
men and then trying to decipher information about lifestyle/environmental factors that impinge on these
their early development to see if this is related in any susceptibilities. As this ‘vicious storm’ is human-
way to their adult reproductive function. This intro- specific, it can be difficult to model or address via
duces two large constraints. First, it encounters the experimental animal studies, so that recourse to
variability issue described above with regard to adult human studies is necessary, with all of the attendant
men. Second, where data from pregnancy or lactation issues that this then brings, as highlighted in my intro-
are concerned, there are obvious issues related to accu- ductory remarks.
racy of recall. For some aspects, such as birth weight or There are three main areas of susceptibility of the
whether breast-fed or bottle-fed, accurate data or recall human testis, and these exert their influence on testis
is likely to exist. However, if the study aim is to evalu- function independently (Figure 17.1); this means they
ate if a particular lifestyle or dietary factor has had any can each add to the overall susceptibility. The first, and
Figure 17.1 Key events in testis development and function over the life course and their potential susceptibility to adverse effects of
lifestyle, dietary and chemical exposure.
261
most fundamental, is the development of the testis and its relevance is that how well the testis develops deter-
the impact that early life (mainly perinatal) events may mines how it will function in adulthood and/or how
have on lifelong testis function; this can impact both susceptible it may be to exogenous influences such
the steroidogenic and spermatogenic functions of the as diet and lifestyle, whether during perinatal life or
testis, unlike the other two aspects, which primarily during puberty and adulthood. Viewed another way,
affect spermatogenesis. The second relates to the orga- the question is whether a testis that develops sub-
nization and efficiency of spermatogenesis in human optimally, or abnormally, has its function compro-
males. The third relates to the absolute need for sper- mised in ways that make it more susceptible to adverse
matogenesis to operate at a lower temperature than effects of lifestyle and environmental factors in adult-
core body temperature, which is achieved via scrotal hood, or that cannot be reversed by postnatal com-
cooling. pensatory changes or even interventions. Of course
These three aspects are considered separately it is largely impossible to answer this question for
below, the aim being to outline how they make the men, because the function of the developing testis is
testis susceptible and then to consider what aspects unknown until adulthood, for other than certain spe-
of our modern diet, lifestyle and environment may cific cases [7]. However, there are one or two pieces of
impact this susceptibility (Figure 17.1). How such human data and animal experimental data that pro-
impacts might be prevented or attenuated is briefly vide some insight into this, as discussed below.
considered, where possible. In this regard, particu- A refinement of the TDS hypothesis that has
lar attention is paid to diet, for the following rea- emerged as the result of animal experimental studies
sons. Change in human diet (to a Western-style diet) is that there is a critical period – the masculinization
is arguably the biggest change to have happened to programming window (MPW) – during which andro-
human males over the past 50 or more years, and gens from the foetal testis play a critical role in setting
there is abundant evidence for how this has impacted up normal development of the male reproductive tract
health and wellbeing, especially in relation to obe- [8, 9]. Anything that disrupts androgen production
sity, metabolic dysfunction and cardiovascular disease or action during this period is likely to have perma-
[6]. It is remarkable that, until relatively recently, lit- nent adverse effects on the development and/or func-
tle attention was paid to the idea that the major alter- tion of the male reproductive system (see chapter 16).
ations to our diet could have impacted male repro- This is arguably the most vulnerable period in male
ductive function, an attitude that seems very much reproductive development as deficiencies in androgen
head-in-the-sand. As discussed below, the (still lim- action within the MPW appear to be nonrecoverable
ited) evidence that a Western-style diet can affect thereafter, based on rodent studies [9]. In humans,
testis function and several aspects of sperm develop- the MPW is reckoned to be within the period 8–14
ment and function has grown considerably in recent weeks of gestation [8, 9]. Current thinking on the TDS
years, even if the mechanisms that underlie such effects hypothesis and the MPW is that it is maldevelopment
remain largely unknown. Where dietary effects on of the testis that leads to androgen deficiency, rather
testis/sperm function appear negative, there is consid- than the other way around, so exactly how andro-
erable room for reversing such effects. Therefore, there gen deficiency within the MPW might lead to reduced
is a special section devoted to how diet may impact the sperm production in adulthood, as proposed by the
susceptibilities that are the focus of this chapter. hypothesis, is unclear. But if the hypothesis is accurate,
then it seems reasonable to suspect that any maldevel-
Importance of Perinatal Life in Setting opment of the testis during the foetal period is likely to
make it more vulnerable to exogenous adverse effects
Up Adult Male Reproductive Function that might occur later in life.
There is now widespread acceptance that some of Low birth weight is known to predispose offspring
the commonest male reproductive disorders, perhaps to a range of cardiometabolic disorders in adulthood,
including low sperm count in adulthood, may have but most studies show that low birth weight does not
their origin in foetal or perinatal life – the so-called appear to compromise adult sperm production [10,
testicular dysgenesis syndrome (TDS) hypothesis [7]. 11]. However, one interesting study has shown that
This is covered in chapter 16 and will not be discussed birth weight is positively related to blood testosterone
in detail here. In the context of the present chapter, levels in adulthood, and that this relationship extends
262
across the full birth weight spectrum [12]. Moreover, sured testis growth (by ultrasound) longitudinally in
this positive relationship remains even taking into male infants from birth to either 6 or 18 months
account factors known to influence testosterone lev- of age, thus before and after mini-puberty. All three
els directly in adulthood, such as obesity. As low birth show testis growth over this period, although the mag-
weight is a risk factor for obesity and obesity-related nitude of reported increase in size in one of the stud-
disorders in adulthood, and all of these disorders are ies [16] is different from that in the other two [17, 18].
associated with lower blood testosterone levels [13], it In the two Nordic studies, involving infants born at
is interesting to speculate whether foetal programming term, a 2-fold increase in testis size was found in
of adult testosterone levels might be a factor in deter- Finnish boys [17, 18] and a 1.5-fold increase in Dan-
mining an individual’s predisposition to obesity and its ish boys [17]. This contrasts with a reported 30-fold
associated disorders. increase in testis size in U.S. boys from birth to 3–6
How foetal life can affect adult testosterone levels months [16]. It seems unlikely that such a difference
is unclear but represents something of a conundrum in testis growth really occurs in U.S. boys, although
because the cells responsible for producing testos- this study did use more detailed ultrasound measure-
terone in adulthood, the adult generation of Ley- ments than in either of the other two studies, so it is
dig cells, do not differentiate within the testis until possible that it may be more accurate. Nevertheless,
puberty. However, one experimental study in rats and it appears in all three studies that the majority of the
mice has shown that this might occur by alteration perinatal testis growth occurs during the first three
of either the numbers of the stem/progenitor cells for months, corresponding with the period of the high-
adult Leydig cells or by epigenetic alteration of the est gonadotrophin and testosterone levels, although
steroidogenic function of these cells when they ulti- growth beyond this period may still occur at a lower
mately differentiate into adult Leydig cells [14]. The level.
same study showed that the adult Leydig stem cells are When testis and penis growth have been compared
androgen targets in the foetal testis and that experi- over 0–6 months for boys born prematurely or full-
mental lowering of foetal intratesticular testosterone term, it was found that premature infants had signifi-
levels caused changes to the stem cells that resulted in cantly smaller testes and penises than full-term babies
adulthood in compensated Leydig cell failure. Interest- at birth but showed catch-up growth for these organs
ingly, in young adult human males, compensated Ley- over the first six months of life, presumably as a result
dig cell failure is commonly associated with low sperm of the supranormal elevation of gonadotrophin and
counts (see chapter 16), providing at least circumstan- testosterone levels during mini-puberty in preterm
tial evidence that the two disorders may be interlinked, boys compared with full-term infants [18]. This could
with a possible origin in foetal life. If true, then this mean that mini-puberty is a check point and compen-
adds weight to the view that early development of the sation phase for reproductive organ growth in males as
foetal testis is critically important for adult reproduc- well as being a time of normal growth for these organs.
tive function and is thus an important period of sus- This could also mean that it is a window of opportu-
ceptibility. However, what factors might impact this nity for intervention to correct deficiencies in genital
vulnerability remains unknown. growth that may have arisen during foetal develop-
ment [19]. Indeed, several studies have shown at least
short-term benefits of hormone treatment of individu-
The Potential Importance of ‘Mini-puberty’ als with congenital hypogonadotrophic hypogonadism
Mini-puberty seems to be specific to species, such during the neonatal period [20], although long-term
as primates, that have a period of testicular quies- follow-up to adulthood is currently lacking.
cence (childhood) between birth and puberty. Dur- Chapter 16 has compared the incidence of repro-
ing mini-puberty in human males (0–6 months), FSH, ductive disorders in newborn males and young men
LH and testosterone levels are all substantially ele- from Finland and Denmark and shown that the Danes
vated and Sertoli cell proliferation is ongoing, likely have a higher incidence of disorders at birth as well
driven by both FSH and androgens [3, 15]; increase as lower sperm counts in adulthood, irrespective
in Sertoli cell number will account for most of the of their fertility status. This difference could repre-
increase in testis size that is seen during the mini- sent different environmental impacts on reproductive
puberty period (Figure 17.1). Three studies have mea- development in the two countries or it could be a
263
consequence of greater susceptibility of Danish boys to Impacts of Lifestyle and Environment on

such influences because of differences in development
of baby boys in the two countries. There is evidence Testis Development and Function
to support the latter possibility from a population- Chapter 16 has detailed the evidence for the perina-
based study that compared testis size and develop- tal origins of TDS disorders in human males and has
ment over the first 18 months of life in a total of reviewed the evidence that environmental chemical
⬎1,600 boys [17]. This showed that testis size (deter- exposures or lifestyle choices, such as smoking, may
mined by ultrasound) at birth and at 3 and 18 months have a role in causing TDS disorders. This will not
was significantly greater in the Finnish boys and that be reiterated here, but certain aspects will be com-
inhibin-B levels (reflective of Sertoli cell number) and mented on that are especially relevant to the topic
FSH levels (which is a driver of Sertoli cell prolifer- of this chapter. First, most of the evidence for envi-
ation) were correspondingly higher at three months ronmental impacts, based on association studies link-
in the Finns than in the Danes. The most dramatic ing maternal exposures to ‘effects’, is restricted to
difference, however, was in the growth in size of the disorders that are detectable around birth (hypospa-
testes from birth to three months, which was three- dias, cryptorchidism) [7]. Whether such exposures or
fold higher in the Finns. It is presumed that this growth lifestyle/dietary choices by the mother might affect
difference mainly reflects increase in Sertoli cell num- perinatal testis development with consequences for
ber (Figure 17.1) and although, in theory, further spermatogenesis in adulthood (Figure 17.1) is essen-
increase in Sertoli cell number can occur during onset tially unknown – for study design reasons outlined ear-
of puberty and perhaps during childhood quiescence, lier. There is one notable exception, as four large stud-
it is considered that the most important period is up to ies have all reported a significant association between
the end of mini-puberty (3–5 months) [3]. heavy maternal smoking in pregnancy and reduced
It seems likely that the Danish–Finnish testis size and/or sperm count in adulthood (see
population-based difference in sperm counts in chapter 16 and Ref. 21 for details); moreover, the mag-
adulthood (chapter 16) is directly connected to the nitude of decrease reported was up to 40%, which is
early postnatal differences in testis growth/increase substantial. What sets these studies apart is that it is
in Sertoli cell number [17]. This raises the question accepted that there is accurate recall of smoking dur-
of what determines this difference? It could reflect ing pregnancy, which is what enabled these retrospec-
differences in perinatal exposures (chapter 16) and/or tive studies to be undertaken and credible results to
differences in genetic makeup between the two pop- be obtained. However, if the same were to be done for
ulations [7]. If it is the latter, it would point to Danes maternal dietary factors, in particular specific dietary
being inherently more susceptible to the adverse components such as the types of fats consumed, it is
impacts of exogenous factors that might reduce sperm unlikely that accurate (and credible) results would be
production, as they start with a lower ceiling of sperm obtained. This is briefly discussed further in the sec-
production (? = lower Sertoli cell number). What tion on dietary effects (below).
is certain is that it is the number of Sertoli cells per Mini-puberty might be a period of susceptibil-
testis that determines the ceiling of sperm production, ity to outside influences that could perturb the nor-
and thus both testis size and sperm count, in an mal hormone-driven reproductive organ growth that
individual. The (limited) available evidence indicates occurs during this period, for example, breast-feeding
that in adult men Sertoli cell number can vary by up or bottle-feeding, as the former may contain accu-
to two orders of magnitude between individuals [3]. mulated lipophilic environmental chemicals. One U.S.
This astonishing variation almost certainly explains group has evaluated this prospectively in a group
the extremely high interindividual variation in sperm of 120 boys who were exclusively breast-fed, bottle-
count (after allowing for abstinence period) in men. fed with cow’s milk formula, or bottle-fed with soy
However, it is completely unknown what determines milk formula (which is rich in plant oestrogens). They
these huge differences in Sertoli cell number, whether found that at age four months, testis size (measured by
these are genetic/epigenetic factors or whether they ultrasound) was significantly smaller (by 13–20%) in
reflect differences in lifestyle, diet or exposures of the bottle-fed infants than in those that were breast-fed
mothers during pregnancy and/or of babies in the [22]. However, a follow-up of this same cohort of boys
immediate postnatal period. (N = 101) at age five years found no difference in testis
264
size according to infant feeding method [23]. This fits mals, both mother and foetus, do not show any major
with data from follow-up of adult men from a Dan- elevation in exposure to a range of common chem-
ish birth cohort, which showed no difference in semen icals known to be present in the sewage sludge, so
parameters or reproductive hormone levels according the increased exposure is at very low levels. Despite
to whether the men had been breast-fed or bottle-fed this, it has significant adverse effects on development
in infancy [24]. of the foetal testis, including reduction in Sertoli cell
There are two animal studies relevant to the ques- number at day 110 of gestation [27]. When such ani-
tion of susceptibility of the developing testis in the mals were followed into adulthood, it was found that
immediate postbirth period, although they come from 7 out of 12 sewage-sludge exposed males had normal-
rats and sheep, neither of which could be considered sized testes and normal spermatogenesis, whereas the
as having a distinct mini-puberty period as in humans. other 5 animals had much reduced testis size and var-
The study in rats involved exposure to daily high doses ious abnormalities of spermatogenesis [26]; indeed,
of the plasticizer dibutyl phthalate (DBP) during ges- the range of abnormalities that the latter group of 5
tation, which resulted at birth in 40% reduction in showed looked very much like the range seen in men
Sertoli cell number [25]. However, as Sertoli cell pro- in infertility clinics. Of course, it is not possible to con-
liferation continues until over halfway through lacta- clude from this that similar effects could, or do, occur
tion in the rat, the DBP-induced deficit in Sertoli cell in human males, but they illustrate the potential for
number evident at birth was corrected during lacta- this to occur at low levels of multichemical exposure
tion by elevated Sertoli cell proliferation, i.e., a com- that are human-relevant. The other important take-
pensatory change [25]. However, if DBP exposure (of home message from this study is that only certain indi-
the lactating mother) was continued after birth or the viduals are vulnerable to such effects, as more than half
mother was exposed to the anti-androgen flutamide, of the sewage-sludge-exposed sheep were unaffected in
then the compensatory increase in Sertoli cell prolifer- adulthood. In this respect, sheep, which are outbred,
ation in her male pups was attenuated or prevented. can be considered an accurate model for humans, and
Although this study involved exposure of the lactat- it is notable that they show more than a twofold range
ing mother to chemicals at levels far higher than are in testis size in adulthood between normal individuals
ever likely to occur in humans, it nevertheless illus- of the same strain [26].
trates the importance, and the potential vulnerabil-
ity, of the immediate postnatal period/mini-puberty
to breast-milk-derived compounds that might affect
Organization and Efficiency of Human
the testes. In this regard, two other points should be Spermatogenesis
noted. First, exposures of the mother during preg- Although the process of spermatogenesis is highly
nancy are likely to still be present after birth, so conserved in all mammals and most vertebrates, its
that continued exposure of the infant occurs, whether organization and efficiency show important species
directly or via breast milk. Second, breast-feeding can differences [1, 2], and the human seems to repre-
deliver lipophilic chemicals to the male infant during sent the worst case (Figure 17.2). In comparison to
mini-puberty that have been mobilized from mater- rodents and most domestic species, and indeed even
nal fat stores but which are not present in the infant’s most primates, the efficiency of human spermatogen-
environment. esis, measured as the number of germ cells supported
The second study involved exposure of sheep to a per Sertoli cell or as the number of sperm generated
complex mixture of environmental chemicals during per gram of testis tissue per day (daily sperm pro-
gestation and lactation via the use of sewage sludge duction, DSP), is far lower than in any other species
treatment of grazing pasture [26]. This followed EU studied (Figure 17.2). Considering the huge evolu-
recommendations on the disposal/recycling of sewage tionary selection pressures on reproductive efficiency,
sludge, which is routine practice across Europe. The this scenario may seem baffling, as it would have
human relevance of this study may seem obscure, but been expected that such poor efficiency would have
in fact it is highly relevant because the sewage sludge been vigorously selected against. Whatever the expla-
is derived predominantly from humans and therefore nation (see below), this poor efficiency probably plays
captures our complex daily chemical exposures, albeit an important determining role in the relatively low
in a rather crude way. Sewage-sludge-exposed ani- couple fertility in humans and the high incidence of
265
Figure 17.2 Comparative efficiency of spermatogenesis in human males in comparison to laboratory and domestic animals and nonhuman
primates [1, 2]. Data are shown for three different measures, daily sperm production (DSP) per unit weight of testis (top), number of germ cells
(spermatids) supported per Sertoli cell (middle) and number of spermatogonial generations/divisions during spermatogenesis (bottom).
Dashed lines show the mean value for humans.
couple infertility, as is discussed in depth elsewhere in aspects of spermatogenesis (Figure 17.2 and Ref. [2]).
this book. However, even if this thinking is correct, it should
In the context of the present chapter, the poor effi- not make us complacent about human male semen
ciency of human spermatogenesis can be viewed as a quality/infertility; rather it should make us more con-
very important susceptibility factor, because it leaves cerned, because in the modern context of aging female
little safety margin for coping with any exogenous fac- partners, this puts men in a highly vulnerable position
tors that impinge negatively on spermatogenesis. For that evolution has not been able to take into account.
example, in rats, which have super-efficient spermato- Moreover, if exogenous lifestyle/environmental factors
genesis when compared with man (Figure 17.2), it is are making human sperm production even worse, for
readily accepted that sperm production can be reduced which there is substantial evidence (chapter 16), then
(experimentally) by 50% or more without impinging the only possible outcome is increasing couple infertil-
in any major way on their fertility. A reduction of ity [28, 29].
such magnitude in a man would be likely to negatively Of the various measures of spermatogenic effi-
affect fertility, at least measured as time to pregnancy, ciency depicted in Figure 17.2, the fundamentally most
although whether it would result in infertility per se important is the small number of germ cells sup-
is likely to depend on what the ceiling for sperm pro- ported by each Sertoli cell, because ultimately this is
duction was in that individual to begin with (which the main determinant of sperm output/sperm counts.
will have been predetermined by his final Sertoli cell This appears to be due to the small number (two) of
number). There is also another, increasingly impor- spermatogonial divisions in humans compared with
tant, angle to low sperm production by human males other animals/primates (Figure 17.2). This means that
that relates to the later age at which couples are now the clones of spermatogonia (interlinked by cytoplas-
planning to try for children [28]. In most Western- mic bridges) that develop as a result of these divisions
ized countries, couples are not trying for a pregnancy are small, when compared with animals that have six
until the female partner is in her thirties, when her such cell divisions [1]. It is probably this that results
fertility is on the decline (which becomes precipitous in the different appearance of seminiferous tubule
beyond age 40). This scenario places greater emphasis cross-sections in humans (2–5 stages of the sper-
on the male partner having a high sperm count with matogenic cycle evident) compared with rats and pri-
good-quality sperm if the couple are to achieve a preg- mates/domestic species [30] that have 5–6 spermato-
nancy, but, as discussed in chapter 15, close to 20% gonial divisions (only one stage of the spermatogenic
of young men in Northern Europe now have a sperm cycle evident in seminiferous tubule cross-sections).
count low enough to impair fertility [7]. Although the It is possible, although unproven, that these differ-
effect of a lower sperm count may be only to prolong ent morphological arrangements may also contribute
the number of menstrual cycles that it will take for to the generation of more variable-quality sperm in
them to impregnate their partners, time is not on the humans, because the function of Sertoli cells varies
side of couples in which the female partner is in her according to the stage of the spermatogenic cycle [1];
thirties or older, which is becoming the new norm [28]. thus where neighbouring Sertoli cells are at different
Moreover, recent data argue that the fertility of women stages of the cycle they are probably functioning dif-
beyond age 30 may have declined [29]. ferently. In contrast, when all Sertoli cells are at the
An important question is why spermatogenesis is same stage of the spermatogenic cycle, and are thus all
so inefficient in humans. Indeed, we can also ask in this ‘doing the same thing’, this may result in a more con-
context why, in a normal man, only a small minority sistent (perhaps optimal?) environment for the devel-
of human sperm are morphologically normal, whereas oping germ cells.
in most species ⬎80–90% of sperm are usually clas-
sifiable as being morphologically normal. There are a Scrotal Cooling and Testis Temperature
number of evolutionary, longevity and mating strategy
theories that provide plausible reasons that there may Regulation
have been less selection for sperm number/quality in From a susceptibility perspective, the absolute need
humans than in other animals/primates, and it seems for spermatogenesis to take place at a temperature 3–
likely that one or more of these may account for human 4°C lower than core body temperature can be viewed
males being at the bottom of the league table for most as an inherent vulnerability of every man, if such a
267
Figure 17.3 Diagrammatic representation

of the processes via which testicular
temperature is maintained 3–4°C lower than
core body temperature. Factors that can
affect the process of testicular cooling are
shown to the left and impact either scrotal
heat loss or the heat-exchanger system in the
pampiniform plexus. Note the numerous
artery–vein anastomoses within the
pampiniform plexus.
temperature difference within the testes is not main- viding some mixing of arterial and venous blood, thus
tained. Under normal conditions, it is achieved by helping in the cooling process (Figure 17.3); addition-
having the testes in a scrotal sack outside of the ally, this arrangement reduces the pulsatility of arte-
body cavity and thus in a position to achieve heat rial blood that arrives in the testis. However, one con-
loss via evaporation from scrotal skin. This arrange- sequence of this exchange system is that the oxygen
ment, combined with a heat-exchanger system in load of incoming arterial blood to the testis is partly
the pampiniform plexus to maintain this temperature reduced, which is one reason that the testis is consid-
differential, is the mechanism via which cool testes ered to be poised on the brink of hypoxia. As the sem-
are ensured (Figure 17.3). This heat exchanger (the iniferous tubules have a high energy demand, because
pampiniform plexus) takes the form of a complex net- of spermatogenesis, but do not have a direct blood sup-
work of veins wrapped around the single spermatic ply, this may mean that the degree of hypoxia is exac-
artery, within which there is a network of artery–vein erbated within the seminiferous epithelium. Although
anastomoses. The latter are important because they this arrangement is normal, it means that any elevation
effectively siphon off 50% of warm incoming arterial of testicular temperature as a consequence of impaired
blood into outgoing venous blood [31] as well as pro- cooling (see below) will increase oxygen/metabolic
268
demand and could thus push the testis and spermato- and/or being overweight/obese (see below) will exac-
genesis into overt hypoxia, which would be detrimen- erbate any impact of a sedentary lifestyle on semen
tal to the developing germ cells. Hence, if scrotal cool- quality.
ing ability is impaired, for example, by using a sauna, Despite the clear evidence for a harmful effect of a
then reversible impairment of sperm number, quality sedentary lifestyle on scrotal temperature, there is no
and DNA integrity is found [32]. strong evidence that this significantly impacts men’s
fertility [35, 37, 40, 41]. However, it seems intuitively
Impacts of Lifestyle and Environment on sensible that men who are planning to become fathers
should modify their lifestyle to minimize any impact of
Scrotal Cooling impaired scrotal cooling, as this can only have a bene-
The essential need for scrotal cooling applies to all ficial effect on spermatogenesis. Indeed, where this has
mammalian species in which adult males have scrotal been done in a properly controlled way, it has been
testes. However, the critical difference between non- proven to be beneficial in men [42]. Furthermore, in
human mammals and man is that man is the only studies of men presenting with infertility, those with a
species that wears clothes around the scrotum, which sedentary lifestyle are overrepresented [43], and when
will inevitably interfere with the efficiency of heat loss semen quality has been evaluated in relation to physi-
via scrotal skin. This cooling applies equally to the cal activity levels of men, there is unanimity in showing
testis and epididymis, although there is still debate as superior sperm counts in physically active men com-
to how important epididymal cooling is for sperm at pared with those in men with low levels of physical
this stage. activity [41, 44]; higher physical activity is likely to
Most men are aware of the scrotal cooling issue to result in more effective scrotal cooling.
some extent, either via the issue of tight underwear or There are additional reasons that men should min-
wearing a kilt with no underwear [33], or the use of imize lifestyle-related impairment of scrotal cooling,
heat-generating laptops [34], and such topics are reg- and these relate to DNA damage to the sperm. Exper-
ularly joked about. However, few if any will think that imental studies in laboratory rodents have shown
impaired scrotal heat loss is an issue for them person- that even moderate scrotal temperature elevation by
ally, as nontight underwear (e.g. boxer shorts) is now immersion in a water bath at 40–42°C (equivalent to
the most common form of underwear in young men a moderately hot bath) for a reasonably short period
and most men now take showers, which do not inter- (30 min) is sufficient to cause a cascade of changes
fere with scrotal cooling, rather than hot baths, which in developing germ cells and in sperm themselves,
interfere with scrotal cooling. The issue of scrotal heat- including increased apoptosis [45]. Of more concern
ing via laptop use has been raised (Figure 17.3), but is that this heating also induces oxidative stress and
does not appear to be an important factor. However, DNA damage, which leads to both impaired male fer-
the reality is that impaired scrotal cooling is a poten- tility and altered embryo development when fertiliza-
tially important issue for many young men in modern tion is achieved [45, 46]. Such effects are equally evi-
societies, not because of the clothing issue but because dent in nonhuman primates [47] and are likely to apply
of our modern sedentary lifestyles. also to humans [48]. The extent to which a sedentary
Using continuous scrotal temperature monitoring lifestyle, which induces more modest scrotal temper-
devices, it has been shown that with increased time ature elevation than hot baths, but for longer periods,
spent seated there is a small but time-related increase might induce DNA damage and its downstream con-
in scrotal temperature [35–37]. Thus, those whose sequences is unclear, as this cannot really be studied in
occupations are sedentary (e.g. office/online workers, animal models.
drivers) are at risk of their scrotal temperature being Changes in men’s lifestyle that potentially inter-
significantly elevated for a substantial part of their fere with scrotal cooling are arguably one of the most
working day [38, 39]. Such effects are exacerbated by important susceptibility factors to have changed dra-
the fact that a proportion of such men also have a matically in recent decades, and one that is read-
sedentary lifestyle outside of work, whether watch- ily amenable to correction. However, one factor
ing TV, being online or playing computer games. It that has changed even more dramatically is men’s
seems likely that wearing tight underwear/clothing diet.
269
Dietary Effects on the Testis and Their eral. There is abundant human evidence that obesity,
especially visceral obesity, is associated with reduced
Interplay with Susceptibility Factors testosterone levels in both young [49] and old men [50]
Most of us will have been told by our parents that ‘you as well as with reduced semen quality [51]. Despite
are what you eat’, and although this may be an over- the wealth of such association data, it is not possible
simplification, it has an element of truth to it. This to draw conclusions from these studies as to whether
is evidenced by the inexorable increase in incidence specific dietary components are responsible for the
of human obesity and its associated metabolic dys- adverse reproductive changes or whether they result
functional disorders (and downstream health conse- from the overall metabolic changes triggered by obe-
quences) that has followed in the wake of our adopt- sity. As discussed below, it seems likely that both diet-
ing a Western-style diet involving overconsumption specific and metabolism-related changes play a role
of refined sugars, saturated fats, and calories in gen- (Figure 17.4). Perhaps more importantly, studies of
Figure 17.4 Diagrammatic summarization of the different levels at which the diet of an adult male can potentially affect testis/epididymal
function and/or sperm. Some of these effects may be general, for example, as a consequence of obesity, and some may relate to specific
components of the diet. Details are given in the text.
270
diet-specific effects, especially on sperm function, sug- greater were the resulting reductions in DSP and epi-
gest that Western-style changes to men’s diet might didymal sperm counts (up to 33% reduction) as well as
affect fertility in some individuals. in testosterone levels. These changes were associated
with significant increases in blood leptin. While this
study appears to indicate that gestation-only exposure
Effects of Diet on Development of the Testis to HFD may not be detrimental, this may not mean
It is well established that altered growth or pace of that no effect occurred; it could mean that an effect
development of the foetus can influence health in occurred that was subsequently compensated for in the
adulthood, including predisposition to obesity and early postnatal period, as described earlier for rats in
cardiovascular disease [52]; the growth changes in which an experimentally induced 40% decrease in
the foetus may be attributable to obesity or metabolic Sertoli cell number at birth was compensated for after
dysfunction in the mother, and perhaps to specific birth [25].
dietary factors. A few studies have investigated if
maternal obesity can influence subsequent male repro-
ductive development (e.g. congenital disorders such as Effects of Diet on Hypothalamic–
hypospadias, chapter 15) or reproductive function in Pituitary–Testis Function and Interplay
adulthood, although there is a paucity of studies on the
latter because of the considerable challenges involved
with Metabolism
in trying to link together events that are two or more Obesity in adult men exerts quite profound effects on
decades apart in humans. The largest study was in a the HPT axis (Figure 17.4), the net effect of which is to
Danish birth cohort (N = 347), but this did not pro- lower testosterone levels irrespective of co-morbidities
duce convincing evidence that maternal obesity was such as type 2 diabetes or cardiovascular disease [57–
associated with any dramatic adverse change in semen 59]. The most common mechanism for this decrease is
quality in resulting sons [53, 54]. In another Danish a reduction in LH levels, indicating secondary hypo-
birth cohort, the authors examined the influence of gonadism, rather than a compensatory increase in LH
maternal BMI on timing of pubertal landmarks (acne, levels due to reduced testosterone negative feedback,
shaving, voice breaking, timing of first nocturnal emis- which is primary hypogonadism and is less common
sion) in resulting sons, and found some evidence for [59]. Thus obesity results in abnormal hypothalamic-
earlier occurrence of these in sons of obese mothers pituitary function, probably for a range of reasons
[55]. There are no studies that have looked at whether including increased aromatization of testosterone to
specific aspects of women’s diet during pregnancy are estradiol in adipose tissue, peripheral and central
associated with changes in semen quality or testos- insulin resistance and pro-inflammatory cytokine pro-
terone levels in adulthood in resulting sons. The only duction (TNF-␣ and IL-6) from adipocytes [59, 60].
relevant information comes from animal studies. Indeed, all of the obesity-related cardiometabolic dis-
An interesting experimental animal study in rats orders are associated with lowered testosterone levels,
evaluated when in development exposure to a high and there is a vigorous ongoing debate as to whether
fat diet (HFD) was most damaging to male reproduc- the low testosterone levels are a cause or a consequence
tive function in adulthood [56]. They exposed devel- of these disorders [61]; probably the best interpreta-
oping males to HFD (20% vs. 4% in controls) either tion of the huge amount of evidence is that it is a bidi-
during gestation, during gestation + lactation, from rectional relationship [13, 61]. In the context of the
weaning to adulthood, from lactation to adulthood, present chapter, the key fact is that obesity and modern
from gestation to adulthood or just in adulthood, and Western diseases are all associated with reduced testos-
then evaluated daily sperm production (DSP) by the terone production by the testis, irrespective of whether
testis and epididymal sperm counts and blood levels this is a primary or secondary effect.
of testosterone and leptin in adulthood. They found
no effect of gestation-only exposure on the endpoints Effects of Diet on the Gut Microbiome and
studied, but some effect of exposure during gestation
+ lactation on DSP (17% reduction) and epididymal Its Testicular Consequences
sperm counts (8% reduction). The main finding was It has become increasingly evident that changes in the
that the longer the exposure to HFD after birth, the gut flora of individuals can exert major effects on their
271
physiology and wellbeing, playing important roles in during pregnancy [64]. One recent study suggested
immune function, nutrition, metabolic function and that a normal gut microbiome is important for nor-
insulin sensitivity, among others [62]. The main fac- mal development of the blood–testis (immune) barrier
tor affecting the microbiome is diet, although sporadic in mice [66].
factors such as exposure to antibiotics can also exert
effects, though probably more transiently than diet. It Effects of Diet on Scrotal Cooling
may therefore be that eating a diet rich in saturated fat,
Although there is no evidence that specific dietary
for example, induces some of its adverse health effects
components might affect scrotal cooling, there are
via alteration of the gut microflora [62]. In this con-
several ways in which adult obesity might impair it
text, a recent detailed study showed that exposure of
(Figure 17.4). Most obviously, fatter men tend to be
mice to emulsifiers that are commonly added to pro-
less active and to devote more of their time to seden-
cessed food altered the gut microbiota, resulting in col-
tary habits, which, as already discussed, will lead to a
itis and metabolic dysfunction [63]. These findings are
degree of elevation of scrotal temperature. Extremely
mentioned in the present context because of the close
obese men may have abdomens that literally flop over
interrelationship between altered metabolic function
their external genitalia when seated, which is likely to
and male reproductive function, especially steroidoge-
impair scrotal cooling even more. In addition, there is
nesis, as outlined above.
some evidence that obesity leads to increased deposi-
There is sporadic evidence that the gut microbiome
tion of fat around the spermatic veins and within or
and its metabolites can affect aspects of male repro-
underneath the scrotal skin in association with infer-
ductive function [64], although there have not been
tility [67], changes which would be expected to provide
enough systematic studies to yield a clear picture of
a degree of insulation for the testis and to impede scro-
how important this might be (Figure 17.4). However,
tal heat loss. A recent study that used 24-h scrotal tem-
their potential importance is highlighted by a convinc-
perature monitoring in obese (BMI ⬎ 30) vs. control
ing study which showed that altering the gut micro-
men (BMI ⬍ 25) found a significant increase in scro-
biota of mice by feeding them from eight weeks of age
tal temperature in the obese men, although the magni-
(young adulthood) with a particular probiotic species,
tude of elevation was only in the range 0.5–0.8°C [68];
Lactobacillus reuteri, was able to completely prevent
nevertheless, this was associated with poorer semen
age-related development of impaired spermatogene-
quality. Of particular interest was the finding that, in
sis, reduced testis weight and reduced testosterone
control men, scrotal temperature showed quite marked
levels [65]. These preventive changes by the probi-
variations during the 24-h period, probably indicative
otic treatment were induced equally in mice main-
of effective scrotal cooling, whereas these fluctuations
tained on a standard diet and those maintained on a
were absent in the obese men, as well as in nonobese
Westernized diet. The same authors had shown ear-
men who had a varicocele [68].
lier that the same probiotic feeding regime was pro-
tective against diet-induced age-related obesity and
also helped maintain much healthier fur. Most impor- Effects of Diet on Sperm Function
tantly, a potential mechanism of effect was identi- There is a reasonably large literature documenting that
fied, as it was shown that the beneficial reproduc- men’s diet, in particular their consumption of fats and
tive effects of the probiotic treatment could be repli- the types of fats consumed, is associated with various
cated by administration to the mice of antibodies to adverse changes to semen quality (Figure 17.4). The
the pro-inflammatory cytokine interleukin-17A [65]. most basic evidence demonstrates that obesity is asso-
As low testosterone levels and obesity are intrinsically ciated with adverse changes in sperm concentration,
linked with a more pro-inflammatory cytokine pro- sperm motility and total sperm/motile sperm count
file in the blood [60], the possibility that this could [69, 70], and the greater the obesity the more severe
be ameliorated via dietary change or probiotics is an are the adverse semen quality changes [51]; the lat-
intriguing area for future research. It is also clear that ter study is based on a meta-analysis of 21 studies and
the gut microbiome can have important developmen- a total of 13,077 men both from the general popula-
tal effects, although how this might affect the devel- tion and from infertility clinics. In a cohort study of
oping reproductive system remains to be explored in 43 men with a BMI ⬎ 33 it was shown that a residen-
detail, especially the role of the maternal microbiome tial weight loss programme, resulting in a mean weight
272
loss of 15%, was associated with an increase in total gate for a broader change in diet (i.e. a Western-style
sperm count and semen volume [71]. In an IVF setting, diet). However, there are a growing number of exper-
male obesity is associated with reduced rates of clini- imental animal studies which strongly support the
cal pregnancy and live births as well as with increased idea that a HFD, especially a high-saturated-fat diet,
pregnancy loss [72], perhaps due to altered (slower) leads to adverse changes in sperm and semen quality
embryo development, which is also well described in (Figure 17.4), and such studies have begun to identify
animal models of male obesity [73]. potential mechanisms of effect. Thus a small study in
Other studies have explored the association rabbits showed that feeding males for 12 months on
between specific components of diet and semen qual- a HFD, rich in saturated fats, resulted in reductions in
ity, based on validated food frequency questionnaires. sperm counts and sperm motility and increased sperm
For example, higher cheese intake, although not morphological abnormalities [81]. All of the HFD-
full-fat dairy intake per se, was associated with lower induced changes in semen quality could be reversed
sperm concentration in men from infertile couples by co-feeding the rabbits with olive oil plus the HFD
[74]. The same study showed that consumption of for 4 months; feeding with olive oil for 12 months on
low-fat dairy foods, predominantly low-fat milk, its own had no beneficial effect on semen quality in
was positively associated with sperm concentration this study [81]. These findings suggest that it is not
and with sperm motility. In a similar cohort of 99 a HFD per se that causes adverse sperm changes but
men, total fat intake (mainly saturated fats) was rather the types of fats/fatty acids that are available in
associated with a reduction in sperm concentration the diet.
[75]. Interestingly, this study also showed that the Two studies in transgenic mouse models sup-
levels of saturated fatty acids in sperm themselves port this interpretation. The first used the liver X-
were negatively correlated with sperm concentration receptor-deficient mouse, which exhibits cholesterol
[75]. A similar dose-dependent association was found ester accumulation because of this deficiency, and
between saturated fat intake and reduction in sperm showed that feeding these mice a cholesterol-enriched
concentration and total sperm count in a relatively diet led to severe post-testicular sperm changes that
large cohort study of young Danish men (N = 701) resulted in infertility [82]. The authors observed that
from the normal population [76]; the highest quartile within the epididymides of the infertile mice there
of saturated fat intake was associated with a reduction was the formation of interstitial ‘foam cells’, which
of 41% in total sperm count. In a smaller group of then migrated across the epithelium to the epididymal
similar young Spanish men (N = 209), it was found lumen, a change akin to the process of atherosclerosis.
that intake of trans fatty acids (mainly manmade from The second study used mice deficient in the enzyme
vegetable fats) was inversely related to total sperm (Fads2) responsible for synthesis of docosahexaenoic
count [77]. In the same group of men, the authors acid (DHA), an important omega-3 fatty acid. These
showed that consumption of a Mediterranean-style Fads-null mice show a failure of acrosome develop-
diet was associated with a higher total sperm count ment in spermatids due to lack of fusion of the pro-
than consumption of a Western diet [78]. However, acrosomic vesicles [83]. Remarkably, dietary supple-
such an association was not found in a U.S. study mentation of DHA to these mice results in restoration
of young men (N = 188), although this did find a of an intact acrosome. Considering the pivotal impor-
positive association between eating a Mediterranean- tance of the acrosome in fertilization, the fact that
style diet and progressive sperm motility [79]. Finally, acrosomal development can be influenced by dietary
an Iranian case-control study (N = 107 vs. 235) fatty acids is quite compelling.
of asthenozoospermic men found that increases in The animal findings just described may have rel-
intake of saturated fats or trans fatty acids were both evance to observations in humans which show that
associated with increased risk of asthenozoospermia, higher intake of omega-3-rich fats is positively related
whereas intake of omega-3 fatty acids was associated to good sperm morphology [75], although it is also
with reduced risk [80]. clear that omega-3 fatty acids play an important role in
The association studies outlined above cannot maintaining fluidity of the sperm plasma membrane,
prove cause and effect for fat intake and semen qual- which is important for fertilization [84]. In this regard
ity changes in men, nor do they exclude the possibil- the thinking is also that lower cell membrane con-
ity that measures of fat intake may simply be a surro- tent of omega-3 fatty acids renders the sperm more
273
susceptible to oxidative damage from free radicals Concluding Remarks and Future
[84]. As these authors point out, a modern Western
diet results in a roughly 10:1 intake of n-6 to n-3 Prospects
polyunsaturated fatty acids (PUFAs), the n-3 being the A question that arises from the foregoing overview
omega-3 form, whereas in a primitive human diet the of the inherent vulnerabilities of the testis to adverse
amounts ingested are roughly equal. effects is how this recognition might be used to
Direct evidence that the ratio of n-6 to n-3 PUFAs improve male reproductive health, in particular testis
in sperm may actually result in functional changes function. It is right that we should place emphasis
comes from another animal study, this time in boars. on the negatives, such as the horrendously high inci-
This study looked at differences in sperm motility in dence of young men with low sperm counts (⬍20 mil-
a relatively large group (N = 106) of adult boars, and lion/ml), as discussed elsewhere, but focusing on the
divided them into two groups on the basis of either vulnerabilities also offers the possibility of consider-
high (⬎60% motile sperm; N = 53) or low (⬍60% ing how to use such understanding/recognition pos-
motile sperm; N = 53) sperm motility [85]. The mean itively. As this chapter has hopefully shown, there are
sperm motility in the two groups was 82.6% ver- inherent vulnerabilities of the testis that are a conse-
sus 30.6%, a substantial difference. They found that quence of the way it is set up to work normally (i.e.
sperm from the high-sperm-motility group had a more cooler temperature and organization of spermatoge-
favourable n-6:n-3 PUFA ratio and a higher overall nesis), neither of which fit very well with the cur-
content of PUFAs, including 50% higher DHA (an n- rent lifestyles of young men (and their female part-
3 PUFA) content, than sperm from the animals with ners). Added to this is the growing recognition that
low sperm motility. These changes were also associ- early development in foetal, and perhaps early post-
ated with levels of total antioxidant levels in sem- natal, life is a critical determinant of later testis func-
inal plasma, which were higher in the group with tion, meaning that anything that can negatively impact
high sperm motility [85]. It must be kept in mind this developmental process is likely to have lifelong
that this is an association study, so therefore it does effects. While we remain rather ignorant about what
not prove that it is low n-3 PUFA levels that actually factors can exert such effects, there is reasonable evi-
cause lower sperm motility in boars, but by measur- dence that these unknown factors are taking their toll
ing levels directly in sperm it provides persuasive evi- (chapter 15 and Ref. [7]). If the factors can be identified
dence that this is likely. Combining a feeding study, then there is the possibility of avoiding them, which
involving different amounts of n-3 PUFAs, with direct would help ensure that testis development occurs opti-
sperm measurements should be able to resolve this mally; this is a difficult research area but an obvious
issue. priority.
Bearing in mind the various animal studies out- With particular regard to final Sertoli cell number
lined above, especially the study in Fads-null mice, (and thus sperm count), far better understanding is
it is tempting to conclude that the change in relative needed of the importance of mini-puberty as a period
amounts of PUFAs that occurs with a Western diet may in which compensatory increase in Sertoli cell num-
be bad news for human sperm development and func- ber can occur, as it is theoretically possible that some
tion. If so, the good news is that it is potentially fixable form of intervention could be possible at this time to
via dietary change or supplementation. enhance Sertoli cell proliferation and thus to correct
One of the concerns about sperm vulnerability to any deficiency that has occurred during foetal life; this
oxidative damage is the potential damage to DNA presupposes that individuals in need of intervention
that free radicals can induce [84]. DNA damage has can be correctly identified, and an appropriate inter-
the potential to affect subsequent embryo develop- vention is feasible [19].
ment and perhaps cause lifelong health/susceptibility Regarding the inherently poor organization of
changes for the resulting person. Whether diet influ- spermatogenesis in men, there seems to be nothing
ences these processes is vigorously debated [86], but that can be done to improve upon this other than
the use of antioxidant-rich dietary supplements (e.g. focusing on minimizing any extraneous factors that
vitamin C + E) is actively being investigated in infertile may erode sperm production further. Evolution may
men, so far with some, if limited and variable, success not have needed to apply the same ‘survival of the
[87]. most fertile’ pressure to human males as for many
274
other species, but this was nevertheless designed for lined above. In this regard, we will need to be vigilant
a scenario in which female pregnancy occurred rel- as data emerge, because if supplementation proves to
atively early after menarche, not halfway or more be a useful way forward, it will attract the full range
through a woman’s reproductive lifespan, as occurs of monetary sharks that lurk around the human fertil-
now. Societal changes have rewritten this scenario in ity/infertility arena, and their eyes are invariably more
such a way that it now places demands on male fer- focused on the opportunity to make money than on
tility that a significant proportion of men struggle actual evidence of benefits.
to meet. Assuming that the situation regarding preg-
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Chapter
Mouse Genetics – How Does It Inform Male
18 Fertility Research?
Laura O’Hara and Lee B. Smith
Male Infertility: The Role of Genetic or affect the transit of sperm through the male repro-
ductive tract up to and including ejaculation; sper-
Background matogenesis is usually present. Contributing disorders
include congenital absence of vas deferens, inflamma-
Causes of Infertility tion of the reproductive tract from bacterial infection,
It is estimated that infertility affects between 4% and retrograde ejaculation and ejaculatory failure. In some
17% of couples who are trying to conceive [1]. Approx- cases the number of sperm in the ejaculate is normal,
imately 1 in 7 heterosexual couples in the United King- but their morphology is abnormal (teratozoospermia).
dom seek advice about difficulties in getting pregnant This may be caused by malfunction of the epididymis.
[2], and a male factor is estimated to be involved in Men with testicular and post-testicular infertility may
more than 40% of these cases [3]. not be diagnosed until they attempt to start families,
Physiological causes of male infertility can result as infertility is their only presenting symptom. A spe-
from dysfunction at several different points of the cific cause of infertility is not found in a fourth of cases,
male reproductive axis. Pretesticular factors include and it is then defined as ‘idiopathic’ [4]. This diagnosis
disorders of the hypothalamus or pituitary resulting is in part a result of poor understanding of the basic
in impaired production of gonadotrophins (hypogo- mechanisms regulating male fertility.
nadotrophic hypogonadism). Some causes of hypog-
onadotrophic hypogonadism are due to genetic dis-
orders such as Kallmann syndrome and Prader–Willi Genetic Lesions in Infertility
syndrome; other causes include abuse of anabolic From 15 to 30% of male infertility cases are thought
steroids resulting in repression of endogenous luteiniz- to have a genetic cause, the etiology of which can
ing hormone or damage to the pituitary gland or range from aneuploidy to a single nucleotide base
hypothalamus from an insult such as surgery, injury change [5]. Deletions or duplications of large regions
or infection. Because of the abnormal hormone of genetic material in autosomes will generally result
profiles in men with these disorders, they are often in a severe developmental phenotype, but lesions that
diagnosed when puberty fails to progress. Testicu- concern the sex chromosomes generally have a sub-
lar factors are associated with failure of normal sper- tler phenotype, often connected with infertility. Thir-
matogenesis when the pituitary hormone support is teen percent of men who present with azoospermia
adequate. Spermatogenesis may be completely absent or severe oligozoospermia are found to have Kline-
(Sertoli-cell-only syndrome), arrested at an imma- felter’s syndrome, 47 XXY aneuploidy caused by mei-
ture stage or present but with a significant reduction otic nondisjunction in either the maternal or pater-
in sperm production. Primary spermatogenic failure nal germ cell, in which spermatogenesis usually arrests
can result from genetic disorders such as Klinefelter’s at the primary spermatocyte stage (reviewed in [6]).
syndrome, testicular cryptorchidism, trauma or dam- The human Y chromosome contains over 200 genes,
age sustained during diseases such as mumps. Post- many of which code for proteins involved in spermato-
testicular factors include conditions that either prevent genesis [7]. From 11 to 18% of azoospermic men are
University Press.
280
Chapter 18: Mouse Genetics – How Does It Inform Male Fertility Research?
found to have a microdeletion of part of the azoosper- The Mouse as a Model Organism
mia factor (Azf) region on the long arm of the Y chro-
The house mouse, Mus musculus, has been growing
mosome (reviewed in [8]). Mutations in an individ-
in popularity as a mammalian model organism for
ual gene can cause a spectrum of effects on its protein
human disease since the beginning of the twentieth
function, from a complete loss of function (null allele),
century due to its anatomical and physiological sim-
to a partial loss of function, to no effect on function, to
ilarity to humans. Its small size, basic needs (food,
a change in function that is not deleterious, to a gain
water, bedding, controlled temperature and light reg-
of function.
imens and a small amount of environmental enrich-
Even though hundreds of genes are known to
ment), short generation time (approximately 10 weeks
be involved in human reproduction, only a few are
from being born to giving birth) and large litter num-
investigated clinically on presentation of infertility,
ber (an average of 5–10 pups) allow rapid generation
and this is usually only if the presentation is con-
of large numbers of experimental animals [11]. The
nected to a particular genetic syndrome. An exam-
availability of congenic inbred strains generated from
ple would be obstructive azoospermia caused by con-
at least 20 generations of sibling intercrosses which
genital bilateral absence of the vas deferens (CBAVD).
are essentially genetically identical and homozygous
The most likely cause of CBAVD is a mutation in the
at all loci limits any influence of genetic variation
cystic fibrosis transmembrane conductance regulator
on experimental outcome [12]. The mouse genome
(CFTR). Ninety-seven percent of men with clinical
sequence was first published in 2002 [13] after an inter-
cystic fibrosis (characterized by chronic lung infection
national collaborative effort. Subsequent analysis dis-
and inflammation, pancreatic insufficiency and defec-
covered that there are mouse homologues of 99% of
tive electrolyte and fluid transport) also have CBAVD,
human genes [13]. More recently, the generation of
but a high frequency of CFTR mutations is also found
transgenic mice by oocyte pronuclear microinjection
in men with CBAVD in the absence of other clinical
and gene targeting in embryonic stem (ES) cells has
manifestations of cystic fibrosis [9].
proven highly successful in mice but less adaptable in
When so many genes are thought to be involved
other species, resulting in an exponential increase in
in infertility (and more are yet to be characterized),
the number of transgenic mouse lines being produced
and of those known, little is understood of the under-
and available [14].
lying mechanisms, it is likely that many cases that
in fact result from genetic lesions are simply labelled
as ‘idiopathic’ due to a lack of understanding, and How Can Mouse Genetics Inform Human
not investigated further. When many cases of infertil-
ity can be treated with intracytoplasmic sperm injec- Male Fertility Research?
tion (ICSI), diagnosing and understanding the cause Studies of human genetic disease are often compli-
is often not seen as necessary for treatment to pro- cated by difficulties in accessing tissues or by genetic
ceed. Although the American Society for Reproduc- heterogeneity in families inheriting these disorders.
tive Medicine has concluded that ICSI appears to Also, unlike other genetic conditions, the inherent dif-
be a safe and effective therapy for the treatment of ficulty in determining genetic causes of infertility is
male-factor infertility, it also notes that certain con- that the inability to produce offspring makes classical
ditions may carry an increased risk for transmission pedigree analysis challenging or impossible. Because
of genetic abnormalities to offspring via ICSI [10]. of these limiting factors, model organisms have been
Whether the increased prevalence of genetic abnor- utilized for research into the genetics of fertility. The
malities observed in ICSI offspring relates to the pro- most commonly used model organism for male repro-
cedure itself or to the characteristics of couples that ductive research is the mouse. The anatomy and phys-
require ICSI to conceive is unclear. It is clear, how- iology of the male mouse reproductive system are very
ever, that for ICSI to be safe and efficient, a more similar to those of the human male reproductive sys-
detailed knowledge of the parents’ genomes is desir- tem, although there are some differences such as the
able. And, of course, ICSI, which relies on germ cell organization of spermatogenesis within the seminif-
progression to at least the round spermatid stage, is erous epithelium and the relative size and organiza-
not a suitable treatment option in many cases of male tion of some of the components of the male repro-
infertility. ductive tract. However, the genetic, endocrine and
281
physiological similarities are close enough so that this, the resulting types of genetic models that are pro-
the mouse has been a vital model organism in iden- duced and databases that archive these models for use
tifying important genes and elucidating their roles by researchers.
in the development and function of human male
reproductive processes. In 2011 it was estimated that Techniques in Reverse Genetics
over 400 genes essential for male fertility have been
revealed from analysis of mouse models, and this Genetic Manipulation of the Early Mouse Embryo
figure increases year after year [15]. Mouse genet- Genetic manipulation of the early mouse embryo
ics has provided us with several important tools for produced the first germline-transmissible transgenic
the advancement of research into male infertility. It mice in 1976. Mouse embryos were transduced with
can be used to increase fundamental knowledge, both Moloney leukaemia retrovirus, and this was shown to
to identify causal genes and importantly to dissect be stably integrated and transmitted to the mouse’s
gene/protein function and the mechanisms underlying offspring [16]. A shift away from transduction-based
infertility (something very challenging in humans). It methods occurred with the advent [17] and develop-
can be used to develop new methodologies and also as ment (reviewed in [18]) of pronuclear microinjection
a preclinical test bed for new therapies. Finally, it can in the early 1980s. Most transgenic mice are now pro-
be used to identify targets for novel male contracep- duced by this technique, in which DNA is microin-
tives; defining the cause of infertility in one man opens jected into a pronucleus of a single-cell mouse embryo
up the ability to subvert this in all men. Without these and integrated randomly into the genome. Typically,
methods it would take a lot longer for any potential multiple copies of the transgene will insert into a sin-
new therapies to reach the clinic. In this review we will gle chromosomal locus in the embryo. The size of the
discuss techniques in mouse genetics and give specific DNA insert can range from smaller than 1 kb up to
examples of how they have contributed to an increase bacterial artificial chromosomes (BACs) that are 150–
in knowledge of male infertility, either by identifying 350 kb. After injection, the single-cell embryos are
new genes involved in fertility pathways or by the gen- transferred to the oviducts of pseudo-pregnant foster
eration of specific models of human diseases. mothers. The offspring resulting from injected eggs
may or may not carry the transgene. On average, about
10–20% of the mice born will test positive for inheri-
Gene-Driven Models (Reverse Genetics) tance of the transgene.
‘Reverse genetics’ is the process of manipulating a More recently, transgenic animals have been cre-
known gene of interest in order to investigate the ated by transducing early embryos with lentiviruses
resulting phenotype. The manipulation can take the containing transgenes [19]. Even though this tech-
form of a knock-out, a knockdown, an overexpression nique is technically less demanding than pronuclear
of an endogenous gene or a knock-in of an exoge- microinjection, its disadvantages are the increased
nous gene. In most cases, there is a requirement for workload required for the production of lentiviral vec-
the genomic change to be throughout the body and tors, the vector only taking transgenes that are 10
also transmissible to offspring, so the genomic change kb or smaller and the increased number of copies
must be introduced at the germ cell or early embryo and dispersed integration sites in the host genome.
stage and be stably integrated into the genome. The Because lentiviral transgenes are dispersed across dif-
most important aspect of reverse genetics is that it ferent chromosomes, onward breeding results in seg-
relies on some prior knowledge, understanding or pre- regation, reducing transgene copy number and poten-
cognizance that the gene you are interested in is likely tially the resultant phenotype with each generation;
to be involved in the promotion of male fertility. Of in addition, each offspring from a transgenic parent
course in many cases this is impossible, and many must be considered to be a unique founder. Although
genes important in male fertility have been revealed genetic manipulation of early mouse embryos can
serendipitously because researchers interested in other result in fast generation of transgenic lines, the trans-
body systems discovered that their genetically mod- gene is randomly integrated into the genome and can
ified mice were infertile. Here we discuss the tech- be prone to epigenetic or heterochromatic silencing,
niques in mouse genome manipulation that facilitate depending on where it inserts in the genome.
282
Direct Germline Manipulation all or part of its open reading frame with a transgene.
Generation of transgenic mice by early embryo trans- Homologous recombination is an endogenous process
genesis is a time-consuming method that requires sig- used by cells to repair deleterious double-strand breaks
nificant numbers of mice due to the breeding required using a similar or identical intact DNA molecule as
and inherent inefficiency of the technique. Direct a template (reviewed in [27]). In somatic cells the
manipulation of the male germline of a mouse to pro- homologous chromosome is used as a template, so the
duce transgenic progeny aims to reduce time, waste sequence may not be identical. It also occurs in mei-
and costs. otic cells to produce chromosomal crossover between
Initial attempts to modify the male germline non-sister chromatids to produce genetic variation in
focussed on the isolation and transgenic modification gametes. Gene targeting involves introducing a tar-
of the diploid spermatogonial stem cell (SSC) pop- geting vector via electroporation into embryonic stem
ulation. It had been difficult to purify this cell pop- (ES) cells growing in culture (Figure 18.1). The tar-
ulation until in 1994 an assay in mice reported that geting vector usually consists of two regions homol-
SSCs could be identified by their ability to repopulate ogous to portions of the endogenous gene, flanking
the seminiferous tubules of a germ-cell-deficient male a replacement sequence which can consist of a com-
after transplantation [20]. Identification of these cells bination of a selection marker (such as a ‘neomycin
led to attempts at long-term culture, and a regimen resistance’ aminoglycoside phosphotransferase gene),
was eventually described that allowed their long-term a lineage tracer (such as ␤-galactosidase or green flu-
proliferation and high rate of survival in the presence orescent protein) or a modified version of the endoge-
of a combination of growth factors [21], while ensur- nous sequence itself (reviewed in [28]). The cells will
ing that cells remained viable following transplanta- then use their own homologous recombination sys-
tion. Long-term cell-line culture of SSCs has made tem to replace their endogenous gene with the trans-
it possible to genetically manipulate them in vitro gene. The ES cells are selected for transgene incorpo-
before transplantation and colonization of a recipient. ration by growth with an antibiotic such as geneticin
Manipulation of the rodent SSC genome has been per- (G418). ES cells are then clonally expanded and tested
formed using retroviruses in vitro [22] and in vivo for specific incorporation at the site of interest, ini-
[23], although both approaches resulted in a poor suc- tially by the PCR, with confirmation by Southern blot-
cess rate, and lentiviruses in vitro, with slightly bet- ting. Transgenic ES cells can then either be injected
ter success rates [24]. More recent attempts have been into the blastocoel of a diploid blastocyst-stage embryo
made to deliver the transgene directly to the germ cells (3.5 dpc) or aggregated with diploid morula-stage
in vivo by electroporation [25] or lentiviral injection embryos (2.5 dpc), which will then develop into a blas-
into the intertubular space [26]. Although more effi- tocyst. The transgenic ES cell will then contribute its
cient than in vitro SSC manipulation, they still strug- lineage to the developing embryo. ES cells used are
gle to reach the efficiency of more established meth- from a mouse line with a different coat colour to the
ods of transgenesis, and the technique is still currently blastocysts to generate chimeric offspring. If the trans-
at the experimental stage and has not been used to genic cell is incorporated into the germline, then off-
generate available lines of transgenic mice. However, spring of these chimeric mice should reflect the coat
it is likely that future modifications to the technique colour of the ES cell donor line rather than the recipi-
will further increase efficiency, and the intratesticu- ent blastocyst and be heterozygous for the transgene in
lar injection technique is cost-effective, simple and fast every cell. More recently, the use of tetraploid embryos
to perform, making it a potential option for future has been exploited to circumvent the chimera step.
transgenesis. Tetraploid cells can form only the extra-embryonic tis-
sue (e.g. the placenta), as such introduction of diploid
ES cells into a tetraploid embryo ensures that all cells
Gene Targeting by Homologous Recombination in in the pup are derived from the transgenic ES cells
Embryonic Stem Cells (reviewed in [29]). The massive contribution of gene
The process of homologous recombination has been targeting to producing mouse models of disease was
exploited by geneticists in gene targeting, a method of recognized when the 2007 Nobel Prize for Physiol-
modifying or ablating the action of a gene by replacing ogy or Medicine was awarded to Capecchi, Evans
283
a
Gene of interest
b
Selection marker
c
d Selection marker
Selection marker
Gene of interest
e
h
g
Figure 18.1 Gene targeting by homologous recombination. (a) Embryonic stem (ES) cell lines are derived from the inner cell mass of
blastocysts of a particular mouse strain. (b) A transgene consisting of homologous regions of the gene of interest flanking a selection marker
is transfected into the ES cell line. (c) The cells’ endogenous homologous recombination mechanism will replace the gene of interest with the
transgene. (d) Treatment of the ES cell line with your selection antibiotic ensures that only cells that have integrated the transgene will
survive. (e) Meanwhile, blastocysts are obtained from a second mouse line, with a different coat colour. (f) Transfected ES cells can be injected
into the ICM of these blastocysts. (g) The blastocysts are transferred into the fallopian tubes of a pseudopregnant mouse. (h) If the ES cells
contribute to the resulting offspring, they will be visibly chimeric from the contribution of two different coat colours. If the ES cells contribute
to the germline, then further breeding will result in completely transgenic offspring.
and Smithies for the development of its fundamental syndrome, an X-linked monogenic defect of purine
techniques [30]. metabolism [31]. Since then, creating a mouse knock-
out of a gene has been an essential part of defining its
function and phenotypic significance.
Infertility Models Generated by Reverse An example where targeted complete knock-
Genetics outs have been informative about male fertility is
that of ‘cation channel of sperm’ (CatSper). CatSper
Complete Knock-Outs is a weakly voltage-dependent, Ca2+ -selective, pH-
The first knock-out mouse line achieved by gene tar- sensitive ion channel that controls the entry of posi-
geting was produced by Evans et al. in 1987. It was tively charged calcium ions into sperm cells. CatSper
a knock-out of hypoxanthine phosphoribosyltrans- is a hetero-tetrameric Ca2+ channel composed of four
ferase (HPRT), which is defective in Lesch–Nyhan separate pore-forming ␣ subunits (CatSper1–4 and
284
three additional auxiliary subunits: CatSper ␤, ␥ and locus of the promoter of interest in an ES cell line,
␦). It is expressed only in the testis and localized to or by ‘random insertion’ lines where a vector contain-
the principal piece of the sperm flagellum (reviewed ing both Cre and the cloned promoter that will drive
in [32]). In mice, knock-out models of any one of the it are inserted at a random locus in the genome by
four CatSper ␣ subunits [33–35] lead to male infertil- pronuclear microinjection. Both methods have their
ity because sperm cannot penetrate the zona pellucida limitations [40]. Knock-ins can produce an insertional
of the oocyte due to failure to achieve Ca2+ -dependent deletion of the gene controlled by the promoter to be
hyperactivated motility. Based on the information used, which may result in a haploinsufficiency pheno-
gleaned from the creation of these mouse knock- type. However, the insertional deletion can be avoided
outs, a search for infertile men with CatSper muta- if the construct uses an internal ribosome entry site
tions identified patients with mutations in CatSper1 (IRES) followed by the transgene placed between the
[36] and CatSper2 [37], but no mutations in 3 and 4 stop codon and the polyadenylation signal of the tar-
have yet been described. Men with these mutations get gene, resulting in production of a bicistronic tran-
have reduced sperm motility (asthenozoospermia) script. Random insertions may be significantly influ-
and abnormal sperm morphology (teratozoospermia), enced by the local environment at the integration site,
but not necessarily a low sperm count. It is likely which can lead to the ectopic expression or silencing of
that more patients with asthenozoospermia and ter- the transgene due to modification of the specificity of
atozoospermia will have CatSper mutations that can the promoter, or a more severe phenotype due to dis-
explain their infertility, but screening is not normally ruption of an unknown gene by insertion of the trans-
offered. gene (insertional mutagenesis).
The corresponding ‘floxed’ line is created so that
Conditional Knock-Outs Using the Cre-loxP System the sequence to be recombined is surrounded with
It is estimated that at least 30% of all KO strains direct loxP repeats. If cell-specific ablation of a gene of
will die during embryonic or perinatal periods. In interest is required, then this sequence can be a gene
these knock-outs it is therefore impossible to study or part of a gene, such as an exon that is critical to the
the effects of the gene of interest in adulthood [38]. gene’s action. An appropriate breeding strategy must
These problems can be circumvented by making a con- then be defined to produce mice containing both alle-
ditional knock-out using the Cre-loxP system, where les of the gene of interest ‘floxed’ and one copy of the
the gene of interest is specifically ablated in the cell type Cre transgene in every cell. When the cell-specific pro-
of interest, rather than throughout the body. moter is activated, Cre is transcribed and translated
The Cre-loxP system is a genetic tool used to cre- and acts as a recombinase at the loxP sites to catalyze
ate conditional knock-out mice by exploiting a system removal of the intervening section of DNA. The struc-
of site-specific recombination occurring in P1 bacte- ture and therefore the function of the gene are then
riophage [39]. Cre recombinase is an enzyme that cat- permanently altered in that cell and its lineage. A fur-
alyzes the recombination of DNA between two loxP ther level of control to the conditional gene ablation
sites. The 34 bp loxP site consists of a directional ele- can be introduced by fusing Cre to a ligand-inducible
ment surrounded by palindromic binding sites for Cre. nuclear translocation signal such as that found in the
The directionality of the loxP sites affects the result of nuclear hormone receptor family. Under the control of
recombination: recombination between two inverted a cell-specific promoter Cre is generated, but will not
loxP sites will cause inversion of the DNA between be transported to the nucleus to execute recombina-
them and recombination between two repeated loxP tion unless a ligand to the hormone receptor of choice
sites will cause deletion of the DNA between them. If is applied [41].
loxP sites are on different molecules of DNA, a translo- Examples where conditional knock-outs have
cation can occur. Exploiting this system for the gener- allowed analysis of cellular processes that cannot be
ation of conditional knock-out mice requires the gen- analyzed in complete knock-outs are dicer and andro-
eration of two transgenic lines (Figure 18.2). One line gen receptor (AR). Dicer is an RNase III endonuclease
must contain a transgene consisting of Cre recombi- that processes microRNAs (miRNAs) and small
nase driven by a cell-specific promoter chosen for the interfering RNAs (siRNAs). A knock-out of Dicer1
cell type of interest. This line can be constructed either is embryonic lethal, as the development of mutants
by ‘knock-in’ targeting Cre itself to the endogenous is arrested before the body plan is configured during
285
a
b
Promoter Cre
Gene
X LoxP
Gene
Gene
LoxP
Cre Floxed
Gene LoxP LoxP
line line
c d e
Promoter Cre Promoter Cre Promoter Cre
LoxP LoxP
Gene
F1
Gene Gene Gene
Figure 18.2 Conditional gene targeting using the Cre loxP system. (a) Mating of a mouse from a line carrying a copy of a transgene with Cre
recombinase downstream of a cell-specific promotor to (b) a mouse from a line with both copies of a gene of interest targeted with transgenic
loxP sites results in (c) F1 offspring that carry both the Cre recombinase transgene, one copy of the floxed gene of interest (from the floxed
parent) and one wild type copy of the gene of interest (from the Cre parent). (d) In cells in which the promotor is active, Cre is transcribed and
translated, binds to loxP sites flanking the gene of interest and induces recombination and ablation of the section of DNA that lies between
the loxP sites, resulting in (e) a population of cells that are heterozygous for a knockout of the gene of interest. Further breeding or alternative
breeding strategies can be used to obtain a homozygous knockout of the gene of interest.
gastrulation [42], so its role in spermatogenesis cannot from the complete knock-out. Although Cre-loxP
be assessed. But conditional knock-outs of Dicer1 specific lines cannot directly model genotype-to-
have shown that its action in both Sertoli [43] and phenotype associations in human disease, they have
germ cells [44] is essential for normal spermatogenesis been essential for increasing our knowledge of the
and male fertility. As another example, male mice basic mechanisms of male reproductive processes.
with a complete knock-out of AR have feminized
external genitalia, no male reproductive tract organs Knock-In Mouse Models
and cryptorchid testes with no spermatogenesis [45]. Although much less commonly produced than knock-
Since spermatogenesis is incomplete and several out models, mouse models where xenogeneic genes
testicular, hypothalamic and pituitary cell types both have been expressed or endogenous genes have been
contribute to spermatogenesis and express AR, it is ectopically expressed have also been important in
impossible to ascertain what androgen signalling in elucidating basic mechanisms of male fertility control.
each of these cell types is contributing to the process An example of a model that transgenically expresses
of spermatogenesis. To address this issue, conditional an endogenous gene in a spatiotemporal ectopic cell
knock-outs have been produced that have revealed type is the transgenic Sertoli cell androgen receptor
specific testicular roles for AR in Sertoli cells [46], (TgSCAR) mouse line [50]. A transgene consisting of
Leydig cells [47], PTM cells [48], and vascular smooth the human AR sequence driven by the rat androgen
muscle cells [49], which could not be elucidated binding protein (Abpa) promoter and incorporated
286
by pronuclear microinjection was expressed in SHBG. These mice allowed the localization and tran-
Sertoli cells before the onset of endogenous AR scriptional control mechanisms of SHBG to be eluci-
expression. This was found to accelerate Sertoli cell dated [53].
maturation and spermatogenic development, which
ultimately reduced the Sertoli cell population and International Mouse Phenotyping Consortium
testis size. The International Mouse Phenotyping Consortium
Examples of xenogenic genes that have been (IMPC) is a collaboration between 18 research institu-
knocked in to produce novel mouse models are the tions and 5 national funders that aims to characterize
panel of genes that have been knocked into the con- the roles of every gene in the mouse genome by gen-
stitutively expressed ROSA26 locus as a transgene erating and systematically phenotyping 20,000 knock-
downstream of the floxed stop codon. Recombination out mouse strains. The IMPC is generating a knock-out
between the loxP sites by Cre results in excision of the mouse strain for every protein coding gene using the
stop codon and expression of the transgene. This strat- embryonic stem cell resource generated by the Inter-
egy has been used for expression of ‘reporter genes’ national Knock-Out Mouse Consortium (IKMC), the
that produce a fluorescent protein (e.g. the A. victoria members of which are working together to mutate all
green fluorescent protein, GFP) or a substrate that can protein-coding genes in the mouse using a combina-
be acted upon to produce a chromogenic product (e.g. tion of gene trapping and gene targeting in C57BL/6
E. coli ␤-galactosidase). It has also been used to express mouse ES cells. These knock-out ES cell lines are made
a floxed simian heparin-binding EGF-like growth fac- available to the research community via public repos-
tor (HBEGF) gene, [51]. As well as its role as an impor- itories. The groups funded to produce IKMC knock-
tant growth factor, HBEGF is also the receptor for out alleles use targeting constructs that have differ-
diphtheria toxin. Although it is widely expressed in ent and complementary properties, but an impor-
both humans and mice, diphtheria toxin binds the tant approach that is available through its contribu-
mouse variant of HBEGF with low affinity, as such tors is the ‘knock-out first’ allele (Figure 18.3). Target-
mice are naturally resistant to diphtheria toxin. Trans- ing starts with the introduction of a cleverly designed
genic expression of simian HBEGF in a specific pop- transgenesis cassette containing an IRES:lacZ trapping
ulation of mouse cells by mating the floed-stop codon cassette, a loxP site and a floxed promoter-driven neo
HBEGF line to a cell-specific Cre line makes these cells cassette, all flanked by FRT site-directed recombina-
specifically susceptible to apoptosis when diphtheria tion sequences upstream of a floxed exon specific to the
toxin is injected. This can be used to experimentally gene of interest. This is targeted to replace the endoge-
ablate specific cell populations. When this system was nous exon of interest and some of the upstream intron.
used to specifically ablate the adult Sertoli cell popula- Cell-specific expression of Cre can act on this allele
tion, it proved that, as well as sustenance of germ cells, to delete the promoter-driven neo and floxed exon of
Sertoli cells maintain the differentiated phenotype of the tm1a allele to generate a lacZ-tagged knock-out
peritubular myoid cells (PTMCs) in prepubertal life, reporter allele (tm1b). In an alternative strategy, cell-
the adult Leydig cell progenitor population in the post- specific expression of Flp recombinase can convert the
natal testis and development of normal adult Leydig ‘knock-out-first’ allele to a conditional allele, restor-
cell numbers [52]. ing gene activity, and then further expression of Cre
Knock-in techniques are also often used to produce deletes the floxed exon of the tm1c allele to ablate
‘humanized’ mouse models in which the expression exon 2 and result in a nonfunctional transcript [54].
of a human gene can be followed and influenced in a Phenotyping of mouse lines generated from these ES
murine model. Human sex hormone-binding globulin cell lines is performed by each IMPC centre using the
(SHBG) transports sex steroids in the blood and regu- standardized protocols prepared by the International
lates their access to target cells. Although the main site Mouse Phenotyping Resource of Standardised Screens
of production is the liver in humans, it is not produced (IMPReSS).
there in mice, and as a result, the amounts of SHBG in Other than the IMPC, the Jackson Laboratory
the blood of mice is much lower than those in other Mouse Genome Informatics (MGI) Website maintains
species. Pronuclear microinjection of a transgene con- a database of all genes which have one or more pub-
taining human SHBG sequence into mouse embryos lished knock-out or conditional knock-out alleles (this
resulted in production of mice transgenic for human includes knock-outs generated by laboratories outside
287
a tm1a
1 lacZ neo 2 3
FRT loxP FRT loxP loxP
Cre Flp
b tm1b c tm1c d tm1d
Cre
1 lacZ 3 1 2 3 1 3
FRT loxP FRT loxP loxP FRT loxP
Figure 18.3 IKMC ‘knockout-first’ allele generation. (a) Targeting starts with the introduction of a cleverly designed transgenesis cassette
containing an IRES:lacZ trapping cassette, a loxP site and a floxed promoter-driven neo cassette all flanked by FRT site-directed recombination
sequences upstream of a floxed exon specific to the gene of interest. This is targeted to replace the endogenous exon of interest itself and
some of the upstream intron. (b) Cell-specific expression of Cre can act on this allele to delete the promoter-driven neo and the floxed exon of
the tm1a allele to generate a lacZ-tagged knockout reporter allele. (tm1b). (c) In an alternative strategy, cell-specific expression of Flp
recombinase can convert the ‘knockout-first’ allele to a conditional allele, restoring gene activity. (d) Further expression of Cre deletes the
floxed exon of the tm1c allele to ablate exon 2 and result in a nonfunctional transcript.
of the IMPC). Each gene symbol is linked to its respec- 1. you are guaranteed a phenotype to study, that is, you
tive MGI Gene Detail page; each allele is linked to its know that whatever the gene, its perturbation leads
MGI Phenotype and Allele Detail page; and a link is to infertility; and 2. the investigation can be made a
provided to the International Mouse Strain Resource priori – no pre-cognizance of the nature of the under-
(IMSR) strain if a repository holds mice carrying one lying genetic lesion is necessary. This therefore pro-
or more of the listed knock-out alleles. vides a great opportunity to identify genes and path-
ways essential for fertility that would never have been
empirically chosen for study.
Phenotype-Driven Models (Forward
Genetics) ENU Mutagenesis
The low frequency and unpredictable nature of spon-
Premise of Forward Genetics taneous mutations mean that it is impractical to
‘Forward genetics’ is the process of determining the attempt to curate a database covering every gene
genetic basis of an observed change in phenotype. It is in the genome purely from spontaneous mutations
a powerful technique for the assignment of a function alone. Several different mutagens have been utilized to
to a previously uncharacterized gene or for the iden- attempt to increase the mutagenesis frequency, includ-
tification of new pathways involved in a disease pro- ing x-rays and chlorambucil (reviewed in [55]), but the
cess. Forward genetics has classically been used to map most commonly used is N-ethyl-N-nitrosourea (ENU,
causal mutations underlying spontaneous phenotypes reviewed in [56]), an alkylating agent that causes point
arising in mouse colonies. More recently, this approach mutations (mostly A-to-T base transversions) at a rate
has been enhanced through the use of mutagens to of approximately one mutation every 1–1.5 Mb in sper-
increase the numbers of random mutations in mouse matogonial stem cells. After a brief period of steril-
colonies. The phenotype of the resulting mice and their ity, the stem cells divide and repopulate the testis with
offspring is noted; then the mutation is mapped to the mature spermatozoa. The rate of mutations induced
gene responsible for the phenotypic change using clas- by a standard ENU treatment regimen is estimated to
sical Mendelian genetics, supported by more sophis- be one loss-of-function mutation in a specific gene for
ticated molecular genetic approaches. The process of every 700 spermatozoa.
determining the gene and mutation responsible for the The first stage of a genomewide mutagenesis
phenotypic variation seen in a mutant can be labori- screen for recessive mutations is breeding to char-
ous, but has been greatly facilitated by the increase in acterize the inheritance of the phenotype. An ENU-
computing resources and bioinformatics tools as well treated G0 male is crossed with a wild type female.
as increased accuracy of mouse genome annotation. First generation offspring (G1 ) from these matings
The major advantages of this approach are as follows: are estimated to contain 30–50 potential functional
288
a G0: mutagenesis, outcross Figure 18.4 ENU mutagenesis-three generation

screening strategy. (a) An ENU-treated G0 male is crossed
ENU with a wild type female from a different strain. (b) The G1
progeny can be screened to identify dominant mutations.
To determine if the mutation is recessive, G1 males must be
mated with wild type females to generate a pool of G2
progeny, which are genetically heterogeneous due to
recombination. (c) The G3 individuals can be obtained by
crossing the G1 male to the G2 daughters to obtain
homozygous mutant progeny, (d) which can then be
phenotypically screened for recessive traits.
b G1: dominant screen,
outcross
c G2: back-cross
d G3: recessive screen
mutations throughout their genomes. The G1 progeny the genes present in that region. To identify the causal
can be screened to identify dominant mutations; how- gene, this information can be used to sequence the
ever, dominant mutations resulting in infertility are exons of every gene in a candidate region and compare
challenging to identify because it is impossible to sus- mutants with wild types. If functional or expression
tain a mouse line with such a mutation (unless it is data are available for the genes in the candidate region,
restricted to one sex). Identification of recessive muta- they can be prioritized for further analysis accord-
tions is more common. To determine that the muta- ing to the likelihood that they are causing the pheno-
tion is recessive, G1 males can be mated with wild type type. Genes with expression patterns consistent with
females to generate a pool of G2 progeny. By cross- the disease phenotype, showing a (putative) function
ing the G1 male to the G2 daughters, it is possible to related to the phenotype or homologous to another
obtain homozygous G3 mutant progeny (Figure 18.4). gene linked to the phenotype are all priority candi-
The second stage is determining the chromosome and dates; but even if a mutation is found, all exons must
general chromosome region of the mutation by linkage still be sequenced to ensure that there is not more than
analysis, the analysis of the inheritance of the muta- one mutation present in the region. Very recently, and
tion with a previously mapped trait and, in recent as we move into the future, it has become economically
times, genotype analysis of SNPs that differ between viable to simply sequence the entire mouse genome
the strain of the G0 treated male and the females it to identify candidate mutations, something inconceiv-
is mated to. When the candidate region is defined, a able even five years ago. Once candidate genes are
database such as Ensembl1 is searched to determine identified, researchers will conduct in vitro functional
289
analysis comparing the mutant gene with its normal gous regions of DNA derived from the initial ENU-
counterpart to prove that gene function is indeed treated C57BL/6J founder in 10 infertile males. This
impacted by the mutation. resulted in identification of a single genomic locus cov-
One adjunct to the use of ENU mutagenesis is its ering 2 Mb of distal chromosome 5 common to all
employment in so-called gene-driven screens [56]. In mutant males, which contained 17 candidate genes.
this case ⬎10,000 male mice are treated with ENU and DNA sequencing of every exon from all 17 candi-
sperm from their male offspring are stored as a cen- date genes in this region in both fertile and infer-
tral resource, with DNA taken for genotyping. A sin- tile males identified a single-homozygous-point muta-
gle gene or exon of interest is PCR amplified from tional change of thymine to guanine within exon seven
all animals, and high-throughput technologies such of the gene, encoding the novel microtubule-severing
as denaturing high-performance liquid chromatogra- protein KATNAL1. Katnal1 was found to be expressed
phy (DHPLC) are used to identify individuals carry- in testicular Sertoli cells, and the mutation was found
ing mutations in that gene or exon. Sperm from these to result in a reduced number of stable microtubules
individuals can then be used in IVF to recover lines in Sertoli cells and a premature sloughing of immature
that definitely carry a mutation in the gene of inter- germ cells from the seminiferous epithelium.
est. This technique can also be performed in ES cells. The Reproductive Genomics Program at The Jack-
One advantage of this over straightforward knock-outs son Laboratory2 screens ENU mutants for infertil-
is the possibility of identifying multiple mutations in a ity phenotypes. Their basic phenotyping pipeline for
gene, some of which may prove hypomorphic or pro- G3 males that fail to impregnate wild type females
vide a gain of function; thus it is possible to generate involves first checking to see if the mice are engag-
a so-called allelic series of mice with differing muta- ing in mating by checking their female partners for
tions in the same gene that work in concert to support vaginal plugs. The mice are then culled, their repro-
mechanistic dissection of function. It is also possible ductive organs weighed, one testis and epididymis
to use sensitized screens in which animals harbour- fixed for histology and the other epididymis used
ing a mutation in a key gene are themselves exposed for sperm count, morphology and motility analy-
to ENU in order to identify genes that modify (i.e. sis. In addition, the sperm are used for in vitro fer-
increase or decrease) the severity of the phenotype. tilization of wild type oocytes to determine their
This is an excellent way of dissecting an interacting competence for development of two-cell and blasto-
network. cyst stage embryos. Mutants are archived and avail-
An example of a male fertility gene discovered able to any researcher who requires them. Further
from an ENU mutagenesis study is Katnal1 [57]. databases of ENU mutant tissue includes the MRC
C57BL/6J males were treated with ENU as part of an Harwell archive where sperm have been archived from
MRC Harwell mutagenesis screen and then outcrossed more than 10,000 G1 males generated as part of
to C3H/HeH females for two generations. Females their ENU mutagenesis programme. The G1 sperm
from the second generation were backcrossed to their are paired with DNA extracts and form the Har-
‘founder’ father. If the second generation female car- well DNA archive, which is available for mutation
ried a recessive mutation, backcrossing to her father screening [56].
would result in a one in four chance that offspring were
homozygous. As part of a screen for developmental
mutants a recessive screen for male fertility was under- Naturally Occurring Mutants
taken. Males with an autosomal recessive infertility Spontaneous mutations occur at a low frequency in the
trait were obtained from one founder. The testes of genome (approximately 5 × 10–6 per locus) [55]. Mice
homozygous mice were 60% of the weight of wild types with naturally occurring mutations will be more likely
from early adulthood onwards. Histological analysis of to be identified in large breeding colonies such as those
testis sections revealed this was because of a reduction at the Jackson Laboratories3 or MRC Harwell4 . Mutant
in the number of postmeiotic germ cells. To identify phenotypes are often observed long before having any
the genetic lesion responsible for the observed reduc- idea which gene is responsible, which can lead to
tion in testis weight and infertility, a genomewide SNP genes being named after their mutant phenotypes. Two
linkage analysis was employed to identify homozy- examples of naturally occurring mutants that have
290
been used as models of human disease in male fertil- of the gene was identified that leaves intact the prox-
ity research are the testicular feminization (Tfm) and imal half of the gene, which can drive normal tran-
hypogonadal (hpg) models, described below. scription but not normal translation. The hpg mouse
The Tfm mouse is a naturally occurring mutant line is an important animal model of hypogonadotrophic
first identified at MRC Harwell in 1970 by Lyon and hypogonadism, which presents as delayed puberty in
Hawkes [58]. Males carrying the X-linked mutation human patients [65].
are characterized by a lack of androgen-dependent The Mutant Mouse Resource (MMR)5 at the
organ differentiation: a feminized external genital Jackson Labs is the world’s largest collection of
appearance, a blind-ended vagina, inguinal testes with novel strains of mice carrying spontaneous genetic
absent spermatogenesis and no Wolffian-duct-derived mutations. The MMR characterizes the spontaneous
structures. Experiments performed during the 1970s mutants that arise within the large breeding colonies
indicated that a problem with the androgen recep- of the Jackson Labs, maintains and distributes these
tor in the Tfm mouse was responsible for the phe- mutant strains and associated information to the sci-
noytpe: organs usually known to be responsive to entific community and encourages use of these unique
testosterone were shown to be unreseponsive in Tfm disease models. It also cryopreserves spontaneous
mice. When Tfm mice were treated with radioactively mutation-bearing strains for future researchers. Over
labelled testosterone it was not found in cell nuclei, and 700 established mutant stocks are maintained in the
full-length androgen receptors could not be detected Mouse Mutant Resource, and 90–100 new mutations
in Tfm cells (reviewed in [59]). But it was not until are at various stages of characterization. New mutant
1991 that the genetic basis of the phenotype was elu- mice are made freely available to the scientific com-
cidated, when it was discovered that male Tfm mice munity following preliminary characterization, and
have a single nucleotide deletion in exon 1 of their Ar details are uploaded.6
gene and the resulting frameshift introduces a prema-
ture termination codon [60, 61]. The Tfm mouse is an
animal model of complete androgen insensitivity syn- Gene Traps
drome (CAIS), a human disorder of sex development Gene trapping (reviewed in [55]) is a technique used
in which people with an XY genotype and a null muta- to induce mutations in the genome with an insertion
tion in androgen receptors are unresponsive to testos- vector consisting of a promoterless reporter gene such
terone. Patients are born with completely feminized as ␤-galactosidase fused to a neomycin phosphotrans-
external genitalia and are assigned a female gender at ferase resistance gene flanked by an upstream splice
birth. They are usually diagnosed either when puberty acceptor site and a downstream polyA sequence. When
does not result in menarche, or with inguinal swellings transfected into an ES cell line, the gene trap vector
in infancy that are found to be testes after investigation will randomly insert into the genome. If it inserts into
[62]. a genomic intron, the reporter gene will be transcribed
The hpg mouse model also arose spontaneously from the endogenous promoter of that gene in the
in the mouse colonies at MRC Harwell [63]. The form of a fusion transcript in which the exons of the
name hpg was given because the phenotype was endogenous gene upstream of the insertion site are
that of hypogonadotropic hypogonadism: homozy- spliced in the frame to the reporter gene. Since tran-
gous hpg mice have hypoplastic genitalia and gonads scription is terminated prematurely due to the gene
and low circulating sex steroids and gonadotropins. trap’s polyA sequence, the processed fusion transcript
Direct measurements of hypothalamic GnRH con- will encode a truncated version of the cellular protein
tent demonstrated that hpg mice lacked GnRH. Tis- and the reporter. Because of this, most of the genes that
sue grafting experiments determined that the fault was the vector inserts into are knocked down or out. The
at the level of the GnRH neuron and not upstream in reporter gene provides both a visual representation of
the pathway. Gnrh1 was investigated as the most logical gene expression (from X-gal staining, used to iden-
candidate gene where the mutation might be found in tify cells that express ␤-galactosidase) and a sequence
hpg mice. The wild type mouse Gnrh1 gene was cloned tag generated by 5’RACE PCR that can be used to
and sequenced and then compared with the sequence search online genome databases to identify the gene
in hpg mice [64]. A deletion affecting the distal half that the vector has inserted into, circumnavigating the
291
time-consuming process of genetic mapping that char- The Human Genome Project published its first
acterizes mutagenesis methods that rely on the intro- draft of the human genome sequence in 2001 [67]. Fur-
duction of point mutations or small deletions. ther refinement and annotation still continue today.
Bclw is an example of a gene that has been iden- Follow-on projects have built upon this knowledge and
tified as contributing to male fertility through a gene the significant technological advances and reduction
trapping study [66]. A line of mice designated as in costs of whole genome next-generation sequencing
ROSA41 were generated using the ROSA ␤-gal retro- to provide a genetic baseline supporting diagnosis and
viral gene trap and found to exhibit recessive male treatment [68]. The 1,000 Genomes project seeks to
infertility. On investigation, their testes and seminal completely sequence the genomes of 1,000 anonymous
vesicles were found to be reduced in size compared individuals, against which a patient’s genome can be
with those of controls, and testicular histology showed compared (69), while other projects, e.g. the Inter-
an increase in apoptotic and degenerating sperma- national Hapmap project [70] and the Human Vari-
tocytes and a reduction in spermatid number com- ome project [71], aim to characterize sites of genetic
pared with controls. Bclw was identified as the gene variation, such as SNPs, that are commonly associ-
harbouring the mutation by cloning and sequencing ated with human conditions and disease. With cur-
the genomic DNA flanking the viral integration site rent advances in technology, demand facilitating the
and then searching genetic databases. X-gal staining of economy of scale, a critical mass of genome infor-
heterozygous ROSA41 testes was used to localize the mation and advances in computing power and the
expression of Bclw1 to the elongating spermatids and ready availability of cheap ‘off the shelf’ replacement
some Sertoli cells. genes, available in a wide range of vector delivery sys-
Previously, gene trap studies were used as a forward tems from competing commercial suppliers, the time
genetics technique to identify new genes in mutage- for personalized genetic medicine is now. In the near
nesis studies in the same way that a chemical muta- future (5–10 years) we can expect a situation where
gen can be used. More recently, the International Gene a patient presenting with infertility has his or her
Trap Consortium (IGTC) has been working to gener- genome sequenced, and any genetic lesion pinpointed
ate a public library of mutated murine ES cell lines. (via sequence comparison with thousands of ‘control’
Gene trap cell lines are available on a noncollabora- genomes) and then corrected by gene therapy, for less
tive basis for a nominal handling fee. Researchers can than $2,000, followed by natural conception or assisted
search and browse the IGTC database for cell lines reproduction to produce a healthy baby.
of interest using accession numbers of IDs, keywords, To illustrate this process, we use a theoretical
sequence data, tissue expression profiles and biological example of a patient presenting with idiopathic male
pathways. The IGTC is a reverse genetics resource for infertility. First, a physical examination and semen and
researchers to obtain gene trap lines for the particular hormone analysis would be used to narrow down the
genes that they are interested in. possible physiological causes of infertility. The patient’s
genome would be sequenced and initially compared
with a list of candidate genes widely associated with
How Knowledge Gained Can Be Used to the patient’s presentation, looking for mutations not
present in reference genomes. If none of the candidate
Correct Male Infertility genes appear to be responsible, a genomewide associa-
Historically, genetic mutations causing male infertility tion study (GWAS) would be performed. GWAS work
are not treated or corrected, and ICSI is performed if by comparing SNPs in the patient’s genome with those
any sperm can be obtained from the testis. This results of a reference genome (potentially a relative with-
in the prospect of the genetic lesion that causes infertil- out infertility) either using a SNP array, or by whole
ity and any associated disorders being passed to the off- genome sequencing (reviewed in [72]). Once candi-
spring. A large amount of useful information about the date mutations were identified, the predicted function
genetics of male fertility pathways has been obtained of these genes would be explored, taking data from
through the creation and analysis of transgenic mice. the published literature, and in particular, mouse mod-
Ideally, this information should contribute first to the els with mutations in the gene of interest (although
diagnosis and second, where possible, to the treatment not essential, were the mouse to exhibit fertility
of male infertility in humans in a clinical setting. problems this would provide additional supporting
292
evidence for a causal link). If no mouse model were effects is required before they are applied to germline
available, viral-mediated transgenesis [73] or access to genetic editing.
the IMPC database of ES cells could be used to develop
a novel model. In addition, gene function in the patient
can be assessed using cultured skin cells [74], either Summary
assessed directly or via conversion to induced pluripo- One in seven couples trying to conceive will struggle,
tent stem (iPS) cells [75]. Alternatively, site-directed and male factors contribute to approximately 40% of
mutagenesis might be used to introduce the candidate infertility cases. A specific cause of infertility is not
mutation into a cell line for analysis of how it impacts found in one-fourth of cases, and it is then defined
gene function [76]. as ‘idiopathic’. This diagnosis is in part a result of
Once the genetic lesion has been identified, and poor understanding of the basic mechanisms regu-
its method of action characterized, the next step will lating male fertility and the incomplete characteriza-
be to correct it. For somatic cell mutations resulting tion of the genes and cellular pathways responsible.
in infertility, delivery of new transgenes has already Genetic mutations are thought to contribute and may
been demonstrated in mouse models [73]. Contro- in fact prove causal when the diagnosis is idiopathic.
versy arises from consideration of genetic modifica- If we are to attempt to treat genetic causes of male
tion of the germline, which is currently prohibited in infertility and prevent their transgenerational trans-
the vast majority of countries. The concept of gener- mission, then repairing the genetic lesions responsi-
ating transgenic humans raises significant ethical and ble in the germline should be our aim. Research using
moral questions, but is not in itself technically chal- mutant and transgenic mouse models has increased
lenging. An important limitation of the majority of our knowledge of male fertility. Using mice as a model
current technologies is that they leave some signa- organism is applicable to human research because
ture behind in the genome that would be propagate their anatomy and physiology are very similar to those
through the generations, be it a new transgene, a novel of humans and they have a sequenced and anno-
microRNA or even just a 35 bp loxP site. All would tated genome with a high homology to the human
be deemed ethically unacceptable. The recent devel- genome that can be manipulated relatively easily using
opment of the clustered regularly interspaced short a toolkit of genetic modification techniques. Mouse
palindromic repeat (CRISPR)/Cas9 system of genome mutant models have revolutionized our insight into
editing (reviewed in [77]) provides significant promise possible genetic causes of male infertility by identify-
in this regard. CRISPR/Cas9 is a transgenic tech- ing genes involved in fertility pathways and dissecting
nique that uses a modified version of a prokaryotic their functions. Gene-driven models involve the intro-
immune response. Among its many possible applica- duction of transgenes or targeted mutations to disrupt
tions, it can be used to replace a section of mutated the function of a gene and then the characterization
DNA by homology-directed repair by introducing a of the resulting phenotype. Examples include complete
‘guide RNA’ that targets a genomic location of inter- knock-outs, conditional knock-outs using the Cre-
est together with a modified version of the Cas9 DNA loxP system, knock-ins and gene traps. Conversely,
endonuclease and a ‘repair template’ into the target phenotype-driven models involve the introduction
cells. Its advantages are that the plasmids and proto- of random mutations into the genome, either natu-
cols required to perform it are open access (and can rally occurring or induced with N-ethyl-N-nitrosourea
be found at http://www.addgene.org), it can be engi- (ENU). Mice are first screened for infertility pheno-
neered so that it is highly specific and importantly it types and the gene harbouring the causative mutation
leaves no trace behind other than the DNA sequence can then be mapped.
it has corrected. The ideal application of this genome The combined knowledge gained from the devel-
editing system would be to introduce it into spermato- opment and use of mutant mouse models in concert
gonia in vivo to correct the mutation so that healthy with human sequence data has opened the gateway to a
sperm could be produced by spermatogenesis and a new era of personalized medicine. In the coming years,
child conceived without further medical intervention. these combined approaches will provide new methods
However, both CRISPR/Cas9 technology and in vivo both of identifying the genetic causes and of correcting
germline transgenesis are new and evolving technolo- male infertility, as well as supporting the development
gies. More basic research into their transgenerational of novel male contraceptives.
293
comparative analysis of the mouse genome. Nature

Notes 2002; 420: 520–62.
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accessory sex glands, 90 Anopheles, 91 blastocysts, 99

acephalic spermatozoa, 46–9 Anopheles gambiae, 95 blood-testis barrier, 11
acetylation, 148 anosmin, 217 body mass index, 250–1, 272
acetylome, 148 antibiotics, 13 BPY2 gene, 214
Acp36DE, 91, 93, 96 antiepileptics, 13 brain-derived neutrotrophic factor
acridine orange, 27–8, 195–6 antioxidants, 251 (BDNF), 64
acrosomal hypoplasia, 44, 45 antisperm antibodies, 15 bulbourethral glands, 90
acrosome, 193–4 aplasia, 43 busulfan, 7
anomalies, 45, 52, 53, 273 apoptosis, 29–30
Ca2+ signalling, 129–30 apoptotic markers, 29–30 Ca2+
development, 40–1 Arbacia punctulata, 133 cytosolic, 177
equatorial segment, 157, 159 artiicial gametes. See stem cells intracellular signalling, 177, 178–9
globozoospermia, 14, 218 artiicial insemination, 80, 113 intracellular stores, 131–2
pathologies, 43–5 Ash1-like (ASH1l), 234 ionophores, 187
sperm head, 42 assisted reproductive technology pumps, 130–1
sperm perinuclear theca proteins, (ART). See also sperm motility regulation, 128–9
161–2 intracytoplasmic sperm sperm signalling, 129–30
spermatid diferentiation, 39–40 injection (ICSI) Ca2+ channels, 130–1
spermiogenesis, 13, 132 indications for, 68 ligand-activated, 130
teratozoospermia, 63–4 safeguarding and improvement of, second messenger operated, 130
acrosome reaction (AR), 154 170–1 store-operated, 130
actin related proteins T1 and T2 scope of, 197–8 voltage operated, 130
(ARPT 1 and 2), 160 success rate, 66 cafeine intake, 250
acute lymphoblastic leukemia, 7 asthenozoospermia, 14–15, 62–3, 66, calcineurin, 147
ADAM2 gene, 215 67, 273, 285 calcium binding tyrosine-(Y)-
adenosine 5’-triphosphate (ATP), alpha␣-tocopherol, 114 phosphorylation regulated
109–10, 111, 113 atransglutaminase (Tgm4) mutation, (CABYR), 63, 148
translocation, 112–13 91 calmodulin, 146–7
adenylyl cyclase soluble (SACY), 91, Austin, Colin, 144 cAMP, 129, 144, 147
144 autosomes, 197, 211 cAMP responsive element modulator
adjunin, 15 autocrine, 6 (CREM), 222
Aedes aegypti, 95 axoneme, 49–51 Cancer treatment and chemotherapy,
agenesis, 43 azoospermia, 10, 82, 280 177
Aitken, John, 29, 30 AZFa, 197, 213 cancers
AKAP3, 146, 148 AZFb, 197, 213–15 breast, 169
AKAP4, 52, 146, 148 AZFb+c, 213–15 childhood, 6–7
A-kinase anchoring protein 4, 146 AZFc, 197, 213 germ cell, 3–4
alcohol intake, 250 deletions, 2 testicular, 240, 242
aldehyde dehydrogenase, 234 obstructive, 1 capacitation, 144
alkylating agents, 7 discovery, 144
aminoglycoside phosphotransferase, bacterial artiicial chromosomes glycosylation, 147–8
283 (BACs), 282 post-translational modiications,
ampullary glands, 90 Basal cells, 74 143–4
anaphase, 11 Bclw, 292 protein acetylation, 148
androgen insensitivity syndrome, 291 Bedford, Michael, 144 proteomics of, 143–8
androgen receptor (AR), 285 beta-nerve growth factor (ß-NGF), 96 PTMS of proteins, 146–7
aneuploidy, 11–12 bicarbonate, 144 signalling cascade, 144–5
animacula, 36 Binder of Sperm (BSP), 92–3, 94 Cas9, 293
anogenital distance (AGD), 242 bisphenol A (BPA), 240, 247 catalase, 117
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CatSper, 16, 130–1, 134–6, 153, 217, cumulus oocyte complex, 152–3 docosahexaenoic acid (DHA), 273
284–5 cunulus motility, 128 Donut Loop model, 26
CatSperKO, 146 cyclic adenosine monophosphate double strand break (DSB), 212
caudal manchette, 161, 165 (cAMP), 129, 144, 147 DP71F-like, 162
CD55, 98 cyclic-adenosine diphospho-ribose drepanocytosis, 7
CD59, 77, 98 (cADPR), 131 Drosophila, 88, 90, 91, 93, 95–6, 97, 98,
CDY gene, 214, 215 cyclophilin, 147 99, 100
cell pathology, 36 cyclophosphamide, 7 dyneins, 128
cell phones, 252 C-YES, 162 dysplasia of the ibrous sheath (DFS),
cellular respiration, 109–10 CYORF15 gene, 213 51, 52
centrioles, 40, 45–6, 47 cystic ibrosis transmembrane receptor dystrobrevin alpha, 220
Centrobin, 48 (CFTR), 170, 215–17, 281 dystrophin, 162
cervix, 92 cytogenetic anomalies, 208–9
chemiluminescence, 115–19 cytosine phosphate guanine E2F transcription factor 1 (E2F1), 220
chemoattractants, 2 dinucleotides (CpGs), 230–1 ECM1, 82
chemotaxis, 136 5’-cytosine-phosphate-guanine-3’ Edward’s syndrome, 209
chemotherapy, 12 (CpG), 221 egg activation, 177
chlorambucil, 7 cytosol, 158–60 EIF1AY gene, 213
CHO cells, 184 ejaculated spermatozoa, 15. See also
chromatin, 15, 222 daily sperm production (DSP), 271 spermatozoa
abnormalities, 41–3 DAZ gene family, 3, 197, 214–15 apoptotic markers, 29–30
compaction, 40–1, 143 De Graaf, Regnier, 73 DCXR in, 82
chromatin-associated RNAs (CAR), 60 DEAD box polypeptide 4 (DDX4), 62 glycosylation, 147
chromomycin A3, 24 deafness-infertility syndrome, 217 ICSI, 200–3
chromosomal markers, 196 DEFB126, 92, 93, 98 RNAs, 62
chromosome Del Castillo syndrome. See Sertoli cell signal transduction pathway, 144
aneuploidy, 209 only syndrome ejaculatory bulb, 90
anomalies, 208–9 deltamethrin, 147 embryogenesis, 25, 60, 64, 234–5, 236–7
inversions, 211–12 DFP71D, 162 embryonic stem cells, 283–4, 287
reciprocal translocations, 211 diacylglycerol (DAG), 183 endocrine disrupting chemicals
Robertsonian translocations, 211 diakinesis, 11 (EDCs), 240
small supranumerary marker dibromochloropropane (DBCP), 240 classiication, 246
chromosomes (sSMC), 210 dibutyl phthalate (DBP), 265 deinition of, 243
structural anomalies, 210 dicer, 285 exposure routes and sources, 245
translocations, 210–11 diet, 251 exposures over time, 248–9
clusterin, 77, 89 gut microbiome and, 271–2 non-persistent, 247–9
colony-stimulating factor 1 (CSF1), 6 high-fat, 271, 273 persistent, 245
comet assay, 28–9, 30, 196 sperm function and, 272–4 sources, 246
complete androgen insensitivity testis development and, 271 endoplasmic reticulum, 109, 177
syndrome (CAIS), 291 di-ethylhexyl phthalate (DEHP), 249 Ensembl, 289
complete asthenozoospermia, 14–15 dihydroethidium (DHE), 118–19 ENU mutagenesis, 288–90
complete knock-outs, 284–5 di-iso-butyl phthalate (DiBP), 249 environmental chemicals, 9, 12–13
conditional knock-outs (Cre-loxP di-iso-nonyl phthalate (DiNP), 249 epiblast, 2
system), 285–6 di-n-butyl phthalate (DnBP), 249 epididymal luid, 74–6, 77, 81, 110–11
congenital absence of vas deferens dioxin, 245 epididymal maturation, 76, 77, 81, 83,
(CBAVD), 199, 215–17, 281 diplotene spermatocytes, 11 147
connecting piece, 45–6, 47 direct germline manipulation, 283 epididymal necrozoospermia, 15
connexin-43, 220 discoidin domain receptor 1 (DDR), epididymal proteome, 76–7
contraception, 15–16 222 epididymal transit, 14–15, 76, 81, 143,
controlled time intercourse (TIC), 66 DNA breaks, 26, 27–9, 30 145
copulatory plug, 91, 94 DNA fragmentation index, 27–8 epididymal tubules, 74
Cre-loxP system, 285–6 DNA methylation, 221–2, 230–3 epididymis, 13–14, 89
CRISP1, 16, 82 DNA methyltransferase (DNMT), anatomy of, 73–4
CRISPR, 293 231–2 deined, 73
c-ros protooncogene, 74 DNA packaging, 25–6, 219 epididymosomes, 77
cryopreservation, 7 DNAH5 gene, 218 function, 73
cryptorchidism, 264 DNMT1, 231–2 histology of, 73–4
cryptozoospermia, 14 DNMT3a and DNMT3b, 231–2 imprinting and, 81
c-SRC, 145 DNMT3l, 231–2 luminal composition, 74–6
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male infertility and, 81–2 chromatin compaction, 222 high DNA stainability (HDS), 24
markers, 82 histone modiication, 222–3 high fat diet (HFD), 271, 273
miRNAs in, 77 hypermethylation, 221 histone linker H1 domain,
patency, 82 microRNAs (miRNAs), 220 spermatid-speciic 1 (HILS1),
proteome, 76–7 Pawp, 170 63
role in sperm maturation, 73–83 species-speciic, 215 histones, 25, 161, 222–3, 233–4
sperm maturation and, 80–1 gene knockout technology, 62 homeobox, 217
sperm motility, 134 gene polymorphisms, 215 homologous recombination, 283–4
sperm reservoir, 83 gene traps, 291–2 horseradish peroxidase, 116–17
sperm transit, 76 geneticin, 283 hpg mouse, 291
thickness, 74 genome-wide association study HSF2 gene, 219
transcriptome, 77–80 (GAWS), 292 HSFY gene, 213
vasectomy, 82–3 germ cell markers, 4, 89 Human Genome Project, 292
vasovasostomy, 82–3 germ cell neoplasia in situ (GCNIS), Human Variome Project, 292
epididymosomes, 77, 80 240 Hunter, William, 73
EPPIN, 16, 89 germ line, 1, 3, 120, 197, 234 hyaluronic acid, 153
estrogen, 244 glands of Littre, 90 HYDIN gene, 218
eurochromatin, 222 glial cell line derived neurotropic 8-hydroxy-2’-deoxyguanosine
exercise, 251–2 factor (GDNF), 6 (8OHdG), 120
globozoospermia, 14, 43, 45, 218 20-hydroxy-ecdysone (20E), 95
F-actin hoops, 43 gluconeogenesis, 110–11 hyperactivation, 126–7
Fenton reaction, 115 glucose transporters (GLUTs), 110–11 hypermethylation, 230–1
fertilization, deined, 177 glycolysis, 110–12 hyperpolarization, 144
ibrinolysin, 90 glycolytic enzyme, 89 hypospadias, 264
ibroblast growth factor 2 (FGF2), 6 glycoprotein beta-defensin 126 hypothalamic-pituitary-testis function,
ibroblast growth factor receptor 1 (DEFB126), 92, 93 271
(FGFR1), 217 glycosylation, 147–8 hypoxanthine
ibrous sheath, 49–51 glycosylphosphatidylinositol, 81 phosphoribosyltransferase
dysplasia, 51, 52 Gnrh1, 291 (HPRT), 284
lagellum, 14, 49–51 Golgi apparatus, 13, 109
abnormalities, 51–3 Golgi complex, 40–1 idiopathic hypogonadotrophic
Ca2+ regulation, 130 gonadotoxic treatment, 12 hypogonadism (IHH), 217
CatSper, 130, 134 during adulthood, 12 idiopathic infertility, 292
development, 49–51 during puberty, 6–9 idiopathic, 280
hyperactivation, 126–7 gonadotropin-releasing hormone ifosfamide, 7
sperm motility, 128 (GnRH), 217 IGF2/H19 gene, 221
FLB1 protein, 81 gonocytes, 2, 4 Immobilized Metal Ainity Column
luorescence activated cell sorting gonosomes, 211 (IMAC), 146
(FACS), 26 gossypol, 15 immotile cilia syndrome, 52
luorescence in situ hybridization guanine monophosphate synthase in situ translation assay, 26
(FISH), 196, 210 (GMPS), 65 in vitro fertilization, 80, 81, 113, 187,
lux transfer chains, 112–13 gut microbiome, 271–2 235
focal adhesion kinase (FAK), 145 indenopyridine, 15
follicle stimulating hormone (FSH), 6, Haber-Weiss reaction, 89, 115 induced pluripotent stem cells (iPS), 1,
208 Halo cells, 74 293
forward genetics HE1 protein, 77 infertility, 1, 59
ENU mutagenesis, 288–90 HE4 secretory protein, 77 causes of, 280
gene traps, 291–2 HE6, 16 deinition of, 193, 208
naturally occurring mutants, 290–1 head-neck attachment, alterations of, genetic lesions in, 280–1
premise of, 288 48 idiopathic, 280
FOXJ2 gene, 220 head-tail junction, abnormalities, 46–9 inner acrosomal membrane (IAM),
free radicals, 114–15, 273 heat shock proteins, 62, 213, 219 157, 170–1
heat-shock factor Y (HSFY), 213 1, 4, 5-inositol trisphosphate (IP3 )
2-gamendazole, 15 HECW1, 165 signalling pathway, 177, 178
gamete intrafallopian transfer (GIFT), HECW2, 165 inositol trisphosphate receptor (IP3 ),
197 heparin-binding EGF-like growth 131, 167
gametogenesis, 234 factor (HBEGF), 287 insertional mutagenesis, 285
gene expression, 61, 65, 77–80, 81, 83 heterochromatin, 222 interchromosomal efect (ICE), 210
ASH1l, 234 hexokinases, 111 interleukin inhibitory factor (LIF), 98
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internal ribosome entry site (IRES), Limulus Polyphemus, 148 mouse genetics, 280–93
285 long intergenic non-coding RNAs oligozoospermia, 62
International Gene Trap Consortium (lincRNAs), 60 oocyte activation failure and, 187
(IGTC), 292 loop domains, 25–6 phospholipase C zeta, 187, 188
International Knock-out Mouse loose tails, 47 physiological causes of, 280
Consortium (IKMC), 287 low birth weight, 262, 263 primary ciliary dyskinesia, 218
International Mouse Phenotyping loxP system, 285–6 pyschological stress, 252, 253
Consortium (IMPC), 170, 287, lucigenin, 115–16 research, 292–3
293 luminol, 116–17 research challenges, 253–4
intracytoplasmic sperm injection luminol-dependent Robertsonian translocations, 211
(ICSI), 36, 53, 163, 198–204 chemiluminescence, 116–17 screening for, 194
acrosome anomalies, 45 luteinizing hormone (LH), 6, 208 semen analysis, 208
Ca2+ oscillations, 178 Lytechinus pictus, 133 single gene mutations, 218–20
clinical results, 200–3 small supranumerary marker
deinition of, 198 male fertility, epigenetic regulation of, chromosomes, 210
dysplasia of the ibrous sheath (DFS), 220–3 smoking, 249–50
52 male germ line, 115–19 sncRNAs and, 64
failure rate, 187 male infertility, 1, 59, 240–54 syndromic genetic causes, 215–17
fertilization rate, 199 alcohol intake, 250 teratozoospermia, 63–4
globozoospermia, 14 altered sperm parameters, 64–6 Y chromosome microdeletion,
indications for, 199–200 aneuploidy of the X chromosome, 212–13
infertility treatment, 281 209–10 mammalian spermatogenesis, 61
mir-34C, 235 antioxidant supplementation, 251 Mann, haddeus, 119
non-obstructive azoospermia, 1 assisted reproduction and, 193–204 marijuana use, 250
popularity of, 198–9 asthenozoospermia, 62–3 masculinisation programming window
pregnancy rate, 199 body mass index, 250–1 (MPW), 262
prevalence, 198 cafeine intake, 250 matrix attachment regions (MARs),
protocols, 80–1 causes of, 62–6 25–6
recommendations, 66 cell phones, 252 maturation arrest, 11–12, 213
safety of, 203–4 chromosomal translocations, 210–11 MEG3 gene, 221
sperm retrieval methods, 200 chromosome aneuploidy, 209 meiosis, 37
when not to use, 203 chromosome anomalies, 208–9 aneuploidy, 11–12
intrauterine insemination (IUI), 66 chromosome structural anomalies, errors, 11–12
inversions, 211–12 210 maturation arrest, 11–12
in-vitro spermatogenesis, 10 clinical evaluation of, 208 phases of, 11
ionomycin, 187 coding RNAs and, 62 physiology of, 11
irradiation, 12 combined phenotypes, 64 melanoma antigen-A4 (MAGE-A4), 4
isolated oligozoospermia (OS), 209 congenital bilateral absence of vas melphalan, 7
deferens, 215–17 mesoderm speciic transcript (MEST),
Jackson Laboratories, 290 deafness-infertility syndrome, 217 221
juvenile hormone (JE), 95, 99 diet, 251 messenger RNAs, 83
environmental factors, 240–54 metaphase, 11
Kallmann syndrome, 217 epidemiology of, 193 methylation determining regions
Kartagener syndrome, 218 epididymis and, 81–2 (MDRs), 221
karyopherins, 162 exercise, 251–2 methylenetetrahydrofolate reductase
Katnal1, 290 gene polymorphisms, 215 (MTHF), 222
KDM5D gene, 213 genetic and epigenetic basis of, 197 microRNAs (miRNAs), 60–1, 64, 77,
kisspeptin, 217 genetic basis, 208–23 83, 220–1, 285
Klinefelter syndrome, 10, 62, 197, globozoospermia, 218 microsurgical epididymal sperm
209–10, 280 idiopathic, 280, 292 aspiration (MESA), 199, 201
knock-in models, 286–7 integrated analysis of mRNAs and microsurgical testicular sperm
sncRNAs, 64–6 extraction (micro-TESE), 199
lactate dehydrogenase C chain inversions, 211–12 microtubules, 11, 45–6, 51, 52, 128
(LDHC), 89 Kallmann syndrome, 217 microvesicles, 77
lactation, 265 Klinefelter syndrome, 209–10 miglustat, 16
Lactobacillus reuteri, 272 lifestyle factors, 249–54 Min Chueh Chang, 144
lactotransferrin, 89 marijuana use, 250 mini-puberty, 263–4
leptotene spermatocytes, 11 markers of, 195 mitochondria, 14, 40
Leydig cells, 6, 7, 9, 208, 241 medical recommendations, 254 mitochondrial RNAS (mRNAs), 64–6
300

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mitochondrial sheath, 37 omega-3 fatty acids, 273 phospholipase A, 182

mitoSox red (MSR), 118–19 oocytes, 14 phospholipase C zeta (PLCZ1)
Moloney leukaemia retrovirus, 282 oolemma, 198 C2 domain, 182
mouse genetics, 280–93 ooplasm, 161, 162, 164, 167, 169 discovery of, 178–9
forward genetics, 288–92 oscillin, 179 EF-hands, 181–2
gene-driven models, 282–8 outer dense ibers, 49–51, 146, 147 fertilization, 177–88
in male fertility research, 281–2 outer periacrosomal layer (OPL), 157, localization of, 182–3
mouse as model organism, 281 159 male infertility, 187
phenotype-driven models, 288–92 oviduct luid, 94 as male infertility indicator, 188
reverse genetics, 282–8 ovulation, 96–7 molecular properties of, 180
MRC Harwell, 290 ovulin, 91 oocyte activation factor, 45, 167
MSH5 gene, 213 oxidative phosphorylation (OXPHOS), oocyte activation failure, 187
mump orchitis, 9–10 110, 112 PIP2 binding, 181–2
mutagenesis, 288–90 oxidative stress, 119–20 putative egg factor, 184–5
Mutant Mouse Resource (MMR), 291 8-oxoguanine DNA glycosylase 1 regulation, 184
mutants, 290–1 (OGG1), 120 SOAF, 169
species-speciic diferences in
Na+ -bicarbonate co-transporter, 144 P2 protamine, 120 activity, 185–7
Na+ -Ca2+ exchangers (NCXs), 130 P34H, 81, 83 sperm factor candidates, 179–80
natural antisense transcripts (NAT), P34H secretory protein, 77 sperm factor hypothesis, 178–9
60 palindromes, 213 sperm quality biomarker, 168–9
naturally occurring mutants, 290–1 pampiniform plexus, 268 structure of, 180
necrozoospermia, 15 paracrine, 6 targeting within eggs, 183–4
neuraminidases, 147 partial zona dissection (PZD), 198 as therapeutic option for egg
neuroendocrine cells, 90 PAS-PT, 162 activation failure, 187–8
neurotrophic tyrosine kinase receptor Patau syndrome, 209 X and Y catalytic domains, 180–1
type 1 (NTRK1), 64 patency, 82 XY-linker, 181, 185–7
Next Generation Sequencing (NGS), paternal pronucleus (PPN), 163, 170–1 phosphorylation events, 144, 145, 146
59, 66 PAWP protein, 165, 169–70 photoactivated adenyl cyclase (bPAC),
next generation sequencing (NGS), as sperm quality biomarker, 168–9 129
208 Percoll, 94 phthalate syndrome, 241
niche deiciencies, 9–10 percutaneous epididymal sperm phthalates, 9, 240, 249
niche numbers, 9 aspiration (PESA), 199, 201 pinheads, 46
nicotinic acid adenine dinucleotide PERF 15, 161 piwi-interacting RNAs (piRNAs), 61
phosphate (NAADP), 131 perluorinated alkyl substances plasma membrane Ca2+ ATPase
Niemann–Pick disease (NPD), 77 (PFASs), 245 (PMCA), 130
Niemann–Pick type C2 disease perluorooctane sulfonic acid (PFOS), pluripotent stem cells (PSCs), 2–3
protein, 77 245 PMCA4, 130
nitrosoureas, 7 perluorooctanoic acid (PFOA), polyamines, 90
nonobstructive azoospermia (NOA), 245 polychlorinated biphenyls (PCBs), 9,
199, 209 periaxonemal structures, 49–51 245
non-seminomas, 3 peritubular myoid cells (PTMCs), polycyclic aromatic hydrocarbons
nonsystematic lagellar anomalies 287–8 (PAHs), 9
(NSFA), 51 periurethral glands, 90 polyluorinated alkyl substances
NR5A1 gene, 219 persistent organic pollutants (POPs), (PFASs), 245
nuclear factor NFE2L2 RNA, 63 245 polymerase chain reaction (PCR),
nuclear matrix, 25–6 pesticides, 9 212
nuclear proteins, 233–4 PH-20, 153 polyunsaturated fatty acids (PUFAs),
nuclear remodeling, 40–1 phosphatase, 90, 145, 147, 184 274
nuclear vacuoles, 53 phosphatidylinositol 4, 5- postacrosomal sheath (PAS), 157–8,
nucleosomes, 23 bisphosphate, 177 159
phosphatidylinositol-3-phosphate postacrosomal sheath WW-domain
obesity, 13, 272 (PI3P), 182 binding protein (PAWP), 157,
obstructive azoospermia, 1, 199 phosphatidylinositol-5-phosphate 179–80
octapeptide, 181 (PI5P), 182 post-translational modiications
oligoasthenoteratozoospermia (OAT), phosphatidyl-inositol-diphosphate (PTM), 143–4, 148
209 (PIP2), 167, 183–4 preputial gland, 90
oligoteratozoospermia (OT), 209 phosphatidylserine, 182 primary ciliary dyskinesia (PCD), 52,
oligozoospermia, 1, 14, 62, 67, 280 phosphoinositide 3-kinase, 29, 30 218
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primordial germ cells (PGCs), 221 genetic manipulation of early mouse hormones, 94–5
epigenetic reprogramming, 2 embryo, 282 humans, 89–90
gonocytes, 2 homologous recombination, 283–4 insects, 90
migration, 2 infertility models, 284–8 mammals, 89–90
physiology of, 1–2 knock-in models, 286–7 modiication, 91
Sertoli cell only syndrome, 2 techniques in, 282–4 ovulation, 96–7
tumors, 3–4 ribosomal RNAs (rRNAs), 59 pregnancy and, 97–8
Principal cells, 74, 77, 81 ribosomes, 109 prostatic secretions, 90
PRKACA, 144, 148 RNAs, 59–62 sperm capacitation, 93
PRM1 gene, 219 characteristics of, 60 sperm functioning regulation, 93–4
PRM2 gene, 219 chromatin-associated RNAs (CAR), sperm retention, 91–2
probiotics, 272 60 sperm storage, 92–3
procarbazine, 7 coding, 62 uterine contraction, 96
progeny phenotype, 99 elements, 235 seminal proteins
progesterone, 134, 153 long intergenic non-coding RNAs evolutionary dynamics, 100
programmed cell death. See apoptosis (lincRNAs), 60 post-mating behaviors, 99–100
proline-rich tyrosine kinase 2 (PYK2), long non-coding (lncRNAs), 235 progeny phenotype, 99
145 male infertility and, 62 seminal vesicle secretion 2 (SVS2), 94
prostaglandin D synthase, 77 messenger RNAs (mRNAs), 83, 235 seminal vesicles, 90, 99
prostaglandin E (PGE), 94–5, 98, 134 microRNAs (miRNAs), 60–1, 64, 77, seminiferous epithelium, 12, 281–2,
prostasomes, 90 83, 220–1, 285 290
prostate glands, 90 new perspectives, 67–8 seminomas, 3
prostatic acid phosphatase (PAP), 90 paternal pronucleus (PPN), 163 sequence-tagged site (STS), 212
protamination, 222–3 piwi-interacting RNAs (piRNAs), 61, Sertoli cell only syndrome, 2, 280
protamines, 21–5, 63 235 Sertoli cells, 6, 7, 9, 43, 241
protease, 90 in reproductive clinic, 66–7 severe oligozoospermia (SOS), 209
proteasome, 66 ribosomal RNAs (rRNAs), 59 sex hormone-binding globulin
protein acetylation, 148 small interfering RNAs (siRNAs), (SHBG), 287
protein kinase A (PKA), 144, 146 285 sex peptide (SP), 93
protein kinase C, 182 small non-coding RNAs (sncRNAs), SHKBP1 gene, 220
protein phosphatase 2A (PP2A), 145 60–2, 64 short tails, 52
proteome, 76–7 small-nuclear ILF3/NF30 short-stature homeobox (SHOX) gene,
protooncogene, 74 associated-(snaR) RNAs, 60 210, 211
psychological stress, 252, 253 sperm epigenetics, 234–5 sickle cell disease, 7
puberty, gonadotoxic treatment Robertsonian translocations, 211 single gene mutations, 218–20
during, 6–9 RPS4Y2 gene, 213 small interfering RNAs (siRNAs), 285
ryanodine receptor, 131 small non-coding RNAs (sncRNAs),
radiotherapy, 7 60–2, 64
rarefactions, 41–3 sarcoplasmic-endoplasmic reticulum altered sperm parameters and, 64–6
RASGRF1 gene, 221 Ca2+ -ATPase (SERCA), small supranumerary marker
RBM gene, 197 131 chromosomes (sSMC), 210
RBMY1 gene, 214 screening, male infertility, 194 small-nuclear ILF3/NF30
reactive oxygen species (ROS), 29–30, scrotal cooling, 267–9, 272 associated-(snaR) RNAs, 60
63, 110, 112, 114–15 SCYP3 gene, 219 smoking, 249–50
detection in male germ line, 115–19 secretory pathway Ca2+ -ATPase sodium-coupled glucose transporters
oxidative stress, 222 (SPCA), 131 (SGLTs), 110
reciprocal translocations, 211 semaphorin, 217 SOHLH1 gene, 218–19
Reproductive Genomics Program, semen analysis, 194–5 soluble adenylyl cyclase (SACY), 91,
290 semenogelins, 91, 94 144
resact, 133 seminal gel, 92 somatic cells, 1, 2–3, 25–6, 30–1, 59,
residual bodies, 14, 29 seminal plasma components, 88–101 90, 109, 110, 129–30, 131–2,
rete testis, 73 accessory sex glands, 90 195–6, 197
retinoic acid, 15, 234 efects on female, 94–100 sonicated and isolated sperm heads
reverse genetics, 282–8 epididymis, 89 (SSpH), 160
complete knock-outs, 284–8 female immune responses, 97, 98 speract, 133
conditional knock-outs (Cre-loxP female reproductive tract’s molecular sperm behaviours, 126–8, 132–6
system), 285–6 biology, 95–6 marine invertebrates, 132–4
deinition of, 282 fertility and, 88 regulation, 135–6
direct germline manipulation, 283 gut physiology and digestion, 99 regulation by Ca2+ signalling, 132–6
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spatio-temporal Ca2+ patterns, cumulus motility, 128 structure of, 193–4

135–6 hyperactivation, 127–8 in vitro storage, 113
sperm-borne oocyte activating factors mammalian, 134–5 zona pellucida interaction, 153–5
(SOAF), 162, 167–8, 169 regulation, 128–9 spermiation, 14
compensatory efects, 169–70 regulation of, 134–5 spermine, 90
deiciency, 170 sperm neck, 45–6 spermiogenesis, 13–15, 37–40. See also
sperm capacitation, 93, 109 sperm nuclear basic proteins (SNBPs), spermatogenesis
sperm chemotaxis, 136 21 asthenozoospermia, 14–15
sperm chromatin, 21–31 sperm pathology, 36 contraception, 15–16
apoptosis, 29–30 sperm perinuclear theca deined, 37
DNA degradation, 30–1 acrosome, 161–2 globozoospermia, 14
histones, 25 biogenesis, 157–8 oligozoospermia, 14
loop domains, 25–6 cytosol, 158–60 physiology of, 13–14
nuclear matrix, 25–6 deined, 157 steps, 39–40
protamines, 21–5 histones, 161 SPGY gene, 197
sperm DNA damage assays, 26–9 molecular composition, 158–62 STAT4, 162
toroids, 25–6 post fertilization, 162–3 stem cell factor (SCF), 2
sperm chromatin fragmentation protein extraction, 158–60 stem cells
(SCF), 30–1 proteins, 159 induced pluripotent, 1
sperm chromatin structure assay structural/cytoskeletal proteins, pluripotent, 2–3
(SCSA), 24, 27–8, 30, 195–6 160–1 spermatogonial, 1, 4
sperm defects, non-speciic or structure, 157–8 niche, 6
non-systemic, 37 transcription factors, 162 proliferation and diferentiation
sperm DNA damage assays, 26–9 sperm plasma membrane protein of, 4–6
comet assay, 28–9 (SPAM1), 153 types, 4–6
Donut Loop model, 26 sperm reservoir, 83 stereocilin, 217
luorescence activated cell sorting sperm retrieval methods, 200 steroidogenesis, 208
(FACS), 26 sperm RNA elements (SREs), 66–7 steroids, 13
in situ translation assay, 26 sperm RNAs, 196 stromal interaction molecule (STIM),
sperm chromatin structure assay sperm storage, 92–3 132
(SCSA), 27–8 spermatid cytoskeleton, 45 stromal-derived factor 1 (SDF1), 2
terminal deoxynucleotidyl spermatid nucleus, 40–1 Strongylocentrotus purpuratus, 134
transferase (TdT), 26 spermatogenesis, 37–40, 109. See also stump tails, 52
TUNEL assay, 26–7 spermiogenesis subacrosomal layer (SAL), 157–8, 159,
sperm epigenetics, 230–5 environmental and lifestyle efects 170–1
diiculty in studies of, 236 on, 12–13 SubH2Bv, 161
DNA methylation, 230–3 organisation and eiciency of, 265–7 sub-zonal insemination (SUZI), 198
histones, 233–4 phases of, 37 sulhydryl compounds, 143
nuclear proteins, 233–4 in-vitro, 10–11 superoxide dismutase (SOD), 115
potential downfalls, 236 spermatogonial stem cells (SSCs), 1, 4, swim-up method, 197–8
RNAs, 234–5 208 synaptotagmin, 182
sperm factor hypothesis, 178–9 gonadotoxic treatment, 6–9 Sytox green, 118
sperm head, 42 modiication of, 283
sperm maturation, 80–1 niche, 6 TCAM1P gene, 215
sperm metabolism, 98 deiciencies, 9–10 TCP10 gene, 215
cellular respiration, 109–10 niche numbers, 9 television watching, 252
detection in male germ line, 115–19 proliferation and diferentiation of, telophase, 11
glycolysis, 110–12 4–6 teratocarcinoma, 3
modulation, 113 types, 4–6 teratoma, 3
oxidative phosphorylation, 112 spermatozoa teratozoospermia, 53, 63–4, 280
pathological aspects, 113–20 acephalic, 46–9 altered transcripts, 67
physiological aspects of, 109–13 apoptosis, 29–30 CatSper mutations, 285
reactive oxygen species, 114–15 ATP translocation, 112–13 irst account of, 36
sperm mid-piece Ca2+ signalling, 129–30 terminal deoxynucleotidyl transferase
development, 49–51 cumulus oocyte complex interaction, (TdT), 26
mitotic multiplication/ 153 testicular dysgenesis syndrome (TDS),
diferentiation of, 37 ejaculated. See ejaculated 240, 262
structural anomalies, 51 spermatozoa anogenital distance and, 242
sperm motility, 119–20 oxidative stress, 119–20 manifestations of, 242
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testicular dysgenesis syndrome (cont.) transgenic Sertoli cell androgen vasectomy, 82–3
rodent counterpart of, 241 receptor (TgSCAR), vasovasostomy, 82–3
symptoms, 240 286 vesicular glands, 90
testicular ine-needle aspiration, 199, transition nuclear proteins, 63 Virchow, Rudolph, 36
201 transposable elements, 60 vitamin E, 114
testicular luid, 76 trichostatin-A, 234 voltage operated Ca2+ channels
testicular germ cell carcinoma triclosan, 248 (VOCCs), 130
(TGCC), 240, 242 Tripneustes gratilla, 129 voltage-dependent anion channel 2
testicular hyperthermia, 16 trisomy 21, 197 (VDCA2), 63
testicular percutaneous biopsy, 201 TR-KIT, 162
testicular sperm extraction (TESE), tubulin, 146 WBP2, 165–7
199 TUNEL assay, 26–7, 30, 196 WBP2NL, 165
testis, 260–75 tyrosine kinase, 222 WW-domain binding proteins (WBP),
daily sperm production, 271 pathway, 145 163–4
dietary efects, 270–4 sperm capacitation, 145 as cancer biomarkers, 169
functions of, 260–1 YES, 162 oocyte activation factor, 167–8
gut microbiome and, 271–2 tyrosine phosphorylation substrates, 164–5
hypothalamic-pituitary-testis CaM inhibition, 147
function, 271 CatSperKO, 146 X chromosome
lifestyle/environmental factors, c-Yes, 145 aneuploidy, 209–10
susceptibility to, 261–2, 264–5 IMAC, 146 inactivation, 230–1
mini-puberty, 263–4 oocyte activation, 164 Klinefelter syndrome, 10, 197
perinatal development, 262–3 sperm capacitation, 145 rearrangement of chromosomal
scrotal cooling, 267–9, 272 materials, 211
spermatogenesis and, 265–7 ubiquitin ligases, 164 Xenopus, 167
testis-speciic serine kinase 6 (TSSK6), ubiquitin-conjugating enzyme E2B XKRY gene, 213
195 (UBE2B), 62 XY catalytic domains, 180–1
testosterone, 244, 260–1 ubiquitin-proteasome system, 42 XY-linker, 181, 185–7
TEX101, 82 United Nations Environment
TEX101 gene, 215 Programme (UNEP), 243, Y chromosomes, 280
TEX11 gene, 219–20 244 azoospermia factor region of, 2
Tfm mouse, 291 uterine contraction, 96 deletions, 62
thioredoxins, 52 microdeletion, 212–13
topoisomerase, 29–30 van Leeuwenhok, Antoni, 36 Yes-kinase associated protein (YAP),
toroid linker regions, 26, 30 vas deferens 164
toroids, 23, 24 ampullary glands, 90
Toxic Substances Control Act, 242–9 congenital bilateral absence, ZDBF2 gene, 221
transcription factors, 162 215–17 ZMYND10 gene, 218
transcriptome, 77–80 luminal composition, 74–6 zona pellucida (ZP), 153–5, 197–8
transforming growth factor ␤1 vasectomy reversal, 80 zona-drilling (ZD), 198
(TGF␤1), 1, 98 vasa eferentia, 73 zytogene spermatocytes, 11
304

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The Sperm Cell

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The Sperm Cell

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1 Spermatogenesis: Clinical and 9 Proteomics of Capacitation 143

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16 Environmental Factors and Male 18 Mouse Genetics – How Does It Inform

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Mahmoud Aarabi, MD, PhD Clémence Belleannée, PhD

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Hanne Frederiksen, PhD Dolores J. Lamb, PhD

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Richard M. Sharpe, PhD Peter Sutovsky, PhD

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subjects are let unclear and compelling, what next

Ryuzo Yanagimachi, PhD

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1 Clinical and Experimental Considerations

Introduction can be obtained through testicular biopsies, how-

ectoderm (epiblast) in the second week after concep- Sertoli-Cell-Only Syndrome

Differentiation Self-renewal ity [36]. For radiotherapy, recovery depends on the

Digestion Organ culture

Patient needing gonadotoxic treatment

In addition to testicular targets for male contra- Concluding Remarks

2 Susceptibility to Damage in Relation to

Introduction sperm DNA is packaged, what we do know has many

Mouse Sperm Chromatin Folding

Solenoid Schematic Intermediate Doughnut

MAR MAR MAR MAR

Nuclear Matrix Filaments

Protamine DNA Fibers

least accessibility to sperm chromatin of all the assays A. TUNEL Assay

Introduction matozoon to be microinjected. It then became clear

Figure 3.6 Neck region of a normal human

References after Annexin V columns in patients with severe

4 Meritxell Jodar, Ester Anton and Stephen A. Krawetz

Introduction ning as a primordial germ cell differentiates into a sper-

Male infertility GO biological altered

3′ 5′ TRANSLATION INHIBITION/ mRNA DEGRADATION/ mRNA DEADENYLATION

Pathways altered in teratozoospermia and asthenozoospermia

hsa-miR-370 Sperm motility

PSMC5, PSMD14, GABRB1

account for only 5% of the ejaculate, while the remain-

ing roles in intercellular interaction and determina-

5 Robert Sullivan and Clémence Belleannée

Introduction more information on epididymal functions obtained

The epididymal epithelium in humans increases in

A variable, ranging from 1 to 21 days [25]. More recent

There are only a few reports describing morpholog-

70 concentration of 3.4 mg/mL [31] and reaches nearly 30

Log 2 miRNA expression

Log 2 mRNA expression

Caput Corpus Cauda

Figure 5.6 Fertility following vas deferens

cholesterol transport and mediates sperm cholesterol

control the reproduction of insects that transmit seri-

[Ca2+ ] 5 nM), [Ca2+ ]i fell rapidly and oscillations