A publ i cat i on of t he

I ndi an Soci et y f or t he St udy of Reproduct i on and Fer t i l i t y

September, 2014 Editor-in-Chief: Prof. N. K. Lohiya Editors: Dr. Dheer Singh & Dr. P. K. Mishra
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Message
Dr. Harsh Vardhan
Minister of Health & Family Welfare, Government of India, New Delhi
Under Millennium Development Goals (MDG 5) we have made extensive efforts to provide universal access to reproductive healthcare but the
progress has been uneven and inequitable. With over 1.21 billion people, sexual and reproductive health in our country is regarded a social phenomenon
often influenced by contextual factors. Interplay of five main structural determinants: economic status, gender, education, social stratification and age
have severely affected reproductive health services imposing large burden on individuals and society. Therefore, plan and policies needs to judiciously
target and help decide what needs to be done where, for whom, and when to reach equitable progress toward improved reproductive health for one and -
for all. To overcome this burden associated with reproductive health needs, we have two choices: continue on the current path or pursue swift
strategies. To stimulate substantial improvement, collaboration among key organizations to spread greater public awareness is an imminent need.
The Indian Society for the Study of Reproduction & Fertility (ISSRF) established in the year 1988 comprising of members representing distinguished
scientists, public health executives, program managers and clinicians from the field of reproductive sciences is now entering the 25th year of its
inception. A retrospective analysis of society's accomplishments during the course of this journey is necessary but to plan the future discourse will also
be highly imperative. ISSRF is uniquely positioned to foster collaborative efforts among academicians, scientists, clinicians, health care workers and
policy makers involved in the area of Reproductive Health Research. I am sure; along with public, private and non-profit organizations ISSRF in
coming years will plan new strategies that facilitate efforts to achieve Reproductive Health for All.
I am happy to note publication of 15th edition of ISSRF Newsletter that is designed to highlight advances in the field of Reproductive Epigenetics,
an important area of reproductive health research.
(Harsh Vardhan)
Message
Dr. V. M. Katoch
Secretary-Department of Health Research & Director General , New Delhi , Indian Council of Medical Research
I am happy to congratulate the Indian Society for the Study of Reproduction & Fertility (ISSRF), in publishing the 15th edition of the ISSRF-
Newsletter with focuses on Epigenetics: an emerging paradigm in reproductive health.
Emerging knowledge in the area of molecular biology of reproduction has surpassed our understanding of what constitutes heritability and the
acquisition of phenotypic traits across generations. While particulate genetic inheritance was considered the hallmark for heritability of traits in the
past, presently epigenetic modifications have been included to the genomic backbone as a primary mechanism in the intricate series of molecular
interactions which ultimately coordinates development and cell fate. By large, it has been realized that epigenetic code is highly essential in efficiently
regulating gene expression patterns that must occur for a cell to leave its pluripotent state and become fully differentiated to then function in adaptive
homeostasis processes of the organism. Therefore, it can be unambiguously stated that ones epigenetic signatures are the net outcome of genotype,
developmental lineage, and gene-environmental interaction.
Communicating the importance of epigenetically-driven change as an influence on reproductive outcomes is likely to have important role to play in
helping the academia, move towards a more refined understanding of how these factors influence the patterns of health and disease. With technology
for genome-wide interrogation of both chemical modifications to nucleotides and the binding and composition of nucleosomes in place, we will gain
knowledge to explain more complex reproductive traits in times to come. I am optimistic that new knowledge of epigenetics will help us in
understanding the basis of a variety of human ailments. This is a long journey plenty of opportunities to identify the basis of adaptations and may be
some day also effectively interfere after gaining solid understanding of these phenomena.
I take this opportunity to greet all the readers and wish them good luck.
(V. M. Katoch)
2
ISSRF
Message
Prof. N. K. Lohiya
President-Indian Society for the Study of Reproduction and Fertility &
Emeritus Medical Scientist, University of Rajasthan, Jaipur
As we edge ever-closer to the 2015 deadline of the Millennium Development Goals where official negotiations will begin in the U.N.
General Assembly in September, greater attention to strategically focus on sexual and reproductive health and rights are an essential
priority. Sexual health is fundamental to the physical and emotional health and well-being of individuals, couples and families, and to
the social and economic development of communities and countries. Therefore, universal access to comprehensive information
about sexuality, knowledge about the risks they face, their vulnerability to the adverse consequences of sexual activity, their access to
good-quality sexual health care, available contraceptive choices, are the key determinants for promoting sexual health within a range
of country settings. For a country like ours with diverse social and cultural values, it is important for us to re-engage in multi-sectoral
reforms on five crucial domains: laws, policies and human rights; education; society and culture; economics; health systems for
promoting sexual health and wider implementation of our planned strategies. We at ISSRF strongly believe that it is high time to
encourage more comprehensive and multidisciplinary integration of sexual and reproductive health and rights into public health
education. Collective efforts to incorporate sexual and reproductive health and rights in our university teaching curriculum,
formalising research groups that might increase credibility, attract students and facilitate fund raising for research, especially in
collaboration with other inter-sectoral groups (both at local or international level) is required to build a public health workforce that
has the skills to be more responsive to current national reproductive health challenges. Although sexual and reproductive health
issues are viewed often through a critical lens, but now required to be addressed through biomedical and behavioural research as
these would have highly significant health consequences for individuals and communities. If we fail, these in turn, will not only have
serious health burden but social and economic implications as well, for our country at large.
Although our National Health Agencies have made significant contributions to advancing sexual and reproductive health, there is
need for a focused, reinvigorated research agenda. Given the interrelationships among sexual and reproductive health problems,
greater public emphasis to some of the intricate, fundamental and interesting questions and problems is necessary to increase
understanding of and improve provision of needed health services that still persist in our society. The time is long overdue to improve
prevention efforts for limiting size of our population growth. It is clear that more complex and multilevel research, including new and
innovative designs and methodologies at societal and community levels (in terms of policies, programs, norms and values); family,
partner and peer influences; and the individual level (demographic, socioeconomic and psychosocial factors) must be assigned
highest priority. Knowing the magnitude and importance of the problems that might result from all these overlapping issues and
damaging consequences associated with failing to resolve them, it is time for us to address the problems within the field of sexual and
reproductive health through a strong and concerted national effort.
With our strong commitment to 3Ds: discuss, deliberate and disseminate knowledge and information, we have made concerted
efforts to invite your timely attention to several priority areas in reproductive health science and medicine. I am sure this volume of
newsletter that primarily focuses on epigenetics will potentially revolutionize your understanding of the structure and behaviour of
reproductive processes. Selected articles published in this edition might help provide a readily understandable introduction to the
foundations of reproductive epigenetics. As we realize that epigenetic programming of the genome by DNA methylation, histone
modification and chromatin remodelling during gametogenesis and early embryogenesis sets the agenda for developmental origins
of adult diseases, an in-depth understanding of these processes is also of high clinical utility and translational significance. Co-
incidentally, work from the laboratory of Dr. Lanlan Shen from Baylor College of Medicine (USA) published recently in Journal of
Clinical Investigation (July 25, 2014) delineating first straight forward involvement of epigenetic modifications in tumorigenesis
opens up the door for a whole new biological paradigm.
On behalf of the Executive Committee of ISSRF, I congratulate the Editors of this volume: Dr. Dheer Singh, Principal Scientist,
National Dairy Research Institute, Karnal & Dr. Pradyumna Kumar Mishra, Associate Professor, Department of Biological
Sciences, Central University, Sagar for their excellent job.
(N. K. Lohiya)
ISSRF
It's Not All in Our Genes
Pradyumna Kumar Mishra 4
Epigenome Dynamics in Early Embryo & Germline
Development
Sonam Mehrotra, Sanjeev Galande 6
Epigenetic Modulation and Reproduction: A Link between
Environment and Genetics
Jens Vanselow 8
Male Infertility and Epigenetic Modulation
Swetasmita Mishra, Rima Dada 11
Dynamics of DNA Demethylation and Epigenetic
Reprogramming in Stem Cells
Swayamsiddha Kar, Samir Kumar Patra 15
Assisted Reproductive Technology (ART) and Imprinting
Disorders: Epimutation in Gametes as a Functional Cause
Shaoni Bhattacharjee, Jana Chakrabarti 19
Epigenetics: A Paradigm in Germ Cells Differentiation and In
Vivo Development
S. D. Kharche, S. K. Agarwal 22
The Expanding Role of Epigenetics in Reproductive Health
M. Ankolkar, N.H. Balasinor 24
Emerging Role of Epigenetics in Ovarian Cancer
Seema Sharma 26
Genomic Imprinting in Male Germ-Line Stem Cells
Pallavi Pushp, Hoon Taek Lee, Mukesh Kumar Gupta 31
Post Translational Histone Modifications in Ovarian
Epithelium and their Plausible Implications in Reproductive
Plasticity and Health
G. V. Raghuram 33
Polycystic Ovary Syndrome: Plausible Epigenetic Distress
and Role of Environmental and Endogenous Modulators
Pooja Sagvekar, Srabani Mukherjee 37
Epigenetic Changes and Human Assisted Reproductive
Technologies
Rajvi Mehta 40
MicroRNA in Testis An Overview
Panneerdoss Subbarayalu, Manjeet K. Rao 43
Epigenetics: A Key Paradigm in Reproductive Health
Varij Nayan, Suneel Kumar Onteru, Dheer Singh 45
Contents
3
About the ISSRF
Dr. R. S. Sharma
Secretary-Indian Society for the Study of Reproduction and Fertility &
Sr. Deputy Director General, Indian Council of Medical Research, New Delhi
The Indian Society for the Study of Reproduction and Fertility (ISSRF) was established in the year 1988 with the aim to provide a unique
platform to academicians, scientists, clinicians, public health experts, program managers and policy makers engaged in reproductive health
research to interact and disseminate their findings and also to generate collaborations for optimization of resources and abilities. Over these
years, the society has grown exponentially and is very well represented with 1186 current life memberships. The society encourages active
participation and diverse presence spanning from national to international scale that include almost each national institute, universities,
international agencies and regulatory bodies in events organized under its umbrella/banner. Since inception the society has successfully
organized 43 international/national conferences, including 24 annual meetings of the Society.
Embarking upon an endeavour to reach beyond national limits and exert a global influence, ISSRF began publication of society Newsletter
in July 1999. Since then the society has successfully published 14 Newsletters covering broad range of issues from biology of reproduction
to reproductive health. To offer broad academic coverage and draw maximum attention, the published Newsletters are increasingly
circulated to ISSRF members, national and international funding agencies, institutes and universities.
ISSRF is now entering the 25th year of establishment. We must take cognizance of the historical moment through which we made this
journey despite all thick and thins. Therefore, it is befitting to plan an academic event commemorating this occasion. National Institute for
Research in Reproductive Health, a premier institute under the umbrella of Indian Council of Medical Research (ICMR) is pleased to host the
25th Annual Meeting of the Indian Society for the Study of Reproduction and Fertility (ISSRF) and International Conference on
Reproductive Health at Mumbai during 14-17 February, 2015. The scientific program will include plenary lectures, symposia sessions,
panel discussions, debates, short communications and poster presentations. It will address a broad range of key areas with contributions from
various disciplines including basic, clinical, veterinary, operational and socio-behavioral research in sexual and reproductive health.
As a part of ISSRF mandate, we have also planned a National Seminar on Reproductive Health Awareness at the IIS University, Jaipur
during September 12-13, 2014 and we are delighted to release this newsletter during this mid-term activity of the Society.
A Word from the Editor
It's Not All in Our Genes
Pradyumna Kumar Mishra
School of Biological Sciences
Central University
Sagar
pkm_8bh@yahoo.co.uk
That there is a heritable element of susceptibility to
chronic human ailments is well established, but there is
compelling evidence that few components of such
heritability are transmitted through non-genetic factors.
Due to an overly complex reproductive process,
dissecting into inheritance patterns of these factors
though not been easy but little doubt exists that besides
the genomic backbone, a range of epigenetic cues affect
our genetic programme. The inter-generational
transmission of epigenetic marks is believed to operate
via four principal means that dramatically differ in their
information content: DNA methylation, histone
modifications, miRNAs and nucleosomal positioning.
Alone or cohesive interaction between these epigenetic
signatures, influence the cellular machinery through
positive and negative feedback mechanisms. To
understand some of the basics of how this mechanism
work and activate and deactivate parts of our genetic
program not only on day-to-day basis but actually over
generations is an important area of reproductive health
research.
DNA methylation, the most well characterized
epigenetic modification is a heritable covalent
modification and binary in nature. Most methylation
occurs at the number five carbon of the cytosine
pyrimidine ring (5-methyl-cytosine) and represents less
than 5 % of all cytosines in our genomes. Genomic
methylation patterns are propagated during cell division
by DNA methyl transferases (DNMT1, DNMT3A/B).
One of the most vital sites of gene regulation by DNA
methylation are CpG-enriched regions associated with
promoters called "CpG islands. DNA methylation
pat t erni ng fi rst est abl i shed duri ng embryoni c
development by DNMT3A/B, is preserved in succeeding
cell divisions by DNMT1 maintenance. Based on the
presence of methylation in the CpG dinucleotide in the
complementary template strand, the specificity of
DNMT1 for hemi-methylated CpG dinucleotides
provides a machinery whereby CpG in the newly
synthesized DNA strands are methylated. Along with
other enzymes, DNA methylation can orchestrate gene
silencing and maintain a repressive chromatin state.
Conventionally, DNA methylation is inversely related to
both the expression of developmentally regulated genes and
the strength of cells. Besides, DNA methylation is also
involved in regulating a myriad of cellular processes that
includes chromatin structure and remodelling, X-
chromosome inactivation, genomic imprinting and
chromosomal stability.
While nucleosomes represent the primary step in the
construction of higher-order chromatin structures,
histones, the globular proteins undergo post-
translational modification and alter regulation of gene
expression, DNA replication, recombination, and repair.
Histones have a protruding charged 15-38 amino acid N-
terminus (histone tail) that influences nucleosome
assembly into higher order chromatin structure. In
condensed state, chromatin remains in a folded
configuration so the nucleosomes are stacked, hence not
readily accessible to gene activation. However, covalent
modifications such as acetylation, methylation,
phosphor yl at i on, pol y- ADP r i bosyl at i on and
ubiquitination at long tails of H3 and H4 alter histones-
DNA interaction and higher order chromatin folding.
These post-translational covalent modifications regulate
the contact between the octamer core and DNA, and
determine DNA accessibility to transcription factor
complexes. The capability to accumulate information
appears to dwell in the amino-terminal tails of the four core
histones which are exposed on the nucleosome surface and
are subject to enzyme-catalyzed post-translational
modifications of select amino acids, including lysine
acetylation, lysine and arginine methylation, serine or
threonine phosphorylation, lysine ubiquitination, lysine
sumoylation, or glutamine ADP ribosylation. Epigenetic
modification of histone tail have key roles in transcriptional
regulation, DNA repair, DNA replication, alternative
splicing and chromosome condensation. With reference to
transcriptional state, human genome can be approximately
compartmentalized into actively transcribed euchromatin
and transcriptionally inactive heterochromatin.
Euchromatin is characterized by towering levels of
acetylation and trimethylated H3K4, H3K36 and H3K79.
On the other hand, heterochromatin is categorized by low
levels of acetylation and elevated levels of H3K9, H3K27
and H4K20 methylation.
miRNAs are single-stranded RNAs of approximately 21-
23 nucleotides in length that are transcribed from DNA
but not translated into proteins. miRNA genes primarily
reside between genes (intergenic) or within introns
(intronic) of genes and are transcribed to a primary
miRNA (pri-miRNA) mediated by polymerase II or III.
The pri-miRNA is processed within the nuclear
compartment to a precursor miRNA ( pre-miRNA) by
Drosha, a class 2 RNase III enzyme. Subsequently, the
transport of pre-miRNAs to the cytoplasm is arbitrated
by exportin-5 (EXP-5). In the cytoplasmic region, they
are processed further to develop into mature miRNAs by
Dicer an RNase III type protein and loaded onto the
Argonaute (Argo) protein to generate the effector RNA-
induced silencing complex. While majority believes that
miRNAs restrain translation, evidence that miRNAs can
actually augment translation through alterations in the
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
ISSRF
4
Argo component of the RISC also has been reported.
Thus, while miRNAs appear to police translation in an
inhibitory fashion, they may also might boost translation in
defined biological settings.
DNA packaging in nucleosomes might affect all stages
of transcription, thereby regulating gene expression. The
precise position of nucleosomes around the transcription
start off sites has an essential control on the initiation of
transcription. Nucleosome positioning not only decides
accessibility of the transcription factors to their target
DNA sequence but has also been reported to take part in
shapi ng t he met hyl at i on l andscape. Besi des
transcription regulation, nucleosome occupancy also
participates in directing meiotic recombination events.
The precise function of nucleosomes is influenced by the
incorporation of different histone variants that are
incorporated into chromatin independently, outside the
S-phase. Often linked with specific histone modifications,
nucleosome remodeling machinery is also influenced by
DNA methylation. Thus, the interaction among diverse
epigenetic partners is evident more often.
While alterations in the epigenomic landscape are
required for regular growth and development, they can
also be accountable for some disease states. The
significance of epigenetics in maintaining typical
development and biology is reflected by the observation
that many diseases build up when the wrong type of
epigenetic marks are introduced or are added at the
wrong time or at the wrong place. Disrupting any of the
four systems that contribute to epigenetic alterations can
cause abnormal activation or silencing of genes.
Epigenetic control systems generally involve three
types of proteins: writers, readers, and erasers.
Writers attach chemical marks, such as methyl groups
(to DNA) or acetyl groups (to the histone proteins that
DNA wraps around). So-called readers bind to
these marks, thereby influencing gene expression;
erasers remove the marks. The marks are passed down
as cells divide, providing a sort of cellular memory to
ensure t hat cell proliferation is effectively regulated. The
reversibility of epigenetic marks provides the possibility
that the activity of key genes and pathways can be regulated
as a therapeutic approach. Recent technological advances
are allowing the study on a more genome-wide scale which
is facilitating a more systems biology based approach to
understand disease aetiology. The epigenome has truly
arrived as a drug development target as pharmaceutical
companies and biotech giants have programmes aimed
squarely at proteins that operate in the epigenetic space.
It was often argued that most epigenetic modification, by
whatever mechanism, is erased with each new
gener at i on, dur i ng gamet o- genesi s and af t er
fertilization. However, one of the more startling
experiments conducted by Skinner published in Science
(2005) challenged this belief and suggested that
epigenetic changes may endure in at least four
subsequent generations. These findings provided a new
paradigm for disease aetiology and basic mechanism in
evolution not previously appreciated before. Today,
epigenetic aberrations have been conjectured to be
highly relevant to sexual and reproductive health as
these accounts for interactive relationship among
genomic landscape, gene-environment interactions
and di sease phenot ype. Novel i nsi ght s i nt o
aetiologies of complex non-Mendelian disease traits
have burgeoned interest in the field of reproductive
epigenetics. How range of epigenetic mechanisms
can differentially influence the male and female germ
lines and developmental process is closely monitored. Of
late, the subtle and elegant modulation of fidelity of
transmissible heritable characteristics through epigenetic
reprogramming has also received wider scientific attention.
Developmental activation and deactivation of epigenetic
signatures at pre-implantation phase provides putative links
between assisted reproductive technologies and imprinting
disorders. Occurrence of imprinting errors also disrupts
placental growth and development in assisted conception
procedures. Besides, epigenetics has potentially helped the
livestock industry to find part of the missing causality and
heritability of complex economic traits (milk yield and
fertility) and animal diseases as well.
With a focus to provide biological nuances and depth on
this key determinant of reproductive function, the 15th
edition of ISSRF Newsletter brings you leading edge
articles on "Epigenetics : an emerging paradigm in
reproductive health". We have made best possible efforts
to provide you a compendium of contributions from
leading experts working in premier institutions of our
country on various aspects of reproductive epigenetics.
It is our pleasure to introduce this new issue of the ISSRF
newsletter. Not only does it bring to readers various
contributions linked to the topics of epigenetics, but
it also presents some important translational
perspectives of reproductive health research, an area
that especially excites us. Hope, this new issue of the
ISSRF Newsletter will provide you with challenging
thoughts and contribute to broadening the debate on
how epigenetic factors can play a significant role in
our reproductive function and well-being.
What makes us who we are ? - Begin before birth and it's not all in our genes
ISSRF
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
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Epigenome Dynamics in Early Embryo &
Germline Development
Sonam Mehrotra, Sanjeev Galande*
Centre of Excellence in Epigenetics
Indian Institute of Science Education and
Research
Pune
*sanjeev@iiserpune.ac.in
Summary
The fertilization of two highly differentiated haploid cells:
the oocyte and the sperm results in the zygote, a cell with
complete developmental potential and capable of producing
any differentiated cell of the adult organism. The success of
the embryonic developmental program is determined by a
series of dynamic epigenetic changes that allow
configuration of parent-specific epigenetic states in the
gametes and support totipotency of the zygote. During early
embryogenesis, the cells undergo critical irreversible cell
fate decisions. The developmental potential of these cells at
different stages is manifested through a combination of
multiple epigenetic changes including DNA methylation,
modification of histones, incorporation of histone variants,
RNA mediated silencing and chromatin modeling. These
mechanisms underlie execution of specific functions by
different cells carrying identical genetic information
through selective activation/repression of specific gene
subsets thus creating unique transcription profiles.
Epigenetics Changes in Early Embryonic Development
The series of epigenetic programming and/ or
reprogramming events that dominate the early embryonic
development culminate in the zygote just after fertilization,
with erasure of DNA methylation marks from the paternal
pronuclear genome (Figure 1). DNA-methylation marks
from 5 position of cytosine residues from most of the CpG
dinucleotides are erased through Tet-catalyzed oxidation
followed by decarboxylation or through DNA repair
pathways (1, 2). This is followed by another passive wave of
DNA demethylation during cleavage /mitotic stages of
embryogenesis and finally extensive chromatin modeling to
silence pluripotency genes in the inner cell mass and
promote differentiation. Chromatin remodeling at this stage
is also associated with re-activation of the inactive X
chromosome in the female embryos. Subsequently, de novo
methylation mediated by DNA cytosine-5 methyl
transferase (DNMT) family enzymes allows reacquisition of
methylation signatures to establish expression patterns of
essential developmental genes in the embryo (3, 4).
A minority of sequences such as imprinted genes escapes
this reprogramming and maintains their parent of origin
germ-cell specific epigenetic patterns into adulthood.
Genetic imprinting is an epigenetic phenomenon that
regulates certain genes such that only one allele is expressed
based on the parental origin. This phenomenon may also
ensure epigenetic silencing of transposable elements such as
Line1 elements, hence critical in maintaining genomic
integrity (5, 6).
The second major reprogramming event occurs in germ cell
precursors known as primodial germ cells (PGCs) of the
developing embryo. For both male and female
gametogenesis involves establishment of cells with germ
line specific chromatin signatures which are distinct from
the surrounding somatic cells (Fig. 1). These primordial
germ cells are direct derivatives of the pluripotent cells of
the inner cell mass of the blastocyst that migrate to
urogenital ridges during gastrulation. Since these cells
consist of essential epigenetic information required for
development of the somatic embryo, the reprogramming at
this stage involves erasure of all pre-existing epigenetic
modifications including those that were maintained during
pre-implantation stage. These result in repression of genes
involved in somatic differentiation and/or activation of
genes involved in maintenance of specific germ cell identity
(7). These phenomena therefore establish sex-specific
epigenetic profiles and transcriptional networks essential for
normal development of the germ-line as well as regulation of
epigenetic inheritance mechanisms such as genomic
imprinting (8).
The Distinct Epigenomes of the Male and Female Germ
Cells
The two mature gametes comprising of maternal and paternal
DNA respectively present distinct epigenetic information at
the time of fertilization. At this stage the two parental
genomes remain physically separate and undergo distinct
programs of chromatin remodeling. The maternal DNA
contained in the oocyte is bound by histones acquired during
oocyte growth comprising of post-translational
modifications associated with stalled metaphase II. At
fertilization the maternal genome undergoes only few
epigenetic changes, these involve euchromatin marks
associated with activation of DNA replication and
transcription such as H3 and H4 acetylation and
argininemethylation. The major difference between the
chromatin of oocyte and somatic nuclei is the absence of H1
linker histones in the oocyte which are substituted by a
specific H1 variant (9). The role of these H1 histone variants
in the oocyte chromatin during early embryogenesis remains
to be understood.
In case of the male gamete, the highly compacted paternal DNA
residing in the sperm head undergoes extensive remodeling
via many decondensation cycles. During spermatogenesis
the paternal DNA in the sperm is compacted by replacement
of histones with protamines, which are then replaced again
by histones when the paternal genome begins to decondense
after fertilization. Recent studies suggest that chromatin of
mature spermatozoa retain small amounts of histones. Such
histone enrichment has been observed on developmentally
regulated genes which are important for early development
and differentiation as well as priming of the zygote (10).
The male pronucleus also exhibits high levels
histoneacetylation which supports higher transcription from
the S-phase in zygote stage and thereafter (11).
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
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Following fertilization, incorporation of different histone
variants also contributes to the differences between the two
parental genomes. For example, incorporation of H3.3
histone variant is observed only in the male pronucleus. It is
possible that the incorporation of H3.3 variant plays a role in
directing or preventing other paternal specific histone
modifications. Modification ofH3.3 has also been linked
with paternal pericentric heterochromatin and mutations in
lysine 27 of H3.3 leads to developmental arrest (12).
Incorporation of other histone variants has also been shown
to be important for early mammalian development. For
instance, the phosphorylation of the C-terminus of SQEY
motifs in H2A.X variant occurs in response to DNA double
strand breaks in somatic cells and is also enriched
duringformation of male pronucleus in Xenopus as well as
mouse embryos (13) Although the exact functions of H2A.X
and H2A.Z variants are not known in early development,
their deletion causes developmental arrest and failure of
implantation. During decondensation cycles the male
pronuclear genome lacks several modifications such as
H3K9me2, H3K9 me3, and H3K27me3 heterochromatin
marks which are detectable in the female pronucleus
genome (14).
The epigenetic asymmetry between the two parental pronuclei is
also detected by differences in the global levels of DNA
methylation. Initially both parental nuclei exhibit high
levels of DNA methylation. However, specific targeting of
DNA demethylation to the male pronucleus may be
regulated by asymmetries in the chromatin template and
lack of repressive histone modification marks. The
specificity of DNA demethylation is also directed by
maternally inherited Stella protein (15). Differences in the
global DNA methylation levels are detected only until the
two-cell stage, which are diluted due to passive DNA
methylation loss in the combined embryonic genome. The
significance of these epigenetic asymmetries in the parental
genomes and their persistence in the early stages of zygotic
development and how they affect early embryonic
development still remains unclear.
Implications of Epigenetic Inheritance
It has been speculated that alterations in the environment may
influence changes in the germ-line epigenome and these
modifications may provide mechanisms that allow
evolutionary adaptations. Recent studies provide evidence
that certain mutations in epigenetic modifiers may lead to
paternal inheritance of epigenetic changes. For example,
haploinsufficiency of Dnmt1 and Smarca5 in male mice is
sufficient for altering gene expression of maternal Avy gene
in an otherwise genetically wild-type animal (16). Studies in
C.eleganshave also demonstrated involvement of non-
coding RNAs such as piRNAs in mechanisms of paternal
epigenetic inheritance (17). Studies involving identification
RNA sequencing of sperm RNA also suggest the possibility
that these RNAscould also contribute to epigenetic states in
the early embryo (18).
Additionally, the epigenetic errors during spermatogenesis in
humans have been identified which lead to reduced
competency of the sperm and fertility in the males (19). In the
females, factors such as maternal nutrition and exposure to
certain toxins have also been associated with epigenetic
changes in the oocyte that have been linked with neonatal
developmental and gestational defects (20). Therefore, even
in a genetically normal organism, environmentally induced
epimutations in the parents germ-line may be transmitted to
the offspring and result in phenotypic variation, range of
diseases and/or developmental disorders. Therefore,
understanding how epigenetic information is established in
the parental germ-line and mechanisms underlying
epigenetic inheritance is of special interest. This knowledge
is crucial in determining the relationship between
environmental influences on epigenetic changes and
eventually towards the development of the organism.
Figure 1: Summary of Epigenetic Programming and
Reprogramming Events During Mammalian Development.
The two mature gametes present genomes in distinct epigenetic
states at the time of fertilization. With fertilization, the materal
genome (depicted in purple) completes meiosis and undergoes
relatively fewer epigenetic changes, involving euchromatic
marks. In contrast, the paternal genome (depicted in light
blue) undergoes several cycles of decondensation,
demethylation and replacement of protamines by histones
and histone variants. The formation of the zygote is followed
by a wave of demethylation of the genome with every
cleavage till the formation of the sixteen-cell stage. The
imprinted regions however, escape this demethylation and
maintain their parent of origin specific epigenetic states.
Following blastocyst formation the diploid genome
undergoes global remethylation, which results in erasure of
imprinting. Few cells in the inner cell mass acquire
epigenetic marks that distinguish these cells from the
surrounding somatic cells. These cells migrate to the
urogenital ridges in the developing gonads of the embryo,
where they undergo paternal and maternal specific
epigenetic changes during the development of mature sperm
and ovum (21).
References
1. Wu SC, Zang Y (2010). Active DNA demethylation: many roads lead
to Rome. Nature Reviews Molecular Cell Biology; 11: 607-20.
2. Hajkova P, Jeffries SJ, Lee C, et al (2010). Genome wide
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7
reprogramming in the mouse germ line entails the base excision
repair pathway. Science; 329: 78-82.
3. Wossidlo M, Nakamura T, Lepikhov K, et al (2011). 5-
hydroxymethylcytosine in mammalian zygote is linked with
epigenetic reprogramming. Nature Communications; 2: 241.
4. Rivera RM, Ross JW (2013). Epigenetics in fertilization and
preimplantation embryo development. Progress in Biophysics
and Molecular Biology; 113: 423-32.
5. Okada Y, Yamagata K, Hong K, et al (2010). A role for the
elongator complex in zygotic paternal genome demethylation.
Nature; 463: 554-8.
6. Weaver JR, Susiarjo M, Bartolomei MS (2009). Imprintind and
epigenetic changes in the early embryo. Mammalian Genome;
20: 532-43.
7. Hemberger M, Dean W, Reik W (2009). Epigenetic dynamics of
stem cells and cell lineage commitment: digging Waddington's
canal. Nature Reviews Molecular Cell Biol; 10: 526-37.
8. Burton A, Torres-Padilla EM (2010). Epigenetic
reprogramming and development: a unique heterochromatin
organization in the pre-implantation embryo. Briefings in
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9. Tanaka M, Kihara M, Meczekalski B, et al (2003). H1oo: a pre-
embryonic H1 linker histone in search of a function. Molecular
Cell Endocrinol; 202: 5-9.
10. Hammoud SS, Nix DA, Zhang H, et al (2009). Distinctive
chromatin in human sperm packages genes for embryo
development. Nature; 460: 473-8.
11. Mayor W, Smith A, Fundele R, et al (2000). Spatial separation of
parental genomes in preimplantation mouse embryos. J Cell
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12. Santenard A, Ziegler-Birling C, Koch M, et al (2010).
Heterochromatin formation in the mouse embryos requires
critical residues of Histone variant H3.3. Nature Cell Biology;
853-62
13. Dimitrov S, Dasso MC, Wolffe AP (1994). Remodelling sperm
chromatin in Xenopus laevis egg extracts: the role of core
histone phosphorylation and linker histone B4 in chromatin
assembly J Cell Biol; 126: 591-601.
14. van der Heijden GW, Dieker JW, Derijck AA (2005).
Asymmetery in Histone H3 variant and lysine methylation
between paternal and maternal chromatin of the early mouse
zygote. Mechanics of Development; 122: 1008-22.
15. Nakamura T, Arai Y, Umehara H, et al (2007). PGC7/Stella
protects against DNA demethylation in early embryogenesis.
Nature Cell Biol; 9: 64-71.
16. Chong S, Ratnam S, Ding F, et al (2007). Modifiers if epigenetic
reprogramming show paternal effects in the mouse. Nature
Genetics; 39: 614-22.
17. Gu SG, PJ, Guang S, et al (2012). Amplification of siRNA in
Caenorhabditis elegans generates a transgernerational
sequence-targeted histone H3 lysine 9 methylation footprint.
Nature Genetics; 44: 157-64.
18. Sendler E, Johnson GD, Mao S, et al (2013). Stability, delivery
and functions of human sperm RNAs at fertilization. Nucleic
Acids Research; 41: 4104-17.
19. Houshdaran S, Cortessis VK, Siegmund K, et al (2007).
Widespread epigenetic abnormalities suggest a broad DNA
methylation erasure defect in abnormal human sperm. PLoS
One; 2.
20. Stringer JM, Barrand S, Western P (2013). Fine-tuning
evolution: Germ-line epigenetics and inheritance.
Reproduction; 146: 37-48.
21. Cantone I, Fisher AG (2013). Epigenetic programming and
reprogramming during development. Nature Structural and
Molecular Biology; 20: 282-9.
Epigenetic Modulation and Reproduction: A
Link Between Environment and Genetics
Jens Vanselow
Leibniz Institute for Farm Animal Biology (FBN)
Dummerstorf
Germany
vanselow@fbn-dummerstorf.de
Epigenetic Mechanisms Modulate Gene Expression
The term epigenetic was firstly used by C.H. Waddington,
actually years before the molecular basis of inheritance has
been established. He used this term in the context of
epigenetic landscape as a conceptual model of how genes
might interact with their surroundings to produce a
phenotype (1). Currently we understand epigenetics as
mitotically and/or meiotically heritable changes in gene
function that cannot be explained by changes in DNA
sequence. (2). However, despite the exploding knowledge
of the molecular nature and function of genes as well as their
role in heredity the tremendous importance of epigenetic
mechanisms for reproduction, development, behavior and
health was recognized only recently. Now it is clear that
epigenetic mechanisms are crucially involved in regulating
the activity of genes (i.e. gene expression) and thus the
phenotypic shape. The physical substrates of genes are DNA
molecules, which are tightly associated with histone
proteins to form chromatin and consolidated within
chromosomes. In the most stringent way, epigenetic
modulation means the chemical modification of the DNA
and associated histone proteins. Histone modifications are
posttranslational covalent modifications to histone
tailsexhibiting either activating or repressive functions.
DNA methylation is the covalent binding of methyl groups
to cytosinesin the context of CpG dinucleotides. DNA
methylation is typically associated with gene silencing (3).
Generally, histone modification is considered as a more
transient mechanism, whereas DNA methylation patterns
are usually stably inherited across mitotic divisions.
Surprisingly, the connection between histone modification
and DNA methylation was not discovered earlier than 1998
with the discovery of the methyl-CpG-binding protein
MeCP2, which brings together methylated DNA, histone
deacetylation and transcriptional silencing (4).
Epigenetic Marks Determine the Cellular Fate and
Parental Imprinting of Genes
Developmental epigenetic marks are established during
gametogenesis and early embryogenesis (5). Particularly in
mammals, during oogenesis and spermatogenesis gender
specific differential DNA methylation marks are established
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on a subset of about 150 genes, thus persistently silencing
expression of these genes in a parent-specific manner. This
mechanism called genomic imprinting was discovered in
1984 (6). Genomic imprinting is responsible for the non-
equivalence of the maternal and paternal genomes in
mammals thus allowing only biparental embryos to develop
and survive. During early embryogenesis another wave of
genomic methylation does occur, which is responsible for
initiating tissue-specific differentiation of previously
pluripotent and omnipotent cells. Methylation marks are
established, which stably determine the cell fate during
subsequent ontogenetic development. Corresponding to the
term genomic imprinting this process can be referred to as
somatic imprinting. Recently however, it could also be
shown that DNA methylation marks can change during
ageing, pathological processes like cancer (7) or infectious
diseases (8), but also during iterative processes of
differentiation like folliculogenesis in adults (9, 10).
Assisted Reproductive Technology can Provoke
Epigenetic Alterations
Nowadays, assisted reproductive technology (ART)
including artificial insemination (AI), superovulation, in
vitro maturation (IVM), in vitro fertilization (IVF), embryo
culture and transfer, and intra-cytoplasmic sperm injection
(ICSI) is an indispensable component of animal breeding.
But also for human reproduction ART is becoming
increasingly important. Currently, ART accounts for 1 to 3%
of annual births in industrialized countries and continues to
expand rapidly (11). Except for an increased incidence of
premature births, these technologies are considered safe.
However, the timing of two key techniques commonly used
during ART, ovarian stimulation and in vitro culture,
coincide with important epigenetic programming events that
naturally occur during gametogenesis and early embryonic
development (12). Accordingly, this gives reason to expect
interference with epigenetic alterations.Indeed,
experimental evidence published during the past decade has
suggested a growing incidence of imprinting disorders in
children conceived by ART. Among children with
BeckwithWiedemann syndrome or Angelman syndrome
caused by an imprinting defect several clinical studies have
reported an increased frequency of ART conceptions (12).
BeckwithWiedemann syndrome (BWS) is an overgrowth
disorder caused by aberrant genetic and epigenetic
regulation at the KCNQ1OT1 and H19 imprinted domains.
ART-conceived BWS children commonly experience
maternal loss of methylation (LOM) at KCNQ1OT1 and
maternal gain of methylation (GOM) at H19. Angelman
syndrome (AS) is a neurological disorder that is caused by
genetic and epigenetic disturbances at the SNRPN-
imprinted domain. ART-conceived AS patients often
possess maternal SNRPN LOM (3). Further evidence comes
from the observation that a high proportion of these ART-
associated imprinting disorders appears to be caused by
epimutations that 1) are not commonly found in the general
population and 2) a majority of which occur on the maternal
allele (12). However, the actual incidence of imprinting
defects in oocytes and embryos from superovulated females
appears to be low and stochastic (13, 14).
Also, one has to consider that human embryos produced via
ARTs are the product of underlying infertility/subfertility
problems. This has led to questions regarding the origin of
epigenetic instability, i. e. whether underlying
infertility/subfertility compromises epigenetic integrity in
gametes/embryos, whether gamete/embryo manipulations
cause epigenetic instability, or whether a combination of
subfertility and ARTs leads to epigenetic disruption. The
relationship between impaired fertility, ARTs, and
epigenetic stability is unquestionably complex. However,
the possibility exists that ARTs and infertility may disrupt
the same biological pathways that lead to epigenetic
instability. If this is the case, perturbations induced by
infertility/subfertility may be deteriorated by gamete or
embryo manipulation, similar to combined ART treatments
(3). Other studies suggest that in humans the increase in the
incidence of imprinting disorders in individuals born by
ART may be due, in some cases, to the use of sperm with
intrinsic imprinting mutations. An important consideration
is to determine if the implicated association between ART
and imprinting disorders is actually related to the procedures
or to infertility itself. Several lines of evidence, however,
suggest that multiple aspects of the ART process, including
the gonadotropin stimulation of folliculogenesis, embryo
culture and/or embryo transfer, have the potential to induce
epimutations in offspring produced by these methods (15).
In animal breeding it is well known that reproductive
techniques, and in particular somatic cell nuclear transfer
(SCNT), but also other procedures connected with ART, can
cause severe abnormalities like large offspring syndrome
(LOS). Interestingly, LOS and BWS show phenotypic and
epigenetic similarities (16) thus suggesting similar
molecular mechanisms including epigenetic dysregulation
of growth associated genes. Data of several studies indicate
that imprinting marks are erased during the reprogramming
of the somatic cell nuclei during early development,
indicating that such epigenetic anomalies may play a key
role in mortality and morbidity of cloned animals (17).
Environmental Factors can Induce Transgenerational
Epigenetic Alterations
From epidemiological studies it was first discovered in
humans that unfavorable nutritional conditions during
specific sensitive life periods in particular during the
prepubertal slow growth period can increase susceptibility
to cardiovascular and metabolic diseases and may
significantly influence longevity even during subsequent
generations (18, 19, 20, 21). These statistical association
studies led to the formulation of the developmental origins
of health and disease hypothesis, which suggests that many
adult-onset diseases can be attributed to altered growth and
development during early life (22). However, underlying
molecular mechanisms were not revealed by these studies.
In contrast, studies in animal models not only demonstrated
deleterious, transgenerational effects of toxic substances
and endocrine disruptors, but also clearly demonstrated that
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persistent epigenetic alterations were responsible for these
effects. Exposure of gestating female rats to the agriculture
fungicide vinclozolin during gonadal sex determination
revealed in their offspring altered expression profiles of
about 400 testicular genes over three generations and
demonstrated epigenetic transgenerational inheritance as an
important component of the molecular etiology of male
infertility (23). Also exposure to dioxin during fetal day 8 to
14 induced prostate disease, ovarian primordial follicle loss
and polycystic ovary disease for at least three generation. In
this study 50 differentially DNA methylated regions could
be identified in specific promoters thus pointing to an altered
DNA methylation pattern as a possible cause for dioxin
induced transgenerational effects (24). Similar studies were
performed with the endocrine disruptor compounds
bisphenol-A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and
dibutyl phthalate (DBP), which demonstrated epigenetic
transgenerational inheritance of adult onset disease and
associated DNA methylation epimutations (25). Molecular
mechanisms were most convincingly demonstrated in a
mouse model showing that maternal exposure to endocrine
disruptors BPA or Genistein can stably affect DNA
methylation levels within the agouti locus and thus the coat
color of offspring. In addition, also metabolic parameters
can be negatively affected in these mice (26, 27).
Conclusions
A plethora of studies published during the last decade
demonstrated that environmental factors as nutrition, toxins
or endocrine disruptors, but also procedures associated with
assisted reproduction technologies can essentially affect the
formation of the phenotype. Data from animal models
indicate that persistent alterations of DNA methylation at
specific loci might be responsible. Surprisingly, these
epimutations and associated phenotypic alterations can be
passed transgenerationally. Thus, the emerging field of
environmental epigenomics clearly deserves our greatest
attention and research efforts not least because of our
responsibility for future generations.
References
1. Wadington CH. The epigenotype. Endeavour. 2042;1:18-20.
2. Russo VEA, Martienssen RA, Riggs AD (1996). Epigenetic
mechanisms of gene regulation. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
3. Denomme MM, Mann MR (2012). Genomic imprints as a
model for the analysis of epigenetic stability during assisted
reproductive technologies. Reproduction;144: 393-409.
4. Nan X, Ng H-H, Johnson CA, et al (1998). Transcriptional
repression by the methyl-CpG-binding protein MeCP2 involves
a histone deacetylase complex. Nature; 393: 386-9.
5. Reik W, Walter J (2001). Genomic imprinting: parental
influence on the genome. Nat Rev Genet; 2: 21-32.
6. Surani MA, Barton SC, Norris ML (1984). Development of
reconstituted mouse eggs suggests imprinting of the genome
during gametogenesis. Nature; 308: 548-50.
7. Kondo Y (2009). Epigenetic cross-talk between DNA
methylation and histone modifications in human cancers.
Yonsei Med J; 50: 455-63.
8. Vanselow J, Yang W, Herrmann J, et al (2006). DNA-
remethylation around a STAT5-binding enhancer in the alphaS1-
casein promoter is associated with abrupt shutdown of alphaS1-
casein synthesis during acute mastitis. J Mol Endocrinol; 37:
463-77.
9. Vanselow J, Frbass R (2010). Epigenetic control of
folliculogenesis and luteinization. Animal Reproduction; 7: 134-
9.
10. Vanselow J, Spitschak M, Nimz M, et al (2010). DNA
Methylation Is Not Involved in Preovulatory Down-Regulation of
CYP11A1, HSD3B1, and CYP19A1 in Bovine Follicles But
May Play a Role for Permanent Silencing of CYP19A1 in Large
Granulosa Lutein Cells. Biol Reprod; 82: 289-98.
11. Eroglu A, Layman LC (2012). Role of ART in imprinting
disorders. Semin Reprod Med; 92-104.
12. deWaal E, McCarrey JR (2010). Effects of exogenous endocrine
stimulation on epigenetic programming of the female germline
genome. Animal Reproduction; 7: 154-64.
13. Sato A, Otsu E, Negishi H, et al (2007). Aberrant DNA
methylation of imprinted loci in superovulated oocytes. Hum
Reprod; 22: 26-35.
14. Market-Velker BA, Zhang L, Magri LS, et al (2010). Dual effects
of superovulation: loss of maternal and paternal imprinted
methylation in a dose-dependent manner. Hum Mol Genet; 19:
36-51.
15. deWaal E., Yamazaki Y, Ingale P, et al (2012). Gonadotropin
stimulation contributes to an increased incidence of epimutations
in ICSI-derived mice. Hum Mol Genet; 21: 4460-72.
16. Chen Z, Robbins KM, Wells KD, et al (2013). Large offspring
syndrome: a bovine model for the human loss-of-imprinting
overgrowth syndrome Beckwith-Wiedemann. Epigenetics;
591-601.
17. Smith LC, Suzuki J, Jr., Goff AK, et al (2012). Developmental
and epigenetic anomalies in cloned cattle. Reprod Domest Anim;
47 Suppl 4: 107-14.
18. Pembrey ME, Bygren LO, Kaati G, et al (2006). Sex-specific,
male-line transgenerational responses in humans. Eur J Hum
Genet; 14: 159-66.
19. Whitelaw E (2006). Epigenetics: sins of the fathers, and their
fathers. Eur J Hum Genet; 14: 131-2.
20. Kaati G, Bygren LO, Pembrey M, et al (2007). Transgenerational
response to nutrition, early life circumstances and longevity. Eur J
Hum Genet; 15: 784-90.
21. Kaati G, Bygren LO, Edvinsson S (2002). Cardiovascular and
diabetes mortality determined by nutrition during parents' and
grandparents' slow growth period. Eur J Hum Genet; 10: 682-8.
22. Dorey ES, Pantaleon M, Weir KA, et al (2014). Adverse prenatal
environment and kidney development: implications for
programing of adult disease. Reproduction; 147: R189-98.
23. Guerrero-Bosagna C, Savenkova M, Haque MM, et al (2013).
Environmentally induced epigenetic transgenerational
inheritance of altered Sertoli cell transcriptome and epigenome:
molecular etiology of male infertility. PLoS One; 8: e59922.
24. Manikkam M, Tracey R, Guerrero-Bosagna C, et al (2012).
Dioxin (TCDD) induces epigenetic transgenerational inheritance
of adult onset disease and sperm epimutations. PLoS One; 7:
e46249.
25. Manikkam M, Tracey R, Guerrero-Bosagna C, et al (2013).
Plastics derived endocrine disruptors (BPA, DEHP and DBP)
induce epigenetic transgenerational inheritance of obesity,
reproductive disease and sperm epimutations. PLoS One; 8:
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26. Dolinoy DC, Huang D, Jirtle RL (2007). Maternal nutrient
supplementation counteracts bisphenol A-induced DNA
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A; 104: 13056-61.
27. Dolinoy DC, Jirtle RL (2008). Environmental epigenomics in
human health and disease. Environ Mol Mutagen; 49: 4-8.
Male Infertility and Epigenetic Modulation
Swetasmita Mishra, Rima Dada*
Department of Anatomy
All India Institutes of Medical Sciences
New Delhi
*rima_dada@yahoo.co.in
Epigenetics is the study of heritable changes in gene activity
that are not caused by changes in the DNA sequence; it also
can be used to describe the study of stable, long-term
alterations in the transcriptional potential of a cell that are
not necessarily heritable. Examples of mechanisms that
produce such changes are DNA methylation and histone
modification, each of which alters gene expression without
altering the underlying DNA sequence. Gene expression can
be controlled through the action of repressor proteins that
attach to silencer regions of the DNA. These epigenetic
changes may last through cell divisions for the duration of
the cell's life, and may also last for multiple generations even
though they do not involve changes in the underlying DNA
sequence of the organism (1); instead, non-genetic factors
cause the organism's genes to behave (or "express
themselves") differently. Specific epigenetic processes
include paramutation, bookmarking, imprinting, gene
silencing, X chromosome inactivation, position effect,
reprogramming, transvection, maternal effects, the progress
of carcinogenesis, many effects of teratogens, and
regulation of histone modifications and heterochromatin.
Chromatin Remodelling
Since DNA methylation and chromatin remodeling play
such a central role in many types of epigenetic inheritance,
the word "epigenetics" is sometimes used as a synonym for
these processes. However, this can be misleading.
Chromatin remodeling is not always inherited, and not all
epigenetic inheritance involves chromatin remodelling (2).
Because the phenotype of a cell or individual is affected
by the genes that are transcribed, heritable transcription
states can give rise to epigenetic effects. There are several
layers of regulation of gene expression. One way that genes
are regulated is through the remodeling of chromatin.
Chromatin is the complex of DNA and the histone proteins
with which it associates. If the way that DNA is wrapped
around the histones changes, gene expression can change as
well. Chromatin remodeling is accomplished through two
main mechanisms, post translational modification of the
histones and methylation of the DNA.
Histone Modifications
Histone proteins are made up of long chains of amino acids.
If the amino acids that are in the chain are changed, the shape
of the histone might be modified and hence the expression of
the genes is changed. These histone proteins carry specific
epigenetic marks on them. DNA is not completely unwound
during replication. The parental histones are carried into each
new copy of the DNA during replication. These histones may
act as templates, initiating the surrounding new histones to be
shaped in the new manner. By altering the shape of the histones
and the epigenetic marks on them these modified histones
cause a lineage-specific transcription program which is
maintained after cell division. The epigenetic marks on the
histone proteins are heritable and play an important role in
regulation of spatial and temporal expression of the genes.
Although histone modifications occur throughout the entire
sequence, the unstructured N-termini of histones (called
histone tails) are particularly highly modified. These
modifications include acetylation, methylation,
ubiquitylation, phosphorylation, sumoylation, ribosylation
and citrullination. Acetylation is the most highly studied of
these modifications. For example, acetylation of the K14 and
K9 lysines of the tail of histone H3 by histone
acetyltransferase enzymes (HATs) is generally related to
transcriptional competence. Methylation of lysine 9 of histone
H3 has long been associated with constitutively
transcriptionally silent chromatin (constitutive
heterochromatin). It has been shown that the histone lysine
methyltransferase (KMT) is responsible for the methylation
activity in histones H3 & H4. This enzyme utilizes a
catalytically active site called the SET domain (Suppressor
of variegation, Enhancer of zeste, Trithorax). The SET
domain is a 130-amino acid sequence involved in
modulating gene activities. This domain has been
demonstrated to bind to the histone tail and causes the
methylation of the histone (3).
Differing histone modifications are likely to function in
differing ways; acetylation at one position is likely to
function differently from acetylation at another position. Also,
multiple modifications may occur at the same time, and these
modifications may work together to change the behavior of the
nucleosome. Multiple dynamic histone modifications regulate
gene transcription in a systematic and reproducible way and
this is called the histone code.
DNA Methylation
Methylation of the DNA is the addition of methyl groups to
the CpG sites, to convert cytosine to 5-methylcytosine. 5-
Methylcytosine performs much like a regular cytosine, pairing
with a guanine in double-stranded DNA. However, some areas
of the genome are methylated more heavily than others, and
highly methylated areas tend to be less transcriptionally
active, through a mechanism not fully understood.
Methylation of cytosines can also persist from the germ line of
one of the parents into the zygote, marking the chromosome as
being inherited from one parent or the other (genetic
imprinting).
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DNA methylation frequently occurs in repeated
sequences, and helps to suppress the expression and
mobility of ' transposable elements' (4). As 5-
methylcytosine can be spontaneously deaminated
(replacing nitrogen by oxygen) to thymidine, CpG sites are
frequently mutated and become rare in the genome, except at
CpG islands where they remain unmethylated. Epigenetic
changes of this type thus have the potential to direct
increased frequencies of permanent genetic mutation. DNA
methylation patterns are known to be established and
modified in response to environmental factors by three
independent DNA methyltransferases, DNMT1, DNMT3A,
and DNMT3B, (5). DNMT1 is the most abundant
methyltransferase in somatic cells localizes to replication
foci has a 1040-fold preference for hemimethylated DNA
and interacts with the proliferating cell nuclear antigen
(PCNA).
By preferentially modifying hemimethylated DNA,
DNMT1 transfers patterns of methylation to a newly
synthesized strand after DNA replication, and therefore is
often referred to as the maintenance' methyltransferase.
DNMT1 is essential for proper embryonic development,
imprinting and X-inactivation. Furthermore, in addition to
the maintenance and transmission of methylated DNA
states, the same principle could work in the maintenance and
transmission of histone modifications and even cytoplasmic
(structural) heritable states (6). Mechanisms of heritability
of histone state are not well understood, however much is
known about the mechanism of heritability of DNA
methylation state during cell division and differentiation.
Heritability of methylation state depends on certain
enzymes (such as DNMT1) that have a higher affinity for 5-
methylcytosine than for cytosine. If this enzyme reaches a
"hemimethylated" portion of DNA (where 5-methylcytosine
is in only one of the two DNA strands) the enzyme will
methylate the other half.
It has been suggested that chromatin-based transcriptional
regulation could be mediated by the effect of small RNAs.
Small interfering RNAs can modulate transcriptional gene
expression via epigenetic modulation of targeted promoters
(7). Various studies on different animal models and humans
have est abl i shed t he concept t hat epi genet i c
programming/reprogramming are essential for proper
spermatogenesis and embryo development. Any defect in
the process leading to epigenetic errors may lead to sperm
disorders and infertility. This review is mainly focussed on
the epigenetics of the sperm genome especially sperm DNA
methylation and the role of altered methylation of sperm
DNA on fertility.
Sperm Cell Epigenetics
During spermiogenesis the round spermatids with active
nuclear activities like transcription and translation are
converted into inert flagellated cells with a compact nucleus.
DNA compaction is a characteristic feature of male germ
cells and a fundamental process required for the
transmission of paternal genome to the next generation (8).
This specific event of sperm DNA compaction is believed to
convey essential epigenetic information to the developing
embryo (9, 10). In the early stages of spermatogenesis the
spermatids have a less compact genome in the form of DNA
around nucleosomes. Nucleosome is comprised of DNA
coiled around an octamer of histones H2A, H2B, H3, and
H4. Then as the spermatogenesis process proceeds further
there occurs significant compaction of the sperm genome by
the replacement of histones by nonhistone proteins.
Histones are first replaced by transition proteins (TNP1 and
TNP2) and ultimately by protamines (P1 and P2) (11). The
protamine bound sperm genome structure is 6-20 times
more compact than the histone bound nuclear structure (12).
After the protamine bound compact sperm genome structure
is reached the sperm cells become transcriptionally and
translationally inert. The mature sperm cells contain only the
mRNA and small RNAs which was present in the spermatids
at the early stages of spermatogenesis. Previously it was
believed that there is no involvement of sperm transcript in
the embryo development but now several studies report the
involvement of sperm genome organisation and paternal
transcriptome in early embryo development (9, 10).
Methylation and Male Fertility
The molecular basis of male infertility remains largely
unknown (13). A number of candidate gene studies revealed
that abnormal sperm DNA methylation patterns are
associated with reduced sperm count and function (14, 15,
16, 17, 18 ) as well as outcome of assisted reproductive
technologies (ART) (19). Accumulating evidence suggests
that the spermatozoa contribute more to the embryo than the
paternal genome (20, 21). Epigenetic factors in the sperm
cells may regulate the expression of various genes necessary
for proper embryogenesis.
Methylation establishment in germ cells is a basic process
which is necessary for proper spermatogenesis (22). It is a
well studied phenomenon in mouse germ cells. There is a
global demethylation remethylation process that takes place
during embryogenesis. This process involves erasure of
somatic DNA methylation and subsequent establishment of
sex-specific de novo DNA methylation (23). Demethylation
takes place in the developing embryo between 8.013.5 days
postcoitum (dpc) and all the methylation marks are removed
(24). Then a specific remethylation process begins at 15.5
dpc (25). This methylation remethylation process during
embryonic development gives rise to a phenomenon called
genomic imprinting.
Genomic Imprinting
It is an inheritance process independent of the classical
Mendelian inheritance, an epigenetic phenomenon ensuring
the expression of certain genes in a parent-of-origin-specific
manner (26).
Diploid organisms possess two copies of the genome in the
somatic cells. Each gene is represented by two copies or
alleles (one copy inherited from each parent at fertilisation).
For majority of autosomal genes expression occurs from
both alleles simultaneously. However, in mammals some
genes (<1% of genes) are imprinted so that gene expression
occurs from only one allele. The expressed allele is
ISSRF
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
dependent upon its parental origin. For example, the gene
encoding Insulin-like growth factor 2 (IGF2/Igf2) is only
expressed from the allele inherited from the father.
In humans, imprinted alleles are silenced such that the genes
are either expressed only from the non-imprinted allele
inherited from the mother (e.g. H19 or CDKN1C), or in
other instances from the non-imprinted allele inherited from
the father (e.g. IGF-2). Genomic imprinting can involve
DNA methylation and histone modulation in order to
achieve monoallelic gene expression without altering the
genetic sequence. These epigenetic marks are established in the
germline that are maintained through mitotic divisions.
Appropriate expression of imprinted genes is important for
normal development. There are several diseases pertaining to
imprinting defects like BeckwithWiedemann syndrome,
SilverRussell syndrome, Angelman syndrome and
PraderWilli syndrome and many more.
Imprinting marks are erased in primordial germ cells
(PGCs) and a specific remethylation occurs in
spermatogonia and type I spermatocytes, before their entry
into meiosis, so that all spermatozoa transmit the correct
paternal imprint (27). Only four imprinted loci are
methylated in the male germ line Igf2/H19, Rasgrf1, Dlk1-
Gtl2, and Zdbf2 (28). DNA methylation is carried out by a
family of enzymes called DNA methyltransferases (DNMTs).
DNMT1 is responsible for methylation maintenance (29), and
DNMT3A, 3B, and 3L specifically allow the methylation
process in germ cells. The activities of these
methyltransferases are essential for completion of
spermatogenesis. It has been demonstrated that Dnmt3a
knockout in germ cells causes germ cell apoptosis and
ultimately impairs spermatogenesi (30). Knocking out
Dnmt3l gene induced meiotic arrest in spermatocytes.
Genomic imprinting is relatively less studied area in human
developmental biology. Only two studies have reported
methylation marks of imprinted genes in germ cells of adult
men. Studies has confirmed the methylation of the H19 in
spermatogonia and the demethylated state of MEST/PEG1
in spermatogonia and type I spermatocytes (31). In humans,
methylation marks are acquired before entry into meiosis but
it is still unknown whether this process occurs at the fetal
developmental period, perinatal period or at puberty.
Several studies have investigated the relationship between
DNA methylation levels and male fertility in humans.
Methylation defects of the sperm DNA has been
demonstrated as a cause of infertility. High methylation
levels of mature sperm DNA has been reported to be
associated with high pregnancy rates (32). Several studies
have been conducted to study global methylation patterens
with sperm quality and hence infertility. Elevated levels of
DNA methylation of imprinted and nonimprinted genes and
repetitive elements was reported in poor-quality semen samples
(33). Improper erasure of methylation marks was elucidated as a
cause of elevated methylation levels in cases of
oligoasthenoteratozoospermia (OAT) rather than de novo
methylation. They also suggested that not only imprinted genes,
but also broad epigenetic defects are involved in causing sperm
defects. Similar kind of study by Aston et al. reported a genome-
wide altered DNA methylation pattern in men with altered
semen parameters (34) giving a connection of global
methylation levels of sperm with fertility status of men.
Marques et al. studied methylation patterns in some
important imprinted genes in different sperm populations.
Loss of methylation of H19 DMR was found in men with
OAT, and also they reported an association of reduced
methylation of H19 DMR with decreased sperm count (14).
Besides H19 DMR they also found altered methylation
levels in MEST DMR in oligozoospermic infertile patients.
In a different study loss of methylation in H19 and GTL2
DMRs and altered methylation acquisition at PEG1, LIT1,
ZAC, PEG3, and SNRPN loci were reported in cases of
oligozoospermia (35). Improper imprinting and methylation
in the sperm has been found to be associated with ART
failures. Using bisulfite conversion and cloning-sequencing
technique it has been observed that imprinting errors in the
spermatozoa resulted in spontaneous abortions in ART (35).
Boissonnas et al. performed a quantitative analysis of
methylation levels at each CpG position included in IGF2
and H19 DMRs (47 different CpGs) in a population of
normozoospermic and oligozoospermic men (36). Loss of
methylation in IGF2 DMR2 (primary IGF2DMR) and
H19DMR was correlated with the severity of
oligozoospermia.
Various other groups also demonstrated the association of
improper methylation with sperm quality and fertility
outcomes (18, 34, 37). Some other groups studied the
methylation status of the promoter of important genes
involved in sperm formation. The promoters of DAZL (17),
CREM (38) and MTHFR (39) genes were studied for
methylation and in all of these genes altered methylation
patterns were found in the OAT patients. Hypermethylation of
the MTHFR promoter was also reported in nonobstructive
azoospermia and in idiopathic infertility (40).
By functional analysis it was reported that there is a gain of
methylation in spermatogenesis-related genes and a loss of
methylation in inflammation- and immune response-related
genes (41). By targeting members of the PIWI-associated
RNA (piRNA) processing machinery altered DNA
methylation patterns has been observed in these genes. Male
i nfert i l i t y was associ at ed wi t h t he promot er
hypermethylation-associated silencing of PIWIL2 and
TDRD1. It was also shown that methylation errors in these
genes resulted in a defective production of piRNAs and a
hypomethylation of the LINE-1 repetitive sequence in the
patients. This study proves the role of altered DNA
methylation in PIWIL2/TDRD1 has a role in the control of
gene expression in spermatogenesis and hence may have an
important role to play in infertility (42). Chunlin et al
detected the testis and epididymis-specific methylated
promoters in human cfsDNA. This finding may be of great
clinical significance and may be used for diagnosis of male
infertility by employing these methylated promoters as
noninvasive epigenetic biomarkers (43).
All the studies till date on epigenetics of sperm suggests the
13
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
14
fact that sperm DNA methylation patterns are essential for
normal sperm function, good fertility outcomes and embryo
development. However, the underlying causes of these
epigenetic faults (methylation errors), and the timing of their
occurrence remain poorly investigated. When are these
methylation errors acquired? It is to be still answered that
whether these methylation errors occur during fetal or early
postnatal development? Epigenetic abnormalities are linked
to abnormal DNA packaging and configuration during
spermiogenesis where histones and protamines play a role in
the maintenance of the methylation marks of male gametes
(44).Studies are ongoing in our lab to correlate free radical
levels and methylation pattern and if oxidative stress apart
from causing non specific DNA damage, accelerated
telomere shortening also cause methylation errors and thus
affect not only the normally imprinted loci but also cause
genome wide methylation defects.
Remarks
As discussed in this review epigenetic or methylation errors
in both imprinted and non imprinted genes in the sperm
DNA are responsible for the sperm defects, the major cause
of male infertility and impaired embryonic development
which manifests as recurrent spontaneous abortions and
congenital malformations. Many studies provide evidence
that epigenetic effects are induced by ART procedures which
use epigenetically unstable and immature germ cells and are
also impacted by the hormonal mileu. There are many
confounding factors, such as parental subfertility, age and
the interaction between epigenetic responses and genetic
variations. All of these factors should be considered in
investigations carried out to elucidate the effects of ART on
epigenetic marks. More research is needed to evaluate the
risk of transmission of epigenetic abnormalities by gametes
of infertile men and women and in our lab we are trying to
study correlation between oxidative stress and methylation
errors. Genetic defects explain only a minor part of male
fertility problems (13). The role of epimutations should be
investigated. Epigenetics provides the most liable molecular
mechanism for gene environment interactions in the male
germ line that may adversely affect semen quality and
embryonic development. As is well understood that
epigenetics is the result of gene environment interactions. It
largely depends on the micro environment in which the gene
has to be expressed. In our lab studies are ongoing to assses
the alterations in methylation patterns in cases with
oxidative stress and understand if epigenetic abnormalities
are the underlying cause of infertility, congenital
malformations and cancers in cases with oxidative stress.
We have initiated a three year study on impact of yoga and
meditation on oxidative stress levels, DNA fragmentation
index , telomere length and telomerase levels and alterations
in methylation levels. Infertility is beleiveed to be an early
marker of cancer. We hypothesise that seminal oxidative
stress, sperm DNA damage of both nuclear and
mitochondrial genome, rapid telomere attrition and
epigenetic abnormalities may be the underlying cause of
cancer in both the infertile person ans well as the offspring.
Methylation errors in the form of hypermethylation of
tumour suppressor genes and hypomethylation of
oncogenes may explain for this link. Yoga and meditations
are actually therapeutic and cause decline in free radical
levels, upregulation in telomerase activity even 10 days
post meditation/yoga and study is ongoing for longer
periods.Yoga caused a significant decline in levels of
inflammatory markers like interleukins and upregulation in
the levels b endorphins. Studies should be conducted to
investigate the role of environmental factors and lifestyle
habits like dietary intake on the sperm epigenome and the
consequent effect on the fertility outcome. Since epigenetic
changes are reversible, a better understanding of epigenetic
dysregulation in the male germ line may help in establishing
new strategies to reinstate fertility in male infertility cases by
modulating DNA methylation by dietary regulations and
lifestyle modifications.
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Dynamics of DNA Demethylation and
Epigenetic Reprogramming in Stem Cells
Swayamsiddha Kar
Samir Kumar Patra*
Department of Life Science
National Institute of Technology
Rourkela
*skpatra_99@yahoo.com
Introduction
The molecular hallmarks guiding the smooth transformation
of a one-celled yet all-powerful, totipotent zygote into a
multicellular entity with over 220 specialized unipotent cell
types are essentially scripted by synchronization between
genetic and epigenetic programs in the stem cells. While
genetic content of the stem cells acts as the template for
generating varied transcriptional outcomes and remains
basically unchanged, the epigenetic choreographers
interpret and translate this information into cell-specific
functional destinies (1, 2). Stem cells, with their ability to
differentiate into any mature cell type and capacity to
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
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undergo indefinite replicative cycles are considered to be
strategically ideal vessels for enactment of the
developmental evolution (3). Epigenetic reprogramming of
the stem cell transcriptome is the ultimate control
mechanism that endows the stem cells with the adaptability,
flexibility and versatility to modify their gene expression
profile in response to developmental cues and differentiate
into any cell type in the adult body (4, 5). This review
focuses on the epigenetic determinants participating in
reconfiguration of the genome towards cellular
differentiation with emphasis on the role of DNA
demethylation in this scheme. Exploring the machinery
behind epigenomic regulation of stem cell fate will provide
better understanding of the intricate system supervising the
stem cells as they execute the molecular floor plan of cellular
development.
Epigenetic Reprogramming is the Master Architect in
Designing Stem Cell Destiny
The mammalian developmental scheme entails the gradual
conversion of a small group of genetically homogeneous
cells into structurally and functionally heterogeneous
organism. As cellular differentiation proceeds towards
terminally specialized states, cells with higher potency
gradually lose their potential and become committed for
specific lineages (6, 7). In terms of transcriptional states,
while totipotent and pluripotent stem cells promote the
expression of pluripotency-associated factors and
transiently restrict the expression of regulatory genes that
lead to lineage-speciation; the onset of lineage-orientation
witnesses gradually stripping off the restrictive marks on
lineage-devoted progenitor and lineage-committed genes,
thus allowing them to reassert their identity (8). The decision
of the stem cell to either maintain its pluripotency or
differentiate into a specialized cell is influenced
predominantly by epigenetic events that deliver heritable
and stable instructions for the specification of chromatin
organization and differential transcriptional outcomes. In
fact, the epigenetic signature of any cell is a sensitive
indicator of its past and current developmental standing and
may ultimately decide its future phenotype and functional
character (9). Thus, epigenetic restructuring of the genomic
domain is an essentiality during transition of a totipotent
zygote into pluripotent stem cells, subsequent
channelization towards multipotent lineage progenitors and
final acquisition of lineage-specific identity of unipotent
somatic cells.
A wide number of epigenetic enforcers converge in the
chromatin, modify and manipulate the nuclear architecture
to yield varied transcriptional consequences in the stem
cells. Of the many agents, the most notable ones are
reversible DNA-methylation and covalent post-
translational histone modifications. These two
modifications work in a stratified manner wherein short-
term flexibility imposed by transient histone modifications
on the lineage-committed genes during early development is
reinforced by induction of long-term or permanent
repression of pluripotency genes by DNA-methylation,
nucleosomal rearrangement and higher-order chromatin
reorganization at the onset of differentiation (4, 10).
Maintenance of such a fine-tuned and efficient arrangement
is ensured via co-ordinations among numerous enzymatic
players responsible for deposition (writers) and removal
(erasers) of the epigenetic marks/tags, as well as various
protein complexes that recognize these modifications
(readers) and convert them into functional capacity of the
cell (11). Alongside these factors, a rigidly coordinated
network of four master transcriptional regulatorsOCT4,
SOX2, NANOG and KLF4 stringently guard stem cell
pluripotency and smoothly maneuver transition between
differential gene expression states (12, 13). The
congregation of numerous epigenetic factors, all working
synchronously towards a single goal, guarantees robustness
at the level of individual cells and warranties high fidelity of
the entire differentiation program.
DNA-Demethylation Plays Godfather Role During
Reorganization of Stem Cell Fate
DNA-methylation and demethylation are considered to be
significantly more important players participating in the
epigenetic scheme behind regulation of development and
differentiation (14, 15). Transcriptional plasticity and
versatility of the stem cells is achieved as a result of a
delicate balance between the processes that add and remove
the epigenetic labels especially the methyl tags on the
cytosine bases. While DNA methylation integrates the
transient restrictive marks previously enforced by histone
modifications to permanently turn off unnecessary genes
during early embryonic divisions and lineage speciation,
DNA-demethylation purges off repressive marks from the
genomic database for rapid reactivation of silenced genes
leading to assertion of individual cell fate (16, 17). Cellular
differentiation and lineage-commitment pathways witness
both genome-wide and gene-specific erasure of methyl
marks via active and passive mechanisms, thus emphasizing
the part played by DNA-demethylation in this scheme (18).
Although, DNA-demethylation cant be held responsible for
all the differentiation related changes but a hypomethylated
state may facilitate transcriptional changes essential to erase
the existing epigenomic memory and generate somatic cell-
destiny. In a nutshell, dynamic changes in the pattern of
methyl marks both at the global, individual chromosome and
gene-specific level by DNA-methylation and demethylation
represents a fundamental mechanism in developmental
progress.
DNA-demethylation arbitrated erasure of epigenetic
barriers appears consistently at every crucial juncture during
mammalian development. Starting at the onset of
fertilization, DNA-demethylation activates the quiescent
transcriptional machinery in the totipotent zygote, facilitates
extensive germ-line reprogramming in the primordial germ
cells and ultimately steers the pluripotent stem cells into
lineage-restricted paths. Just after fertilization and before
the initial zygotic divisions, DNA demethylation mediates
remodeling of the paternal genomes via both active and
passive mechanisms. Before pronuclear fusion and first
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
cleavage division, the paternal pronucleus experiences
genome-wide active demethylation (19, 20). During the
progress towards the 16-celled morula, the maternal
pronucleus is passively deprived of its methylation load via
passive, replication-dependent loss of methylation (21). At
the blastocyst stage, the delineation of trophoectoderm (TE)
from the inner cell mass (ICM) is also aided by promoter
hypomethylation of transcription factor E-74 like Factor 5
(Elf5) (6, 22). Thus, DNA-demethylation stimulates the
onset of pluripotency in the zygote leading to expression of
lineage-determining transcription factors and segregation
into the three embryonic germ layers.
The second critical phase of DNA demethylation mediated
epigenetic reorganization is seen during germ-line
reprogramming of PGCs. In the first round, the early PGCs
acquire the ability to generate pluripotent embryonic germ
cells (EGCs) by transcriptional redirection and activation of
the germ cell genome from a somatic cell fate (23). In the
second round, demethylation in the PGCs results in the
expression of a number of genes involved in myriad aspects
of germ-line genome activity. For example, activation of
Deleted in Azoospermia-Like (Dazl) and Synaptonemal
Complex Protein 3 (Sycp3) is necessary for gametogenesis
(24), hypomethylation of germ-line genome-defense
specific genes such as Testis expressed gene (Tex19.1,
Tex19.2), Mili (also called Piwi-like 2) is necessary to
suppress any unmasked retrotransposon activity and ensure
genome integrity (25) whereas demethylation assisted
removal of parent-of-origin-specic imprinting marks
including the maternally methylated small nuclear
ribonucleoprotein N (Snrpn), Lit1, insulin-like growth
factor 2 (Igf2) and the paternally methylated H19 and Ras
protein-specific guanine nucleotide-releasing factor 1
(Rasgrf1) leads to imprint erasure (26). Hence, DNA-
demethylation induced erasure of epigenetic barriers in the
PGCs results in rebirth of the totipotent zygote in the next
generation.
Finally, as the pluripotent ESCs gear up for lineage-
commitment, DNA-demethylation assists in deciding their
final destination. Hypomethylation mediated over-
expression of lineage-determining transcription factors,
lineage-specific and lineage-restricted genes leads to a
satisfying end to the process of organogenesis in the form of
terminally differentiated mature cell types (27). Promoter
DNA-demethylation helps in hematopoietic speciation in
three concurrent stepsfirstly, conversion of ESCs into
hematopoietic stem cell (HSC) population by
hypomethylation of HSC-specific genes such as Runt-
Related Transcription Factor 1 (Runx1) and Vasorin (Vasn),
followed by over-expression of hematopoietic lineage-
specific genes that creates myeloid (Myeloperoxidase
(Mpo), -globin, Platelet Glycoprotein 6 (GP6/GPVI)) and
lymphoid (Lymphocyte-Specific Protein Tyrosine Kinase
(Lck), ADP-ribosylation factor-Like 4C (ArL4c), Smad7)
lineages from the multipotent HSCs and finally,
demethylation of hematopoietic-restricted genes in the
lineage-specific progenitors to form the mature cells such as
B-cells, T-cells, Natural Killer (NK) cells, myeloid cells, red
blood cells, platelets, etc.(28, 29, 30). Similarly,
dissemination of the mesenchymal stem cells (MSCs) into
different functional states such as myogenic, osteogenic,
chondrogenic or adipogenic lineages (31, 32) and sequential
differentiation of neural stem cells (NSCs) into the three
major neural lineages-neurons, astrocytes and
oligodendrocytes is also mediated by global and gene-
specific hypomethylation (33). Thus, DNA-demethylation
is a major factor instigating epigenetic manipulation of the
pluripotent stem cell niche into functional specialization in
the adult.
Histone Modifications and Nuclear Remodeling also
Regulate Stem Cell Chromatin Landscape
Post-translational covalent reversible histone modifications
are crucial agents directing epigenetic regulation of stem
cells. Specific histone modification profiles are responsible
for generating differential transcriptional outcome.
Depending on the presence and abundance of histone marks,
three different states of chromatin arrangement is seen in
case of ESCs (34). Firstly in the totipotent zygote and
pluripotent ESCs open, active euchromatin is associated
with permissive marks such as histone H3 lysine 4
trimethylation (H3K4me3), histone H3 lysine 9 acetylation
(H3K9ac) and histone H4 acetylation (H4ac). As
differentiation advances towards somatic cells, lineage-
control genes are stably silenced by repressive histone
marks such as histone H3 lysine 9(H3K9me3) and histone
H3 lysine 27 (H3K27me3) trimethylation. The final type of
histone mark arrangement is a unique feature known as
bivalent domains. Bivalent chromatin is ESCs and
multipotent neural progenitors, MSCs and hematopoietic
stem and progenitor cells. These bivalent chromatin
domains contain active H3K4me3 and repressive
H3K27me3 along with non-methylated CpG DNA regions
(non-mCpG) and repressive H2AK119Ub1 marks (35, 36).
The presence of these domains on key developmentally
regulated genes indicates that the lineage-specific gene
expression programs in these loci are repressed or poised,
yet are primed for rapid induction of expression upon
receiving differentiation cues (11). Thus, histone
modifications act as a gateway for smooth conduct of
differentiation traffic along the right direction during
lineage-commitment.
Nuclear architecture also undergoes extensive changes such
as level of chromatin compaction, its accessibility and
positioning within specialized nuclear domains and changes
in chromatin organization components heterochromatin
and centromere positioning when ESCs progress along the
differentiation axis. As gradual decrease in cellular potency
sets in, chromatin landscape witness a drastic
metamorphosis, i.e. from an open and globally accessible
state in the undifferentiated ESCs permitting stem cell
pluripotency to a rigid, transcriptionally restrained state of a
lineage-committed cell (34). Also, deviations in relative
positioning of epigenetic enforcers and transcription factors
between the transcriptionally-restrictive nuclear periphery
17
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
18
and transcriptionally-permissive nuclear interior promote
cellular differentiation. Thus, the different degree of
condensation state of chromatin and changes in nucleosomal
organization allows various transcriptional profiles to be
established during the course of cell commitment (11).
Epigenetic Makeover of Stem Cells via DNA-
Demethylation A Breaking Dawn in
Biomedical Research
Molecular reorganization of the
epigenetic determinants in stem
cells for cellular therapy, tissue
repair and regeneration is
now considered to be
enterprising option in the
field of biomedical
r e s e a r c h . I n t h i s
scenario, stem cell based
p h a r m a c o l o g y
spearheaded by DNA-
demethylation can fulfil
the dream of achieving individually-
tailored patient-specific remedies. In vitro
alteration of MSCs via DNA-demethylation have
been considered for cartilage tissue engineering,
remodelling, repair, and rejuvenation of
bone tissues and gene therapies
for local or systemic
pathologies (37). In a
similar approach, DNA-
demethylation induced
in vivo differentiation of
NSCs i nt o ne ur a l
pr ecur s or cel l s i s
c u r r e n t l y
investigated for futuristic
treatment of brain cancer,
ischemic spastic paraplegia,
chronic spinal cord injury and
chronic stroke. Treatment of a number of hematological
malignancies can also be envisioned by applying the same
principle of DNA-demethylation of lineage-unrelated genes
such as that will lead to normal haematopoietic
differentiation (38). Along with prognostic and treatment-
related benefits, DNA-demethylation arbitrated molecular
reorganization of stem cells also offers possibilities for
resolving many frustrating problems in the field of human
reproductive health. Germ-line reprogramming via global
hypomethylation can be employed for solving human
infertility problems, treatment of sex-linked genetic
disorders and improving the efficiency of mammalian
embryo cloning. Addressing the issue of aging by
rejuvenating older somatic cells has by far been the
proverbial Pandoras Box. Reversing the course of the aging
somatic cells and transforming them into younger ones via
global demethylation is now thought to be a likely solution
to this dilemma. In a nutshell, epigenomic renovation of
stem cells via DNA-demethylation is the ultimate weapon in
the area of healthcare management with promising answers
to mysteries in the field of developmental biology.
Concluding Remarks
Epigenetic modifications are increasingly implicated to be
participating in cellular homeostasis, preservation of
genome integrity and of course disease progression;
hence, it becomes necessary to script an
epigenetic instruction manual highlighting
the molecular details of cellular
commitment and differentiation.
Elucidation of intricacies of
epi genomi c r egul at or y
mechanism will help
actively manipulate cell
fate conversion and
coax stem cells to
a d o p t a d e s i r e d
phenotypic outcome.
Under s t a ndi ng t he
dynami cs of DNA-
demethylation will offer
answers to problems faced
during cellular programming events
such as induced stem cell pluripotency,
transdifferentiation and nuclear reprogramming of adult
stem cells. Exploring and establishing the
founding principles upon which cell
lineages are defined and maintained by
hypomethylation facilitated changes
will go a long way in shaping the
molecular floor plan of cellular
development.
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(2007). Genetic and epigenetic
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3. Collas P (2009). Epigenetic states in stem cells. Biochem
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4. Lunyak VV, Rosenfeld MG (2008). Epigenetic regulation of
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5. Zhao X, Ruan Y, Wei CL (2008). Tackling the epigenome in the
pluripotent stem cells. J Genet Genomics; 35 (7): 403-12.
6. Hemberger M, Dean W, Reik W (2009). Epigenetic dynamics of
stem cells and cell lineage commitment: digging Waddington's
canal. Nat Rev Mol Cell Biol; 10(8): 526-37.
7. Li M, Liu GH, Belmonte JC (2012). Navigating the epigenetic
landscape of pluripotent stem cells. Nat Rev Mol Cell Biol;
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8. Atkinson S, Armstrong L (2008). Epigenetics in embryonic
stem cells: regulation of pluripotency and differentiation. Cell
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9. Meissner A (2010). Epigenetic modifications in pluripotent and
differentiated cells. Nat Biotechnol; 28(10): 1079-88.
10. Cedar H, Bergman Y (2012). Programming of DNA methylation
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Figure 1: Molecular Scheme Regulating Cellular
Reprogramming of Embryonic Stem Cells into
Functionally Specialized Adult Cells.
patterns. Annu Rev Biochem; 81: 97-117.
11. Tollervey JR, Lunyak VV (2012). Epigenetics: judge, jury and
executioner of stem cell. Epigenetics; 7(8): 823-40.
12. Egli D, Birkhoff G, Eggan K (2008). Mediators of
reprogramming: transcription factors and transitions through
mitosis. Nat Rev Mol Cell Biol; 9(7): 505-16.
13. Patra SK, Deb M, Patra A (2011). Molecular Marks for
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Cells. Clin Epigenet; 2: 27-53.
14. Patra SK, Bettuzzi S (2008). Epigenetic DNA-(Cytosine-5-
carbon) Modifications: 5-Aza-2-deoxyctyidine and DNA
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15. Seisenberger S, Peat JR, Hore TA, et al (2013). Reprogramming
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breaking epigenetic barriers. Philos Trans R Soc Lond B Biol
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16. Patra SK, Patra A, Rizzi F, et al (2008). Demethylation of
(cytosine-5-Cmethyl) DNA and regulation of transcription in the
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17. Meissner A, Mikkelsen TS, Gu H, et al (2008). Genome-scale
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18. Arney KL, Erhardt S, Drewell RA, et al (2001). Epigenetic
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3295-313.
Assisted Reproductive Technology (ART) and
Imprinting Disorders: Epimutation in
Gametes as a Functional Cause
Shaoni Bhattacharjee
Jana Chakrabarti*
Department of Biotechnology
Presidency University
Kolkata
*jana_chakrabarti@yahoo.co.uk
According to the CDC's (Centres for Diseases Control and
Prevention) definition (based on 1992 Fertility Clinic
Success Rate and Certification Act) 'Assisted Reproductive
Technology' (ART) includes all fertility treatments in which
both eggs and sperms are handled (1). Generally procedures
included in ART treatments are, In vitro Fertilisation (IVF),
Intracytoplasmic Sperm Injection (ICSI), Frozen Embryo
Transfer (FET), Gamete Intra-Fallopian Transfer (GIFT),
Assisted Hatching etc. (2). But It should also be remembered
that they do NOT include treatments in which only sperm are
handled (i.e., intrauterine - or artificialinsemination) or
procedures in which a woman takes medicine only to
stimulate egg production without the intention of having
eggs retrieved (1).
Assisted reproductive technology (ART) has grown by leaps
and bounds in the last few years in India. In a latest survey,
19
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
based on the number of applications received for National
ART Registry, Indian Council of Medical Research
(ICMR) puts the number of such clinics as 125 in the
capital city of India. Officials however believe that the
actual figures are around 250-300 (3). In the year 2000,
only 5500 cycles of IVF-ICSI were performed in our
country, but this number increased to 21,500 in 2006 and
in 2011 it became 110,000 (4). However, still ART has
some safety problems and risks that need to be described and
evaluated so that current clinical policies and laboratory
procedures can be revised, if necessary.
The term 'Imprinting' corresponds to a specific epigenetic
regulation leading to expression of only one parental allele
of a gene. Some imprinted genes exhibit paternal
expression whether others exhibit maternal expression.
The best-characterized mark of gene imprinting is DNA
methylation / unmethylation (5, 6). Usually, methylated
DNA sequences are transcriptionally inactive, whereas
unmethylated DNA sequences are transcriptionally active
(7). There are two mechanisms by which DNA
methylation inhibits gene transcription: the first one is the
interference of the methyl group with the binding of particular
transcription factors to the DNA (8). On the other hand the
second one involves the methyl-binding domain proteins
mediating transcriptional repression through binding to
the DNA (9).
Animals studies on epigenetic reprogramming in the oocyte
suggest that imprints are established in growing oocytes
during primordial to antral follicle transition (10, 11) and
are not completed for some genes until just prior to
ovulation. For this reason, maternal imprinting
mechanisms could be vulnerable to ovarian stimulation.
Because of these concerns, it is important to scrutinize the
strength of current evidences regarding the association
between ART and imprinting. But the rigorous analysis of
the issue becomes complicated by the variability in ART
protocols and the rarity of imprinting disorders.
Table 1: Selected Human Disorders Linked to an
Imprinting Defect that has been Reported after ART
The word Epimutation means a heritable change in gene
expression that does not affect the actual base pair
sequences of DNA. It is thought that an epimutation in the
germ line would produce a phenotype equivalent to that
resulting from an inactivating germ line mutation in the
same gene.
Evidences accumulated from the literatures published
during last few years that gamete progressively acquire
epigenetic information, it is possible that immature
gametes do not have the full and correct complement of
epi genet i c i nformat i on necessary for normal
development. Round spermatids, (largely used in ICSI),
are transcriptionally active, and introducing their RNAs
into the oocyte via ICSI may lead to altered gene
expression, either directly or through RNA interference
mechanisms (12). Ejaculated spermatozoa, elongated
spermatids and round spermatids all have the correct
paternal imprint on chromosome 15q11-13, suggesting
that this specific paternal imprint is established in
immature testicular spermatids. However, other imprinted
regions must be assessed before round spermatid injection
may be considered safe (13).
Analysis of epimutations should include ontogenetic
specificity of epigenetic processes at the different stages of
individual development. Most significant changes of the
epigenetic organization of the genome occur during
maturation of germ line cells and at early stages of pre- and
post implantation development of mammals (14).
Inheritance of genetic alterations is undoubtedly among the
central issues of genetics that can be addressed to the
inheri t ance of epi mut at i ons over generat i ons.
Investigation of inheritance of epimutations can be taken
into account at least two circumstances: first, epigenetic
genome modifications are reversible and second, there are
ontogenetically determined periods of total epigenetic
genome reprogramming that provide elimination of
aberrant epigenotypes. In this circumstance, a special
interest is driven to inheritance of epimutations of
imprinted genes, since their phenotypic manifestation
depends on sex of the parent that transmitted the
epimutation to the offspring.
In most of the cases of mutation in Imprinting Centers (IC)
with Angelman Syndrome (AS) and Prader-Willi Syndrome
(PWS) are familial. Since in Angelman Syndrome Shortest
Region of deletion Overlap (AS-SRO) mutations involve
only maternal imprinting, they are transmitted via male
germ line without phenotypic expression. Likewise, IC-
SRO mutations involving paternal imprinting are not
expressed when t ransmi t t ed t hrough mat ernal
gametogenesis. This explains the unusual mode of
inheritance of IC mutations in generations. For instance,
mutation in IC-SRO, appearing on chromosome 15 in
grandmother, will lead to any phenotypic consequences
neither in her nor in her son (Fig.1). However, passing
through spermatogenesis, this mutation will cause PWS in
her grandchildren. Likewise, to cause AS, the IC mutations
must pass through maternal gametogenesis (15).
20
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Disorders Candidate Chromosomal
Location
BWS (Beckwith-Wiedemann syndrome) 11p15
AS (Angelman Syndrome) 15q11-13
PWS (PraderWilli syndrome ) 15q11-13
SRS (SilverRussell syndrome) 7
Isolated hemihyperplasia 11p15
Autism 15q11-13
Bipolar disorder 18p11.2
Schizophrenia 18p11.2
Retinoblastoma 13q
[Source: Carter M. Owen and James H. Segars Jr. Imprinting Disorders and
Assisted Reproductive Technology. Semin Reprod Med.; 2009 September;
27(5): 417-428. Doi: 10.1055/s-0029-1237430.]
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
21
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Figure 1: Hypothetic Pedigrees Explaining the Inheritance of Chromosome 15 IC Mutations Fixing Maternal (a) or
Paternal (b) Imprinting in PraderWilli and Angelman Syndromes. When Fixed Paternal Imprinting Passes Through
Oogenesis, the Patient has the Maternal Homolog with the Paternal Epigenotypemat (pat), which Results in the
Formation of AS
[Source: N. Lebedev and E. A. Sazhenova. Epimutations of Imprinting Genes in the Human Genome: Classification, Causes,
Association with Hereditary Pathology. Russian Journal of Genetics; 2008; Vol. 44, No. 10, pp. 11761190. DOI:
10.1134/S1022795408100062]
In a another study, to examine the origin of chromosome 15
with AS-SRO in 18 AS patients, it was found that seven
patients inherited the mutation from the grandmothers and
eleven patients, from the maternal grandfathers (16). This
mode of inheritance might be explained by the appearance of
the imprinting defect after the erasure of the parental
epigenotypes and possibly even after fertilization. An
additional support of the post zygotic appearance of the
epimutations is provided by the above-mentioned (Fig-1)
somatic mosaicism for IC methylation, revealed in
approximately 30% of AS patients. The mechanisms
underlying this mosaicism are unclear, but they are likely
associated with IC demethylation in some somatic cells.
Moreover, in addition to inheritance of epimutations of
imprinted genes, reports on possible transmission to offspring
of aberrant epigenetic modifications of non-imprinted
genomic loci is also described in two patients with colorectal
cancer, who had mosaic hypermethylation of the promoter
region of the mismatch repair gene MLH1(17). An
epimutation was also present in sperm of one of the patients,
indicating a defect in the embryonic germ line and possible
transmission to the offspring. The available reports therefore
attest to a potential contribution of the conformation
(epigenotype) of DNA in determining the functional activity
of the gene that can also be inherited, thus causing the
development of diseases in certain cases. This epigenetic
inheritance may be characterized by transmission of the
epimutation over several generations and possible
restoration of the normal epigenetic status in the pedigree.
However, it is still unclear whether the repair of resistance of
epimutations is determined by sex (spermatogenesis or
oogenesis), which can be of special importance in case of
imprinted genes, whose activity depends on the sex of the
parent having transmitted them.
Therefore, to date the possibility of transmission of primary
epimutations and their repair in generations is still under
debate. No convincing molecular evidence is available to
explain such mechanisms of epigenetic inheritance. Diseases
like Autism, Bipolar disorder, Schizophrenia, Retinoblastoma
(Table 1) are still in the light of further research to get linked
with assisted reproductive technologies. However, in the
present discussion, an attempt has been made to mention in
short about the diversity of imprinted genes because
investigation on their causes, phenotypic effects, and
inheritance patterns seems to be a very promising line of
research in the modern human genetics, to enhance our
understanding of etiological basis of hereditary diseases.
References
1. http://www.cdc.gov/art
2. http://www.rtc.org.au/glossary
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
3. Chandra N (2013). When you cannot produce a baby, design it!
Over 500 babies born each month in Delhi fertility clinics; Mail
Today
4. Pratap RK (2011). IVF Baby Boom. Outlook Business; 10.
5. Li E, Beard C, Jaenisch R (1993). Role for DNA methylation in
genomic imprinting. Nature; 366: 3625.
6. Kaneda M, Okano M, Hata K, et al (2004). Essential role for de
novo DNA methyltransferase Dnmt3a in paternal and maternal
imprinting. Nature; 429: 9003.
7. Dennis C (2003). Epigenetics and disease: altered states. Nature;
421: 6868.
8. Iguchi-Ariga SM, Schaffner W (1989). CpG methylation of the
cAMP-responsive enhancer/promoter sequence TGACGTCA
abolishes specific factor binding as well as transcriptional
activation. Genes Dev; 3: 6129.
9. Swales AK, Spears N (2005). Genomic imprinting and
reproduction. Reproduction; 130: 38999.
10. Hajkova P, Erhardt S, Lane N, et al (2002). Epigenetic
reprogramming in mouse primordial germ cells. Mech. Dev;
117(12): 1523.
11. Obata Y, Kaneko-Ishino T, Koide T, et al (1998). Disruption of
primary imprinting during oocyte growth leads to the modified
expression of imprinted genes during embryo-genesis.
Development; 125 (8): 155360.
12. Borghol N, Blachere T, Lefevre A (2008). Transcriptional and
epigenetic status of protamine 1 and 2 genes following round
spermatids injection into mouse oocytes. Genomics; 91: 415
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13. Huntriss J (2011). Epigenetics and Assisted Reproduction in Kay
Elder and Brian Dale (with contribution from Harper J, Huntriss
J) ed. Book on In-Vitro Fertilization: Third Edition;;
Cambridge University Press; 252-267 [ISBN: 978-0-521-
73072-3 (Paperback)]
14. Li E (2002). Modification and Epigenetic Reprogramming in
Mammalian Development.;
Nat Rev Genet; 3: 66273.
15. Lebedev N, Sazhenova EA (2008). Epimutations of Imprinting
Genes in the Human Genome: Classification, Causes,
Association with Hereditary Pathology. Russian Journal of
Genetics; 44(10): 117690.
16. Buiting K, Gross S, Lich C, et al (2003). Epimutations in
PraderWilli and Angelman Syndromes: A Molecular Study of
136 Patients with an Imprinting Defect. Am J Hum Genet; 72:
5717.
17. Suter CM, Martin DI, Ward RL (2004). Germ line Epimutation of
MLH1 in Individuals with Multiple Cancers. Nat Genet; 36:
497501.
Epigenetics: A Paradigm in Germ Cells
Differentiation and In Vivo Development
S. D. Kharche*, S. K. Agarwal
PR & SM Division
Central Institute for Research on Goats
Makhdoom
*kharche62@gmail.com
Epigenetics has evolved very quickly from the study of an
obscure collection of diverse phenomena to become one of
the most exciting topics in contemporary biology. The
traditional view that gene and environment interactions
control disease susceptibility can now be expanded to
include epigenetic reprogramming as a key determinant of
origins of human disease. It is a rapidly expanding field of
study in which the molecular mechanisms of unrelated
normal processes, such as paramutation in maize, position
effect variegation (PEV) in the fruit fly, and genomic
imprinting and X-chromosomal inactivation in mammals,
are now recognized as evolutionarily conserved epigenetic
processes. Currently, epigenetics is defined as heritable
changes in gene expression that do not alter DNA sequence
but are mitotically and transgenerationally inheritable. The
epigenetic regulation of gene expression is essential for the
normal growth, development, and aging of higher organisms
(1). Epigenetics also underlies genomic imprinting,
programming, and reprogramming in early life and the
increased susceptibility to disease in later life. Epigenetic
reprogramming is the process by which an organisms
genotype interacts with the environment to produce its
phenotype and provides a framework for explaining
individual variations and the uniqueness of cells, tissues, or
organs despite identical genetic information. The main
epigenetic mediators are histone modification, DNA
methylation, and non-coding RNAs. They regulate crucial
cellular functions such as genome stability, X-chromosome
inactivation, gene imprinting, and reprogramming of non-
imprinting genes, and work on developmental plasticity
such that exposures to endogenous or exogenous factors
during critical periods to permanently alter the structure or
function of specific organ systems. Aberrant changes in
linear DNA sequences result in mutations, deletions, gene
fusion, tandem duplications, or gene amplifications causing
dysregulation of gene expression that lead to the genesis of
disease (2). Recently, however, it has become clear that
epigenetic disruption of expression plays an equally
important role in the development of disease (3) and this
process is more susceptible as compare to environmental
modulation.
Germ cells and somatic cells have the identical genome.
However, unlike the mortal fate of somatic cells, germ cells
have the unique ability to differentiate into gametes that
retain totipotency and produce an entire organism upon
fertilization (4). The processes by which germ cells
differentiate into gametes, and those by which gametes
become embryos, involve dramatic cellular differentiation
accompanied by drastic changes in gene expression, which
are tightly regulated by genetic as well as epigenetic
mechanisms (5). Epigenetic regulation refers to heritable
changes in gene expression that are not due to changes in
primary DNA sequence. It alters chromatin structure by
changing the position, organization and composition of
nucleosomes, while preserving primary DNA sequence and
contributes significantly to cellular memory, which
maintains a particular cell fate through both mitotic and
meiotic cell divisions (6). Epigenetic regulation of the
genome involves factors such as histone modifications (i.e.
acetylation and methylation) and DNA methylation that
directs chromatin structure and gene transcription. Among
all known cell types, germ cells are unique due to their
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
abilities to produce the next generation of an entire organism
upon fertilization. Because of their immortal nature, it is
critical for germ cells to not only undergo proper cellular
differentiation within one generation, but also retain
accurate information to initiate the next generation.
Specifically, epigenetic mechanisms may play roles in: (1)
ensuring meiosis and terminal differentiation program
during gametogenesis; (2) reliably retaining information in
gametes for the next generation; (3) erasing improper
features in zygotes before initiating a new life cycle, in order
to prevent inheritable epimutations.
In many species, gametogenesis is initiated by germ cells,
which undergo transit-amplificationbefore committing to
meiosis and terminal differentiation. The chromatin
structure of germ cells acts as an intrinsic mechanism to
maintain their unique self-renewal ability and block
differentiation. In addition to chromatin remodeling,
dynamic regulation of his tone modifications is also required
to maintain germ cells identity. Meiosis is a process unique
to germ cells. It is also thought that histone modifying
enzymes that remove and generate these histone
modifications mustact cooperatively to ensure the changes.
Indeed, histone modifying enzymes with similar or distinct
activities act in an orchestrated manner in regulating germ
cell differentiation. Another dynamic epigenetic regulation
during mammalian gametogenesis is the establishment of
differential DNA methylation at imprinted genes, which are
differentially expressed according to their parental origin
and important for embryonic development. In females,
genomic imprinting occurs after birth in oocytes arrested at
diplotene stage of meiotic prophase I, and the de novo
methylation process is complete by the fully-grown oocyte
stage (7).
A recent report has revealed that de novo DNAmethylation
in mouse male germline occurs mainly in spermatocytes at
early meioticprophase, in addition to spermatogonial cells
(8). During spermiogenesis in post-meiotic germ cells
histones are displaced by the transition nuclear proteins
(Tnps) followed by protamines (Prms), allowing for
extensive chromatin condensation and DNA packaging in
sperm nuclei. This process is regulated by a variety of
epigenetic mechanisms, including histone modifications,
chromatin remodeling, and histone variants deposition. The
DNA methylation pattern of the genome becomes
reprogrammed following de-methylation and re-
methylation processes after fertilization and during early
embryonic development. This epigenetic reprogramming
during early embryonic cell differentiation transmits a
unique DNA methylation pattern to developing organs in the
offspring. An additional epigenetic reprogramming event
(i.e. DNA methylation) occurs later in development in the
germ line during sex determination. A small subset of
imprinted genes is transmitted to subsequent generations
through the male or female germ line. Imprinted genes have
an allele specific DNA methylation pattern and expression
that is maternally or paternally transmitted between
generations. Clearly a number of different epigenetic
mechanisms (e.g. histone modifications, chromatin
structure and DNA methylation) will be involved in
programming the germ line. Alterations in the epigenetic
reprogramming of the germ line can promote heritable
changes on transcription and disease (9, 10).
Parthenogenesis, which is a successful development of
unfertilized eggs, is observed in many vertebrate species
and, if it were possible in mammals, would provide a way to
produce clones of livestock animals. However, imprinting is
a major barrier for parthenogenetic embryo development in
mammals because expression levels of the imprinted genes,
which include many important developmental genes, are
unbalanced in such embryos. However, by modifying
imprinted genes by genetic engineering and developmental
manipulation, it was possible to derive adult female mice
with two maternal genomes and no paternal complement
(11, 12). As the method involves genetically engineered
animals and highly complex nuclear-transfer technologies,
its direct application to livestock seems difficult. Similarly,
genomic imprinting and expression of developmental genes
in parthenogenetic goat embryos also play an important role
during in vivo embryo development. Hence the
parthenogenetic goat embryos could not developed in vivo
beyond thirty four days following embryo transfer in to
surrogate mother (13).
References
1. Feinberg AP (2007). Phenotypic plasticity and the epigenetics
of human disease. Nature; 447: 43340.
2. Garg V (2006). Insights into the genetic basis of congenital
heart disease. Cell Mol Life Sci; 63: 11418.
3. Dolinoy DC, Weidman JR, Jirtle RL (2007). Epigenetic gene
regulation: linking early developmental environment to adult
disease. Reprod Toxicol; 23: 297307.
4. Cinalli RM, Rangan P, Lehmann R. (2008). Germ cells are
forever. Cell; 132: 55962.
5. Kimmins S, Sassone-Corsi P (2005). Chromatin remodelling
and epigenetic features of germ cells. Nature; 434: 5839.
6. Ringrose L, Paro R (2004). Epigenetic regulation of cellular
memory by the Polycomb and Trithorax group proteins. Annu
Rev Genet; 38: 41343.
7. Hiura H, Obata Y, Komiyama J, et al (2006). Oocyte growth-
dependent progression of maternal imprinting in mice. Genes
Cells; 11: 35361.
8. Oakes CC, La Salle S, Smiraglia DJ, et al (2007).
Developmental acquisition of genome-wide DNA methylation
occurs prior to meiosis in male germ cells. Dev Biol; 307:
36879.
9. Tarozzi N, Bizzaro D, Flamigni C, et al (2007). Clinical
relevance of sperm DNA damage in assisted reproduction.
Reproductive BioMedicine Online; 14: 74657.
10. Yang J, Yang S, Beaujean N et al (2007). Epigenetic marks in
cloned rhesus monkey embryos: comparison with counterparts
produced in vitro. Bio Reprod; 76: 3642.
11. Kono T, Obata Y, Wu Q, et al (2004). Birth of parthenogenetic
mice that can develop to adulthood, Nature; 428: 8604.
12. Kuwahara, M, Wu Q, Takahashi N, et al (2007). High-
frequency generation of viable mice from engineered bi-
maternal embryos. Nat Biotechnol; 25: 104550.
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13. Kharche SD, Goel AK, Jindal SK, et al (2014). Developmental
potency of parthenogenetic goat embryos following in vivo
transfer in caprahirus. International conference on reproductive
health: Issue and strategies under charging climate scenario
held on 6-8 February, IVRI, Izatnagar Bareilly.
The Expanding Role of Epigenetics in
Reproductive Health
M. Ankolkar, N. H. Balasinor*
Neuroendocrinology Division
National Institute for Research in
Reproductive Health (ICMR)
Mumbai
*balasinorn@nirrh.res.in
Epigenetics: An Emerging Concept
Ever since the theory of Genes and Mendelian
inheritance has been proposed by legendary personality
Gregor Mendel in the early 19th century, and the work of
other eminent Nobel laureats, Thomas H. Morgan, Barbara
Mclintock; our perception of genetic hereditary and
inheritance has been that it is written in the language of
DNA and the nucleotide backbone. For decades we have
assumed how genetic mutations and changes in the
nucleotide sequence can predict and explain the observed
phenotypic traits. Although genetics plays a large role in
defining the physique and physiology of organisms, it is a
combination of genetics with the organisms' experiences
(a.k.a. environment) that determines the ultimate phenotype.
One of the mechanisms by which this can be achieved is by
controlling the expression of genes depending on need.
Although, genetic mutations are a known mechanism of
evolution in respect to the change in environment, it is a very
slow process that occurs over thousands of years and is thus
insufficient to explain the continuous adaptation of an
organism to its constantly changing environment. Around
1942, a concept was introduced by a noted developmental
biologist, Sir Conrad H. Waddington, to explain this
phenomenon called "The theory of Epigenesis". He coined
the term "Epigenetics" which was defined as the interactions
of the genes with the environment that brings the phenotype
into being (1). In modern biology, it is defined as the changes
in gene expression without affecting the DNA sequence. The
concept of epigenetics is capable of explaining the ability of
the cells to adapt to the dynamic environment despite having
the same genetic constitution. It is brought about by 3 basic
mechanisms DNA methylation at the 5th Carbon atom of
Cytosine residue at CpG dinucleotides, Histone tail specific
residue modifications and heritable RNAs.
Epigenetics is involved in two main aspects of reproductive
health, namely, gametogenesis and embryogenesis. One
such epigenetic phenomenon is genomic imprinting,
whereby expression of some genes or chromosomal regions
depends on the parental origin of the allele. Genomic
imprinting is important for embryo development; however
its establishment is during gametes formation. The parental
specific expression is due to differential marking on the
DNA on the two parental alleles by DNA methylation during
gametogenesis. In addition to establishment of imprinting
marks during gametogenesis, another significant epigenetic
modification happens during spermatogenesis, when round
spermatids undergo cyto-differentiation to form mature
spermatids with condensed chromatin, which is due to the
replacement of histones by protamines, thereby modifying
the necleosomal chromatin structure into toroidal.
During early embryogenesis in mammals, following the
union of the two unipotent gametes, a wave of genome-wide
DNA methylation reprogramming takes place. Firstly, the
paternal and maternal genome undergoes demethylation
leading to totipotent zygote. After implantation, there is
global de novo methylation, which is maintained throughout
life in the somatic cells. It has been hypothesized that these
process of DNA demethylation and remethylation may be
affected during assisted reproductive technique, where
early embryo development till the blastocyst takes place in-
vitro.
Epigenetic Alterations in Assisted Reproductive
Techniques
ARTs account for 1-4% of births in the developed countries.
During recent years ARTs have been found to be associated
with in developmentally associated disorders related to
genomic imprinting. One such problem is the overgrowth
syndrome called the Beckwith-Wiedemann syndrome
(BWS). It occurs in approximately 1 in 13700 births. A case-
control study showed that 1 out of 148 healthy children in the
control group were born after IVF, whereas 4 out of 37 BWS
children were born from IVF procedures (2). BWS has been
associated in some cases, with the imbalanced expression of
genes in the 11p15.5 region, either due to trisomy in case of
chromosomal relocalization or imbalanced expression of the
paternally expressed IGF2 gene or H19 gene due to DNA
methylation aberration. Similarly, other imprinting
disorders like Angelman Syndrome, Prader-willi syndrome
have been found to be associated with IVF procedures (3, 4,
5). However, studies linking ART to imprinting disorder is
controversial with some studies showing no association and
hence systematic multinational long-term follow-up studies
are needed (6).
Based on the results from animals and humans studies,
epigenetic errors related to ARTs seem to be caused by
gamete and embryo manipulations procedures carried out at
a critical time when old epigenetic marks are erased, new
marks established and maintained during gametogenesis
and read during embryogenesis. Manipulations include
ovarian stimulation, superovulation and in-vitro
preimplantation embryo culture. In-vitro culture in animal
models has been found to lead to reduced viability and
growth, developmental abnormalities, behavioural changes
and loss of imprinting. Gonadotropins used for maturing
many oocytes simultaneously could also cause the
premature release of oocytes that has not completed the
imprinting process leading to errors in imprinting (7).
Abnormal methylation patterns in 2-cell embryos from
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
superovulated females as compared to non-superovulated
females have been reported in rodents (8). In vitro
maturation of cultured embryos might induce epigenetic
aberrations due to components of the culture medium (9).
Alternatively, it is possible that the imprinting error is
inherent in the gametes and could be a cause of infertility,
which is unmasked with assisted reproduction technology.
Association of Epigenetics with Primary Infertility and
Embryonic Death
As mentioned above it is possible that the epigenetic
aberrations might already be underlying the very infertility
for which people undertake ART. For example, the first
study of genomic imprinting marks at H19 gene showed
significant DNA hypomethylation in 17% moderate and
30% severe oligozoospermic men. Subsequently, many
studies showed that such DNA methylation aberrations are
present in other genes like the PEG1, LIT1, ZAC, PEG3 etc.
as well, in oligozoospermia (10, 11). Similarly,
superovulation of oocytes in infertile women has been found
to be associated with DNA hypomethylation in PEG1 gene
which, in normal oocytes, is methylated (7). The association
of DNA methylation aberration is not only present in
oligozoospermia but also in some other reproductive
disorders. Study done in our laboratory demonstrated DNA
methylation aberrations at imprinted genes in spermatozoa
of couples with idiopathic recurrent spontaneous
miscarriages (RSM) (12). Also it has been observed in one
study that certain genetic polymorphisms at imprinted genes
might predispose an individual towards recurrent
spontaneous abortions. They observed a higher genotypic
frequency of the ApaI polymorphism in the embryo
mitogenic IGF2 gene, in idiopathic RSM cases as compared
to Control group of proven fertility (13). Similar to
Recurrent Spontaneous Miscarriges, but much more
frequent are cases of single spontaneous abortions and peri-
natal fetal death. In a study of 38 cases of spontaneous
miscarriages/ fetal deaths, the paternally imprinted
PHLDA2 gene was found to be over expressed in a number
of cases in the first 12 weeks of gestation (14). Another
study analyzing spontaneous abortions and induced
abortions (of 12-24 weeks gestation) observed extreme
methylation values in multiple imprinted genes in tissues
obtained from such material (15).
Environmental Factors and Epigenetic Aberrations in
Adverse Pregnancy Outcome
Another adverse embryonic complication is the
teratogenic effects of growing embryo due to either
parenteral or in utero alcohol exposure which is associated
with a wide range of neurobehavioural and physical
abnormalities collectively referred to as Fetal Alcohol
Spectrum Disorders (FASD). Ever since its recognition, a
lot of research in the causal mechanism has been focusing
on myri ad of perspect i ves i ncl udi ng genet i c,
biomolecular, cellular and morphological (16), however,
the epigenetic factors are also slowly getting realized. One
of the main implications of the epigenetic perspective has
realized that FASD is not only limited to in utero exposure
of alcohol but also parenteral consumption of alcohol during
the preconception period. Alcohol is known to affect one-
carbon-metabolism that controls the supply of the methyl
donor (S-adenosyl methionine-SAM) and thus, perhaps,
DNA methylation (17). Research has also focused on role of
many histone modifications and histone and modifying
enzymes like histone acetyl transferases as well as DNA
methyltransferase in the pathophysiology of chronic alcohol
exposure (18). An increased levels of MIR9 (miR-9) miRNA
(a calcium- and voltage-activated potassium (BK) channel
were observed after exposure of rat neurons to 20 mM
alcohol suggest the role of the epigenetic factor- the small
non-coding RNAs being one of the targets of alcohol
exposure. In summary, ethanol is a known inhibitor of one-
carbon metabolism and DNA methyltransferase, and it
interferes with various epigenetic factors, including DNA
As a result of the Industrial Revolution, many potentially,
though subtly, harmful chemicals are released in the
environment and are encountered by humans in day-to-day
life. Many of these compounds, like Bisphenol A (BPA-
found in plastics), Vinclozolin, Pthalates, etc. have
endocrine disrupting properties. Since the growth of the
embryo, is very intricately modulated and controlled by
hormones, endocrine disruption in pregnant mothers would
not only lead to fetal malformations but also affect
transgenerationally. Mounting evidence reveals that
endocrine disruption causes alterations in the male germ line
and leads to transgenerational effects by epigenetic
mechanisms (19). Studies in our laboratory on the use of
tamoxifen, a Selective Estrogen Receptor Modulator
(SERM) revealed significant post implantation implantation
loss in female rats upon mating with adult male rats treated
with tamoxifen for 60 days, owing to epigenetic aberrations
of imprinted genes in the spermatozoa (20).
Conclusion
Reproductive Health, that was once analyzed through wide
parameters like fertility, sperm analysis, genetic
polymorphisms etc. is being viewed in the light of
epigenetics, which promises to give a mechanistic
understanding to the dynamics of reproductive physiology.
The role of assisted reproduction in mediating epigenetic
alterations; though looks possible, still needs to be
ascertained by more focused studies in non-confounding
model systems. At the same time, another avenue of research
has also sprouted which shows that epigenetic alterations
could well be associated with the infertility for which people
are resorting to ARTs. Similarly, a lot of research in
environmental influence and endocrine disruption on
reproductive health is coming to the fore. In our experience,
although ART procedures could be playing a significant role
in altering reproductive epigenetics, there is a lot of scope in
elucidating epigenetic variability in infertility cases. Since
epigenetic alterations are so sensitive to hormonal
imbalance, we presume environmental pollutants could be
playing a significant role in fluctuating reproductive
outcome transgenerationally and needs to be stressed upon.
All in all, epigenetics indeed, is an emerging paradigm and a
novel perspective is studying reproductive health.
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methylation, histone modifications, and small ncRNAs (21).
References
1. Speybroeck VL (2002). From epigenesis to epigenetics: the
case of C. H. Waddington. Ann N Y Acad Sci; 981: 61-81.
2. Hal l i day J, Oke K, Br eheny S, et al ( 2004) .
BeckwithWiedemann syndrome and IVF: a case control
study. Am J Hum Genet; 75: 5268.
3. Maher ER, Brueton LA, Bowdin SC, et al (2003).
BeckwithWiedemann syndrome and assisted reproduction
technology (ART). J Med Genet; 40: 624.
4. Maher ER, Afnan M, Barratt CL (2003a). Epigenetic risks
related to assisted reproductive technologies: epigenetics,
imprinting, ART and icebergs? Hum Reprod; 18: 250811.
5. Allen C, Reardon W (2005). Assisted reproduction technology
and defects of genomic imprinting. BJOG; 112: 158994.
6. Paoloni-Giacobino A (2007). Epigenetics in reproductive
medicine. Pediat Res; 61: 51R-57R.
7. Sato A, Otsu E, Negishi H, et al (2007). Aberrant DNA
methylation of imprinted loci in superovulated oocytes. Hum
Reprod; 22(1): 26-35.
8. Shi W, Haaf T (2002). Abnormal methylation patterns at the
two-cell stage as an indicator of early developmental failure.
Mol Reprod Dev; 63: 329-34.
9. El Hajj N, Haaf T (2013). Epigenetic disturbances in in vitro
cultured gametes and embryos: implications for human assisted
reproduction. Fertil Steril; 99: 632-41.
10. Kobayashi H, Sato A, Otsu E, et al (2007). Aberrant DNA
methylation of imprinted loci in sperm from oligospermic
patients. Hum Mol Genet; 16: 254251.
11. Marques CJ, Costa P, Vaz B, et al (2008). Abnormal methylation
of imprinted genes in human sperm are associated with
oligozoospermia. Mol Hum Reprod; 14: 6774.
12. Ankolkar M, Patil A, Warke H, et al (2012). Methylation
analysis of idiopathic recurrent spontaneous miscarriage cases
reveals aberrant imprinting at H19 ICR in normozoospermic
individuals. Fertil Steril; 98: 1186-92.
13. Ostoji S, Pereza N, Volk M, et al (2008). Genetic
predisposition to idiopathic recurrent spontaneous abortion:
contribution of genetic variations in IGF-2 and H19 imprinted
genes. Am J Reprod Immunol; 60(2): 111-7.
14. Dria S, Sousa M, Fernandes S, et al (2010). Gene expression
pattern of IGF2, PHLDA2, PEG10 and CDKN1Cimprinted
genes in spontaneous miscarriages or fetal deaths. Epigenetics;
5: 444-50.
15. Pliushch G1, Schneider E, Weise D, et al (2010). Extreme
methylation values of imprinted genes in human abortions and
stillbirths. Am J Pathol; 176(3): 1084-90.
16. Goodlett CR, Horn KH (2001). Mechanisms of alcohol-induced
damage to the developing nervous system. Alcohol Res Health;
25: 175-84.
17. Cravo ML, Gloria LM, Selhub J, et al (1996).
"Hyperhomocysteinemia in chronic alcoholism: correlation
with folate, vitamin B-12, and vitamin B-6 status". American J
Clin Nutrit; 63(2): 2204.
18. Shukla SD, Velazquez J, French SW, et al (2008). Emerging role
of epigenetics in the actions of alcohol. Alcohol Clin Exp Res;
32(9): 1525-34.
19. Skinner M (2011). Environmental epigenetic transgenerational
inheritance and somatic epigenetic mitotic stability;
Epigenetics 6(7): 838-42.
20. Pathak S, Kedia-Mokashi N, Saxena M, et al (2009). Effect of
tamoxifen treatment on global and insulin-like growth factor 2-
H19 locus-specific DNA methylation in rat spermatozoa and its
association with embryo loss. Fertil Steril Vol 91 (Suppl): 2253-
63.
21. Haycock PC (2009). Fetal alcohol spectrum disorders: the
epigenetic perspective. Biol Reprod; 81: 607-17.
Emerging Role of Epigenetics in Ovarian
Cancer
Seema Sharma
Department of Obstetrics & Gynecology
Mahatma Gandhi University of Medical Sciences &
Technology
Jaipur
drseemadsharma@gmail.com
Introduction
Ovarian cancer ranks as the fifth leading cause of cancer-
related deaths among women, and the leading cause of death
from gynecological cancer (1). Difficulty to diagnose the
disease at an early stage and the persistence of dormant,
drug-resistant cancer cells that cause relapse, are the primary
reasons for the high mortality rate in ovarian cancer patients
(2). Lack of early detection strategies and unfavourable
anatomical situation make it difficult to diagnose in early
stages. By the time most ovarian cancers are diagnosed, they
are already at stage III or IV. Ovarian cancer screening with
transvaginal ultrasound and CA125 was evaluated in the
Prostate, Lung, Colorectal, and Ovarian (PLCO) trial; and it
was revealed that the predictive value of both tests was
relatively low (3). Increasing evidence indicates that
epigenetic mechanisms may play a major role in the
development of ovarian cancer. Administration of therapies
that reverse epigenetic Silencing of tumor suppressors and
other genes involved in drug response cascades could prove
useful in the management of drug-resistant ovarian cancer
patients. Moreover, when used in combination with
conventional chemotherapeutic agents, epigenetic-based
therapies may provide a means to resensitize ovarian tumors
to the proven cytotoxic activities of conventional
chemotherapeutics (4).
Epigenetic Modification in Ovarian Cancer
Altered epigenetic states are intimately associated with
ovarian tumorogenesis. Based on morphology, genetics, and
site of origination, ovarian cancers of epithelial-cell origin
have been categorized into two groups. The type I group are
those that are strictly confined to the ovary and are low-
grade serous, endometrial, mucinous, and clear-cell type.
These tumors are genetically more stable, have few to rare
p53 mutations, are easily diagnosed and have a good
prognosis. The Type 2 group contains tumors are aggressive
and compr i se hi gh- gr ade ser ous car ci nomas,
undifferentiated carcinomas and carcinosarcomas. These
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tumors constitute 75% of the ovarian cancers with a 90%
death rate and the site of origin stems from tissues other than
the ovary. These tumors exhibit genetic instabilities with a
higher percentage of p53 mutations (5, 6, 7).
Histones and DNA are primary targets of epigenetic
regulation (8). Genes that harbor CpG islands are
susceptible to epigenetic modifications, consisting
primarily of DNA methylation (9). Both types, histone-
based and DNA-based methylation marks, are largely
known to inhibit gene expression through transcriptional
inhibition. The well-studied acetylation modifications
primarily affect the exposure of the DNA to the
transcriptome machinery. In some cases, non-histone
proteins like p53 when acetylated affect its cellular functions
through its DNA binding interactions and stability (10).
Increased methylation of tumor suppressor genes have also
been observed in ovarian tumors. Increase in promoter
methylation of O-methylguanine DNA methyltransferase
(MGMT), paired box 5 (PAX5), Cadherin 13, H-Cadherin
(Rose et al.) (CDH13), Wilms tumor 1 (WT1),
Thrombospondin 1 (THBS1), and GATA5 have been
observed in endometroid ovarian cancer as compared to
serous ovarian cancer (11). Tumor suppressors cyclin-
dependent kinase inhibitor 2B (CDKN2B), CDH13and
RASSF1, a gene that encodes Ras association domain-
containing protein 1 have significant hypermethylation and
CDKN2B promoter hypermethylation was observed in clear
cell carcinomas as compared to other histological types (12).
Hypermethylation of BRCA1 has been shown to be frequent
in spontaneous breast and ovarian cancers. Demethylation
of BRCA1 appears to decrease chemosensitivity of
platinum-sensitive cells associated with partial increase of
BRCA1. Thus BRCA1 hypermethylation favors treatment
sensitivity and has been shown to function independently of
PI3K-Akt pathway (13). Methylation analysis of ovarian
tumors of genes involved in the Wnt pathway demonstrated
that the naked cuticle homolog 1 (NKD1) and disheveled
homolog (DVL1) methylation increased risk of disease
progression. Hypermethylation of members of SHh
pathway, zinc finger protein 1 (ZIC1), results in poor
progression free survival (PFS) (14). The relationship
between DNA hypermethylation generally favors reduced
gene expression. Ovarian cancer cell lines tend to have
higher methylation patterns as compared to primary tumors.
Adult stem cells are found in the ovaries and may provide a
key link into how tumors arise in the ovary (15). Linking the
methylation patterns to these adult stem cells, normal
epigenetic marks may undergo a change mediated by
environmental cues (external/internal) e.g., parity,
inflammation, toward a more tumorigenic phenotype by the
suppression or loss of tumor suppressor genes.
Hypoacetylation of H3 and H4 in association with GATA4
and 6 transcription factors have been found in a variety of
ovarian cancer cells (16).
Micro RNAs (miRNAs) are tightly controlled in normal
cells but become highly deregulated in cancer cells. They are
single stranded non-coding RNA molecules about 22
nucleotides in length and regulate the levels of gene
expression by performing silencer-like type functions,
degrading the mRNA to which they bind (17). They bind
either to certain sequences within the mRNA or to the 3-
untranslated region of the gene. The post-transcriptional
modification of genes by miRNA and the presence of varied
miRNA expression levels within solid tumors provides a
map of miRNA signatures for specific cancers. Formulating
drugs against these miRNAs may provide for a therapeutic
approach.
Epigenetic Pathways in Ovarian Cancer
Several pathways and genes are found to be involved in the
biogenesis of ovarian cancers. Tumor suppressor proteins
play a pivotal role in the cell cycle process and these proteins
are highly deregulated in ovarian cancers. Epigenetic
changes in p53, p21 and p27 has been most commonly
observed in ovarian malignancy.
p53 : Tumor suppressor gene p53 controls the regulation
and function of cyclin D1, p16INK4a, p27 and p21 and also
predominantly mutated in high grade ovarian cancers. The
changes in p53 could be both at the gene and protein level.
Point mutations, missense mutations and truncations have
been observed, and over expression of the p53 protein has
been detected in many of the immunohistochemical studies .
The aberrations in p53 result in the accumulation of the
altered protein within the cell that has a negative effect on
BAX, a transcriptional target of p53 (18). It seems likely
that p53 alterations in high grade tumors reduce BAX
expression allowing the progression of solid tumors.
The BCL family of apoptotic proteins along with p53 can
serve as tools for histotyping (19). The expression of p53-
BCL-2 and BCL-2-BAX have been shown to have a strong
association with tumor grade and histopathological sub-
typing, factors that could be vital for identifying the specific
epithelial ovarian carcinoma for adjuvant or combined
therapies. Ovarian tumors are initially very responsive to
treatment but later become chemoresistant. Possibly the
treatment itself may cause a few cells within the tumor mass
to harbor mutations in p53 that, in addition to epigenetic
silencing of promoter regions of apoptotic favorable genes
such as p16 or Rb, may account for the relapse and
progression of the tumor.
p21 and p27
Cyclin dependent kinase (CDK) inhibitors are major co-
regulatory proteins in the cell cycle along with the p16, p53
and retinoblastoma (Rb) pathways. p21 is a direct
transcriptional target of p53 (20). In the presence of wild-
type (WT) p53, p21 induction ensues followed by the
inhibition of cyclin E/CDK2 preventing the G1-S transition,
encouraging the apoptotic phenotype of cells (21) CDK
inhibitors p21 and p27 control various phases of the cell
cycle based on the cyclin with which they associate. The role
of p27 in ovarian cancer is somewhat contradictory. There
appears to be no correlation between p21 and p27 expression
and in terms of ovarian cancers, as yet these proteins are not
especially useful tools for the prognosis of the disease (22).
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
Cell proliferation regulatory pathways implicated in the
biogense of ovarian cancer are:
p16INK4a and Rb pathway
KRAS/MAPK/ERK pathway
PI3K/PTEN/AKT pathway
PI3K/PTEN/AKT pathway
The PI3K/AKT/mTOR pathway
Hedgehog pathway
Multiple Drug resistant (MDR) pathway
Notch Pathway
Forkhead Box M1pathway (FOXM1)
Breast cancer type 1/2 susceptibility protein
(BRCA1)/(BRCA2) and homologous repair and
nucleotide excision repair pathway
In ovarian cancer cells, cell proferation regulator pathway
p16INK4a is found mutated or its promoter region is
hypermethylated switching off its expression. p16INK4a is
considered to be a direct target of Rb expression (23). Some
studies have contradicted this finding.
Kirsten rat sarcoma oncogene (KRAS) activation triggers a
sequence of events through the RAF/MEK and mitogen
activated protein kinases (MAPK) pathways, and in
conjunction with mammalian target of rapamycin (mTOR),
a target of the protein AK strain thymoma (AKT) pathway,
control cell proliferation (24). Point mutations in KRAS
provide an advantage for the survival and progression of
tumors (25). KRAS mutations have been implicated in the
genesis of low-grade ovarian tumors, inducing an overactive
proliferative phenotype. In addition to breast cancer
associated protein 1 (BRCA1) and 2 (BRCA2) that have
familial roles in sporadic germline-based breast and ovarian
cancers, KRAS is now considered the third player (26).
KRAS expression levels differ based on the histopathological
type of ovarian tissue and the expression levels may help
determine the various type of ovarian epithelial cancers (27).
These signatures have a specific pattern in tandem with the
expression of RAF/MAPK components, phosphatase and
tensin homolog (PTEN) levels and may help differentiate
normal from borderline to early-stage cancers. It is also
probable that in late stage ovarian cancers, the earlier
mutations in KRAS can stimulate and induce the over-
activation of NF-B in cancer stem cells (CSCs) that survive
chemotherapy.
KRAS i n d u c e s mi t o g e n a c t i v a t e d p r o t e i n
kinase/extracellular signal-regulated kinases (MAPK/ERK)
through Serine thrconine protein kinase (BRAF).
Stimulation of KRAS via GTPase activity activates BRAF.
The stimulation can be mediated by cytokines, growth
factors or proto-oncogenes. The downstream target of
BRAF, MEK stimulates ERK when activated. ERK targets
transcription factors that are involved in cell proliferation
such as Myc (28).
Phosphatidylinositide-3 kinase (PI3K) and phosphatase and
tensin homolog (PTEN) proteins influence multiple
pathways within the cell that have many effects on the
cellular phenotype through protein AK strain thymoma
(AKT), also known as protein kinase B (PKB). These genes
are crucially required to transcend the input from external
stimuli (growth factors) and convey them to AKT.
Phosphorylation of key amino acid residues in these proteins
triggers the downstream activation processes. PTEN is a
tumor suppressor gene and either mutations in the gene or
loss of function of the protein is observed in many cancers,
including ovarian cancers (29). Loss of function in this gene
is mediated through epigenetic silencing and its role in
ovarian cancer needs to be further assessed (30).
PI3K is a heterodimeric protein comprised of a regulatory
subunit, p85, and a catalytic subunit, p110 (31). The
PI3K/AKT/mTOR pathway is involved in type I and type II
ovarian cancers. Single mutations of a member of the
pathway coupled with a mutation with members of another
pathway promote ovarian hyperplasia, and double
mutations within the same pathway are necessary for
ovarian tumorogenesis. Mutations in parallel pathways that
are involved in cross-talk are found to be mutated in ovarian
carcinomas. The integrated genomic analysis study of
ovarian carcinomas showed that at least 45% of the cases
contained mutations in the PI3K/RAS signaling pathway,
where PTEN deletions (7%); mutations (<1%), PIK3CA
amplifications (18%); mutations (<1%), AKT isoform
amplifications AKT 1 and AKT 2 (3 and 6 % respectively),
were observed in conjunction with KRAS amplification
(11%) (32).
Hedgehog is a signaling pathway that controls development
and is expressed during embryonal development (33). The
expression of Hh in adult ovarian tissue has not been
observed. The signaling mechanism is likely to be activated
within the stem cell population in the ovarian tissue that is
necessary for the repair of the ovarian surface epithelium
(OSE). Cancers arising from OSE have an epithelial
phenotype. They are thought to arise through mutated Hh
signaling and produce spheroid like structures with cancer
stem cell-like properties (34).
ATP-binding cassette (ABC) transporters are implicated in
multidrug resistance (MDR) of many tumors. Ovarian
cancers by far appear to build up resistance to many
treatments and ABC upregulation is thought to play a major
role in this process (35). Changes in ABC transporters
appear to stem from treatment rather than in situ tumor
development. Abnormal expression of the transcription
factor Gli1, a downstream target of Hh signaling, has been
shown to induce MDR resistance in a subset of ovarian
cancers and that the promoter regions of ABCB1 and
ABCG2 genes contain Gli1 binding specific consensus
sequence (35).
Notch pathway is regulated by ligands such as Jagged1, 2
and Delta-like 1, 3, 4 (36). Elements of the Notch pathway
are expressed in EOCs (37). Notch pathway signaling
appears to be fundamentally important to cell survival,
motility and development of vasculature (38). The
chemoresistance observed to platinum-based therapy of
28
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
ovarian cancers stems from the expansion of cancer stem
cells that have Notch activated (Notch 3) (39).
FOXM1 is a transcription factor that regulates genes that
control the cell cycle and thus proliferation and tumor
progression (40) and its role in angiogenesis has also been
observed (41). Serous ovarian cancers express high levels of
the protein that correlates with tumor progression (42).
FOXM1 is also involved in the induction of breast cancer
type 2 susceptibility protein (BRCA2) a downstream target
that regulates DNA repair (42). FOXM1 is involved in cell
migration/invasion in ovarian cancers via ERK that acts
upstream of FOXM1 (42) and regulates cell proliferation
through a number of elements in the pathway.
BRCA1 and BRCA 2 are tumor suppressors that readily
associate with p53 to serve apoptotic functions. Most of the
mutations seen in ovarian tumors are somatic mutations.
Mutations in BRCA1 and BRCA2 are of heritable germline
type. Only 10% of the ovarian tumors that arise are
hereditable, which involve theBRCA1/2 mutations (43) The
remaining 90% arise through somatic mutations affecting
proteins such as BRCA1/2 or p53 (44) BRCA1 expression is
favored over BRCA2 expression in terms of staging the
cancer. p53 accumulation is more apparent with BRCA1
mutations in late stage or stage III ovarian cancers (45).
BRCA proteins are associated with DNA repair and exist as a
complex with other repair proteins (46). Hereditable ovarian
cancer involving BRCA requires two-hits for tumor
formation. A single BRCA1 affected allele may not be
sufficient to promote tumor formation. However, DNA
stability is affected as DNA repair is affected that can
encourage tumor formation through the loss of function of
the second allele or mutations in genes governing the repair
pathway. The paradoxical role of BRCA in cancers is
apparent (47). Hereditable and somatic mutations in BRCA
are responsible for breast and ovarian cancer formation, and
yet, cells that carry WT BRCA with other pathway
anomalies are less sensitive to treatment or develop
chemoresistance. The observations are controversial as
some studies point out that the BRCA status (proficient
versus absent) of the cells do not have a significant
correlation to treatment outcome, whereas others have
shown that the absence of BRCA1 enhances the sensitivity
to treatment by agents that induce DNA damage, including
ionizing radiations (48) showed that the presence of BRCA1
mutations was associated with better survival outcomes as
compared to those patients that carried WT BRCA1.
Epigenetic Mechanisms in Chemoresistance
Current therapies against ovarian cancer involve cisplatin
treatment (49) Patients that are initially responsive become
resistant to the treatment. Platinum-based cisplatin therapy
involves the induction of DNA damage through inter and
intrastrand crosslinking between purines (50) The 1, 3 and
the 1, 2-intrastrand crosslinks are excised and removed
through nucleotide excision repair (NER) with the former
lesions being easier to remove due to less distortion of the
helix (51). The 1, 3-interstrand lesion are complex and
require homologous recombination where double stranded
breaks (DSB) are involved (52) The NER system is robust in
terms of lesion recognition and requires a host of various
NER components. The up-regulation of these elements by
DNA-induced damage could account for the gain of
resistance to treatment. In terms of DSBs, Rad51
recombinase is required and acts in conjunction with
BRCA2 (53). Therefore, loss of function of BRCA2 or
mutations that silence the expression of the protein favors
sensitivity to treatment and thus women with BRCA1/2
mutations have a better diagnosis and are more responsive to
platinum-based therapy. The direct correlation between the
expression of NER genes or its members and resistance to
therapy does not always hold true. The data obtained from a
study analyzing NER efficiency and cisplatin resistance
showed that altering the HRR pathway, but not NER
member expression, could enhance the sensitivity of
cisplatin-resistant tumors to platinum-based agents (54).
These observations have been corroborated by the Spellman
et al. study that showed that of the samples tested 23%
carried mutations/epigenetic altered states in BRCA1, while
11% carried mutations in BRCA2 (32) and that 51% of the
cases had altered HRR pathway that involved BRCA2 and
Rad51C (32).
Epigenetic and Diagnosis of Ovarian Cancer
So far only two biomarkers of protein origin (CA125 and
HE4) have been approved by the FDA for monitoring
ovarian cancer (55). However, benign gynecological and
medical conditions such as endometriosis, congestive heart
failure, and cirrhosis can also have elevated CA125 levels,
and elevated serum HE4 level was only related to the
advanced stage of epithelial ovarian cancer. More specific
and early detection biomarkers are surely needed. As one of
the basic elements of epigenetic mechanisms, DNA
methylation has been recognized as a potential ideal
biomarker due to its stability compared with RNA and
protein, sensitivity of detection by PCR, the possibility of
localization to a specific gene region and potential of
development as a screening method specific for cancer
detection (56).
Epigenetic mechanisms as main players in cancer
development are emerging as attractive targets for
characterizing reliable biomarkers of ovarian cancer.
Hypermethylation of a number of TSG promoters has been
detected in the serum or peritoneal fluid of a group of
ovarian cancer patients in stage I. These TSGs include
RASSF1A, BRCA1, APC, CDKN2A, and DAPK (57)
Especially for DAPK methylation, there is a tight
association of the methylation status between DNA isolated
from the peripheral blood and primary tumor (58).
Genes that are specifically methylated in ovarian cancer still
wait to be discovered that have the potential to distinguish
ovarian cancer from other cancers and to therefore serve
diagnostic purposes. Epigenomics studies consisting of
methylomic analysis may hold the key in this regard.
However, multicenter-conducted studies and well-
controlled clinical trials will eventually be needed to further
validate these biomarkers.
29
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
Conclusion
Ovarian cancers are lethal diseases as they slip detection and
are far advanced when detected. Research in ovarian cancer
is just in infancy in terms of understanding the pathways
deregulated in the disease. Clearly, there does not appear to
be a strong association between deregulated patterns and the
gene specific expression and subcellular correlation
patterns. Utilizing more quantitative technologies such as
microarray systems, western blots, real-time PCR, or whole-
genome sequence analysis might provide insight into the
etiology and pathology of the disease. Identifying and
utilizing markers of DNA methylation patterns and miRNA
expression patterns governing gene expression in ovarian
cancer and its subtypes may make treatments and therapies
more customized.
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Genomic Imprinting in Male Germ-line Stem
Cells
2
Pallavi Pushp, Hoon Taek Lee
1
Mukesh Kumar Gupta *
1
Department of Biotechnology & Medical
Engineering
NIT, Rourkela
2
Bio-Organ Research Center, Konkuk University
Seoul, South Korea
*guptam@nitrkl.ac.in
Male infertility is a common condition in human and its
etiology remains unknown in a high proportion of cases. It
can be empirically treated with intracytoplasmic sperm
injection (ICSI) as long as at least one sperm per oocyte is
available. In cases wherein sperms are not available for
ICSI, testes-derived male germ-line stem (GS) cells has
been envisaged as future therapeutic module for restoration
of male fertility (1, 2). It has particular application in patients
expecting to undergo radiotherapy or chemotherapies, for
instance, after cancer therapies, which often disrupt
31
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
spermatogenesis. Prior to the start of chemotherapy, GS
cells may be isolated from testicular biopsy samples and
cryopreserved for restoring the fertility by GS cell
transplantation upon successful completion of the cancer
therapy (Figure). This approach is especially useful in
pediatric patients, whose sperms are not available for
preservation. GS cells can also be used for in vitro
spermatogenesis to bypass the pre- or post-meiotic barriers
to sprematogenic process and restoration of fertility (Figure
1). Recent studies have further revealed that, under
appropriate culture conditions, GS cells can acquire
pluripotency to become multipotent GS (mGS; from
neonatal testes) or multipotent adult GS (maGS; from adult
testes) cells, which has the capacity to differentiate into the
cells of three germ layers and therefore, has applications in
regenerative medicine (3, 4). The mGS/maGS cells
originate from the cultured GS cells themselves and
therefore, at any particular time point, in vitro cultured GS
cells may contain some contaminating mGS/maGS cells
which may produce teratoma instead of initiating
spermatogenesis upon testicular transplantation.
Consequently, a molecular marker that can distinguish GS
cells from mGS/maGS cells is essential for their application
in clinics. Recent studies have demonstrated that, the
androgenetic genomic imprinting in mouse GS cells also
changes to embryonic stem (ES) cell-like pattern in
mGS/maGS cells. Thus, genomic imprinting may be used as
molecular signature to distinguish GS and maGS cells.
Genomic imprinting refers to mono-allelic expression of
genes in a parent-of-origin manner. In recent years,
abnormal genomic imprinting in germline cells has been
identified as a possible cause of impaired spermatogenesis
in some men diagnosed with idiopathic infertility. During
germ cell maturation and gametogenesis, male germline
cells undergo widespread erasure of somatic-like patterns of
DNA methylation followed by establishment of sex-specific
patterns by de novo DNA methylation. Incomplete
programming of DNA methylation in the male germline
cel l s may t her ef or e, r esul t i n compr omi sed
spermatogenesis. An association between altered DNA
methylation of imprinted genes in sperm and male factor
infertility has also been established (5, 6). Further, DNA
methylation patterns in spermatogonia and male GS cells
was generally found to be stable in culture and were highly
reproducible (7, 8). Thus, analysis of DNA methylation may
serve as a valuable diagnostic tool in clinical andrology. We
observed that mouse maGS cells were epigenetically stable
for DNA methylation at imprinted Igf2-H19 gene cluster
during in vitro culture and differentiation (8) but re-acquired
GS cell-like growth and differentiation characteristics with
altered DNA methylation pattern when they were re-
cultured in the GS-like conditions (9). During
reprogramming of GS and maGS cells, presence of leukemia
inhibitory factor (LIF) favored the decrease in androgenetic
DNA methylation of paternally imprinted genes (Igf2, H19)
while glial cell line-derived neurotrophic factor (GDNF)
prohibited the reprogramming of paternal imprinted genes
and favored the increase in DNA methylation of maternally
imprinted genes (Peg1). Neither LIF nor GDNF affected the
DNA methylation of non-imprinted Oct4, Nanog and Stra8
genes (9). It was further observed that, the DNA methylation
of mouse GS and maGS cells varied with the degree of
reprogramming in different cell lines and hence, could be a
marker of reprogramming in GS cells (3, 4, 8, 9, 10).
Besides imprinted genes, we also analyzed the imprinted
microRNAs (miRNA) in mouse GS and maGS cells.
Impaired biogenesis of miRNAs was found to disrupt
spermatogenesis in male mice and lead to infertility (11). On
the other hand, miRNAs such as miR-122-transfected
human induced pluripotent stem (iPS) cells formed
spermatozoa-like cells (12). Several miRNAs (e.g. miR-
15b, miR-16, miR-19a, miR-19b, miR-22, miR-34b, miR-
34b*, miR-34c-5p, miR-122, miR-383, miR-449a, miR-
1973, Let-7 etc.) have now been shown to be differentially
expressed in asthenozoospermic and oligoasthenozoospermic
males compared with normozoospermic males (13, 14).
Imprinted miRNAs represent a family of miRNAs that are
mono-allelically expressed in a parent-of-origin manner and
acts in trans, generally outside the genomic region from
where they are encoded. Genes encoding the imprinted
miRNA are mainly clustered in two chromosomal domains
[PWS-AS (also called Snurf-Snrpn) cluster and Dkl1-Gtl2
cluster] in mouse although few singleton imprinted miRNA
are also present at several genomic regions.
Besides, almost all well-characterized imprinted genes
clusters such as Igf2-H19, Peg10, Copg2, Rasgfr1, Gnas-
Nespas, Kcnq1 and Igf2r-Air also encode one or more
imprinted miRNAs whose expression is restricted in a
parent-of-origin manner and is controlled by DNA
methylation at imprinted control regions (ICR) of the
respective gene cluster. These imprinted miRNAs show
distinct temporal- and tissue-specific expression patterns in
different tissues, including ES cells, and control a wide
range of developmental and physiological pathways,
including stem cell pluripotency and differentiation. We
observed that the expression pattern of imprinted miRNAs
in mouse sperm was distinct from those of ES or somatic
cells (10). Sperm showed significantly higher expression of
imprinted and paternally expressed miRNAs (miR-296-3p,
miR-296-5p, miR-483) and lower expression of imprinted
and maternally expressed miRNAs (miR-127, miR-127-5p)
than those observed with somatic cells. Similar to sperm, the
expression of imprinted and paternally expressed miRNAs
(miR-296-3p, miR-296-5p, miR-483) were consistently
higher while those of imprinted and maternally expressed
miRNA (miR-127, miR-127-5p) were consistently lower in
GS cells than in control ES cells. This suggests that,
expression pattern of imprinted miRNA in mouse GS cells is
likely androgenetic and therefore, may possibly form an
epigenetic signature on testes-derived GS cells. DNA
methylation analyses of ICRs, that control the expression of
all imprinted miRNAs in respective gene clusters (Gnas-
Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR),
32
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
confirmed that imprinted miRNAs were androgenetic in GS
cells. On the other hand, DNA methylation of imprinted
miRNA genes in maGS cells resembled those of ES cells but
the expression pattern of the imprinted miRNAs was
intermediate between those of GS and ES cells. These data
confirm the conversion of androgenetic GS cells to
multipotent maGS cells but also suggest that the acquisition
of ES cell-like characteristics in maGS cells was likely
incomplete or partial. Indeed, several studies have shown
that genomic imprinting in maGS cells stand in between GS
and ES cells (4, 9, 15).
Taken together, studies suggest the genomic imprinting and
expression of imprinted miRNAs are androgenetic in GS
cells but changes to ES cell-like pattern upon their
conversion to maGS cells. Differential genomic imprinting
may thus, serve as epigenetic signature or molecular marker
to distinguish GS cells from maGS or ES cells. Since maGS
cells originate from GS cells during their extended in vitro
culture, these markers may have implication in clinical
settings to distinguish GS cell colonies from maGS cell
colonies and thereby, minimize the likelihood of teratoma
formation by contaminating maGS cells generated from the
GS cells.
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derived neurotrophic factor alters the growth characteristics
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the development of sperm abnormalities from human induced
pluripotent stem cells by regulating TNP2 expression. Stem
Cells Dev; 22(12): 1839-50.
13. Abu-Halima M, Hammadeh M, Schmitt J, et al (2013). Altered
microRNA expression profiles of human spermatozoa in
patients with different spermatogenic impairments. Fertil
Steril; 99(5):1249-55 e1216.
14. Gupta MK, Lee HT, editors (2012). Differences between
multipotent adult germline stem cells and germline stem cells
for microRNA: Springer, USA. pp 113-29.
15. Ko K, Tapia N, Wu G, et al (2009). Induction of pluripotency in
adult unipotent germline stem cells. Cell Stem Cell; 5(1): 87-
96.
Post Translational Histone Modifications in
Ovarian Epithelium and their Plausible
Implications in Reproductive Plasticity and
Health
G. V. Raghuram
Clinical Research Centre
Advanced Centre for Treatment, Research
and Education in Cancer
Navi Mumbai
raghuram.venkata@gmail.com
While the early research in past century has defined
particulate genetic inheritance as a primary mechanism
for the heritability of traits, more recent work in past decades
in lower eukaryotes and early mammalian species have
speculated epigenetic (or upon the genome)
modifications to this genetic framework, as a primary
mechanism, for the varied molecular events regulating the
reproductive development (1). Ovarian surface epithelium
(OSE) is a single layer of modified squamous or cuboidal
mesothelial cells covering the surface of the ovary separated
from underlying ovarian stromal tissue by a basal lamina of
collagenous connective tissue. The epithelium is
physiologically involved in follicular rupture and the
subsequent repair of the follicle wall during reproductive
33
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
age. The cyclic ovulation process predisposes ovarian
surface epithelium to genetic alterations if left unrepaired
(2). Given the dynamic nature of the ovarian follicle,
presents it as a best model to study the coordinated activation
and inactivation of genes related to cell growth and
differentiation during ovulations and the surrounding
ovarian epithelium. The gene expression is not only cycle
specific but also reflects the differentiation status of the
follicular granulose and theca cells of the ovary (3). Though,
significant progress has been made in ascertaining factors
that promote the transcription of ovarian genes mediating
gonadotropin action and steroidogenesis; yet clear
understanding is required about the appropriate time at
which these specific genes are turned on and off and also
how these factors influence the chromatin remodeling needs
to be delineated. In this regard, an attempt has been made in
this brief monograph on post translational histone
modifications in ovarian epithelium and their plausible
implications in reproductive plasticity and health.
Last decade, has seen surge in studies focusing epigenetic
regulation of ovarian genes through histone modifications
on a preliminarily aspect (4). Nucleosome represents the
basic repeating unit of chromatin consisting of two copies
each of four histone (H2A, H2B, H3, and H4) proteins.
Histone H1 is outside the core bound to linker DNA. The
core histones have protruding charged amino-terminal tails
which undergo various post-translational covalent
modifications including methylation, phosphorylation,
acetylation, ubiquitnation, sumoylation, ADP ribosylation
and citrullination (5). While, some of these modifications
promote transcription, others repress it. Open chromatin
modifications promoting transcription recruit chromatin
remodeling ATPases enzymes eventually leading to
nucleosome repositioning and increased access to DNA (6).
In addition, transcription factors such as HNF3 and GATA4,
upon mutation, open the compacted chromatin themselves
(7).
Over the past decade, various studies have substantiated the
role of the epigenetic alterations during mammalian germ
cell development through candidate regulatory factors.
Among all the histone modifications, the most studied ones
include the acetylation of lysine (K) residues, the
methylation of arginine (R) and K residues, the
phosphorylation of serine (S) and threonine (T) residues,
and the ubiquitination of K residues.
Histone Acetylation and Deacetylation
Acetylation involves the addition of acetyl groups to lysines
residues of the histone tails in neutralizes positive charges in
presence of histone acetyltransferases (HATs) resulting in
alterations to the chromatin fiber and increase accessibility
to the DNA and facilitate transcription. In the tail region,
acetylation occurs at lysines at amino acid position 9, 14, 18,
and 23 within H3 and at positions 5, 8, 12, and 16 within H4
(8, 9). The coactivators p300 and CREB-binding protein
(CBP) are the histone acetyltransferases which acetylate H3
and H4 tails and recruit additional acetyltransferases such as
p300/CBP-associated factor (pCAF) to specific gene
promoters by transcription factors associated with the DNA
(10).
During folliculogenesis, unique epigenetic and
transcriptional changes occur in germ cells, including the
differentiating and migrating PGCs, oogonia, and the early
mitotic-/meiotic-arrested primary oocytes. Seki et al (11) in
their studies showed that H3K9 acetylation (H3K9ac)
modifications associated with transcriptionally active
chromatin were transiently increased starkly upon their
entry into the genital ridge and further global chromatin
analysis found that H3K9ac levels in PGC precursors were
indistinguishable from those of the PGC cells somatic
neighbours. While, with increasing age and in fully grown
oocytes, the histone of the perinucleolar region is highly
acetylated (H3K9ac, H3K18ac, H4K5ac, and H4K12ac)
than in the growing oocytes suggestive of hyperactive
chromatin pattern. Evaluation of histone acetylation within
the steroidogenic acute regulatory protein (STAR) gene
promoter region (whose protein product regulates the rate-
limiting step in steroidogenesis and active during
gonadotrophin surge) in the ovary, have shown
hyperacetylated H3 in luteinized granulosa cells exhibited a
32-to 206-fold increase in acetylated H3 associated with the
STAR promoter (27 h post-hCG) when compared with non-
luteinized granulosa cells under induced ovulatory scenarios
(12).
Morever, just as histone acetylation is important for
facilitating active gene transcription, deacetylation
catalyzed by histone deacetylases (HDACs) too is important
for turning off genes and maintaining some genes in a
repressed state. Mammalian histone deacetylases fall into
three classes (IIII) based on their homology to yeast
HDACs. Classes I and II HDACs require trichostatin A
(TSA) and Class III HDACs require NAD+ as a cofactor.
HDACs form the multiprotein complexes recruited to the
chromatin by DNA binding proteins (13). However, the
precise functions of many of these HDACs in ovary function
are still unknown and specifically in ovarian follicle
formation are beginning to be understood. Speculation has
been that during genome reprogramming in germ cells
acetylated lysines should be deacetylated to erase cell
memory by histone deacetylation to create undifferentiated
or totipotent zygotes of the next generation. Preliminary
studies have shown that during the oocyte growth phase,
histone H3 was deacetylated at K9, K14, and K18 as the
chromosomes condensed when the fully grown oocytes
began maturing with subsequently disappearing completely
at the time of germinal vesicle (GV) breakdown (14).
Histone Methylation
Histones are methylated to different degrees of mono-, di-,
or trimethylation of K residues leading to gene repression or
activation, whereas R residues can bemono- or dimethylated
methylation (15). Methylation is controlled in a reversible
fashion by methyltransferases and demethyltransferases,
often associating in complexes. With reference to follicle
formation in primordial germ cells (PGCs), the upregulation
of H3K27me3 is complemented by the erasure of H3K9me2
34
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
to maintain a proper repressive chromatin state of the
genome during their migration from E7.5 to E10.5, stages.
However, first sign of chromatin changes in gonadal PGCs
at E11.5 was a rapid loss of linker histone H1 concomitant
with loss of H3K9me3 and H3K27me3, as well as loss of the
repressive H4/H2AR3me2. At about E12.5, the H3K9me3
marks reappeared, and some other chromatin changes also
reverted to the original state of development until at least
E10.5 periods examined (16). During follicle maturation,
histone methylation modifications play important roles in
the regulation of chromatin structure and gene expression,
especially for oocyte meiotic maturation. H3 methylation at
lysines 4 (K4) and 9 (K9) (H3K9me1 and H3K9me2
methylated) by euchromatic histone-lysine N-
methyltransferase 2 (EHMT2) are essential for early meiotic
progression. It has been found that H3K9me3 appeared in
growing oocytes from early antral follicles, increased
thereafter during the growth phase, and was maintained
during oocyte meiotic maturation and activation (14, 17).
However, a different sensitivity of histone methylation
including H3K4, H3K4me, H3K4me2, and H3K4me3 in
granulosa cells of follicles was found at different
developmental stages. Furthermore, oocytes from primary
to antral-stage follicles were positive for H3K4 mono-, di-,
and trimethylation with altered patterns throughout oocyte
development (18).
Histone Phosphorylation
Phosphorylation of histone tails entails a negative charge
altering chromatin accessibility locally. During mitosis and
meiosis, phosphorylation of serines 10 and 28 and threonine
11 of histone H3 is associated with chromatin condensation,
yet serine 10 (S10) phosphorylation has more recently been
implicated in transcriptional activation (19). During follicle
formation, high levels of phosphorylated histone variant
H2A.Z was detected in early E10.5 PGCs, but most of the
signals were lost by E11.5eE12.5. However, levels remained
relatively constant in the neighbouring somatic cells (16).
During follicle maturation, Histone H3 phosphorylation is
altered with changes in chromatin condensation during
mouse oocyte meiosis with both phH3/ser10 and
phH3/ser28 presented in GVBD, meiotic I (MI), or meiotic I
(MII) oocytes (20). Gu et al. (21) showed a stage-dependent
dynamics of phosphorylated H3 (phH3/ser10) and
phH3/ser28 during porcine oocyte meiotic maturation with
no apparent phH3/ser28 signals in GV-, late-GV-, or early-
GVBD-stage oocytes; but high levels were observed in pre-
MI-, MI-, and MII-stage oocytes. Furthermore, mechanisms
of histone phosphorylation during mammalian follicle
development (especially its relationship with granulosa-
cell-mediated reproductive hormones) remain unknown.
Histone Ubiquitination
Ubiquitination and deubiquitination of proteins are
reciprocal events involved in many cellular processes,
including the cell cycle. Monoubiquitination of histone H2B
at lysine 120 (H2Bub1) has been shown to have key roles in
transcription, the DNA damage response and stem cell
differentiation (22). Histone variants and histone
ubiquitination may be other regulatory factors during
follicle development (23) showed that ubiquitinated histone
H2A is associated with transcriptional silencing of large
chromatin regions during meiosis in females (24). This
suggests that orderly and proper epigenetic reprogramming
in premigratory and migratory germ cells might be
necessary for the production of gametes with an appropriate
epigenotype to support subsequent normal development.
Histone Modifications and Ovarian Cancer
In lieu of the fact that the ovarian surface epithelium is
exposed to stressful agents generated during periovulatory
processes, any genetically altered proliferative ovulatory
wound-repair responses could give rise to a transformed
phenotype. Greater than 95% of ovarian cancers originate in
the epithelial cells on the ovary by perturbed ovulation (25).
Recent there has been a consensus on significance of
epigenetic alterations such as global genomic hypo/hyper-
methylation along with changes in the histone-code in the
transformation and carcinogenesis of ovarian epithelial cells
(26).
Ovarian cancer manifests silently, usually revealing no clear
symptoms until disease advances to a malignant metastatic
stage. Histone modifications have been known to facilitate
dysregulated gene expression in such malignant disease.
Recent studies have shown that alike in normal embryonic
stem cells, both normal and transformed neoplastic ovarian
cells/cancer stem cells maintain genetic flexibility or
bivalent epigenetic signature by co-occupancy of opposing
histone marks of transcription activating and/or repressive
modifications (epigenetic silencing). It has been shown that
the tight junction proteins claudin-3 (CLDN3) and claudin-4
(CLDN3) are frequently overexpressed in ovarian cancer
through concurrent hyper-acetylation of histone H3
(H3K9Ac) and H4 (H4K16Ac) and promoter DNA
hypomethylation (27). Also, over expression of the HDACs
I, II and III have been demonstrated to result in overall
aberrant histone deacetylation in ovarian cancers which in
turn correlated with high-grade tumors and poor prognosis
of the disease (28). Besides, other important several genes
the such as GATA family of differentiation-associated
transcription factors and the cell cycle inhibitor p21
appeared to be regulated solely by histone modifications
(29, 30). Convincing results gained through preclinical and
clinical studies using HDAC inhibitors have shown
potential for the treatment of ovarian cancers through
decreased cancer cell growth, apoptosis and increased cell
differentiation. However, further studies are required
towards increased survival and better prognosis (31).
Regarding histone methylation, the best characterized
histone modification in ovarian cancer is the repression of
bivalent mark" of H3K27me3/H3K4me2 in aggressive form
of disease. However, recent studies have revealed a
mul t i val ent hi st one met hyl at i on marks bi nary
H3K9me3/H3K27me3, trivalent H3K4me2/ H3K9me3/
H3K27me3 and tetravalent H3Ac/H3K4me2/ H3K9me3/
H3K27me3 marks. These have further substantiated the
effect of the local microenvironment on the epigenetic
35
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
plasticity in response to the presence two different cellular
conditions of three-dimensional multicellular aggregates
(spheroids) and two-dimensional monolayers ascites and
peritoneal mesothelia, in ovarian malignancies (26, 32).
Recently, overexpression of methyltransferase Enhancer of
Zeste Homolog 2 (EZH2), a catalytic subunit of polycomb
repressive complex 2 (PRC2), has been shown to generate
histone H3 (H3K27me3) mark contributing to the
proliferative ovarian tumor environment (33). In addition,
under stress induced premature senescent conditions,
induction of hypermethylation of H3K9me3 through
increased senescent heterochromatin foci (SAHF)
formation has been noted in ovarian epithelial cells our
recent study (34).
Histone phosphorylation has been implicated in the DNA
damage response given their fine control of chromatin
configuration that determines access by transcription factors
and DNA repair proteins. In this regard, ovarian cancer is
undoubtedly a tumor driven by aberrant DNA damage
signaling. Incidentally, high levels of phosphorylated
histone H2AX isoform (-H2AX) along with histone H3.3
phosphorylation on serine 10 has been observed in high
grade ovarian tumors leading to cell death (35). Therefore
the potential exists to improve the way this pathway is
targeted with current therapies by a greater understanding of
the chromatin landscape.
Among the other histone modifications monoubiquitination
of histones H2A/H2B are clear instances of alternative roles
for ubiquitin in transcription and DNA repair through
controlled by ubiquitin ligases such as the RING finger
proteins RNF20 and RNF40 and deubiquitinases (DUBs).
Loss of global levels of H2Bub1 has been reported in
ovarian cancers. Histone small ubiquitin-like modifiers
(SUMO) through sumoylation in response to genotoxic
stress, and co-localizes at sites of DNA damage with
SUMO1, SUMO2/3 and the SUMO-conjugating enzyme
Ubc9. However, exact mechanistic insights into these forms
of histone marks are still unknown.
Recently, another form of histone modification, histone
citrullination, has been shown to occur. It is catalyzed by an
enzyme called peptidylarginine deiminase 4 (PAD4, also
called PADI4), which converts both histone arginine (Arg)
and mono-methyl arginine residues to citrulline. PAD6 is
found in ovaries, which needs to investgated further for
functional significance. Preliminary studies have found that
histone citrullination counteracts the effect of histone
arginine methylation and functions as a repressive marker to
turn off gene expression (36). However, whether
citrullination is the cause or the consequence of the
pathological alterations such as tumorigenesis needs to be
investigated.
Taking together, the study of histone modifications and their
impact on ovarian epigenetic plasticity and function are
worth investigating as these processes aim to regulate the
transcription activity of genes and foster to establish a new
expression system. The orderly and proper epigenetic
reprogramming in ovary occupies prime importance as germ
cells might be necessary for the production of gametes with
an appropriate epigenotype to support subsequent normal
ovarian development. Future investigations on the histone
post translational epigenetic modifications along with new
epigenetic modifiers could act as surrogate biomarkers of
disease risk that could potentially facilitate tailored
treatment and further refine preventive measures for ovarian
epithelial cancers.
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Polycystic Ovary Syndrome: Plausible
Epigenetic Distress and Role of Environmental
and Endogenous Modulators
Pooja Sagvekar*, Srabani Mukherjee
Molecular Endocrinology Department
National Institute for Research in Reproductive
Health (ICMR)
Mumbai
*sagvekarpooja@gmail.com
Polycystic ovary syndrome (PCOS) is a multifactorial,
chronic disease state of a yet unknown etiology affecting 6-
10% of women of childbearing age (1). It is one of the
leading causes of anovulatory infertility and subfertility
demarcated by an array of ovarian, hormonal and metabolic
derangements and characterized by chronic menstrual
irregularities, folliclular cysts on ultra-sonography,
hyperandrogenemia, hyperinsulinemia and obesity. PCOS
pathophysiology can be outlined starting from
hypothalamo-pituitary-ovarian axis, where persistent,
elevated GnRH release leads to increased LH secretion that
exerts gonadotropic effects through its receptors on theca
and granulosa cells. In PCOS, immature follicles get
prematurely sensitized to LH and facilitate excess ovarian
steroidogenesis. Elevated steroid production coupled with
aromatization defects downstream in these pathways favor
increased synthesis of androgens over estrogens, thus
contributing to hyperandrogenemia. Another important
characteristic of PCOS is insulin resistance (IR), in which
peripheral muscles and fat tissues exhibit low tolerance to
insulin and glucose, further promoting compensatory
hyperinsulinemia. Excess insulin acts synergistically with
LH to augment ovarian steroidogenesis and aggravates the
intensity of androgenic stress. Further, ovarian androgens
coupled with adrenal androgens result in clinical
manisfestation of hyperandrogenism (Textbook Ref, 2).
Also obesity, a commonly observed trait that stems from
inherent or de-novo abnormalities in lipid metabolism is an
added risk factor in development of PCOS, though lean
phenotypes are also prevalent. Therefore, PCOS women
develop co-morbidities like hyperglycaemia, type-2
diabetes mellitus (T2DM), hypertension, cardio-vascular
disorders (CVDs), coronary heart diseases (CADs) and
metabolic syndrome (MS) that predispose them to life
threatening health risks.
Although PCOS bears a well-established genetic
component, it holds equal ground for epigenetic
mechanisms that bring about phenotypic plasticity, fetal
reprogramming, late-life onset of disease and such
phenomena that persist beyond the understanding of gene
sequence abnormalities. Recent information on heritability
of epi-mutations over several generations of progeny
(transgenerational epigenetic inheritance), has raised
concerns among scientists in reproductive health circles (3).
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
Events such as early/late-life exposure to endocrine
disruptors (ECDs) and environmental pollutants,
unfavorable diet and lifestyle malpractices or even altered
circadian or diurnal rhythmicity are known to affect this
inheritance mechanism. Several such factors have been
linked with PCOS etiology in the form of circumstantial as
well as direct evidences on epigenetic modifications in
PCOS (Table 1).
Table 1: Epigenetic Studies in PCOS Women and PCO
Models. (Conclusive Inference Marked in Blue)
However, the lack of solid evidence pertaining to these
changes has necessitated high throughput investigations on
this subject. This article therefore, highlights the
significance of epigenetic distress in PCOS as an outcome of
endogenous remodeling events in response to
environmental contaminants and unfavorable life styles.
Epigenetic programming is achieved by an intricate control
over mechanisms that bring about addition or removal of
chemical groups in the chromatin i.e DNA methylation and
post-translational histone modifications without altering the
genetic code (4). These alterations may interfere with basic
chromatin structure and functions and result in global
epigenetic changes (eg: DNA hypo and hypermethylation in
cancer) and/or differential expression changes in target
genes and alterations in non-coding RNAs (ncRNAs). Such
fluctuations may manifest as easily palpable, rare
phenotypic anomalies like the Angelman syndrome (a gene
imprinting disorder) or subtle and common variations like
obesity, caused by metabolic perturbations (5, 6).They also
confer tissue specificity by controlling the transcriptional
switch responsible for gene expression or repression, and
are therefore associated with cell specific or tissue
associated derangements contributing to disease states.Of
these epigenetic modifications, DNA methylation changes
are less labile, relatively easy to track and are hence more
frequently investigated than the rest. Thus, epigenetic
studies are crucial in understanding the obscure but
significant phenomena related to pathophysiology of
chronic diseases. PCOS is one such disorder that has
recently gained attention owing to the extent of reproductive
dysfunction in women.
Role of Inadvertent Exposure to Hormones, EDCs/drugs
and other Environmental Pollutants in PCOS
Epigenetic modification studies provide a nexus to translate
the emerging effects of environmental exposures to
reproductive morbidities in both men and women and the
extent of incurred damage is determined by both, frequency
and intensity of a given exposure. In PCOS, a significant
portion of its current understanding on reproductive
anomalies comes from data extrapolated from experimental
animal models owing to the limitations discussed earlier.
Untimely or excess exposure of female fetus to uterine
androgens in rodent and rhesus monkey models of PCO has
improved our understanding on fetal origins of this disease.
These prenatally androgenized females developed PCOS
like features in adulthood, which may be explained by
epigenetic priming processes (7, 8). Similarly, fetal estrogen
excess studies in PCO and non-PCO animal models
demonstrate development of PCOS features like
hyperandrogenism, irregular menstrual cycles and impaired
fertility. Interestingly, estrogen deficiency in fetus also
promoted hyperandrogenic, multicystic ovarian
phenotypes, thus emphasizing that optimal estrogen levels
are necessary for normal reproductive functioning (9). Also,
studies on hormone receptor genes eg: X-chromosome
inactivation in androgen receptor (AR) (Laisk et al., 2010),
hypomethylation in LH receptor (LHCGR) (Table.1) in
PCOS and other genes eg: Lamin A/C (LMNA)
hypermethylation 10) in insulin resistant PCOS phenotypes
have encouraged additional studies on role of hormone
induced epigenetic dysregulation. These findings are in
support of Barkers hypothesis of developmental origins of
health and disease (DOHaD) in PCOS (11).
EDCs predominating the external environment are
additional culprits in epigenetic programming and can be
classified as phyto-estrogens, heavy metal toxins,
fungicides, pesticides, pharmaceutical and industrial
wastes, plasticizers, flame-retardants and hydrocarbon
mixtures. Their actions are exerted by modifying estrogen or
androgen dependent steroidogenic pathways with most
endocrine damage incited by estrogenic mimetics that act
via estrogen receptors (ESR). Bisphenol-A (BPA), a
plasticizer with such actions is widely used in the industrial
manufacture of plastic wares, dental resins and food-can
lining has been reported to be high in PCOS (12). High
circulatory BPA levels are proposed to interfere with insulin
signalling mechanisms of glucose metabolism thereby
causing IR (13, 14). They are also associated with other
PCOS features ofincreased testosterone synthesis by theca-
interstitial cells, lowering of hepatic androgen-related
hydroxylases that stall testosterone degradation and
competence with endogenous androgens that bind to sex
hormone-binding globulin (SHBG). Diethylstilbestrol
(DES) is yet another EDC that was exploited for its ability to
mimic estrogen signaling and was prescribed heavily during
1950s-60s to achieve pregnancy (9) Third generation studies
have revealed reproductive anomalies in sons and daughters
of women exposed to DES in utero, more-so in daughters
who showed excess GnRH secretion, hyperandrogenism
38
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
and infertility due to menstrual irregularities that are
suggestive of PCOS (9). However till date, information on
whether epigenetic mechanisms play a role in EDC induced
PCOS features in women is not available.
There are several more undetectable environmental
components namely volatile agents from exhaust fumes,
hydrocarbon emissions, electromagnetic and radio waves or
even unknown constituents that have more pronounced
impact during prenatal stages of organ development (16).
This is because key epigenetic programming in fetal stages
occurs in two phases: (i) in pre-implantation embryo bearing
three germ layers (affecting overall organ development) (ii)
in primordial germ cells (PGCs) during their migration from
extra-gonadal sites to the genital ridge (affecting germline
reprogramming and subsequent progeny) (16). Although,
exposure hazards during adulthood are reported to be
equally debilitating, they are reversible and therefore
manageable.
Effect of Dietary Indulgence
Methylation at 5-carbon (C5) position of cytosine bases in
DNA is brought about by DNA methyl transferases
(DNMTs) while methylation of lysine and arginine residues
in the histone tails is a function of histone methyl
t r a ns f e r a s e s ( HMTs ) a nd pr ot e i n a r gi ni ne
methyltransferases (PRMTs) respectively. Both these
processes are effectively facilitated by S-Adenosyl
methionine (SAM), an active derivative of the essential
amino acid methionine that acts as a methyl donor for most
of the biochemical reactions. These SAM-dependent
methyltransferases release methyl groups by conversion of
SAM to S-adenosylhomocysteine (SAH), which acts as a
feedback inhibitor for SAM dependent methyltransferases.
Thus, an excess of methionine in the system favours
hypermethylation in chromtatin while high amounts of
homocysteine are conducive to hypomethylated states (17).
Systemic depletion of SAM and a concurrent rise in SAH
levels has been attributed to less intake of folate and choline
rich diet. Limited availability of folate restricts the synthesis
of its active counterpart, 5-methyl tetrahydrofolate, thus
depleting SAM and resulting in decreased cytosine
methylation in DNA (18). In PCOS women, high levels of
homocysteine have been reported earlier, which may be
associated with low intake of folate supplements and
resultant defects in DNA methylation machinery (19).
Studies on PCOS associated co-morbidities namely CAD
and MS carried out in leucocytes and visceral adipose tissue
respectively also indicate towards global DNA
hypomethylation in PCOS (20, 21). Adherence to protein
restricted diet and supplementation of folate co-factors,
choline and vitamin B12 has experimentally proven to
facilitate restoration of methylation loss by increasing the
abundance of SAM (22, 23). Therefore, women with PCOS
can be recommended for consumption of food with low
cholesterol, low glycaemic index (GI) and high folate
content.
Another important dietary risk factor for PCOS is advanced
glycation end-products (AGEs), which are reactive
metabolites of non-enzymatic glucose-protein reactions
produced in response to hyperglycemia, oxidative stress or
exogenously ingested carbohydrate-rich food processed at
high-temperatures. They contribute to preeminent risks of
diabetes and cardiovascular disease in the general
population. PCOS women are reported to have high
concentrations of AGEs in their sera, which positively
correlate with elevated serum androgen levels (24). Also,
women with insulin resistance demonstrate heightened
serum AGEs and their respective receptors (RAGEs) on
ovarian theca and granulosa cells (25), suggesting a putative
direct action of AGEs in PCOS.
ART Induced Endocrine Milieu as an Additional
Epigenetic Stressor
Diagnosis of infertility in couples and the surging trend in
delay of maternal age at first childbirth, especially in
metropolitan sectors, has provided impetus to taking support
of assisted reproductive technologies (ART) in child
conception. PCOS, as aforesaid maximally contributes to
anovulatory infertility and thereby oocyte defects in women
who thereby resort to in-vitro fertilization (IVF) programs
for conception. Infertility, as we know, has been already
ascertained as a consequence of erroneous epigenetic
programming in quality-compromised oocytes or sperms,
thereby, making PCOS women more susceptibility to a
whole new set of ART induced epigenetic alterations as well.
IVF encompasses protocols like controlled ovarian hyper-
stimulation (COH) for oocyte retrieval and in-vitro oocyte
maturation (IVM) that precede embryo-transfer (ET) and
intra-cytoplasmic sperm injection (ICSI).With every
proceeding step, the spermatozoa and ovarian cells,
especially granulosa cells and oocytes get exposed to
external environmental agents including the culture media,
laboratory pathogens, temperature fluctuations and so on.
These fluctuations also contribute to errors in epigenetic
mechanisms that are manifested in both early developmental
stages and/or adulthood (26, 27).
These initiatives were based on observations that upto 4% of
the ART-borne infants presented with severely abnormal
phenotypes that matched with BeckwithWeidemann
syndrome while 1 in 15,000 newborns presented with
Angelman syndrome (28). Extensive methylation analyses
of regulatory regions in several imprinted genes have
identified susceptible targets that develop such phenotypes.
H19, Ubiquitin-protein ligase E3A (UBE3A) and insulin-
like growth factor-2 (IGF2) are some of these imprinted
gene targets (29). However, there are no reports thus far,
explaining the outcome of ART in PCOS affected women,
thereby keeping this aspect open for investigation.
To conclude, self-imposed dietary choices, life-style
vagaries and undesired exposure to combinations of
exogenous and endogenous agents discussed thus far, can
lead to development of PCOS. However, additional
tendencies like alcohol consumption, smoking, substance
abuse, lack of physical exercise and prolonged late-night
work shifts, contact with bacteria, viruses etc. cannot be
ignored as the confounding factors while investigating
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
epigenetic effects in such life-style related disease states.
With eminent progress in epigenetic studies, we therefore
speculate that the enigma behind complex disorders like
PCOS can be resolved and would lead to the use of
appropriate epigenetic therapy in alleviation of their health
risks in future times.
References
1. Bu Z, Dai W, Guo Y, et al (2013). Overweight and obesity
adversely affect outcomes of assisted reproductive technologies
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2. Dunaif D, Chang R, Franks S, et al (2008). Polycystic Ovary
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3. Nilsson E, Larsen G, Manikkam M, et al (2012).
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6. Oken E, Gillman MW, Fetal MWG (2003). Fetal Origins of
Obesity. Obes Res; 11(4): 496-506.
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mechanism underlying the fetal origin of polycystic ovary
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9. Abbott DH, Padmanabhan V, Dumesic DA (2006).
Contributions of androgen and estrogen to fetal programming of
ovarian dysfunction. Reprod Biol Endocrinol; RB&E, 4, 17.
10. Ting W, Yanyan Q, Jian H, et al (2013). The relationship
between insulin resistance and CpG island methylation of
LMNA gene in polycystic ovary syndrome. Cell Biochem
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11. De Boo HA, Harding JE (2006). The developmental origins of
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12. Kandaraki E, Chatzigeorgiou A, Livadas S, et al (2011).
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Epigenetic Changes and Human Assisted
Reproductive Technologies
Rajvi Mehta
Trivector Embryo Support Academy
Mumbai
rajvim@gmail.com
Introduction
Assisted Reproductive Technologies [ART] are now an
established modality for treating infertility. The
International Committee for the Monitoring of Assisted
Reproductive Technology (ICMART) has reported that
approximately 350000 ART cycles are performed
worldwide per year; 5 million babies in the world have been
born through ART till 2013; of which nearly 25% have been
born in the last 6 years.
As ART involves many non-natural procedures such as
40
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
ovarian stimulation, physical manipulation of the sperms
and oocytes; embryo culture and cryopreservation there
has always been a concern on whether these procedures
would affect the health of the children born through ART. In
a meta-analysis, Bower and Hansen (1) reported on an
increase in low birth weight as well as small for gestational
age babies born after ART as compared with those born after
natural conception. There is a 2.6-fold increased risk of low
birth weight in term IVF infants, with a greater risk of
prematurity such that 0.4% of all very low birth weight
infants are conceived by IVF. This observation was only for
singleton pregnancies (2, 3) ruling out the possibility of the
low birth babies being due to multiple gestations. Although,
there does not appear to be any significant difference in the
physical growth or hormonal parameters between ART and
naturally conceived children.
The question that now arises is whether the low for gestation
age and low birth weight of babies conceived through ART
would affect the future development of these children?
Barker showed a connection between birth weight and adult
disease expressed later in life (4). He proposed the fetal
origins hypothesis of adult disease. According to this
hypothesis the origins of diseases in adults begin in utero.
This hypothesis has gained credibility through
epidemiologic studies performed in Europe and the United
States (5). If the onset of diseases in adults originates in
utero then could the onset of abnormalities in utero or low
gestational age be a result of early embryonic
developments? With the extensive manipulations on the
gametes and embryos in ART, there is a concern on whether
these could be responsible for the low gestational age or low
birth weight of the babies born through ART.
Abnormalities in Children Born After ART
Birth defects: Studies have been carried out comparing the
prevalence of birth defects in ART babies especially
comparing IVF with babies born following ICSI.
As ICSI is more invasive than IVF, there is a concern on
whether the procedure would have a detrimental effect on
the embryos. Bonduelle et al. in a multi-centric study
compared 540 ICSI and 437 IVF-conceived 5-year-old
children from five European countries and found that major
malformations were more frequent in the ICSI group, in
particular in ICSI boys, beyond the neonatal period with the
majority of these increased defects due to an excess in
urogenital malformations (6). However, in other recent large
studies and meta-analyses, no significant differences were
found between ICSI and IVF children (7, 8).
In one of the largest meta-analysis, Wen et al. reviewed 46
studies and a total of 124468 children born following
IVF/ICSI. They reported on a pooled risk estimation of 1.37
(95% CI 1.261.48) following IVF-ICSI which was not
statistically different when the data of IVF was compared to
that of children born through ICSI. Thus it appears that there
is no increase in birth defects following ICSI (9).
Long Term Development
The growth patterns of ART-conceived children have
attracted the attention of many researchers. The vast
majority of these studies have not found any differences in
the postnatal growth until 12 years of age between ART and
naturally conceived children (7, 10, 11). However, some
studies do suggest that ART children are taller. Miles et al.
(12) found that IVF/ICSI-conceived children aged 56 years
were significantly taller than naturally conceived controls
following adjustment for age and parental height (12). If
these observations are corroborated by other studies than
one needs to determine whether such an outcome is
specifically due to ART manipulations?
Increased Vascular Disorders
Healthy children conceived through ART seem to have
an elevated risk of suffering from cardiovascular
diseases in the future (13, 14, 15). Ceelen et al. (13)
observed that 8 to 18 year olds conceived following ART
had higher blood pressure and fasting glucose levels
compared with age and gender-matched controls (13).
Scherrer et al. (15) compared systemic and pulmonary
vascular function in 65 healthy ART-conceived children
and 57 naturally conceived controls and reported that
ART-conceived children were apparently normal but
may have had generalized vascular dysfunction (15).
They found that flow-mediated dilation of the brachial
artery was 25% smaller ((6.71.6)% versus (8.61.7)%),
carotid-femoral pulse-wave velocity was significantly
faster, carotid intima-media thickness was significantly
greater, and the systolic pulmonary artery pressure at
high altitude was 30% higher in ART-conceived children
than in controls.
What is the cause of the vascular abnormalities remains
unclear. It is hypothesized that these could be due to
epigenetic changes or genomic imprinting disorders.
Methylation of Genomes and Gene Silencing
With the understanding of epigenetics in the 1950s, it was
realized that genetic expression can be altered in response
to the environment. The mechanism by which genetic
expression is altered as well as its role in fetal development
is now being understood. The alteration in the DNA
methylation pattern can cause gene silencing and alter
gene function without any changes in the gene itself (16).
The delivery of a methyl group to the gene spurs a tight
recoiling of the DNA. The contraction makes it harder, or
impossible, for transcription factors to interact with the
gene leading to silencing of the gene Methylation of
the CpG dinucleotide causes gene silencing. 70% to
80% of the genome are methylated during development
and when some of the CpG islands become methylated
then the associated gene gets silenced (17, 18).
Epigenetic Changes During Gametogenesis and
Embryogenesis
During mammalian development, there are various phases
of epigenetic modification.
During gametogenesis, there is genome-wide
demethylation followed by remethylation before
fertilization.
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
41
Early embryogenesis is then characterized by a second
genome-wide demethylation event (19) following
which methylation is re-established early in embryonic
life following implantation.
These post fertilization demethylation and remethylation
phases are likely to play a role in the removal of acquired
epigenetic modifications, particularly those acquired during
gametogenesis (20, 21).
Genes that undergo genomic or parental imprinting are
among the most well-understood examples of epigenetic
transcriptional modification. A subset of approximately 80
genes in humans display mono-allelic expression, i.e.
expression only occurs from a single parent. Genomic
imprinting-induced silencing of one parental allele results in
mono-allelic expression from either the paternal or maternal
copy of a gene. The imprint control regions for genomic
imprinting usually contain a differentially methylated CpG
island in which one parental allele is methylated and the
other unmethylated.
Epigenetic Changes in Response to In Vitro Culture
As the human embryos created by ART are exposed to a
varied environment, there have been concerns on whether
they are subject to epigenetic alterations. There is a concern
that exposure to an exogenous environment could
potentially lead to epigenetic alterations and subsequently
immediate and long-term impacts on the fetus and the health
of the offspring. Although, gene expression studies on
human embryos are rare due to obvious ethical
considerations, development of children born following
ART is being monitored to determine whether there are any
differences in children born after ART as compared with
those born after natural conception; And, changes if any can
be attributed to epigenetic alterations. Extrapolations are
also being made from animal experimentation.
Data from animal studies clearly show that nutrition during
embryonic development does lead to changes in DNA
methylation patterns because mammalian one carbon
metabolism, which is the source of all methyl groups for all
biologic methylation reactions, is very dependent upon
dietary methyl donors and cofactors (22). If the dietary
methyl donors influence embryonic development then it is
hypothesized that the composition of the culture medium in
ART would alter methylation patterns and thereby
expression of imprinted genes (23, 24).
Mouse embryos cultured in Whitten's media showed loss in
methylation of the H19 differentially methylated region that
was not seen in embryos cultured in Biggers KSOM
medium which contained amino acids (25). When mouse
embryos were cultured in fetal cord serum, which for a long
time had been a protein supplement even in human embryo
culture media had reduced viability, reduced body weight,
decreased expression of H19 and IGF2, and increased
methylation of the H19 imprinting control region when
compared with controls (26). All these studies on mice ART
clearly indicate that epigenetic changes do occur when
embryos are cultured in vitro and these alterations do affect
the development of the embryos and development of
embryos into fetuses and live births.
Studies in mice demonstrate that ART alters the methylation
pattern of genes involved in vascular function and induces
vascular dysfunction, a problem that can be prevented by
modification of the culture media used for ART (27).
Abnormalities in ART Conceived Children Specifically
due to Epigenetic Alterations
As crucial imprinting events coincide with the specific ART
procedures raising serious concerns on the epigenetic
changes due to ART. Since 2002, several reports have raised
concerns that children conceived by ART are at an increased
risk of having imprinting disorders, especially some rare and
severe imprinting-related diseases, such as Beckwith-
Wiedemann syndrome (BWS), Angelman syndrome (AS),
and retinoblastoma (28) . However, these disorders are very
rare and therefore it is difficult to determine whether their
occurrence is related to ART, However, some studies have
suggested that the subfertile condition of the parents may
also be responsible for imprinting disorders and not
essentially the in vitro culture conditions (29-30). However,
other studies show no correlation between ART and genomic
imprinting disorders, including BWS and AS (31). Some
epigenetic modifications appear to be dynamic throughout
life and could contribute to adult-onset diseases Thus, ART
could lead to subtle abnormalities in children that could
present later in life.
Katari et al. found a modest change in the methylation level
of CpG sites in about 700 genes from the cord blood samples
from ART-conceived children (32). Many of these altered
genes have been implicated in chronic metabolic disorders,
such as obesity and type II diabetes. When the peripheral
blood cord blood of 18 children conceived through ART
were analysed, it found hypomethylation at specific regions
in 3 of 18 clinically normal children conceived by ART.
(33). Nevertheless, many other studies do not corroborate
these observation on aberrant methylation patterns in
children conceived following ART (34, 35).
Conclusions
Most children conceived following any of the ART are
healthy. There are some risks on poorer perinatal outcome,
low birth weight and small for gestational age in ART
conceived children. However, it is unclear whether these
risks are an outcome of epigenetic changes induced by the in
vitro culture conditions or the infertility per se. Larger
studies with long-term follow-up are needed to answer these
questions. The current data on epigenetic alterations in ART
conceived children are controversial which in turn could be
de to the epigenetic changes being reversible!
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status at differentially methylated regions in IVF-conceived
newborn twins. Fertil Steril; 95(6): 19759.
35. Tierling S, Souren NY, Gries J, et al (2010). Assisted reproductive
technologies do not enhance the variability of DNA methylation
imprints in human. J Med Genet; 47(6): 3716.
MicroRNA in Testis An overview
Panneerdoss Subbarayalu
Manjeet K. Rao*
University of Texas Health Science Center at San
Antonio
San Antonio, Texas
*raom@uthscsa.edu
MicroRNA (miRNA) are naturally occurring small non-
coding RNAs that have withstood years of evolutionary
pressure to be abundantly expressed in animals and humans.
miRNAs are endogenously expressed molecule that are
present either in the intron where they are transcribed as a
part of the genes or in the intergenic region where they are
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
43
transcribed by the independent transcriptional complex.
miRNAs have emerged as important regulators of gene
expression as they bind to 3' untranslated region (UTR) of
the target gene and silence their expression at the
transcriptional and/or translational level. Studies in several
organisms have revealed that miRNAs play important roles
during normal development and differentiation in various
tissues (1, 2).Recent studies have established an equally
important roles for miRNAs in regulating testicular
development and spermatogenesis. One of the first evidence
that miRNAs may be key players in testicular physiology
came from Kotaja et al. (3) when they reported that miRNA
processing machinery is localized in the chromatoid bodies
of the male germ cells and likely play an important role
during germ cell differentiation. Another exciting study
came from Dr. Wei Yan's group claiming that many X-linked
miRNAs escape meiotic sex chromosome inactivation
during spermatogenesis (4). Subsequent studies reported in
vivo model that showed importance of miRNA during
spermatogenesis. In these in vivo models, miRNA
processing enzyme dicer was specifically deleted in the
primordial germ cells resulting in spermatogenic arrest
likely due to compromised spermatogonial proliferation and
differentiation (5). Though insightful, these studies could
not specifically address the importance of miRNAs in post-
meiotic germ cell development, as Dicer conditional
knockout in early germ cell development produced few if
any Dicer-deleted germ cells that entered meiosis (5). To
address these issues, we generated post-meiotic germ cell-
specific Dicer knockout mouse model. Our results revealed
that loss of miRNA expression in haploid germ cells resulted
in impaired spermatid differentiation and compromised
fertility. Specifically, we showed that loss of miRNA-
dependent control of actin-associated protein Arpc5 resulted
indefective chromatin compaction in differentiating post-
meiotic germ cells (6). This was an important finding as
sperm chromatin compaction state is an important
independent prognostic characteristic associated with
human male fertility.
MicroRNA Biogenesis
The majority of miRNA genes located in the non-protein
coding intronic region which is transcribed by RNA
polymerase II/III to produce long primary miRNA (pri-
miRNA), which is approximately 400 bp, that includes
several hairpin loop structures. This pri-miRNA transcripts
are processed in the nucleus by a microprocessor complex
enzyme Drosha III, which generates ~70 bp precursor
miRNA (pre-miRNA). The Pre-miRNA is then transported
to cytoplasm by nuclear membrane exportin5. Cytoplasmic
RNase III enzyme Dicer cleaves this pre-miRNA duplex
into two separate strands. One strand called guide strand
which ultimately target the 3'UTR of gene and induce
mRNA degradation or translational suppression. Another
strand called passenger strand (*) which gets degraded
shortly.
miRNAs are also highly expressed in the Sertoli cells and
like germ cells miRNAs are reported to play an equally
important role in the Sertoli cells. For example, Sertoli cell-
specific knockout of miRNA processing enzyme Dicer
during embryonic stages were shown to result in testicular
dysgenesis due to alteration in Sertoli cell architecture and
infertility (7). Importantly, genes including Gdnf, Kitl,
Man2a2, and Serpina5 that are known to be critical for
spermatogenesis were reported to be significantly down-
regulated in mice lacking Dicer in the Sertoli cells. These
results clearly underscored the importance of miRNAs in the
developing Sertoli cells. Little was known about the role of
miRNAs in adult Sertoli cell until recently when we showed
that miRNAs may control androgen-mediated events in the
adult Sertoli cells. We showed that androgen directly
regulates the expression of several miRNAs in adult Sertoli
cells (8). Importantly, we found that several of these
miRNAs are preferentially expressed in the testis and target
genes that are highly expressed in the Sertoli cells. Examples
of such genes included Foxd1, a forkhead/winged-helix
transcription factor important in Sertoli cell metabolism (9),
and desmocollin-1 (Dsc1), a desmosomal cadherin that
plays a crucial role in establishing cell-cell adhesion and
desmosome formation in epithelial cells (10). These
findings suggested that miRNAs may regulate androgen-
mediated events either directly, by targeting genes in the
androgen receptor-responsive Sertoli cells, or indirectly, by
regulating germ cell-specific genes.
Conclusion and Perspective
Emerging evidence suggests that miRNAs are important
players in male fertility. A concerted effort to identify role of
individual miRNAs during spermatogenesis will pave the
way for establishing whether miRNAs can serve as
important prognostic indicators and/or therapeutic regimens
to diagnose/treat male infertility.
References
1. Bernstein E, Kim SY, Carmell MA, et al (2003). Dicer is
essential for mouse development. Nat Genet; 35: 215-7.
2. Kanellopoulou C, Muljo SA, Kung AL, et al (2005). Dicer-
deficient mouse embryonic stem cells are defective in
differentiation and centromeric silencing. Genes Dev; 15: 489-
501.
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
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3. Kotaja N, Bhattacharyya SN, Jaskiewicz L, et al (2006). The
chromatoid body of male germ cells: similarity with
processing bodies and presence of Dicer andmicroRNA
pathway components. Proc Natl Acad Sci U S A; 103: 2647-52.
4. Song R, Ro S, Michaels JD, et al (2009). Many X-linked
microRNAs escape meiotic sex chromosome inactivation. Nat
Genet; 41: 488-93.
5. Hayashi K, Chuva de Sousa Lopes SM, Kaneda M, et al (2008).
MicroRNA biogenesis is required for mouse primordial germ
cell development and spermatogenesis. PLoS One; 5: e1738.
6. Chang YF, Lee-Chang JS, Imam JS, et al (2012). Interaction
between microRNAs and actin-associated protein Arpc5
regulates translational suppression during male germ cell
differentiation. Proc Natl AcadSci U S A;109: 5750-5.
7. Papaioannou MD, Pitetti JL, Ro S, et al (2009). Sertoli cell
Dicer is essential for spermatogenesis in mice. Dev Biol; 326:
2509.
8. Panneerdoss S, Chang YF, Buddavarapu KC, et al (2012).
Androgen-responsive microRNAs in mouse Sertoli cells.
PLoS One; 7: e41146.
9. Dahle MK, Gronning LM, Cederberg A, et al (2002).
Mechanisms of FOXC2- and FOXD1-mediated regulation of
the RI alpha subunit of cAMP-dependent protein kinase
include release of transcriptional repression and activation by
protein kinase B alpha and cAMP. J BiolChem; 277: 229028.
10. King IA, O'Brien TJ, Buxton RS (1996). Expression of the
skin-type desmosomal cadherin DSC1 is closely linked to
the keratinization of epithelial tissues during mouse
development. J Invest Dermatol; 107: 5318.
End Note from the Editor
Epigenetics: A Key Paradigm in Reproductive
Health
Varij Nayan, Suneel Kumar Onteru
Dheer Singh*
National Dairy Research Institute (NDRI)
Indian Council of Agricultural Research (ICAR)
Karnal
*drdheer.singh@gmail.com
Several evidences have reiterated that reproductive health is
a consequence of interplay between genetic, biochemical,
physiological, pathological, nutritional, environment and
management factors. Hormones, steroids, growth factors
and even the follicular microenvironment (1) have greater
roles in reproductive outcome. Transcription factors
regulated by specific hormone-activated signalling
pathways obviously dictate the activation of target genes of
reproductive events. Recently, the reproductive events also
involve epigenetic changes that impact gene expression and
thus cellular and physiological functions. It is imperative
now that epigenetic regulatory machinery may contribute to
the fertility and reproductive health. Perturbations to these
epigenetic mechanisms may further delineate the intrinsic
causes of infertility or state of fertility. External factors
including environment and food are represented as
examples of the nutriment and nurture-epigenetic-
phenotype relationship. Lately, there is increased realization
that the environment modulates the organism (2), and is
gradually taking shape as one of the basic tenet of modern
biological science.
Definition, Mechanisms and Reproductive Perspective
Historically, Conrad Waddington (19051975) was given
credit for introducing the term epigenetics as equivalent to
experimental embryology. It was assumed as the branch of
biology that studies the causal interactions between genes
and their products, and which bring the phenotype into being
(3). Epigenetics was ascribed as a developmental program,
where genes determine the individuals phenotype by
considering the internal and external environmental cues (4,
5). In spite of the word epigenetic being originally derived
from an older terminology epigenesis - referring to an
embryological concept, it literally gives the sense of being
beyond or above genetics (6). However, for molecular
biologists, a more suitable definition of epigenetics is the
study of mitotically and/or meiotically heritable changes in
gene function that cannot be explained by changes in DNA
sequence (7).
These changes are effected by several molecular
mechanisms now commonly known as epigenetic
mechanisms that include DNA methylation, histone
modi f i cat i ons ( met hyl at i on, acet yl at i on and
phosphorylation) and chromatin remodelling such as
altering the position of nucleosomes, all of which bring
about changes in chromatin structure and function. The
important role of non-coding RNAs (ncRNA) like miRNA
as an additional epigenetic mechanism in this process is also
emerging. All these diverse groups of successive epigenetic
modifications ensure the creation of a healthy being and as a
consequence, the reproductive health. Important epigenetic
reprogramming events occur during germ cell development
and early embryogenesis in mammals.
Figure 1: A General Overview of Dynamics of Epigenetic
Reprogramming Events During DNA Methylation in
Mammalian Female Developmental Time Line in
Mammalian Female [Adapted from (8)]
Any disruption during early embryogenesis or germ cell
development has the potential to alter epigenetic
reprogramming. This is evident from the presence of large
offspring syndrome (LOS) in animals during in vitro embryo
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
45
production (IVEP). Thus any venture of IVEP and assisted
reproductive technology (ART) should take into account of
epigenetic reprogramming considerations.
Epigenetics vis--vis Nutriment, Nurture and
Developmental Origins of Health and Disease
It is worthwhile to realize that epigenetics goes beyond DNA
sequences. Different nutritional and environmental factors
are known to influence developmental plasticity and thus
phenotypes in a wide variety of animals, from yeast, insects
to humans (9, 10). Mounting evidence has re-affirmed the
importance of early environmental conditions for adult
health and disease. Utilizing this idea, about twenty two
years ago, Hales and Barker (11) proposed the thrifty
phenotype hypothesis. It was argued that nutritional
conditions during uterine development have effects much
later in life, and influence the occurrence of adult
metabolism and diseases. They proposed that, under poor
nutritional conditions, the foetal environment could modify
the development of the embryo to prepare the offspring for a
future environment with low resources during adult life (a
thrifty phenotype). Later on, many epidemiological and
animal studies supported the thrifty phenotype theory and
gradually, it has evolved into a more general theory known
as the developmental origins of health and disease
(DOHaD), which proposes that a wide range of
environmental conditions during embryonic development
and early life determine susceptibility to disease during
adult life (10)
Figure 2: Hales and Barker's 'thrifty phenotype'
Hypothesis
It is now recognised that nutrients have the ability to interact
and modulate molecular mechanisms underlying an
organisms physiological functions and has pressed for a
revolution in the eld of nutrition (12). One of the SCFAs,
butyrate apart from participating in metabolism as nutrients,
also functions as histone deacetylase (HDAC) inhibitor and
histone hyperacetylation (13, 14, 15). HDAC is one of the
important epigenetic regulators. Liu et al (16) reported that
transient exposure to NaBu after GVBD improves meiotic
competence, but not later on, through histone acetylation
during oocyte maturation. It was also found that butyrate
induced histone acetylation regulates miRNA expression,
and also, miRNAs may interfere with butyrate induced
modulation of gene expression and cellular functions, which
may be due to the cross-talk of miRNA and histone
acetylation (17). In mice, epigenetic transgenerational
inheritance of ovarian disease states was induced by
environmental compounds and it is suggestive of a role of
environmental epigenetics in ovarian disease etiology (18).
All these results are encouraging and provide us a great
opportunity for exploring and understanding of the role of
environment, management conditions, feed/dietary
components in changing epigenetic landscape and definitely
will have impact on integrated-omics research in humans
and animals alike.
Translational Significance from our Experiences
Recognizing the recent advances in epigenetics, we worked
on its potential in regulation of female fertility. Specifically,
recent works concerning DNA methylation and chromatin
modification of the buffalo CYP19 gene, a key fertility gene,
during folliculogenesis and luteinization confirmed this.
Our laboratory reported a T/C single nucleotide
polymorphism (SNP) in a regulatory region at the exon 2 of
CYP19 gene (19). The C allele adjacent to the putative TATA
binding element could be a putative methylation site.
Methylation analysis of 5 CpG dinucleotides of placenta-
specific promoter I.1 and proximal promoter of the CYP19
gene (PII) showed hypo-methylation in early and term
placenta and hyper-methylation in mid-placenta for PI.1,
whereas PII was found to be hypomethylated in all (20). We
also conducted methylation analysis of the CpG
dinucleotides of two major promoters of CYP19 gene in
granulosa cells and luteinized tissue (21). Methylation
analysis of five CpG dinucleotides of ovary specific
proximal promoter II showed hyper-methylation in corpus
luteum while hypo-methylation in large follicle. It was also
implied that a mechanism other than differential
methylation is involved in the change in Cyp19 gene
expression, where the expression of Cyp19 was found lower
in retained foetal membrane cases and higher in normal ones
and both the major promoters were hypomethylated in the
two tissues. All these support that DNA methylation
provides a heritable, stable and critical component of
epigenetic regulation. While conducting analysis of histone
modifications using ChIP assay, our group also revealed that
the distal promoter (PI.1) of CYP19 gene is more enriched
with acetylated histone H3 in corpus luteum than in the large
follicle.
Concluding Remarks
In summary, reproduction involves a complex series of
endocrinological, biochemical and molecular events during
folliculogenesis and further developmental stages. Fertility,
encompassing the successful fertilization of quality oocytes
and sustained development of viable embryo, set the stage
for successful pregnancy. We have a wealth of information
regarding mammalian reproduction but many fundamental
and emerging questions related to it need further insight and
research. The human and animal phenotypes are summation
of gene-environment-nutrition interactions. The genetic and
epigenetic health of female gamete and its further
development is thus regulated and affected by epigenetic
modifiers such as prenatal nutrition, endocrine disruptors, in
vivo maternal environment and also in vitro culture
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Views and opinions published in ISSRF Newsletter are not necessarily endorsed by the Indian Society for the Study
of Reproduction and Fertility or the institutions and organizations to which the respective authors belong.
DISCLAIMER
The ISSRF President, Executive and Life Members heartily express their appreciation and gratefulness for valuable
contributions being made by the authors and the editors in the publication of this newsletter.
A WORD OF GRATITUDE
ACKNOWLEDGMENTS
The scientific inputs and assistance provided by Dr. A. S. Ansari & Mr. Deepak Jaiswal in compilation of the newsletter
are gratefully acknowledged.
conditions. The epigenetic modifications influence viability
of female gamete and further embryo development. This is
also important in the success of assisted reproductive
technologies (ARTs), embryo transfer programs and recent
initiatives of stem cell therapy and cloning in some animals.
A comprehensive holistic policy and plans comprising the
management, environmental factors, nutrition, and in vitro
embryo production for fertility augmentation and ARTs
should be taken care of with epigenetic perspectives on
horizon.
References
1. Fortune JE, Rivera GM, Yang MY (2004). Follicular
development: the role of the follicular microenvironment in
selection of the dominant follicle. Anim Reprod Sci; 82-83: 109-
26.
2. de Magalhes JP, Wuttke D, Wood SH, et al (2012). Genome-
environment interactions that modulate aging: powerful targets
for drug discovery. Pharmacol Rev; 64: 88-101.
3. Waddington CH (1942). The epigenotype. Endeavour; 1: 18-20.
4. Jablonka E, Lamb MJ (2002). The changing concept of
epigenetics. Ann. NY Acad Sci; 981: 82-96.
5. Holliday R (2006). Epigenetics: a historical overview.
Epigenetics; 1: 76-80.
6. Speybroeck LV (2002). From epigenesis to epigenetics: the case
of C.H.Waddington. Ann NY Acad Sci; 981: 6181.
7. Riggs AD, Martienssen RA, Russo VEA (1996). Introduction. In
Epigenetic mechanisms of gene regulation (Ed. Russo, VEA. et
al.), (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York) pp 1.
8. Nayan V, Onteru SK, Singh, D (2014). Epigenetics: A Promising
Paradigm for Controlling Fertility in Dairy Animals. In:
Contemporary Topics in Life Sciences (Ed. Mathur, PP),
(Narendra Publishing House, New Delhi) pp.53-74.
9. Sadhu MJ, Guan Q, Li F, et al (2013). Nutritional control of
epigenetic processes in yeast and human cells. Genetics; 195:
831-44.
10. Feil R, Fraga MF (2012). Epigenetics and the environment:
emerging patterns and implications. Nat Rev Genet; 13: 97-109.
11. Hales CN, Barker DJ (1992). Type 2 (non-insulin-dependent)
diabetes mellitus: the thrifty phenotype hypothesis.
Diabetologia; 35: 595601.
12. Mutch DM, Wahli W, Williamson G (2005). Nutrigenomics and
nutrigenetics: the emerging faces of nutrition. FASEB J; 19:
1602-16.
13. Davie JR (2003). Inhibition of histone deacetylase activity by
butyrate. J Nutr; 133: 2485S-93S.
14. Myzak MC, Ho E, Dashwood RH (2006). Dietary agents as
histone deacetylase inhibitors. Mol Carcinog; 45: 443-6.
15. Steliou K, Boosalis MS, Perrine SP, et al (2012). Butyrate
histone deacetylase inhibitors. Biores Open Access; 4: 192-8.
16. Liu L, Song G, Gao F, et al (2012). Transient exposure to sodium
butyrate after germinal vesicle breakdown improves meiosis but
not developmental competence in pig oocytes. Cell Biol Int; 36:
483-90.
17. Li CJ, Li RW, Elsasser TH (2010). MicroRNA (miRNA)
expression is regulated by butyrate-induced epigenetic
modulation of gene expression in bovine cells. Genet. Epigenet;
3: 23-32.
18. Nilsson E, Larsen G, Manikkam M, et al (2012).
Environmentally induced epigenetic transgenerational
inheritance of ovarian disease. PLoS ONE 7 e36129.
19. Onteru SK, Sharma D, Singh D et al (2008). CYP19
(cytochrome P450 aromatase) gene polymorphism in murrah
buffalo heifers of different fertility performance. Res Vet Sci; 87:
427-37.
20. Ghai S, Monga R, Mohanty TK, et al (2010). Tissue-specific
promoter methylation coincides with Cyp19 gene expression in
buffalo (Bubalus bubalis) placenta of different stages of
gestation. Gen Comp Endocrinol; 169: 182-9.
21. Monga R, Ghai S, Datta TK et al (2011). Tissue-specific
promoter methylation and histone modification regulate CYP19
gene expression during folliculogenesis and luteinization in
buffalo ovary. Gen Comp Endocrinol; 173: 205-15.
ISSRF
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
Dr. Dheer Singh received Ph.D. degree from National
Dairy Research Institute (NDRI) Deemed University
at Karnal (Haryana) in India. After his doctoral degree
he was selected as Agricultural Research Scientist
(ARS) and joined Animal Biochemistry Division at
NDRI as scientist and subsequently promoted to
Principal Scientist. Awarded BOYSCAST (Better
opportunity for young scientist in chosen area of
science and technology) fellowship from Department
of Science and Technology (DST), Ministry of
Science and Technology, Govt. of India to pursue
higher study abroad and worked in Department of Cell
and Developmental Biology and Anatomy at School of
Medicine, University of South Carolina, USA in the area of transcriptional regulation of
eukaryotic genes. He was a visiting scientist to School of Medicine, Stanford University, USA
and identified novel endocrine genes using DNA microarray, computational tools and RNAi in
functional analysis of genes in granulosa cells. He is Life Member of prominent professional
national scientific societies namely, SBC, India; ISSRF; ISCB etc. Apart from several educational
fellowships such as ICMR Junior research fellowship (JRF), NDRI-JRF and SRF etc, he is

th th
recipient of 9 & 11 AAAP/CAPI Outstanding Research Award of Asian Australian Animal
Production Society and the prestigious Labhsetwar Award-2013 of the Indian Society for the
Study of Reproduction & Fertility. Handled more than 15 research projects funded by various
funding agencies, including Indo-German joint collaborative project (DST-DFG & DST-DAAD)
on epigenetic regulation of genes and published more than 40 research papers in peer reviewed
International Journals. His current research interests include: 1) Identification and analysis of the
regulation of selected trait-affecting (e.g. fertility) genes; 2) Application of RNA interference
tools for livestock functional genomics; 3) Epigenetic regulation of gene expression; and 4)
Development of nano-biosensor.
Dr. Dheer Singh
Principal Scientist
Animal Biochemistry Division
National Dairy Research Institute
Karnal 132 001, India
e-mail: drdheer.singh@gmail.com
Dr. Pradyumna Kumar Mishra is currently working
as Associate Professor in School of Biological Sciences
at Central University - Sagar. Earlier, he served as
Scientist E, Division of Translational Research, Tata
Memorial Centre, Mumbai and Head of Research Wing,
BMHRC, Indian Council of Medical Research, Bhopal.
Dr. Mishra is a recipient of prestigious Fulbright-Nehru
Fellowship (USA) and is a Visiting Faculty Associate to
Weizmann Institute of Science, Israel and Osaka
University, Japan. He is a recipient of Young Investigator
Award from Department of Biotechnology, New Delhi.
For his seminal contribution in developing Dendritic
Cell based therapeutic vaccines, he received JEM Young
Scientist Award in 2008 at Kobe, Japan. His laboratory has received extra-mural funding support
from DBT, DST and CSIR. He has validated a number of biomarkers for human pathologies and has
developed two quantitative PCR based technologies for rapid identification and characterization of
occult hepatitis C virus and latent tuberculosis infection. To his credit, he has one US and one WIPO
patent. He has authored more than 80 peer-reviewed original research articles (cumulative impact
index 245.97) with more than 1225 citations and his publication h-index is 21. Dr. Mishra is
Associate Editor-in-Chief of WJG and serves in editorial board of three highly impact international
journals. He has guided 7 students for their Ph.D. programme and more than 40 M. Phil. & PG
dissertations. Dr. Mishra is a Post-graduate & Doctoral Committee Member of 2 central, 11 state
universities and 4 national institutes. He is life-member of eight scholarly societies and his primary
research focus is molecular and translational medicine. Of late, his laboratory has been engaged in
delineating the epigenetic dimension of oxygen radical injury in spermatogonia which is considered
an inextricable component responsible for genome integrity maintenance in testicular milieu.
Dr. Pradyumna Kumar Mishra
Associate Professor (Sr. Grade)
School of Biological Sciences
Central University
Sagar 470 003, India
e-mail: pkm_8bh@yahoo.co.uk
About Our Editors
President
Dr. N. K. Lohiya, Jaipur
Vice-Presidents
Dr. Suneeta Mittal, New Delhi
Dr. Sudha Salhan, New Delhi
Secretary
Dr. R. S. Sharma, New Delhi
Joint Secretary
Dr. A. H. Bandivadekar, Mumbai
Treasurer
Dr. A. S. Ansari, Jaipur
Members
Dr. Nomita Chandhiok, New Delhi
Dr. S. G. Dastidar, Kolkata
Dr. Sujata Kar, Bhubaneswar
Dr. S. S. Majumdar, New Delhi
Dr. P. B. Seshagiri, Bangalore
Dr. G. Taru Sharma, Bareilly
Dr. Dheer Singh, Karnal
Co-Opted Members
Dr. M. M. Misro, New Delhi
Dr. Roya Rozati, Hyderabad
Ex-Officio Members
Immediate Past President
Dr. C. P. Puri, Mumbai
Immediate Past Secretary
Dr. Smita Mahale, Mumbai
ISSRF Executive
48
Valuable comments of the
readers will serve as the
source of inspiration & also
help us to improve upon future
newsletters of the Society.
49
ISSRF
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
An International Conference on Reproductive Health: Issues and Strategies under Changing Climate Scenario and the
24th Annual Meeting of the Indian Society for the Study of Reproduction and Fertility (RH-ISCS-ISSRF 2014) has been
organized by Dr. G. Taru Sharma, Director, CAFT, Principal Scientist and Head Division of Physiology & Climatology at
the Indian Veterinary Research Institute (IVRI), Izatnagar (UP) under the auspices of the ISSRF during February 6-8,
2014. The conference was held in conjunction with the celebration of 125 years of establishment of IVRI with a glorious
history of serving the country in animal health and production.
The conference focused on the issues and strategies related to reproductive health at a juncture when scientists across the
globe are concerned about the adverse effects of climate change on earth. Optimized nutrient requirement plays a vital role
in reproduction and as per predictions of the Inter-Governmental Panel on Climate Change (IPCC) global earth
o
temperature is likely to increase by 1.8 to 4.0 C by the end of this century, and this may have an adverse effect on food
production. Reproduction is optimized within a narrow range of environmental conditions and this process is
compromised due to change in climate, because nutrients are diverted to maintain homeostasis, which is a priority over
reproduction.
Existence and continuity of all living organisms is ensured only through the process of reproduction. Various reproductive
events involving male-female gametes, fertilization, embryo development, implantation, pregnancy and birth of the
young ones are beautifully orchestrated processes. Yet the strings of these physiological processes are delicately balanced
to maintain good reproductive health and require a perfect harmony, else it leads to disturbances. Therefore, it is
imperative that biologists study the effect of climatic changes on reproductive health and develop suitable strategies to
mitigate its adverse impact.
The inaugural session of the conference was held at 9.00 a.m. on February 6, 2014 at Central Auditorium, Administrative
Block, IVRI, Izatnagar. The conference was inaugurated by the Chief Guest Dr. V.P. Kamboj, Former Director CDRI &
Chairman, Biotech Consortium India Ltd., New Delhi. Dr. G. Taru Sharma, Organizing Secretary gave a conference
briefing and offered the welcome address. Prof. N.K. Lohiya, President-ISSRF and Prof. Gaya Prasad, ADG (AH),
th
ICAR and Former Director, IVRI addressed the inaugural function. The proceedings of the conference and 14 issue of
the ISSRF Newsletter on Challenges of reproductive health due to influence of changing environment and
lifestyle edited by Dr. Savita Yadav, AIIMS New Delhi were formally released on the occasion. The presidential
address was delivered by Dr. R.K. Singh, Director & Vice-Chancellor, IVRI. During inaugural function, Lifetime
Achievement Award was conferred to Dr. V.P. Kamboj. Dr. G. Singh, Sr. Scientist proposed a vote of thanks. More
than 200 delegates and 60 invited speakers from different parts of India and overseas attended this conference.
International Conference on Reproductive Health:
th
Issues and Strategies under Changing Climate Scenario and the 24 Annual Meeting of the ISSRF
(RH-ISCS-ISSRF 2014)
February 6-8, 2014, IVRI, Izatnagar
Inaugural session was followed by the keynote addresses of Dr. M.L. Madan and Dr. S.M. Totey.
Scientific program included keynote addresses, invited talks, oration lectures, senior and young scientist's oral
presentations and poster presentations. Conference had a total of sixteen sessions; six award sessions, two oral and one
young scientists session, seven scientific sessions namely, Gametogenesis, Epigenetics and reprogramming, Climate-
endocrine fertility relationship, Reproductive health, Stem cell biology and regenerative therapy, Upstream
reproductive techniques and Implantation biology besides three poster sessions of one hour each. Tradition of ISSRF
50
ISSRF
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
was followed by inviting reproductive biologists from different organizations to interact and intensify network to
address the issues related to reproductive health in the context of ONE WORLD ONE HEALTH. A parallel session
was avoided, so as to encourage a useful discussion and exchange of ideas with and amidst the scientific resource
persons. Noted scientists from India and abroad in the field of reproductive health delivered their talks.
Amongst the senior colleagues, invited for the meeting were Dr. Vijayaraghavan Srinivasan, Kent State University,
Kent, Ohio, USA; Dr. Dieter Schams, Technische Universitt Mnchen, Freising, Germany; Dr. Pradeep Kumar G., Rajiv
Gandhi Centre for Biotechnology, Trivandrum; Dr. Dheer Singh, National Dairy Research Institute, Karnal; Dr. Savita
Yadav, All India Institute of Medical Sciences, New Delhi; Dr. Polani Seshagiri, Indian Institute of Science, Bangalore;
Dr. S. Shivaji, CCMB/LVP Eye Institute, Hyderabad; Dr. Surendra Sharma, Women and Infants Hospital, Brown
University, Providence, RI, USA; Dr. S.N. Kabir, Indian Institute of Chemical Biology, Kolkata; Dr. Ashutosh
Halder, All India Institute of Medical Sciences, New Delhi; Dr. Rajvi Mehta, Trivector Embryology Support
Academy, Mumbai; Dr. C.V. Rao, Florida International University, Miami, Florida, USA; Dr. Bhanu Prakash
Telugu, University of Maryland, College Park, USA; Dr. Ramesh Bhonde, Manipal Institute of Regenerative
Medicine, Bangalore; Dr. Taruna Madan, National Institute for Research in Reproductive Health, Mumbai; Dr Riaz
Ahmad, Faculty of Veterinary Sciences, SKUAST, Srinagar. There were sixteen oral presentations by younger
colleagues during the meeting.
Young scientists oral session was organized to encourage the junior scientists, total seven presentations were scheduled in
the session. All the presentations were appreciated. However, the panel of judges recommended three best presentations, and
the awardees were presented a certificate, plaque and a cash award of Rs. Three thousands each during valedictory function.
Several awards of ISSRF were given away during this 3-day conference. Labhsetwar award was shared by Dr. Malini
Laloraya and Dr. A. S. Ansari. Dr. S.D. Kholkute received Prof. N. R. Moudgal Memorial Oration Award. Dr. S.
Majumdar received Founder President Dr. T.C. Anand Kumar Memorial Oration Award. Prof. N. R. Moudgal Young
Scientist Award was given to Ms. Swapna Desai. Dr. Jaysree Sengupta received Prof. L.S. Ramaswami Memorial
Oration Award. Prof. G.P. Talwar Gold Medals went to Dr. S. Mondal for middle career scientist, Dr. Pankaj Suman
and Dr. Shikha Saini for young scientist awards. All the awardees delivered their exciting award lectures.
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Day one banquet with the cultural evening was organized at the beautiful lawns of Executive Club, Bareilly. An elegant
Sufi dance performed by the artists to give the fragrance of Northern India, was appreciated by everyone. Cultural evening
witnessed the spark from the delegates too, specially Dr. S.D. Kholkute, Dr. Subeer Majumdar, Dr. Surendra Sharma,
Dr. A. H. Bandivadekar and Dr. Sandeep Goel, who made it very memorable with their lively and energetic
participation. Day two banquet was enjoyed by all at Hotel Awadh Plaza with good mood and cheer.
The Executive Committee Meeting of the ISSRF was held on 7th February, 2014 and that of General Body Meeting on 8th
February, 2014. Prof. N.K. Lohiya, President ISSRF presided over the meetings and the Secretary Dr. R.S. Sharma
presented minutes of the ISSRF EC & GB meetings held at the RGCB, Thiruvananthapuram, and mid-term EC meeting held
at NIRRH, Mumbai along with a brief about the activities of past one year of the Society. The General Body appreciated
joining of large number of scientists as life member of the Society leading to 1176 members on roll. Both, the ISSRF
Executive Committee and General Body complimented organizers of the International Conference on Reproductive
Health: Issues and Strategies under Changing Climate Scenario and the 24th Annual Meeting of the ISSRF for very well
planned scientific programme with participations of leading scientists from different parts of the country and abroad.
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The valedictory function was the last activity of the conference. Padmabhushan Prof. G.P. Talwar, Director, Talwar
Research Foundation, New Delhi was the Chief Guest of the function. Dr. R.K. Singh, Director & Vice-Chancellor, IVRI,
presided over the function. Prof. N.K. Lohiya gave the opening remarks followed by a report of the conference by Dr. G.
Taru Sharma. Prof. G.P. Talwar gave a talk on Multiple Facets of Human Chorionic Gonadotropin. It was followed by
the award ceremony. The meeting concluded with the vote of thanks offered by the Organizing Secretary.
Best poster presentation awards were also given by the Society. A total of 152 abstracts were invited for the poster
presentations, spread over all three days. A four member jury selected three best posters. The best poster awards were given
to following young scientists: Dr. Indrashis Bhattacharya, National Institute of Immunology, New Delhi, Mr. Eswara
Murali S. and Ms. Sarbani Saha, CSIR-Indian Institute of Chemical Biology, Kolkata.
The ISSRF has instituted following awards. The applications/nominations for various awards should to
addressed to the President, Indian Society for the Study of Reproduction & Fertility (ISSRF), Centre for Advanced
Studies, Department of Zoology, University of Rajasthan, Jaipur 302 004. The last date of receipt is November
30, every year.
Labhsetwar Award
Founder President Dr. T. C. Anand Kumar Memorial Oration
Prof. G. P. Talwar Young Scientist Award / Prof. G. P. Talwar Gold Medal for Middle Career Scientists
Prof. L. S. Ramaswami Memorial Oration
Prof. N. R. Moudgal Memorial Oration / Prof. N. R. Moudgal Young Scientist Awards
The Lifetime Achievement Award & The Fellowship Award
Kindly visit ISSRF website for further details. www.issrf.org
CALL FOR ISSRF AWARDS
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
RECENT ACCOMPLISHMENTS OF LIFE MEMBERS OF THE SOCIETY
Dr. Anil Suri is well recognized eminent scientist in the field of
Cancer Biology. Dr. Suri is the Fellow of National Academy of
Sciences and a Fellow of National Academy of Medical Sciences
(India). His outstanding cancer research has resulted in discovery
of unique molecules which are important for early detection and
diagnosis and can be implicated as immunotherapeutic targets for
reproductive tract cancers. Dr. Suri was recently conferred upon a
prestigious oration award which carries Citation and Silver plate for 6th RNC
th
Memorial Oration Lecture award for the year 2014-2015 on 11 August, 2014 at
Post Graduate Institute of Medical Education and Research, Department of
Experimental Medicine and Biotechnology, Chandigarh. Dr. Suri delivered
award lecture on BENCH-TO-BEDSIDE DEVELOPMENT OF CANCER
THERAPY.
Compliments & hearty congratulations, on
behalf of the ISSRF, for taking over as
Director of the National Institute for
Research in Reproductive Health, Mumbai -
a premier institute of the country.
Dr. Smita D. Mahale
February 14-17, 2015
th
25 Annual Meeting of the Indian Society for
the Study of Reproduction and Fertility
and
International Conference on Reproductive Health
"ISSRF Silver Jubilee Celebrations"
Dr. Jayanti Mania-Pramanik & Dr. Nafisa H. Balasinor
Scientist 'E'
National Institute for Research in Reproductive Health
(Indian Council of Medical Research)
Jehangir Merwanji Street
Parel, Mumbai - 400 012, India
Tel.: +91-22-24192000/2025/2039
Fax: +91-22-24139412
E-mail : issrf2015@gmail.com
Website : www.nirrh.res.in & www.issrf.org
st
Deadline for submission of Abstract : 31 October, 2014
th
Deadline for early Registration : 30 November, 2014
IMPORTANT DATES
Organizing Secretaries
THEMES
Infertility
Stem Cells
Fertility Regulation
Reproductive Cancer
RTI/STI including HIV
Reproductive Toxicology
Reproductive Endocrinology
Reproductive Ageing, Maternal and Child Health
Lifestyle, Stress, Occupation and Reproductive Health
Environment, Endocrine Disruptor and Reproductive Health
NEWSLETTER FOR THE ISSRF-2015 EVENT
It is proposed to bring out the 16th special issue of the Newsletter to commemorate Silver Jubilee celebrations of our Society to
be released during ISSRF 2015 event at the NIRRH, Mumbai. The newsletter will cover the following:
attainment of specific objectives relating to reproductive health as per the national scenario
highlights the priorities for setting up an agenda beyond millennium development goals
showcase contributions of Indian Scientists in arena of reproductive health research that translated from bench to bedside.
provides a perspective of ISSRF - journey of 25 years and still counting; and importantly
need to reinvigorate national strategies to achieve 'Reproductive Health for All.'
The thematic focus may be "Perspectives of Reproductive Health Research on a Post - 2015 Development Framework".
For further information please contact the ISSRF secretariat.
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Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
ISSRF
Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v
55
ISSRF
Ocial peer reviewed journal of Indian Society for the Study of Reproducon and Ferlity (ISSRF).
Aims to publish juried original scienc arcles in clinical and laboratory research relevant to reproducve and
developmental biology, andrology, immunology, genecs, contracepon, menopause and inferlity.
Target audience comprises obstetricians, gynecologists, reproducve endocrinologists, physiologists,
pathologists, urologists and sciensts working on human reproducon issues.
Accepts original arcles, reviews, short communicaons, opinions, commentaries, news and leer to the editors.
Naonal Editorial Board
Prof. N. K. Lohiya, Jaipur
Dr. Subeer S. Majumdar, New Delhi
Dr. Rakesh K. Tyagi, New Delhi
Prof. P. B. Seshagiri, Bangalore
Dr. Smita Mahale, Mumbai
Dr. R. S. Sharma, New Delhi
Dr. G. Taru Sharma, Bareilly
Dr. Dheer Singh, Karnal
Prof. K. Muralidhar, New Delhi
Dr. Pradeep Kumar G., Thiruvananthapuram
Dr. Sash Kumar Gupta, New Delhi
Prof. Sujoy K. Guha, Kharagpur
EDITORIAL BOARD
Chief Editors
Prof. Debabrata Ghosh, New Delhi
Prof. Jayasree Sengupta, New Delhi
Internaonal Editorial Board
Prof. David Kennaway, Australia
Prof. Berthold Huppertz, Austria
Prof. Carlos Simon, USA
Prof. Denny Sakkas, USA
Prof. Thomas D'Hooghe, Belgium
Prof. Shigeru Saito, Japan
Prof. C. V. Rao, USA
ISSRF Secretariat: Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur - 302 004, India
Telephone No.: +91-141-2701809 Fax No.: +91-141-2701809 E-mail: lohiyank@gmail.com Website: www.issrf.org
Design & Print at Popular Printers, Jaipur +91-141-2606883