ARCHIVES OF ANDROLOGY 45:215–225 (2000)

Copyright ã 2000 Taylor & Francis

0148-5016 /00 $12.00 + .00

SPERM CHROMATIN

G. FUENTES-MASCORRO
H. SERRANO
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A. ROSADO
Division de Ciencias Biológicas y de la Salud, Universidad
Autónoma Metropolitana, Unidad Iztapalapa, México DF, Mexico

Available data on dry and hydrated nuclear volume of mammalian spermatozoa indicate that available
volume is clearly insufficient to contain sperm chromatin packed in nucleosome-like structures. There-
fore, sperm DNA–protein complexes must be packed differently than somatic DNA–protein complexes.
Packing of DNA in fixed, dehydrated mammalian sperm approaches the physical limits of molecular
compaction, making mammalian sperm chromatin the most condensed eukaryotic DNA known. The
fundamental packaging unit of sperm chromatin is a toroid ~900-Å outer diameter, 200-Å thickness,
and 150-Å diameter hole. Each toroid contains 60 kilobases of DNA and is linked to other toroids by
uncoiled DNA stretches. The factors that contribute to mammalian chromatin structuration are still
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under study. The role of protamines in sperm chromatin condensation and nuclear shaping has been
overstressed to the exclusion of other possible factors. Chromatin organization in sperm nuclei is main-
tained during sperm condensation by tight interactions with the nuclear matrix at fixed sites, inducing
the formation of individual toroid-shaped DNA loop stuctures. Observations that abnormal manchettes
affect sperm head shape and chromatin organization inducing sterility speak about manchette impor-
tance during chromatin organization. The presence in sperm chromatin of regions packaged in specific
ways with several types of protamines or even with histones, indicates that nuclear shaping and chroma-
tin organization must be under DNA control. The structural properties that distinguish sperm DNA from
somatic DNA may play the most important role in chromatin organization.

Keywords chromatin structure, nucleosomes, sperm DNA

Sperm chromatin has been the subject of extensive research. DNA in the sperm of some
animal species, such as the sea urchin [33] and frog [34], appears to be packed in nucleosomes
in a similar way to somatic chromatin. In most fishes, certain insects, echinoderms and mam-
mals, sperm DNA is packed with a special type of small, basic proteins, into a tight, almost
crystalline status, that is at least 6 times more condensed than in mitotic chromosomes. For
practical purposes sperm DNA seems to be biochemically inert. During spermiogenesis round
spermatids undergo dramatic changes giving rise to cells, sperm, completely different from the

We acknowledge the help of Gerónimo Cruz García for line drawing elaboration.
Address correspondence to Gisela Fuentes-Mascorro Departamento de Biología de la Reproducci ón, área de Re-
producción Animal Asistida, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Av. Michoacán y La Purísima
Iztapalapa 09340, México D.F. A.P. 55-535, Mexico. E-mail: protamina@starmedia.co m

215
 216 G. Fuentes-Mascorro et al.

morphological standpoint. These changes are mostly due to the loss of most (if not all) of the
cytoplasm and the development of a motile tail. During these steps, and after the transcriptional
activity of the genome has ceased, the sperm must develop a highly condensed nucleus with a
species-specific shape, and with diminished replicative, translational, and DNA repair activi-
ties. The process involves a complicated series of reactions through which somatic histones and
nonhistone chromatin proteins are replaced during a variable period of time and in a species-
specific way by one or more protamine types [40]. In the rat, mouse, and sheep, this replace-
ment involves a set of special proteins known as “transition proteins.” Histone exchange by
protamines constitutes one of the most remarkable changes in chromatin structure observed in
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eukaryotes and seems to be essential for sperm nuclear organization.

Histone Replacement
Protamines are highly basic proteins about half the size of a typical histone (5–8 kDa).
From 55 up to 79% of the aminoacid residues of protamines are arginines, permitting a strong
DNA binding. Protamines contain a significant number of cystein residues that are very impor-
tant during the final stages of sperm nuclear maturation because they participate in sperm
chromatin compaction by forming multiple inter- and intraprotamine disulfide cross-links [41].
All these interactions contribute to make mammalian sperm DNA the most condensed eukary-
otic DNA [67]. Mature sperm chromatin is notably refractory to in situ hydrolytic enzyme
treatment [1], not easily stainable by the Feulgen method, and interacts scarcely with dyes and
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fluorochromes [60]. This remarkable arrangement explains the resilience of sperm nuclei [38],
and why isolated mammalian sperm nuclei, completely deprived of nuclear envelope, preserve
their volume and shape [4].
While the histone–DNA structure is highly conserved in most eucaryotic organism, prota-
mines show a high degree of species specificity [5]. Not all species replace histones in the
sperm nucleus during spermatogenesis: some species, such as carp [16], retain histones as
typical proteins in the sperm nuclei. Other species, such as humans, retain up to 15% of their
original histones content [22]. Birds completely replace histones in the sperm nucleus [48].
Bulls [5], rams [40], and rats [25] contain only one type of protamine, whereas mice and
humans have two [5] (Table 1).

Table 1. Chromatin structuration on different animal species

Animal Protamine Transition

species Histones type proteins Reference

Rabbit P1 36
Bull P1 25, 49
Ram P1 Yes 25
Stallion P1, P2 25, 39, 63
Bull P1 25, 49
Man P1, P2 Yes
Man Yes, 15% 22
Mice Yes P1, P2 Yes 6, 52, 55
Rat P1 Yes 37
Boar Yes P1 7
 Sperm Chromatin 217

Transition Proteins (TP)

In rats [44], mice [49], and humans [66, 70], nuclear histones are gradually replaced during
spermiogenesis by transition proteins with intermediate basicity, which will themselves be
replaced by protamines. TP1 [35] and TP2 [24] appear during stages 12–15 of rat spermiogen-
esis [47], being replaced by protamines during stages 15–19 [8]. In rat spermatids histones are
replaced by lysine-rich transition proteins TP1, TP2, and TP4, immediately before the sperma-
tid chromatin loses its nucleosome arrangement and transcription ceases [14]. TP1 is a 6-kDa
protein with numerous basic aminoacids distributed randomly throughout the molecule [35].
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TP1 has important DNA destabilizing properties, probably due to the presence of two tyrosine
residues flanked by basic aminoacids [62]. TP2, a 13-kDa protein, contains proline, serine,
arginine, and lysine basic residues [24]. By aminoacid sequence analysis, Baskaran and Rao
[8] identified two potential zinc finger domains in mouse and rat TP1 that may play an impor-
tant role in the initiation of chromatin condensation and/or cessation of transcriptional activity
during mammalian spermiogenesis.
Sperm Nuclear Volume and Chromatin Packing
Sperm epididymal maturation implies a final stage of chromatin organization, involving loss
of water, protamine cross-linking by disulfide bonds formation, and further reduction in the
volume of the sperm nuclei, together with a complete restriction of the facilities for DNA
replication and transcription [31]. DNA in fixed, dehydrated mammalian sperm is packed in a
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way that approaches the physical limits of molecular compression [30]. To reach this degree of
compactness, sperm DNA–protein complexes must first be packaged differently than somatic
DNA–protein complexes. Chromatin in somatic cells is first packed into nucleosomes. These
structures consist of a protein core formed by an octamer of histones with two laps of wrapped
DNA (around base pairs) (Figure 1A). Small (40–50 bp) chains of “linking” DNA associated
with the linker hitone join the cores. Nucleosomes are then further associated “cheek by jowl”
into regular helixes, also called solenoids [64].
Even as late as the 1980s the dominant idea was that sperm chromatin was organized in
nucleosomes or nucleosome-like complexes. Packing the DNA in single, closely packed nu-
cleosomes would require a volume of 9.9 µm3, which is more than twice the volume deter-
mined in an average sperm nucleus [52]. DNA volume in fully hydrated mammalian sperm, as
shown with the atomic force microscope, is about twice the volume indicated previously [3].
Sperm chromatin is organized in a way completely different than the nucleosome structure of
somatic chromatin (Table 2).

CHROMATIN ORGANIZATION
There is significant evidence suggesting that sperm nuclei are essentially crystalline aggre-
gates of nucleoprotein complexes [70]. Freeze fracture studies of spermatozoa in bulls [50],
rabbits [36], and men [37] indicate that mammalian sperm nuclei seems to be composed of
parallel stacks of lammellar sheets oriented to the sperm’s long axis. Circular dichroism experi-
ments [63] and the use of polarized light [39] on stallion sperm heads led two different groups
to support these presumptions, adding the idea that eutherian sperm chromatin behaves as a
cholesteric liquid in which DNA strands are packaged into parallel planes of linear arrays.
Nodular organization of sperm chromatin has been supported by other experimental evi-
dence. Fluorescense in situ hybridization in mouse and human sperm [26] suggests that a
 218 G. Fuentes-Mascorro et al.

H4 H3
H 2b H 2a

H 2a H 2b
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H3 H4

900 ¯
B
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150 ¯

C Polyarginine segment

Minor groove

Figure 1. (A) Somatic nucleosome (from Csordas [15]). (B) Doughnut shape (toroid), the fundamental
packaging unit of bull sperm chromatin (from Hud et al. [28]). (C) Position of protamine in minor groove
(from Balhorn [5]).
 Sperm Chromatin 219

Table 2. Early proposals about sperm chromatin structure

Species Characteristics Reference

Rabbit Lamellae, parallel to the axis of the nuclei 36

Bull Tight network 45
Stallion Cholesteric liquid crystal 63
Bull DNA folded around sperm specific histone, multimers spaced regularly 49
Ram along the fiber generating a linear array of sperm nucleosome
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Stallion connected by short stretches of uncomplexed DNA

Bull Beads on a string configuration 25
Man
Mice The protamine in sperm chromatin do not provide a subunit 52
core around which the DNA is wrapped
Mouse Sperm DNA is packaged side by side in a linear array 5
Rat Lamellae, parallel to the axis of the nuclei 37
Stallion Liquid cholesteric DNA strands are packaged into linear arrays in 39
parallel planes

discrete nodular substructure of ~300 nm diameter may represent the basic packaging unit
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(BPU) of mammalian sperm chromatin. That bull, mouse, and rat sperm chromatin may have
a nodular organization has been shown by atomic force microscopy (AFM) [4]. Chromatin
nodules appear to be ellipsoidal in shape, 15–25 nm thick and 50–100 nm in diameter. The
fundamental packaging unit of bull sperm chromatin is a doughnut shape (toroid) of ~900-Å
outer diameter, 200-Å thickness, and 150-Å diameter hole (Figure 1B), each doughnut contain-
ing around 60 kilobases (kb) of DNA. Toroids in sperm chromatin are linked to each other by
uncoiled DNA stretches [28]. In human sperm chromatin, although the basic packaging unit is
formed by large nodular structures averaging 98 nm in diameter, it is possible to find numer-
ous, smaller, nucleosome-like particles forming linear chains near the nuclear periphery [2].
An adequate chromatin structure is required for normal fertilization and correct embryonic
development. Abnormalities of sperm chromatin structure may have an effect on the fertilizing
ability of males [65] (Table 3). Poor chromatin packaging and/or DNA damage may contribute

Table 3. Importance of abnormal sperm chromatin structure on reproduction

Clinical problem Sperm chromatin alteration Reference

Hypofunction of seminal vesicles High sperm chromatin stability 23

Round-headed sperm Low levels of P1 and P2, unusually high 12
levels of P2 protamine precursor
Male infertility Disturbance of the histone replacement by 56
protamines during spermatogenesi s
Elevated scrotal temperature Reducing sperm chromatin stability in both 32
in bulls epididymal and testicular sperm
Chronic lead exposure Alters sperm chromatin structure 19
Scrotal heat stress Decreases in protamine disulfide bonding 43
 220 G. Fuentes-Mascorro et al.

to failure of the intraoocyte male nucleus decondensation process after ICSI and results in
failures of fertilization [59]. However, observations in transgenic mice indicate that the very
precise packaging of DNA in germ cells may not be essential for the subsequent formation of
the male pronucleus into fertilized eggs and consequently unimportant for subsequent normal
embryonic development [55].
Several components and structures have been proposed as participants in the process of
sperm chromatin organization and subsequent sperm shaping. Proposal A accepts that chroma-
tin organization has to be considered as an integral process together with nuclear shaping
supporting the previously proposed hypothesis that sperm head shaping (and perhaps chroma-
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tin organization) cannot be performed by the spermatid alone and that the ectoplasmic special-
izations (actin) of the Sertoli cells must participate in this process [10]. Proposal B restricts the
problem to chromatin organization, supporting the hypothesis that this process depends on the
spermatozoa themselves being essentially determined by the manner in which all their internal
biochemical components participate in the assembly [51]. We will restrict ourselves to this last
proposal, briefly discussing the suggested role for each component.
Protamines
The role of protamines in sperm chromatin condensation and nuclear shaping has been
overstressed to the exclusion of other possible factors, even though numerous studies do not
support this hypothesis [13]. Protamine participation in the final arrangement of the sperm
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nucleus may be secondary, because (1) DNA-directed protamine interactions [6] must be con-
sidered more significant in many fundamental processes, including chromatin packing [20],
and (2) the role of protamine-induced chromatin condensation is to reinforce the already initi-
ated shape changes due mainly to the action of the microtubular manchette [58] and other
structural elements, such as the perinuclear theca [9] and the calyx [42], acting on the sperma-
tid nucleus.
Protamine interacts with DNA in an extended conformation: The central polyarginine-rich
segment of protamine P1 binds to the minor groove of DNA, cross-linking and neutralizing the
phosphodiester backbone of the macromolecule, while the carboxy and amino terminal resi-
dues of the protein participate in the formation of inter- and intraprotamine hydrogen bridges
and hydrophobic and disulfide bonds [5] (Figure 1C). This model may be adequately applied
to fishes and mammals that have only one protamine type, but problems arise in animals that
contains two, or even three, different types of protamines. In these cases the evidence is contra-
dictory: Protamines P1 and P2 may lie down in the major groove of DNA [51], be attached to
both the minor and the major grooves of DNA [17], or attach to the external surface of the
DNA chain via electrostatic interactions with the phosphate residues with no specific locations
on the DNA grooves [11]. HP2 protamines (P2 family) might perform a unique function in
sperm chromatin organization important for fertility [29]. Some infertile patients have a higher
percentage of large head sperm, with an increase of the P1/P2 ratio associated with lower HP2
amounts than normal, fertile patients [31, 53]. Problems in the sequence of chromatin organi-
zation are reflected by the higher amounts of P2 presursors in these infertile men [18].
Protamines are located in channels between the DNA molecules [54]. In this model, DNA
molecules are arranged hexagonally in the x-y plane, while their relative positions with respect
to the z axis are shifted by 0, 1/3, and 2/3 of the pitch of the double helix. Thus, large cavities
are formed in 3 out of the 6 channels that surround each DNA molecule where the large
 Sperm Chromatin 221

grooves are juxtaposed. Protamine molecules are arranged in such a way that they can fill the
large grooves in all of the three surrounding DNA molecules simultaneously [54].
Nuclear Matrix and the Manchette
Solid experimental evidence supports the fact that the nuclear matrix of hamster sperm
organize the DNA into loop domains having only about half the size of somatic cell loops [66,
68]. The union of the DNA to the nuclear matrix and its interactions with protamines made
individual toroid-shaped DNA loop stuctures in the sperm chromatin called DNA loop doughnout s
[65]. Chromatin organization in sperm nuclei is maintained during sperm condensation by tight
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interactions with the nuclear matrix at fixed sites [57]. In bulls [53] and mice [31] repetitive
and protein-coding sequences are arranged quite orderly in sperm DNA and are related to
specific interations between DNA and sperm nuclear matrix proteins forming domains that are
sequence (or gene) specific. Loop domains and folding patterns must be highly ordered struc-
tures, especially at the centromere to telomere positioning.
During spermatogenesis and spermiogenesis, the manchette is attached to the nucleus at
distinct angles, inducing or, at least, reflecting changes in nuclear shape [46]. Drugs that affect
the manchette, such as taxol, cytoxan, and 5-fluorouracil, also affect sperm head shape and
chromatin organization [58]. Highly altered head shapes and abnormal manchettes characterize
several mutations in the mouse resulting in a reduction in fertility or sterility [13], supporting
the idea that manchette has some function during nuclear shaping [61].
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DNA
Nuclear shaping, and probably chromatin organization, must be under genetic control [30].
Although the progressive oxidation of sulfhydril groups in protamines may assist in sperm
shaping, its participation in the final organization of sperm chromatin may be secondary and
dependent on specific structural characteristics of the DNA in the sperm chromatin complex [5,
20].
Despite some reports [6, 27, 65] indicating that sperm DNA has some important structural
properties that distinguish it from somatic DNA, the possibility that sperm DNA, by itself,
contributes to sperm chromatin arrangement has been studied only recently. These structural
differences may help to explain some of the divergences between somatic and sperm DNA
interactions with nuclear matrix during chromatin building: (1) sperm loop domains are 60%
shorter (average DNA length of 46 ± 7 kbp); (2) sperm DNA is devoid of any detectable
supercoiling [68]; (3) sperm DNA interacts in a characteristic way with the nuclear matrix [67].
Sperm DNA, when submitted to the same nucleosome-reconstitution conditions as liver
DNA, is not capable of interacting adequately with histones to form structures similar to
normal somatic nucleosomes [6, 21]. The secondary structure of certain DNA sequences (par-
ticularly those related with promoters and termination regions) have different affinities for
nucleosomal histones, forming nucleosomal structures that are hypersensitive to nuclease di-
gestion [69]. It would appear that, in addition to the differences in DNA primary structure, the
secondary structure of sperm DNA may have the capacity to interact specifically with prota-
mines during chromatin arrangement.
The presence in human sperm of chromatin regions packaged into nucleoprotamines (85%)
or into nucleohistones (15%) in a sequence specific way [22] indicates the necessary participa-
tion of DNA in the determination of the sites that can be complexed either with protamines or
 222 G. Fuentes-Mascorro et al.

with histones. This DNA-dependent specificity must be even more important in those animals
in which more than one type of protamines participate in sperm chromatin organization.
Repetitive and protein-coding sequences in sperm DNA assembled in an ordered fashion
may play an important role in chromatin arrangement [31]. Analysis by DNA re-association
kinetics showed that both repetitive and protein-coding sequences in sperm DNA occur in
characteristic proportions [6] statistically different than those in somatic cells [27]. This sug-
gests the presence on sperm DNA of specific clusters of highly repetitive nucleotide sequences,
which may be important in determining the sites of specific binding between DNA and prota-
mines. Reassociation kinetics studies performed by cross-incubation between somatic and sperm
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DNA with histones and/or protamines also showed that somatic DNA interacts inadequately
with protamines, while sperm DNA incubated in the presence of histones forms nucleosomes
with important differences in their biophysicochemical characteristics to those found when
histones are incubated with somatic DNA.

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