The Testis as a Conduit for Genomic Plasticity
Organization of chromosomes in spermatozoa: an
additional layer of epigenetic information?
A. Zalensky1 and I. Zalenskaya
The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA 23518, U.S.A.
Abstract
Elaborate non-random organization of human sperm chromosomes at different structural levels, starting
from the DNA packing by protamines up to the higher-order chromosome configuration and nuclear
positioning of chromosome territories, has been discovered. Here, we put forward a hypothesis that the
unique genome architecture in sperm provides a mechanism for orchestrated unpacking and ordered
activation of the male genome during fertilization, thus offering an additional level of epigenetic
information that will be deciphered in the descendant cells.
Introduction
Dramatic reorganization of chromosomes occurs during
mammalian spermatogenesis due to gradual replacement
of histones first with transitional proteins, then with protamines [1]. In mature sperm, DNA is supercondensed,
and genetic activity is completely shut down [2]. Recent
studies demonstrate that, in human sperm, chromatin has an
elaborate multilevel organization from the DNA packaged
by protamines into toroids [3] and up to the higher-order,
‘looped hairpin’ structure of chromosome territories [4].
Furthermore, overall nuclear architecture in the mammalian sperm cell is orderly arranged (Figure 1A). All chromosomes are joined through centromeres that are combined
into internally localized chromocentre [5–8], while chromosome ends are exposed to the nuclear periphery, where two
telomeres of each chromosome interact [9–11].
Another important characteristic of human sperm nuclear
organization is the existence of a preferred chromosome intranuclear positioning [12,13]. This feature has been conserved
throughout mammalian evolution, as has been demonstrated
by studies of sperm from rats [10], pigs [14], marsupials and
monotreme mammals [15,16]. Study of porcine spermiogenesis demonstrated ordered repositioning of some chromosomes to the locations characteristic of mature spermatozoa
[14].
Therefore individual chromosome territories and the genome as a whole are highly organized, with the telomere and
the centromere heterochromatin domains playing decisive
roles in this arrangement. Taken together, all these data point
towards a functional significance of chromosome organization in male germ cells, and it has been proposed that the
unique genome architecture is important in fertilization and
early embryonic development [17–19].
By fertilization, two terminally differentiated cells, egg
and sperm, are combined to create a totipotent zygote.
Key words: centromere, chromosome, epigenetic information, histone, male pronucleus,
spermatozoon.
Abbreviations used: ICSI, intracytoplasmic sperm injection; mPN, male pronucleus.
1
To whom correspondence should be addressed (email [email protected]).
The inactive sperm nucleus is being transformed, in a
co-ordinated fashion, into a functional mPN (male pronucleus) within an activated egg cytoplasm [20]. After incorporation into an oocyte, the sperm nuclear envelope breaks down
and chromatin decondenses. During this time (0–5 h postfertilization), paternal chromosomes interact directly with
ooplasm factors [21]. Very little is known about the mechanisms of sperm chromosome withdrawal, their controlled
decoration with histones, formation of mPN, and its
movement towards the female pronucleus. It is believed
that transformations that take place in the developing mPN
depend, to a large extent, on the machinery provided by
the ooplasm. The role of the sperm nuclear structures and
molecular entities in this process is poorly recognized.
From the current knowledge of genome organization in
sperm it may be predicted that paternal chromosomes are
pulled out in a certain order in space and time and that for
each chromosome there is a stepwise rather than simultaneous exposure of different domains to the ooplasm.
As a result, each chromatin domain is decondensed and
remodelled at a certain time, which provides differential gene
activation patterns in the early embryo [14]. In addition,
timely reading and processing of paternal genomic imprints
and initiation of replication would depend on non-random
spatial organization of the sperm genome.
Below we will discuss how the sperm-specific chromosome
organization may be involved in the mPN development at
fertilization.
Telomeres
In mammalian sperm nuclei, all telomeres are coupled into
dimers [10]. In humans, these dimers are localized at the nuclear periphery and, most probably, interact with the nuclear
membrane [7]. Telomere dimers are formed by association
of the p- and q-telomeres of one chromosome, thus providing
the chromosome with a looped hairpin configuration [4,11].
We propose that such a conformation favours an ordered
withdrawal of chromosomes via telomeres. This suggestion
is supported by an observation of the chromosomal arrangement resembling a cartwheel with the centromeres directed
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Figure 1 Model of genome architecture (A) and intranuclear
positioning of chromosomes (B) in human sperm
In (A), selected chromosome territories, telomeres (green circles) and
centromeres (red circles) are shown. (B) Schematic representation
of the preferred positioning for 11 human chromosomes based on
longitude/radial localization and inter-chromosomal distances.
to the interior and telomeres stretching out towards the
mPN periphery in one-cell mouse embryos [22].
Chromosome withdrawal may be mediated by telomere
interactions with the sperm aster (microtubule) machinery.
The situation is hypothetically analogous to meiotic movement of chromosomes in fission yeast: at this stage of
development, telomeres are joined and lead characteristic
horse-tail nuclear movements [23]. Meiotic telomeres in yeast
are attached to the spindle pole body, the fungal equivalent
of the centrosome, and pulled by the astral microtubules;
mechanical force is provided by cytoplasmic dynein [24].
During fertilization in most mammals, the microtubule
aster extending from the sperm head participates in mPN
decondensation, its migration towards the female pronucleus
and pronuclear centration [20]. Neither association of sperm
telomeres with microtubule machinery nor sperm/ooplasm
proteins that might be involved in such interactions have been
demonstrated yet. Some evidence towards the possibility of
telomere–microtubule interactions can be found in the literature. For example, specific contacts between telomerespecific protein Pin2/TRF1 (telomere-repeat-binding factor
1) and microtubules have been shown in HeLa cells [25];
vimentin specifically binds telomeric DNA in vitro [26].
The importance of the sperm telomeres for fertilization
and embryo development has been clearly confirmed in
mice. Telomerase deficiency disrupts reproductive function,
as has been demonstrated in telomerase-knockout mice [27].
Fertilization of wild-type eggs with telomerase−/− sperm
results in impaired fertilization, with defects in early cleavage
and pre-implantation [28]. Therefore only the progeny of
germ cells with chromosomes having sufficient telomere
length are capable of developing normally.
Finally, if the hypothesis about withdrawal of sperm
chromosomes via telomeres is true, chromosomal domains
starting from subtelomeric regions will be sequentially
exposed to remodelling activities and transcriptional factors
contained in ooplasm.
Centromeres
In sperm nuclei, centromeres aggregate into one or a few
large chromocentres [7]. They are located internally and are
the most resistant to decondensation in vitro [4]. Mature
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human spermatozoa retain centromeric protein A [6], which
is a variant protein that replaces histone H3 in centromeric
nucleosomes [29]. Centromeric sequences in sperm DNA
are associated both with histones and with protamines [30].
Therefore it is possible that sperm DNA in centromere
domains, at least in part, is organized into nucleosomes.
Noteworthily, parts of the telomere domains in human sperm
also preserve nucleosomes [31]. Nucleosomal organization
suggests that these regions of chromosome may not need
to be dramatically remodelled following fertilization. Their
structural features may rather be used as a template dictating
nucleosome positioning and/or spacing of the adjacent regions during histone deposition. In addition, centromeric
heterochromatin that is prone to self-aggregation may define
a set of structural landmarks against which the location of a
given gene or interactions between widely separated loci in
early embryos can be determined [32].
Internal regions of chromosomes
In human sperm nuclei, approx. 15% of the basic proteins
are represented by histones [1]. There are indications that
a fraction of residual sperm histones marks specific DNA
sequences [30,33]. According to the Donut-Loop model
developed by S. Ward’s group [34], histone-bound DNA
stretches are localized at the spacer regions separating
DNA loop domains. This nucleohistone may contain a set
of histones different from that associated with telomeres and
centromeres. During mPN development, internal chromosome regions organized into nucleosomes would be processed by the ooplasm components in a different way
compared with the protamine packaged DNA. These
structures might provide additional marks that affect early
post-fertilization development.
Chromosome positioning
Preferential longitudinal chromosome positioning has been
established for 11 chromosomes in human sperm [4,12,13,35],
their arrangement in the direction from acrosome to tail is:
X, 7, {6, 15, 16, 17}, 1, {Y, 18}, 2, 5 (Figure 1B). This means
that the male chromosomes might be affected by maternal
cytoplasmic environment after fertilization in sequential
order, which could ultimately bring differences in timing of
sperm chromatin domain reorganization [14]. Similar differentiation will be also imposed through radial positioning of
chromosomes. For example, in human sperm, chromosomes
7 and 6 are mostly peripheral in location, whereas chromosomes 16 and 18 are more internal [13] (Figure 1B). As a consequence, chromosomes that are the closest to the nuclear
edge are exposed to ooplasm and remodelled earlier than the
others.
Preferential nuclear positioning of sperm chromosomes
may be a major cause of the increased de novo formation of
anomalies in ICSI (intracytoplasmic sperm injection) babies
[36]. Delayed pronuclei formation, chromatin remodelling,
and DNA synthesis were noted in human [36] and mouse
[37] sperm nuclei after ICSI in comparison with IVF
The Testis as a Conduit for Genomic Plasticity
(in vitro fertilization). In ICSI, injected sperm cells preserve
perinuclear theca, therefore the most affected are the
chromosomes located at the apical region.
Irregularities in chromosome organization
in subfertile sperm
It is generally accepted that a significant number of infertile
males produce sperm with nuclear malformations or chromosome/chromatin defects. Among these are chromosomal
numerical abnormalities, breaks/rearrangements and mutations [38], deficiencies in the basic chromosomal proteins
[39,40] or broadly instituted chromatin condensation defects
[41]. Recently, we proposed that yet another previously
unattended class of sperm chromosome abnormality might
have an impact on fertilization and early development [42].
These aberrations are connected with: (i) atypical packing
of chromosome territories, which affects the higher-order
chromatin and chromosome architecture; (ii) unstable or
aberrant nuclear positioning of chromosomes; and (iii) disturbed telomere and centromere structure or interactions.
This hypothesis is based on a limited set of data obtained
by FISH (fluorescence in situ hybridization) in small groups
of patients with idiopathic male infertility. Several unrelated
phenotypes have been noticed: large and diffuse chromosome
territories, altered intranuclear localization of chromosomes and absence of telomere–telomere interactions characteristic of normal spermatozoa [42]. The molecular
basis for these phenotypes is unknown, and further studies
are needed to uncover an impact of the sperm genome
architecture defects on fertilization.
Summary and perspectives
We put forward a hypothesis that the unique chromosome
organization in sperm provides a mechanism for differential
exposure of chromosomes and/or their domains to the
components of ooplasm at fertilization, thus orchestrating
the unpacking and ordered activation of the male genome.
Multiple levels of the genome structural organization overviewed here have all features characteristic of epigenetic code
that will be deciphered in the descendant cells. Chromosome
organization in sperm would direct reading and processing
both genetic and next level epigenetic information, the latter
being written in histone codes and methylation patterns.
Future experimental approaches to explore the impact of
the peculiar sperm chromosome arrangement are connected
with the use of model animal systems, heterologous ICSI
of human sperm into oocytes of domestic animals [43], and
in vitro remodelling of human sperm in cell-free oocyte
extracts.
This work was supported by National Institutes of Health grant
HD-042748 to A.Z.
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