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- Review -
Relationship between chromatin organization, mRNAs profile and human male gamete quality
Isabelle Galeraud-Denis1,2, Sophie
Lambard3, Serge Carreau2
1UPRES EA 2608-USC INRA, Université de Caen, 14032-CAEN, France
2Biology of Reproduction, CHU-Caen, Avenue G. Clemeanceau, 14033-CAEN, France
3Hospital St Antoine,184, Rue Fg St Antoine, 75571-PARIS, France
Abstract
Spermiogenesis is a complex process leading to the formation of motile spermatozoa characterized by a highly
stable chromatin compaction that transfers the paternal genome into the oocyte. It is commonly held that these
haploid cells are devoid of transcriptional and translational activities and that the transcripts represent remnants of
stored mRNAs. Recently, the chromatin organization of mature spermatozoa has been revisited as a double
nucleoprotamine-nucleohistone structure possessing less-condensed regions sensitive to nuclease activity, which
could be implicated in the expression of genes involved in the early embryo development. The existence of a complex
population of mRNAs in human sperm is well-documented, but their role is not yet elucidated. Evidence for a latent
transcriptional capacity and/or a potential de
novo translation in mature spermatozoa from fertile men are essential for
understanding the last steps of sperm maturation, such as capacitation and acrosome reaction. As such, we have
documented the relationship between sperm quality and the distribution of sperm RNAs by showing divergent levels
of transcripts encoding for proteins involved in either nuclear condensation (protamines 1 and 2) or in capacitation
(eNOS and nNOS, c-myc) or in motility and sperm survival (aromatase) between low and high motile sperm issued
from the same sample. Therefore, analyzing the profile of mRNAs could be helpful either as a diagnostic tool for
evaluating male fertility after spermatogenesis or for prognosis use for
fertilization. (Asian J Androl 2007 Sep; 9: 587_592)
Keywords: chromatin; fertility; gamete quality; man; spermatozoa; transcripts
>Correspondence to: Dr Serge Carreau, EA 2608-USC INRA 2006, Université de Caen, Esplanade de la paix, 14032-CAEN, France.
Tel: +33-231-565-488 Fax: +33-231-565-120
E-mail: serge.carreau@unicaen.fr
Received 2006-12-06 Accepted 2007-05-19
DOI: 10.1111/j.1745-7262.2007.00310.x
1 Introduction
Mammalian ejaculated spermatozoa comprises highly differentiated cells that originate from the complex process
of spermatogenesis, involving three major steps: the proliferation and differentiation of spermatogonia; the meiotic
divisions during the spermatocyte stage; and, finally, the spermiogenesis. The transformation of spermatids into
spermatozoa entails major morphological and molecular changes concerning acrosome and flagellar formation,
cytoplasm elimination and mitochondria rearrangement and, finally, nuclear reshaping. Over the past few decades,
spermatozoa have been considered just as mobile units for transferring the paternal genome from the testis to the oocyte,
demonstrated by the histone-to-protamine exchange and, consequently, the resulting chromatin condensation, which
finally leads to the shutdown of nuclear transcription. Indeed round spermatids store messages as ribonucleoprotein
particles for long periods [1] and the particles are then translated in elongating spermatids [2]. The stability of mRNA
resulting from the package and the severe loss of
spermatozoal cytoplasm during spermiogenesis have resulted
in spermatozoa being considered as biologically inert cells
without transcriptional and translational potencies.
However, recently, in relation to a better understanding
of chromatin organization [3] and a possible de
novo translation of mRNAs in spermatozoa [4], new insights
about putative functions of spermatozoa should be considered.
2 Chromatin organization
2.1 Chromatin condensation players
The basic unit of somatic chromatin is the
nucleosome in which DNA is coiled around a histone octamer,
including H2A, H2B, H3 and H4 histones. Adjacent
nucleosomes are connected by histones of the H1 linker
class. The combination of covalent processes, such as
methylation, acetylation and ubiquination, are thought to
contribute to chromatin organization and gene expression.
The testis-specific histones H1A and H1B are found in
pre-meiotic male germ cells, but a higher number of
histone variants have been identified in male germ cells than
in somatic cells. The displacement of histones is
concomitant to the appearance of more basic nuclear proteins,
such as tH2A, tH2B, H1T, spermatid-specific H2B,
testis specific HMG, histone H1-like proteins in spermatids
(Hils 1) and transition proteins (TNP) (see [5] for review).
Understanding the correct implication of such
molecular variants during the histone/protamine exchange
could help to define the expression pattern of testicular
genes essential for the formation of normal spermatozoa.
A novel histone H1 variant, H1T2, selectively and
transiently expressed in round and elongated spermatids [6],
seems to play a major role in chromatin reorganization
by initiating and directing chromatin condensation. Null
mutation in H1T2 demonstrates the critical role of H1T2
in spermiogenesis, with delayed nuclear condensation and
reduced fertility. The epididymal sperm are
morphologically and functionally abnormal, leading to a deficient
motility and an inability to fertilize eggs under
in vitro fertilization (IVF) conditions [7]. Moreover, Hils1 is a
histone restricted to elongating spermatids and is tightly
implicated in chromatin condensation [8, 9].
TNP1 and TNP2 are the predominant nuclear proteins representing 55% and 40%, respectively, of the total
nuclear proteins in spermatids [10]. Male mice with null
mutations in either TNP1 or TNP2 are subfertile and are
able to produce offspring [11, 12], although male mice
with null mutations in both TNP1 and TNP2 are sterile
[13]. In contrast with histone H1, protamines contain a
very low amount of lysine but arginine is the most
important residue (> 50%), resulting in higher
DNA-binding affinity. Mice, horses and humans express two
structurally distinct forms of protamines: Prm-1 and Prm-2.
Only Prm1 is found in other mammals. Protamine
haplo-insufficient mice are infertile following the absence of
chromatin assembly [14]. Consequently, changes in the
expression of Prm 1 and Prm2 could increase DNA damage in spermatozoa and have been related to human
male infertility (see [15] for a review).
2.2 Chromatin structure
Chromatin condensation is based on the presence of
tightly packed toroids containing up to 60 kb of DNA
resistant to DNA I digestion. The cessation of
transcriptional activity in rat intermediate spermatids is
concomitant with the disappearance of DNAse I-hypersensitive
regions normally present in transcriptionally active genes.
In hamster spermatozoa, each protamine toroid is a single
DNA loop domain interspersed by DNAse I sensitive chromatin segments called toroid-linker regions, which
are also the sites of matrix attachment regions [16]. The
formation of less-condensed regions from this "donut
loop" model leads to the creation of sites of chromatin
digestion by endogenous nucleases present in hamster,
mouse and human spermatozoa [17].
The replacement of histones by protamines is
incomplete in human sperm, which retains roughly 15% of
histones, unlike the sperm of other mammals [18]. The
core histone fraction remaining is enriched in histone H2B,
with a selective distribution of H2B variants in sperm
nuclei [19]. This double structure
nucleoprotamine/nucleohistone could correspond to the differential
rearrangment of chromatin regions in which
histone-enriched regions might possess specific functions, such as
enhanced nuclease sensitivity and, therefore, could be
implicated in the formation of a specific subset of genes
involved in early embryogenesis [20]. This nuclease
activity could be mediated by a nuclear matrix, including
topoisomerase II B (TOP2B) interacting with an
extracellular Mn2+/Ca2+-dependent nuclease to promote DNA
fragmentation and degradation [21].
The relation between function and nuclear
architecture is well-known in somatic cells, but needs to be
clarified in spermatozoa. Classically, chromatin compaction
following spermiogenesis leads to the arrest of active
transcription. Ribosomal 18 and 28S proteins have been
only detected until the elongating spermatid stage in mice.
Therefore, spermatozoa does not have sufficient 80S
cytoplasmic ribosomal complexes, excluding, therefore, the
support of any RNA translational process. However,
new insights into chromatin organization lead us to the
view that potential translational/transcriptional events exist
in some circumstances.
2.3 Evidence for de novo translation and/or latent
transcriptional capacity in mature spermatozoa
The potential involvement of mRNAs in functional
spermatozoal activities is currently subject to debate
[22_25], but the idea of a complete inactive
transcription/translation in mature spematozoa is discussed here in light
of most recent data. An SAGE analysis of ejaculates
from fertile men has revealed 25 functional gene groups:
the most important one includes 96 nuclear protein genes
involved in transcription and a second group comprises
84 ribosomal subunit genes interacting with protein
synthesis [26]. Evidence of sperm endogenous reverse
transcriptase activity [27] and the incorporation of foreign
DNA sequences into sperm issued from reverse transcription of RNA molecules then transmitted to IVF
embryos suggest that reverse transcribed products are fully
active in spermatozoa being translated and expressed in
offspring [28]. Very recent experiments described
either by Pittoggi et al. [29], showing the correct outsplice
of an intronic sequence incorporated in a DNA construct,
or by Shaman et al. [21], who reported the
demonstration of an active topoisomerase TOP2B associated with
nucleases, are in favor of a less inert activity of
chromatin than previously evoked. Despite the presence of a
high degree of chromatin compaction in mature spermatozoa, these new data fit with the existence of
isolated domains in more DNAse-I-sensitive open conformations, indicating a potential transcriptional state
for specific genes involved in early embryogenesis.
Translational repression could be lifted in capacitating
spermatozoa, as demonstrated by the incorporation of
labeled amino acids into polypeptides [4] or the decrease
of c-myc transcripts in capacitated spermatozoa [30].
The first report describes an endogenous inhibition of
the amino acids uptake only by mitochondrial translation
inhibitors targeting mitochondrial 55S ribosomes [4],
whereas the second work shows a restoration of the
amount of c-myc transcripts in the presence of
cycloheximide, a cytoplasmic 80S ribosome inhibitor [30].
Despite the relative absence of ribosomal complexes, the
understanding of the translation mechanisms must be
further clarified. In spermatozoa, the potential
translation of mRNAs into proteins would be essential for the
final events of the sperm maturation, such as
capacitation and acrosome reaction.
The chromatin organization could be also responsible for modifications of the epigenome transmitted to
embryos and involved in the early steps of embryonic
development. Classically, changes in DNA and histone
methylation profiles represent the main process of
epigenetic regulation [31]. Rassoulzadegan
et al. [32] reported an unexpected model of epigenetic inheritance by
zygotic transfer of RNA molecules (RNA-mediated
non-mendelian inheritance), which leads to the modification
of phenotypic expression of the wild type allele of the Kit
receptor gene in the progeny of heterozygote mice,
suggesting a role of RNA molecules in the establishment of
epigenetic states.
3 Significance of RNAs in mature spermatozoa
3.1 RNAs in human sperm
Most of the RNA synthesized is heterogeneous nuclear RNA, U1 and U2 small nuclear
ribonucleoproteins [33, 34]. Until now, the remaining cytoplasmic
mRNAs have been considered as negligible compared to
the intranuclear and mitochondrial mRNAs. New
investigations using various techniques such as reverse
transcription polymerase chain reaction (RT-PCR) [24],
microarray technology [35], in situ hybridization [5],
confocal microscopy, serial analysis of gene expression
[26] have discovered complex and specific populations
of RNAs in mature spermatozoa, including microRNAs
[36]. Whereas the average spermatozoon contains
0.015 pg total mRNA, a somatic cell contains 600-fold
more mRNA [24]. Consequently, mRNA contamination by somatic cells or cytoplasmic droplets should be
excluded by using a double swim-up or centrifugation on
density gradients followed by a hypotonic treatment [35]
and/or analysis of somatic cell markers [30].
Using array-based tests, up to 3 281 and 2 780 RNA
transcripts have been detected from a pool of ejaculate
samples and a single individual ejaculate sample,
respectively [35]. The presence of various transcripts in mature
sperm of rodents and men including, for example,
c-myc, protamines 1 and 2, heat-shock proteins 70 and 90,
β-integrins, phosphodiesterase isoforms, and numerous
receptors have been listed (for reviews see [5, 24, 25]).
Among the complex population of mRNAs in spermatozoa, anti-sense microRNAs have been
identified in human spermatozoa. Their similarity with siRNAs
involved in RNA-mediated regulation suggest that they
could influence early embryonic development [36, 37].
We and others have provided data on the presence of
aromatase and estrogen receptors both in human
immature germ cells and ejaculated spermatozoa [38, 39].
Spermatozoa functions, such as motility, could also be
related to the mRNA profile. Therefore, we have
compared the levels of different transcripts coding for
molecules involved in nuclear condensation (Prm-1 and
Prm-2), capacitation (eNOS, nNOS and c-myc), motility and
sperm survival (P450 arom) using semi-quantitative
RT-PCR in high and low motile fractions from normospermic
patients [30]. Indeed, we have observed differential
mRNAs distribution between the two fractions. No
significant change in the c-myc/Prm-2 ratio between the two
populations of spermatozoa was observed. Conversely,
the amount of Prm-1 mRNA was significantly higher in
low motile than in high motile fractions; in most of high
motile sperm samples analyzed, eNOS and nNOS transcripts were undetectable, whereas they were in low
motile sperm. In contrast, a 30% decrease of aromatase
mRNA amount was observed in immotile sperm fraction,
and was recorded in all samples studied. Moreover, the
aromatase activity determined in vitro was also
diminished by 34%. In addition, we observed amplified
aromatase mRNA using real-time RT-PCR in asthenospermic infertile men and recorded a 44% decrease of
the amount of transcripts as compared to normospermic
controls (Saïd, Galeraud-Denis and Carreau, unpublished
data).
Another study based on the array-predicted
differential expression of genes in the two infertile (impaired
motility) and fertile populations confirmed that spermatozal
RNA profile [40] could provide new information about
the relation between the gamete quality and the
environmental conditions of spermatogenesis and/or
spermiogenesis in testis.
3.2 RNAs and male gamete quality
The existence of a potential translational activity in
mature spermatozoa is obvious. The transcripts present
in ejaculated and uncapacitated spermatozoa might be
remnants from post-meiotically active genes, especially
from round spermatids, which contain numerous RNAs
either produced in the early stages of spermatogenesis
[41] or during spermiogenesis, such as protamines and
transition proteins [2].
Analysis of spermatozoal mRNA could represent the
fingerprint for monitoring past events, especially the
development profile of gene expression during
spermatogenesis or spermiogenesis. In fact, impaired functions
of reproduction have been reported above in knockout
experiments performed on histone variants leading to
abnormal gene expression during meiosis. Consequently,
we can hypothezise the existence of an impaired mRNA
profile in human spermatozoa.
The use of a cDNA microarray technique for
qualitative and quantitative gene expression has shown the
presence of thousands of mRNA species from testis and
from pooled or individual spermatozoa, some of which
are delivered to the oocyte upon fertilization [42]. The
genome-wide approach between fertile and infertile
patients could provide information about testis gene
expression during spermatogenesis and help us to
understand the mechanisms involved in the control of either
normal or pathological spermatogenesis [43].
However, the study of individual mRNAs in fertile
and idiopathic infertile men could also contribute to the
knowledge of pathways playing a role during
capacitation/acrosome reaction and/or fertilization. The
accumulation of high amounts of transcripts, such as eNOS
or Prm1, in low motile spermatozoa could be the
consequence of an altered translation during spermiogenesis
along with either a defective histone/protamine exchange
and/or an impaired chromatin condensation. It would be
interesting to study the putative relation between the
chromatin composition, such as the histone/protamine ratio,
and the wrong translation of some transcripts during
spermiogenesis. Contrary to eNOS or Prm-1 transcript,
aromatase RNA levels are reduced in low motile
spermatozoa fraction. The relationship between the active
synthesis of estrogens in mature spermatozoa and the amount
of aromatase transcripts must be further investigated in
pathological situations (asthenospermic and teratospermic
patients) to elucidate the implication of estrogens in events
such as spermatozoa survey and/or capacitation.
Furthermore, the stability of the wide range of
spermatozoal RNAs seem to be different from each other:
some of them could be degraded following damage occurring during successive freeze_thaw cycles, but
others remain stable during similar treatment [44].
To conclude, all these observations reflect the
complexity and heterogeneity of the RNA transcripts present
in spermatozoa (Figure 1). Further investigations are
necessary to understand the significance and the
differential role of these mRNAs present in ejaculated and
uncapacitated spermatozoa, particularly in relation to
pathological sperms. Some of them might only be the
fingerprint of spermatogenesis and/or spermiogenesis
events. Others could be important for the final events
just before and after fertilization. Analysis of the profile
of mRNAs using a genome-wide approach applying microarray techniques or the evaluation of individual
transcripts using real-time RT-PCR in fertile and infertile
patients could be helpful either as diagnostic tools for
evaluating male fertility [45], for the prognosis of fertilization
[35] or for use in embryo development [44].
Acknowledgment
This work was supported by a grant from the French
Ministry of Education and Research.
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