Haploid male germ cells package their DNA into a volume that is typically 10% or less that of a somatic cell nucleus. To achieve this remarkable level of compaction, spermatozoa replace most of their histones with smaller, highly basic arginine and (in eutherians) cysteine rich protamines. One reason for such a high level of compaction is that it may help optimise nuclear shape and hence support the gametes' swimming ability for the long journey across the female reproductive tract to the oocyte. Super-compaction of the genome may confer additional protection from the effects of genotoxic factors. However, many species including the human retain a fraction of their chromatin in the more relaxed nucleosomal configuration that appears to run counter to the ergonomic, toroidal and repackaging of sperm DNA. Recent research suggests that the composition of this 'residual' nucleosomal compartment, a generally overlooked feature of the male gamete, is far more significant and important than previously thought. In this respect, the transport and incorporation of modified paternal histones by the spermatozoon to the zygote has been demonstrated and indicates another potential paternal effect in the epigenetic reprogramming of the zygote following fertilisation that is independent of imprinting status. In this review, the most recent research into mammalian spermatozoal chromatin composition is discussed alongside evidence for conserved, non-randomly located nucleosomal domains in spermatozoal nuclei, all supporting the hypothesis that the spermatozoon delivers a novel epigenetic signature to the egg that may be crucial for normal development. We also provide some thoughts on why this signature may be required in early embryogenesis.

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

The possible role of apoptosis in spontaneous or induced germ cell death was investigated by treating adult male rats with either a GnRH antagonist (112.5 micrograms kg-1 day-1 for 14 days) or methoxyacetic acid (650 micrograms kg-1; single dose) or sham-treated with either of the vehicles (n = 3 per group). The antagonist virtually abolished gonadotrophin secretion, while methoxyacetic acid reduced serum testosterone concentrations and slightly increased those of FSH (neither significantly). Bands of low molecular mass characteristic of apoptotically degraded DNA were detected by electrophoresis in both treatment groups but not in the controls. Sectioned, Carnoy-fixed testes were screened for degenerating cells with periodic acid-Schiff's base and haemalaun or examined for apoptotic cells using a modified in situ end-labelling procedure. Periodic acid-Schiff's-stained dying cells were found in low numbers in control animals with a distribution and frequency that matched that of apoptotic cells. Degenerating germ cells identified by histology were present at certain stages of spermatogenesis after 2 weeks of antagonist treatment. A comparison of their distribution with that of end-labelled cells identified the cell death as apoptotic. Methoxyacetic acid caused a massive depletion of spermatocytes at stages IX-II, which was also found to be apoptotic. It is concluded that spontaneous germ cell death in adult rats is apoptotic and that both gonadotrophin ablation and administration of methoxyacetic acid can cause apoptosis in the germ cells of adult male rats, but via different routes.

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The concept that mutations can be induced in the male germ-line and result in adverse effects in the offspring has achieved only limited acceptance despite considerable theoretical appeal. This is partly because fetal malformations are generally perceived to be induced solely as a result of maternally mediated events during gestation and partly because the low incidence of the end-points concerned make experimental approaches costly and time-consuming. Nonetheless, a substantial body of work relating to the hypothesis has accumulated in the last 20 years, which has never been reviewed in its entirety. A consideration of the available evidence indicates that preconceptional paternal exposure to mutagens (particularly radiation, cyclophosphamide and ethylnitrosourea) can indeed, under certain conditions, have adverse effects on offspring. The results suggest two principal mechanisms by which such effects may be induced: the induction of germ-line genomic instability or the suppression of germ cell apoptosis.

Spermatogenesis is characterized by the succession in time and space of specific germ cell associations (stages). There can be a single stage (e.g., rodents and some macaques) or more than one stage (e.g., chimpanzee and human) per tubular cross section. We analyzed the organization of the seminiferous epithelium and quantified testicular germ cell production and apoptosis in a New World primate, the common marmoset (Callithrix jacchus). Tubule cross sections contained more than one stage, and the human six-stage system could be applied to marmoset spermatogenesis. Stereological (optical disector) analysis (n = 5) revealed high spermatogenic efficiency during meiosis and no loss of spermatids during spermiogenesis. The conversion of type A to type B spermatogonia was several-fold higher than that reported for other primates. Highest apoptotic rates were found for S-phase cells (20%) and 4C cells (15%) by flow cytometric analysis (n = 6 animals); histological analysis confirmed spermatogonial apoptosis. Haploid germ cell apoptosis was <2%. Marmoset spermatogenesis is very efficient and involves substantial spermatogonial proliferation. The prime determinants of germ cell production in primates appear to be proliferation and survival of spermatogonia rather than the efficiency of meiotic divisions. Based on the organizational similarities, common marmosets could provide a new animal model for experimental studies of human spermatogenesis.

Spermatogenesis is a precisely controlled and timed process comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, and the maturation and differentiation of haploid spermatids. Cell proliferation is controlled by genes involved in the regulation of the cell cycle. Among the principal regulatory proteins are cyclins, which are categorized according to their appearance during the cell cycle. B-type cyclins are mitotic cyclins and function at the G2/M transition of the cell cycle. We have investigated the expression and regulation of cyclin B1 during rat spermatogenesis. Rat cyclin B1 was isolated from a testis cDNA library and further used as a probe to detect mRNA expression. Northern blot hybridization of testis mRNA revealed the presence of a single 1.7-kilobase transcript. In situ hybridization showed stage-specific expression during spermatogenesis with highest expression found in late pachytene spermatocytes and early round spermatids. This pattern was confirmed in fractions of isolated germ cells. Immunocytochemistry displayed highest protein levels in round spermatids. Depletion of gonadotropins did not change the quantitative and qualitative expression pattern of cyclin B1. Therefore, the signals triggering the onset of cyclin B1 expression seem not to originate from the pituitary-gonadal endocrine axis and might therefore be paracrine factors originating within the germinal epithelium. Our observations suggest that cyclin B1 plays a hitherto unknown role in spermatid maturation in addition to its known function in dividing cells.

Using a well-established endonuclease-based chromatin dissection procedure in conjunction with both experimental comparative genome hybridisation (CGH) array profiling and in silico data mining, we show that mouse spermatozoa contain chromatin that is sensitive and resistant to digestion with micrococcal nuclease (MNase). Sequences represented in the micrococcal nuclease digestion solubilised (MNDS) but not the MND insoluble (MNDI) chromatin are strongly enriched in chromosomal regions of high gene density. Furthermore, by fluorescence in situ hybridisation (FISH) analysis, we show that MNDS and MNDI DNAs occupy distinct domains of decondensed mouse sperm nuclei that may also retain abundant histones. More detailed in silico analysis of CGH probe location in relation to known promoters and sequences recognised by CCCTC binding factor (CTCF) shows a significant excess of both in MNDS chromatin. A functional analysis of gene promoters reveals strong ontological signatures for ion transport on methylated promoters associated with CTCF binding sequences in MNDS chromatin. Sensory perception is the only strong ontological signature present in MNDI chromatin, driven by promoters that are not associated with CTCF regardless of their methylation status.