The Association between Long-Term DDT or DDE Exposures and an Altered Sperm Epigenome-a Cross-Sectional Study of Greenlandic Inuit and South African VhaVenda Men.

BACKGROUND: The organochlorine dichlorodiphenyltrichloroethane (DDT) is banned worldwide owing to its negative health effects. It is exceptionally used as an insecticide for malaria control. Exposure occurs in regions where DDT is applied, as well as in the Arctic, where its endocrine disrupting metabolite, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) accumulates in marine mammals and fish. DDT and p,p'-DDE exposures are linked to birth defects, infertility, cancer, and neurodevelopmental delays. Of particular concern is the potential of DDT use to impact the health of generations to come via the heritable sperm epigenome. OBJECTIVES: The objective of this study was to assess the sperm epigenome in relation to p,p'-DDE serum levels between geographically diverse populations. METHODS: In the Limpopo Province of South Africa, we recruited 247 VhaVenda South African men and selected 50 paired blood serum and semen samples, and 47 Greenlandic Inuit blood and semen paired samples were selected from a total of 193 samples from the biobank of the INUENDO cohort, an EU Fifth Framework Programme Research and Development project. Sample selection was based on obtaining a range of p,p'-DDE serum levels (mean=870.734±134.030 ng/mL). We assessed the sperm epigenome in relation to serum p,p'-DDE levels using MethylC-Capture-sequencing (MCC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We identified genomic regions with altered DNA methylation (DNAme) and differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in sperm. RESULTS: Differences in DNAme and H3K4me3 enrichment were identified at transposable elements and regulatory regions involved in fertility, disease, development, and neurofunction. A subset of regions with sperm DNAme and H3K4me3 that differed between exposure groups was predicted to persist in the preimplantation embryo and to be associated with embryonic gene expression. DISCUSSION: These findings suggest that DDT and p,p'-DDE exposure impacts the sperm epigenome in a dose-response-like manner and may negatively impact the health of future generations through epigenetic mechanisms. Confounding factors, such as other environmental exposures, genetic diversity, and selection bias, cannot be ruled out. https://doi.org/10.1289/EHP12013.

Sperm histone H3 lysine 4 tri-methylation serves as a metabolic sensor of paternal obesity and is associated with the inheritance of metabolic dysfunction.

OBJECTIVE: Parental environmental exposures can strongly influence descendant risks for adult disease. How paternal obesity changes the sperm chromatin leading to the acquisition of metabolic disease in offspring remains controversial and ill-defined. The objective of this study was to assess (1) whether obesity induced by a high-fat diet alters sperm histone methylation; (2) whether paternal obesity can induce metabolic disturbances across generations; (3) whether there could be cumulative damage to the sperm epigenome leading to enhanced metabolic dysfunction in descendants; and (4) whether obesity-sensitive regions associate with embryonic epigenetic and transcriptomic profiles. Using a genetic mouse model of epigenetic inheritance, we investigated the role of histone H3 lysine 4 methylation (H3K4me3) in the paternal transmission of metabolic dysfunction. This transgenic mouse overexpresses the histone demethylase enzyme KDM1A in the developing germline and has an altered sperm epigenome at the level of histone H3K4 methylation. We hypothesized that challenging transgenic sires with a high-fat diet would further erode the sperm epigenome and lead to enhanced metabolic disturbances in the next generations. METHODS: To assess whether paternal obesity can have inter- or transgenerational impacts, and if so to identify potential mechanisms of this non-genetic inheritance, we used wild-type C57BL/6NCrl and transgenic males with a pre-existing altered sperm epigenome. To induce obesity, sires were fed either a control or high-fat diet (10% or 60% kcal fat, respectively) for 10-12 weeks, then bred to wild-type C57BL/6NCrl females fed a regular diet. F1 and F2 descendants were characterized for metabolic phenotypes by examining the effects of paternal obesity by sex, on body weight, fat mass distribution, the liver transcriptome, intraperitoneal glucose, and insulin tolerance tests. To determine whether obesity altered the F0 sperm chromatin, native chromatin immunoprecipitation-sequencing targeting H3K4me3 was performed. To gain insight into mechanisms of paternal transmission, we compared our sperm H3K4me3 profiles with embryonic and placental chromatin states, histone modification, and gene expression profiles. RESULTS: Obesity-induced alterations in H3K4me3 occurred in genes implicated in metabolic, inflammatory, and developmental processes. These processes were associated with offspring metabolic dysfunction and corresponded to genes enriched for H3K4me3 in embryos and overlapped embryonic and placenta gene expression profiles. Transgenerational susceptibility to metabolic disease was only observed when obese F0 had a pre-existing modified sperm epigenome. This coincided with increased H3K4me3 alterations in sperm and more severe phenotypes affecting their offspring. CONCLUSIONS: Our data suggest sperm H3K4me3 might serve as a metabolic sensor that connects paternal diet with offspring phenotypes via the placenta. This non-DNA-based knowledge of inheritance has the potential to improve our understanding of how environment shapes heritability and may lead to novel routes for the prevention of disease. This study highlights the need to further study the connection between the sperm epigenome, placental development, and children's health. SUMMARY SENTENCE: Paternal obesity impacts sperm H3K4me3 and is associated with placenta, embryonic and metabolic outcomes in descendants.

Early-Life Exposure to Environmental Contaminants Perturbs the Sperm Epigenome and Induces Negative Pregnancy Outcomes for Three Generations via the Paternal Lineage.

Due to the grasshopper effect, the Arctic food chain in Canada is contaminated with persistent organic pollutants (POPs) of industrial origin, including polychlorinated biphenyls and organochlorine pesticides. Exposure to POPs may be a contributor to the greater incidence of poor fetal growth, placental abnormalities, stillbirths, congenital defects and shortened lifespan in the Inuit population compared to non-Aboriginal Canadians. Although maternal exposure to POPs is well established to harm pregnancy outcomes, paternal transmission of the effects of POPs is a possibility that has not been well investigated. We used a rat model to test the hypothesis that exposure to POPs during gestation and suckling leads to developmental defects that are transmitted to subsequent generations via the male lineage. Indeed, developmental exposure to an environmentally relevant Arctic POPs mixture impaired sperm quality and pregnancy outcomes across two subsequent, unexposed generations and altered sperm DNA methylation, some of which are also observed for two additional generations. Genes corresponding to the altered sperm methylome correspond to health problems encountered in the Inuit population. These findings demonstrate that the paternal methylome is sensitive to the environment and that some perturbations persist for at least two subsequent generations. In conclusion, although many factors influence health, paternal exposure to contaminants plays a heretofore-underappreciated role with sperm DNA methylation contributing to the molecular underpinnings involved.

Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development.

The paternal environment has been linked to infertility and negative outcomes. Such effects may be transmitted via sperm through histone modifications. To date, in-depth profiling of the sperm chromatin in men has been limited. Here, we use deep sequencing to characterize the sperm profiles of histone H3 lysine 4 tri-methylation (H3K4me3) and DNA methylation in a representative reference population of 37 men. Our analysis reveals that H3K4me3 is localized throughout the genome and at genes for fertility and development. Remarkably, enrichment is also found at regions that escape epigenetic reprogramming in primordial germ cells, embryonic enhancers, and short-interspersed nuclear elements (SINEs). There is significant overlap in H3K4me3 and DNA methylation throughout the genome, suggesting a potential interplay between these marks previously reported to be mutually exclusive in sperm. Comparisons made between H3K4me3 marked regions in sperm and the embryonic transcriptome suggest an influence of paternal chromatin on embryonic gene expression.

ChIP-seq protocol for sperm cells and embryos to assess environmental impacts and epigenetic inheritance.

In the field of epigenetic inheritance, delineating molecular mechanisms implicated in the transfer of paternal environmental conditions to descendants has been elusive. This protocol details how to track sperm chromatin intergenerationally. We describe mouse model design to probe chromatin states in single mouse sperm and techniques to assess pre-implantation embryo chromatin and gene expression. We place emphasis on how to obtain high-quality and quantifiable data sets in sperm and embryos, as well as highlight the limitations of working with low input. For complete details on the use and execution of this protocol, please refer to Lismer et al. (2021).

Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring.

A father's lifestyle impacts offspring health; yet, the underlying molecular mechanisms remain elusive. We hypothesized that a diet that changes methyl donor availability will alter the sperm and embryo epigenomes to impact embryonic gene expression and development. Here, we demonstrate that a folate-deficient (FD) diet alters histone H3 lysine 4 trimethylation (H3K4me3) in sperm at developmental genes and putative enhancers. A subset of H3K4me3 alterations in sperm are retained in the pre-implantation embryo and associated with deregulated embryonic gene expression. Using a genetic mouse model in which sires have pre-existing altered H3K4me2/3 in sperm, we show that a FD diet exacerbates alterations in sperm H3K4me3 and embryonic gene expression, leading to an increase in developmental defect severity. These findings imply that paternal H3K4me3 is transmitted to the embryo and influences gene expression and development. It further suggests that epigenetic errors can accumulate in sperm to worsen offspring developmental outcomes.

The genomic distribution of histone H3K4me2 in spermatogonia is highly conserved in sperm†.

Environmental exposures can alter the long-term health and development of offspring. How this environmental information is transmitted via the germline remains unknown, but it is thought to involve epigenetic inheritance. We recently determined that genetic disruption of histone H3 di-methylation at lysine 4 (H3K4me2) in sperm alters gene expression in the embryo and negatively impacts development across generations. However, little is known regarding when in spermatogenesis H3K4me2 methylation is established, and whether specific regions bearing H3K4me2 resist the epigenome remodeling that occurs throughout spermatogenesis. Our objective was to determine what genomic regions bearing histone H3K4me2 in spermatogonia are also present in sperm. Methods: Using transgenic mice expressing Oct4-GFP, we isolated an enriched spermatogonia population and performed ChIP-seq for H3K4me2, followed by downstream bioinformatics analysis. Using our epigenomic data and existing datasets, we compared the genomic distribution of H3K4me2 between spermatogonia and sperm. We also assessed the expression level of genes enriched in H3K4me2 in spermatogenic cell types and at specific embryonic developmental time-points. We observed that many regions of the sperm epigenome bearing H3K4me2 are already present in spermatogonia, suggesting an early establishment of this histone mark in spermatogenesis. Subsets of genes with a high enrichment in H3K4me2 in sperm are strongly expressed in spermatogenesis and others are associated with high gene expression during embryo development. These findings suggest that if epimutations in H3K4me2 are induced in spermatogonia they have the possibility to persist throughout spermatogenesis and may influence fertility by altering gene expression in spermatogenesis and in the embryo.

Inferring and modeling inheritance of differentially methylated changes across multiple generations.

High-throughput methylation sequencing enables genome-wide detection of differentially methylated sites (DMS) or regions (DMR). Increasing evidence suggests that treatment-induced DMS can be transmitted across generations, but the analysis of induced methylation changes across multiple generations is complicated by the lack of sound statistical methods to evaluate significance levels. Due to software design, DMS detection was usually made on each generation separately, thus disregarding stochastic effects expected when a large number of DMS is detected in each generation. Here, we present a novel method based on Monte Carlo sampling, methylInheritance, to evaluate that the number of conserved DMS between several generations is associated to an effect inherited from a treatment and not randomness. Moreover, we developed an inheritance simulation package, methInheritSim, to demonstrate the performance of the methylInheritance method and to evaluate the power of different experimental designs. Finally, we applied methylInheritance to a DNA methylation dataset obtained from early-life persistent organic pollutants (POPs) exposed Sprague-Dawley female rats and their descendants through a paternal transmission. The results show that metylInheritance can efficiently identify treatment-induced inherited methylation changes. Specifically, we identified two intergenerationally conserved DMS at transcription start site (TSS); one of those persisted transgenerationally. Three transgenerationally conserved DMR were found at intra or integenic regions.

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally.

A father's lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.

The histone demethylase KDM1A is essential for the maintenance and differentiation of spermatogonial stem cells and progenitors.

Little is known of the fundamental processes governed by epigenetic mechanisms in the supplier cells of spermatogenesis, the spermatogonial stem cells (SSCs). The histone H3 lysine demethylase KDM1A is expressed in spermatogonia. We hypothesized that KDM1A serves in transcriptional regulation of SSCs and fertility. Using a conditional deletion of Kdm1a [conditional knockout (cKO)] in mouse spermatogonia, we determined that Kdm1a is essential for spermatogenesis as adult cKO males completely lack germ cells. Analysis of postnatal testis development revealed that undifferentiated and differentiating spermatogonial populations form in Kdm1a-cKO animals, yet the majority fail to enter meiosis. Loss of germ cells in the cKO was rapid with none remaining by postnatal day (PND) 21. To gain insight into the mechanistic implications of Kdm1a ablation, we isolated PND 6 spermatogonia enriched for SSCs and analyzed their transcriptome by RNA sequencing. Loss of Kdm1a was associated with altered transcription of 1206 genes. Importantly, differentially expressed genes between control and Kdm1a-cKO animals included those that are essential for SSC and progenitor maintenance and spermatogonial differentiation. The complete loss of fertility and failure to establish spermatogenesis indicate that Kdm1a is a master controller of gene transcription in spermatogonia and is required for SSC and progenitor maintenance and differentiation.

ß-catenin stabilization in gonadotropes impairs FSH synthesis in male mice in vivo.

Although classically considered a WNT signaling intermediary, ß-catenin (CTNNB1) can also mediate GnRH induction of gonadotropin ß-subunit (Fshb and Lhb) transcription in the murine gonadotrope-like cell line LßT2. Here, we assessed CTNNB1's role in gonadotropin synthesis in vivo. We used a Cre/lox approach to introduce both gain- and loss-of-function mutations in the murine Ctnnb1 gene in gonadotrope cells. Gonadotropin production and fertility were normal in Ctnnb1 knockout mice. Similarly, females harboring a deletion of exon 3 of Ctnnb1, which stabilizes the resulting CTNNB1 protein, showed normal fertility and gonadotropin synthesis. Interestingly, males with the activating CTNNB1-Δexon 3 mutation exhibited 50% reductions in FSH synthesis and secretion, without a corresponding change in LH. This selective regulation of FSH suggested an alteration in the activin/inhibin/follistatin system. Indeed, CTNNB1-Δexon 3 males showed a 60% increase in serum inhibin B levels, and in culture, their pituitaries exhibited a greater sensitivity to exogenous inhibin than controls. At the same time, pituitary, but not testicular, follistatin (Fst) expression was increased significantly in these mice. Castration normalized FSH levels in CTNNB1-Δexon 3 males to those seen in castrated controls. Paradoxically, pituitaries from CTNNB1-Δexon 3 males exhibited greater basal and activin-stimulated FSH synthesis in vitro. Similarly, CTNNB1-Δexon 3 overexpression potentiated activin A-induced murine Fshb promoter activity in LßT2 cells. Together, these results indicate that CTNNB1 is dispensable for gonadotropin synthesis in vivo. However, sustained CTNNB1 signaling potentiates activin-induced Fshb expression in gonadotropes, but this effect is overcome in vivo by enhanced inhibin feedback sensitivity and Fst expression.

Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors.

Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.

Specialized distribution of the histone methyltransferase Ezh2 in the nuclear apical region of round spermatids and its interaction with the histone variant H1t2.

The formation of sperm requires tightly regulated gene expression and unique chromatin remodeling. In the present study, we investigated the spermatogenic distribution of the lysine-specific histone H3 methyltransferase Ezh2 in mice. The distribution of Ezh2 was highly regulated with its localization predominantly restricted to round spermatids in the perinuclear acrosome region. This localization is concomitant with the dramatic epigenetic reorganization that occurs during spermiogenesis leading to an extreme compaction of the chromatin. Spermiogenesis involves the incorporation of sperm-specific nuclear proteins, including the testis-specific histone variant H1t2. Using immunofluorescence, Ezh2 was shown to juxtapose H1t2, and an interaction in chromatin was confirmed by immunoprecipitation. These findings suggest that, in the testis, the apical region of the round spermatid nucleus could be a specialized epigenetic region where methylation of histones serves a role in the spermiogenic chromatin remodeling and that Ezh2 might be a key effector of this event.

The dynamic epigenetic program in male germ cells: Its role in spermatogenesis, testis cancer, and its response to the environment.

Spermatogenesis is a truly remarkable process that requires exquisite control and synchronization of germ cell development. It is prone to frequent error, as paternal infertility contributes to 30-50% of all infertility cases; yet, in many cases, the mechanisms underlying its causes are unknown. Strikingly, aberrant epigenetic profiles, in the form of anomalous DNA and histone modifications, are characteristic of cancerous testis cells. Germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition. All are postulated to engender unique epigenetic controls. In support of this idea are the findings that mouse models with gene deletions for epigenetic modifiers have severely compromised fertility. Underscoring the importance of understanding how epigenetic marks are set and interpreted is evidence that abnormal epigenetic programming of gametes and embryos contributes to heritable instabilities in subsequent generations. Numerous studies have documented the existence of transgenerational consequences of maternal nutrition, or other environmental exposures, but it is only now recognized that there are sex-specific male-line transgenerational responses in humans and other species. Epigenetic events in the testis have just begun to be studied. New work on the function of specific histone modifications, chromatin modifiers, DNA methylation, and the impact of the environment on developing sperm suggests that the correct setting of the epigenome is required for male reproductive health and the prevention of paternal disease transmission.

Phthalates impair germ cell development in the human fetal testis in vitro without change in testosterone production.

BACKGROUND: Several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat. OBJECTIVES: Using the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate--mono-2-ethylhexyl phthalate (MEHP)--an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function. METHODS: Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH). RESULTS: Whatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10(-4) M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10(-4) M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3-positive germ cells, without modification of their proliferation. CONCLUSIONS: This is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.

Ontogenesis of testicular function in humans.

The two major functions of the testis, steroidogenesis and gametogenesis, take place during fetal life. These two functions have been extensively studied in rodents and adult humans. However, their onset during fetal life is poorly documented in humans. In the first part of this work we presented both our experimental data and some data of literature concerning the development of the human fetal testis. In the second part of this article, using the organ culture system we previously developed, we have investigated the regulations or perturbations of fetal testis development both in rodent and human models. Our findings provide important insight into the potential role of exposure to environmental pollutants (physical factors, in particular ionizing radiation, cadmium and endocrine disruptors such as phthalates) during fetal testicular development and their potential deleterious effects on male fertility in adulthood. Our results highlight the specificity of the human model compared with rodent models.

Adverse effects of endocrine disruptors on the foetal testis development: focus on the phthalates.

There are great concerns about the increasing incidence of abnormalities in male reproductive function. Human sperm counts have markedly dropped and the rate of testicular cancer has clearly augmented over the past four decades. Moreover, the prevalence rates of cryptorchidism and hypospadias are also probably increasing. It has been hypothesized that all these adverse trends in male reproduction result from abnormalities in the development of the testis during foetal and neonatal life. Furthermore, many recent epidemiological, clinical and experimental data suggest that these male reproductive disorders could be due to the effects of xenobiotics termed endocrine disruptors, which are becoming more and more concentrated and prevalent in our environment. Among these endocrine disruptors, we chose to focus this review on the phthalates for different reasons: 1) they are widespread in the environment; 2) their concentrations in many human biological fluids have been measured; 3) the experimental data using rodent models suggesting a reprotoxicity are numerous and are the most convincing; 4) their deleterious effects on the in vivo and in vitro development and function of the rat foetal testis have been largely studied; 5) some epidemiological data in humans suggest a reprotoxic effect at environmental concentrations at least during neonatal life. However, the direct effects of phthalates on human foetal testis have never been explored. Thus, as we did for the rat in the 1990s, we recently developed and validated an organ culture system which allows maintenance of the development of the different cell types of human foetal testis. In this system, addition of 10-4 M MEHP (mono-2-ethylhexyl phthalate), the most produced phthalate, had no effect on basal or LH-stimulated production of testosterone, but it reduced the number of germ cells by increasing their apoptosis, without modification of their proliferation. This is the first experimental demonstration that phthalates alter the development of the foetal testis in humans. Using our organotypic culture system, we and others are currently investigating the effect of MEHP in the mouse and the rat, and it will be interesting to compare the results between these species to analyse the relevance of toxicological tests based on rodent models.

Sex-specific differences in fetal germ cell apoptosis induced by ionizing radiation.

BACKGROUND: We have previously shown that male human fetal germ cells are highly radiosensitive and that their death depends on p53 activation. Male germ cell apoptosis was initiated with doses as low as 0.1 Gy and was prevented by pifithrin alpha, a p53 inhibitor. In this study, we investigated the radiosensitivity of early female and male fetal proliferating germ cells. METHODS AND RESULTS: Both male and female fetal germ cells displayed a similar number of gamma H2AX foci in response to ionizing radiation (IR). In organ culture of human fetal ovaries, the germ cells underwent apoptosis only when exposed to high doses of IR (1.5 Gy and above). Accumulation of p53 was detected in irradiated male human fetal germ cells but not in female ones. Inhibition of p53 with pifithrin alpha did not affect oogonia apoptosis following irradiation. IR induced apoptosis similarly in mouse fetal ovaries in organ culture and in vivo during oogonial proliferation. Germ cell survival in testes from p53 knockout or p63 knockout mice exposed to IR was better than wild-type, whereas female germ cell survival was unaffected by p53 or p63 knockout. CONCLUSIONS: These findings show that pre-meiotic male and female fetal germ cells behave differently in response to a genotoxic stress--irradiation--with oogonia being less sensitive and undergoing p53-independent apoptosis.

Histone H3 tails containing dimethylated lysine and adjacent phosphorylated serine modifications adopt a specific conformation during mitosis and meiosis.

Condensation of chromatin, mediated in part by posttranslational modifications of histones, is essential for cell division during mitosis. Histone H3 tails are dimethylated on lysine (Kme2) and become phosphorylated on serine (Sp) residues during mitosis. We have explored the possibility that these double modifications are involved in the establishment of H3 tail conformations during the cell cycle. Here we describe a specific chromatin conformation occurring at Kme2 and adjacently phosphorylated S of H3 tails upon formation of a hydrogen bond. This conformation appears exclusively between early prophase and early anaphase of the mitosis, when chromatin condensation is highest. Moreover, we observed that the conformed H3Kme2Sp tail is present at the diplotene and metaphase stages in spermatocytes and oocytes. Our data together with results obtained by cryoelectron microscopy suggest that the conformation of Kme2Sp-modified H3 tails changes during mitosis and meiosis. This is supported by biostructural modeling of a modified histone H3 tail bound by an antibody, indicating that Kme2Sp-modified H3 tails can adopt at least two different conformations. Thus, the H3K9me2S10p and the H3K27me2S28p sites are involved in the acquisition of specific chromatin conformations during chromatin condensation for cell division.