Nucleolar migration regulates meiotic sex chromosome inactivation via phase separation during mammalian spermatogenesis.

Meiotic sex chromosome inactivation (MSCI) is an essential process in the male germline. While genetic experiments have established that the DNA damage response (DDR) pathway directs MSCI, due to limitations to the experimental systems available, mechanisms underlying MSCI remain largely unknown. Here we establish a system to study MSCI ex vivo, based on a short-term culture method, and demonstrate that active DDR signaling is required both to initiate and maintain MSCI via a dynamic and reversible process. DDR-directed MSCI follows two layers of modifications: active DDR-dependent reversible processes and irreversible histone post-translational modifications. Further, the DDR initiates MSCI independent of the downstream repressive histone mark H3K9 trimethylation (H3K9me3), thereby demonstrating that active DDR signaling is the primary mechanism of silencing in MSCI. By unveiling the dynamic nature of MSCI, and its governance by active DDR signals, our study highlights the sex chromosomes as an active signaling hub in meiosis.

BRCA1, Histone H2AX Phosphorylation, and Male Meiotic Sex Chromosome Inactivation

BackgroundIn mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable associations form through homologous chromosome synapsis. In male mouse meiosis, the largely heterologous X and Y chromosomes synapse only in their short pseudoautosomal regions (PARs), and DSBs persist along the unsynapsed non-homologous arms of these sex chromosomes. Asynapsis of these arms and the persistent DSBs then trigger transcriptional silencing through meiotic sex chromosome inactivation (MSCI), resulting in formation of the XY body. This inactive state is partially maintained in post-meiotic haploid spermatids (postmeiotic sex chromatin repression, PSCR). For the human, establishment of MSCI and PSCR have also been reported, but X-linked gene silencing appears to be more variable compared to mouse. To gain more insight into the regulation and significance of MSCI and PSCR among different eutherian species, we have performed a global analysis of XY pairing dynamics, DSB repair, MSCI and PSCR in the domestic dog (Canis lupus familiaris), for which the complete genome sequence has recently become available, allowing a thorough comparative analyses.ResultsIn addition to PAR synapsis between X and Y, we observed extensive self-synapsis of part of the dog X chromosome, and rapid loss of known markers of DSB repair from that part of the X. Sequencing of RNA from purified spermatocytes and spermatids revealed establishment of MSCI. However, the self-synapsing region of the X displayed higher X-linked gene expression compared to the unsynapsed area in spermatocytes, and was post-meiotically reactivated in spermatids. In contrast, genes in the PAR, which are expected to escape MSCI, were expressed at very low levels in both spermatocytes and spermatids. Our comparative analysis was then used to identify two X-linked genes that may escape MSCI in spermatocytes, and 21 that are specifically re-activated in spermatids of human, mouse and dog.ConclusionsOur data indicate that MSCI is incomplete in the dog. This may be partially explained by extensive, but transient, self-synapsis of the X chromosome, in association with rapid completion of meiotic DSB repair. In addition, our comparative analysis identifies novel candidate male fertility genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1501-9) contains supplementary material, which is available to authorized users.

More than a century ago, a densely stained area inside the nucleus of male meiotic cells was described. It was later shown to harbor the sex chromosomes which undergo transcriptional inactivation in conjunction with heterochromatinisation and synapsis to form the XY body. Formation of the XY body is conserved throughout the mammalian phylogenetic tree and is thought to be essential for successful spermatogenesis. However, its biological role as well as the molecular mechanisms underlying XY body formation are still far from being understood. A lot of effort has already been undertaken to characterize components of the XY body and to investigate their functional implications in sex chromatin heterochromatinisation and meiotic sex chromosome inactivation (MSCI). This review gives an overview of those components and their possible implications in XY body formation and function.

Single-Cell RNA Sequencing of the Cynomolgus Macaque Testis Reveals Conserved Transcriptional Profiles during Mammalian Spermatogenesis.

The differentiation of sex chromosomes through evolution has resulted in significant loss of homology and gene content which has profound consequences on gene dosage between the sexes and impacts on their behaviour through meiosis. During spermatogenesis the unpaired DNA, of heteromorphic sex chromosomes becomes transcriptionally silenced and sequestered into a ‘sex body’, a process termed ‘meiotic sex chromosome inactivation’. This essential meiotic event involves an ordered hierarchy of epigenetic changes to the sex chromosome DNA initiated by conserved DNA damage, repair and checkpoint machinery. Although the reason for this repression remains unclear, it is thought to prevent recombination between nonhomologous sex chromosome DNA and to avoid stringent meiotic checkpoints. Recent work has extended our understanding of the evolutionary conservation of meiotic silencing to include the avian ZW sex chromosome system providing new insights into the evolutionary dynamics of meiotic sex chromosome organization in diverse species. Key concepts: The accumulation of mutation and rearrangements on sex chromosomes lead to blocks in recombination, accumulation of sexually antagonistic genes and the differential degradation of sex chromosomes. Heteromorphic sex chromosomes result in gene dosage imbalance between the sexes and unsynapsed DNA in meiosis I. Meiotic sex chromosome inactivation protects unpaired sex chromosome DNA from aberrant recombination and checkpoint surveillance machinery. Sex chromosomes undergo transcriptional silencing during late prophase due to the accumulation of conserved epigenetic changes. Therian mammals have active X‐borne retrogenes on autosomes to compensate for the loss of transcription of sex chromosome genes important for spermatogenesis.

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. The germ cell-specific depletion of ARID1A resulted in a pachynema arrest and failure to repress sex-linked genes, indicating a defective MSCI. Consistent with this defect, mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. By investigating potential mechanisms underlying these anomalies, we identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA Meiotic Recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

X chromosome inactivation is most commonly studied in the context of female mammalian development, where it performs an essential role in dosage compensation. However, another form of X-inactivation takes place in the male, during spermatogenesis, as germ cells enter meiosis. This second form of X-inactivation, called meiotic sex chromosome inactivation (MSCI) has emerged as a novel paradigm for studying the epigenetic regulation of gene expression. New studies have revealed that MSCI is a special example of a more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC), which silences chromosomes that fail to pair with their homologous partners and, in doing so, may protect against aneuploidy in subsequent generations. Furthermore, failure in MSCI is emerging as an important etiological factor in meiotic sterility.

Male carriers of structural chromosomal abnormalities provide a useful model for studying the effects of impaired synapsis on human meioses and male fertility. We used immunofluorescent techniques to examine recombination (MLH1), synapsis (SYCP3/SYCP1) and transcriptional inactivation (BRCA1/gammaH2AX/RNA polymerase II) of meiotic chromosomes in an azoospermic carrier of a t(8;13) reciprocal translocation. Two biopsies were performed 1 year apart and on different testes. Global recombination rates differed between the two biopsies. Although global recombination rates were not altered when compared with control men, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and gammaH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes. Immunostaining for RNA polymerase II provided further evidence that unsynapsed regions are silenced during human meiosis. The fidelity of synapsis is a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

WITHDRAWN: DErratum to “Phosphorylated extracellular signal-regulated kinase 1/2 is localized to the XY body of the meiotic prophase spermatocytes” [Biochem. Biophys. Res. Commun. 346 (2006) 1261–1266

Gametogenesis is a sexually dimorphic process requiring profound differences in germ cell differentiation between the sexes. In mammals, the presence of heteromorphic sex chromosomes in males creates additional sex-specific challenges, including incomplete X and Y pairing during meiotic prophase. This triggers formation of a heterochromatin domain, the XY body. The XY body disassembles after prophase, but specialized sex chromatin persists, with further modification, through meiosis. Here, we investigate the function of DMRT7, a mammal-specific protein related to the invertebrate sexual regulators Doublesex and MAB-3. We find that DMRT7 preferentially localizes to the XY body in the pachytene stage of meiotic prophase and is required for male meiosis. In Dmrt7 mutants, meiotic pairing and recombination appear normal, and a transcriptionally silenced XY body with appropriate chromatin marks is formed, but most germ cells undergo apoptosis during pachynema. A minority of mutant cells can progress to diplonema, but many of these escaping cells have abnormal sex chromatin lacking histone H3K9 di- and trimethylation and heterochromatin protein 1β accumulation, modifications that normally occur between pachynema and diplonema. Based on the localization of DMRT7 to the XY body and the sex chromatin defects observed in Dmrt7 mutants, we conclude that DMRT7 plays a role in the sex chromatin transformation that occurs between pachynema and diplonema. We suggest that DMRT7 may help control the transition from meiotic sex chromosome inactivation to postmeiotic sex chromatin in males. In addition, because it is found in all branches of mammals, but not in other vertebrates, Dmrt7 may shed light on evolution of meiosis and of sex chromatin.

The heteromorphic X and Y chromosomes behave in a special way in mammalian spermatocytes; they form the XY body and synapse only partially. The aim of this article was to study the origin and the role of the special differentiations in the XY pair of the domestic cat during pachytene by analyzing its fine structural characteristics and the immunolocalization of the main meiotic proteins SYCP3, SYCP1, SYCE3, SMC3, γ-H2AX, BRCA1, H3K27me3, and MLH1. The cat XY body shows particularly striking structures: an extreme degree of axial fibrillation in late pachynema and a special location of SYCP3-containing fibrils, bridging different regions of the main X axis, as well as one bridge at the inner end of the pairing region that colocalizes with the single mandatory MLH1 focus. There are sequential changes, first bullous expansions, then subdivision into fibrils, all involving axial thickening. The chromatin of the XY body presents the usual features of meiotic sex chromosome inactivation. An analysis of the XY body of many eutherians and metatherians suggests that axial thickenings are primitive features. The sequential changes in the mass and location of SYCP3-containing fibers vary among the clades because of specific processes of axial assembly/disassembly occurring in different species.

Meiotic Sex Chromosome Inactivation: Compensation by Gene Traffic

In somatic cells of female marsupial and eutherian mammals, X chromosome inactivation (XCI) occurs. XCI results in the transcriptional silencing of one of the two X chromosomes and is accompanied by specific covalent histone modifications attributable to the inactive chromatin state. Because data about repressed chromatin of the inactive X chromosome (Xi) in marsupials are sparse, we examined in more detail the distribution of active and inactive chromatin markers on metaphase X chromosomes of an American marsupial, Monodelphis domestica. Consistent with data reported previously both for eutherian and marsupial mammals, we found that the Xi of M. domestica lacks active histone markers-H3K4 dimethylation and H3K9 acetylation. We did not observe on metaphase spreads enrichment of the Xi with H3K27 trimethylation which is involved in XCI in eutherians and was detected on the Xi in the interphase nuclei of mature female M. domestica in an earlier study. Moreover, we found that the Xi of M. domestica was specifically marked with H3K9 trimethylation, which is known to be a component of the Xi chromatin in eutherians and is involved in both marsupials and eutherians in meiotic sex chromosome inactivation which has been proposed as an ancestral mechanism of XCI.

Spermatogenesis is a hormonally regulated process involving three sequential events: (1) the mitotic amplification of the spermatogonial cell progeny, (2) the completion of meiosis by the spermatocyte progeny, and (3) spermiogenesis, the gradual morphogenesis of the spermatid progeny. Mitosis, meiosis and spermiogenesis coexist in the seminiferous epithelium in association with a post-mitotic stable population of somatic Sertoli cells. Cell components of each spermatogonial, spermatocyte, and spermatid cell progeny remain connected by intercellular cytoplasmic bridges. Intercellular bridges are disrupted upon completion of spermiogenesis leading to the release in the seminiferous tubular lumen of single mature spermatids transported to the epididymal duct for acquisition of fertilizing activity. Several key cell cycle regulators have been shown to operate during the mitotic amplification of the spermatogonial progeny. During meiotic prophase, autosomal bivalents are engaged in prominent ribosomal RNA and non-ribosomal RNA transcriptional activity, in contrast with the transcriptional silencing of the condensed XY chromosomes. An autosomal bivalent is a synapsed (conjoined) chromosomal pair, excluding the sex chromosomes X and Y, observed during meiotic prophase I. Each member of a chromosomal bivalent (autosomes and X-Y) consists of two sister chromatids that will disjoin (separate) upon completion of meiosis to produce a haploid genome (spermatid). During spermiogenesis, gradual genetic inactivation of the spermatid genome correlates with spermatid head shaping. The acrosome-acroplaxome-manchette complex is emerging as a significant player in spermatid head shaping as well as in the assembly of the sperm head–tail coupling apparatus and the development of the outer dense fiber-axoneme-containing sperm tail. The acroplaxome is a cytoskeletal plate bordered by a desmosome-like marginal ring fastening the descending recess of the acrosomal sac to the nuclear envelope of the spermatid. The manchette is a transient microtubular-containing structure developed beneath the acroplaxome and encircling the elongating spermatid nucleus. This chapter is restricted to recent developments in the bioregulation of the spermatogonial stem cell progeny, the process of transcriptional inactivation of the XY bivalent, and the steps leading to spermatid head shaping. These are three relevant aspects that, when disrupted, can lead to male infertility.