Meiotic Chromosome Synapsis and XY-Body Formation In Vitro

To achieve spermatogenesis in vitro, one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, in vitro derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our in vitro culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the in vitro-generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in vivo in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional in vitro, meiotic crossover formation should be closely monitored when mimicking gametogenesis in vitro to prevent generation of aneuploid gametes.

Deficiency of SYCP3-related XLR3 Disrupts the Initiation of Meiotic Sex Chromosome Inactivation in Mouse Spermatogenesis

In mammals, the X and Y chromosomes share only small regions of homology called pseudo-autosomal regions (PAR) where pairing and recombination in spermatocytes can occur. Consequently, the sex chromosomes remain largely unsynapsed during meiosis I and are sequestered in a nuclear compartment known as the XY body where they are transcriptionally silenced in a process called meiotic sex chromosome inactivation (MSCI). MSCI mirrors meiotic silencing of unpaired chromatin (MSUC), the sequestration and transcriptional repression of unpaired DNA observed widely in eukaryotes. MSCI is initiated by the assembly of the axial elements of the synaptonemal complex (SC) comprising the structural proteins SYCP2 and SYCP3 followed by the ordered recruitment of DNA Damage Response (DDR) factors to effect gene silencing. However, the precise mechanism of how unsynapsed chromatin is detected in meiocytes is poorly understood. The sex chromosomes in eutherian mammals harbor multiple clusters of SYCP3-like amplicons comprising the Xlr gene family, only a handful of which have been functionally studied. We used a shRNA-transgenic mouse model to create a deficiency in the testis-expressed multicopy Xlr3 genes to investigate their role in spermatogenesis. Here we show that knockdown of Xlr3 in mice leads to spermatogenic defects and a skewed sex ratio that can be traced to MSCI breakdown. Spermatocytes deficient in XLR3 form the XY body and the SC axial elements therein, but are compromised in their ability to recruit DDR components to the XY body.

TH2BS12P histone mark is enriched in the unsynapsed axes of the XY body and predominantly associates with H3K4me3-containing genomic regions in mammalian spermatocytes

Various studies have focussed on understanding the repertoire and biological function of the post-translational modifications that occur on testis-specific histone variants like TH2B, Transition Proteins etc. In our attempt to decipher the unique functions of histone variant TH2B, we discovered a new modification Serine 12 phosphorylation on TH2B (TH2BS12P) in spermatocytes. Our present study is aimed at understanding the function of the TH2BS12P modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies revealed that TH2BS12P histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body axes associated proteins like Scp3, γH2AX, pATM, ATR etc. We also observe that TH2BS12P is associated with DSB initiator Spo11 and with several recombination related proteins like pATM, ATR, Rad51, γH2AX etc in vivo. This modification was also found to associate with transcription and recombination related histone marks like H3K4me3 and H3K36me3 in the context of mononucleosomes. Genome-wide occupancy studies as determined by ChIP sequencing experiments revealed that TH2BS12P is localised to subset of recombination hotspots, but majorly associated with H3K4me3 containing genomic regions like gene promoters. Mass spectrometry analysis of proteins that bind to TH2BS12P containing mononucleosomes revealed many proteins linked with the functions of pericentric heterochromatin, transcription and recombination related pathways. We propose that TH2BS12P modification could act alone or in concert with other histone marks for recruitment of appropriate transcription or recombination protein machinery at specific genomic loci. This is the first report documenting the role of a post-translational modification of a germ cell specific histone variant in meiotic prophase I related events.

Distinct prophase arrest mechanisms in human male meiosis

To prevent chromosomal aberrations to be transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of all males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. We here unravel two clearly distinct meiotic arrest mechanisms that act during the prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y-chromosome encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63 mediated spermatocyte elimination. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to properly silence the X-chromosome encoded gene ZFX. Discovery and understanding of these meiotic arrest mechanisms increases our knowledge on how genomic stability is guarded during human germ cell development.

The XY Body of the Cat (Felis catus): Structural Differentiations and Protein Immunolocalization

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.