The male pachynema-specific protein MAPS drives phase separation in vitro and regulates sex body formation and chromatin behaviors in vivo.

Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps-/- pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. MapsΔ2-9/Δ2-9 male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps-/- mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.

Sex body: A nest of protein mixture.

During the pachytene stage in mammalian meiosis, the X and Y chromosomes remain largely unsynapsed outside the pseudoautosomal region, while autosomes are fully synapsed. Then, the sex chromosomes are compartmentalized into a "sex body" in the nucleus and are subjected to meiotic sex chromosome inactivation (MSCI). For decades, the formation and functioning of the sex body and MSCI have been subjects worth exploring. Notably, a series of proteins have been reported to be located on the sex body area and inferred to play an essential role in MSCI; however, the proteins that are actually located in this area and how these proteins promote sex body formation and establish MSCI remain unclear. Collectively, the DNA damage response factors, downstream fanconi anemia proteins, and other canonical repressive histone modifications have been reported to be associated with the sex body. Here, this study reviews the factors located on the sex body area and tries to provide new insights into studying this mysterious domain.

Phosphoproteomics of ATR signaling in mouse testes.

The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.

Meiotic sex chromosome inactivation and the XY body: a phase separation hypothesis.

In mammalian male meiosis, the heterologous X and Y chromosomes remain unsynapsed and, as a result, are subject to meiotic sex chromosome inactivation (MSCI). MSCI is required for the successful completion of spermatogenesis. Following the initiation of MSCI, the X and Y chromosomes undergo various epigenetic modifications and are transformed into a nuclear body termed the XY body. Here, we review the mechanisms underlying the initiation of two essential, sequential processes in meiotic prophase I: MSCI and XY-body formation. The initiation of MSCI is directed by the action of DNA damage response (DDR) pathways; downstream of the DDR, unique epigenetic states are established, leading to the formation of the XY body. Accumulating evidence suggests that MSCI and subsequent XY-body formation may be driven by phase separation, a physical process that governs the formation of membraneless organelles and other biomolecular condensates. Thus, here we gather literature-based evidence to explore a phase separation hypothesis for the initiation of MSCI and the formation of the XY body.

A Hypothesis: Linking Phase Separation to Meiotic Sex Chromosome Inactivation and Sex-Body Formation.

During meiotic prophase I, X and Y chromosomes in mammalian spermatocytes only stably pair at a small homologous region called the pseudoautosomal region (PAR). However, the rest of the sex chromosomes remain largely unsynapsed. The extensive asynapsis triggers transcriptional silencing - meiotic sex chromosome inactivation (MSCI). Along with MSCI, a special nuclear territory, sex body or XY body, forms. In the early steps of MSCI, DNA damage response (DDR) factors, such as BRCA1, ATR, and γH2AX, function as sensors and effectors of the silencing signals. Downstream canonical repressive histone modifications, including methylation, acetylation, ubiquitylation, and SUMOylation, are responsible for the transcriptional repression of the sex chromosomes. Nevertheless, mechanisms of the sex-body formation remain unclear. Liquid-liquid phase separation (LLPS) may drive the formation of several chromatin subcompartments, such as pericentric heterochromatin, nucleoli, inactive X chromosomes. Although several proteins involved in phase separation are found in the sex bodies, when and whether these proteins exert functions in the sex-body formation and MSCI is still unknown. Here, we reviewed recent publications on the mechanisms of MSCI and LLPS, pointed out the potential link between LLPS and the formation of sex bodies, and discussed its implications for future research.

Dual mechanism of chromatin remodeling in the common shrew sex trivalent (XY 1Y 2).

Here we focus on the XY1Y2 condition in male common shrew Sorex araneus Linnaeus, 1758, applying electron microscopy and immunocytochemistry for a comprehensive analysis of structure, synapsis and behaviour of the sex trivalent in pachytene spermatocytes. The pachytene sex trivalent consists of three distinct parts: short and long synaptic SC fragments (between the X and Y1 and between the X and Y2, respectively) and a long asynaptic region of the X in-between. Chromatin inactivation was revealed in the XY1 synaptic region, the asynaptic region of the X and a very small asynaptic part of the Y2. This inactive part of the sex trivalent, that we named the 'head', forms a typical sex body and is located at the periphery of the meiotic nucleus at mid pachytene. The second part or 'tail', a long region of synapsis between the X and Y2 chromosomes, is directed from the periphery into the nucleus. Based on the distribution patterns of four proteins involved in chromatin inactivation, we propose a model of meiotic silencing in shrew sex chromosomes. Thus, we conclude that pachytene sex chromosomes are structurally and functionally two different chromatin domains with specific nuclear topology: the peripheral inactivated 'true' sex chromosome regions (part of the X and the Y1) and more centrally located transcriptionally active autosomal segments (part of the X and the Y2).

Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males.

Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3'UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1-ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I.

Meiotic and sperm aneuploidy studies in three carriers of Robertsonian translocations and small supernumerary marker chromosomes.

OBJECTIVE: To study the meiotic behaviour of one carrier of a small supernumerary marker chromosome (sSMC): 47,XY,+mar; one carrier of a Robertsonian translocation (ROB): 45,XY,rob(13;21) (q10;q10); and one carrier of both a sSMC and a ROB: 46,XY,rob(13;21) (q11.1;q11.1),+mar. DESIGN: Case-control study. SETTING: University-affiliated research center and hospital. PATIENT(S): Subfertile men with ROB and sSMC. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The chromosomal origin of the sSMC was assessed by multiplex fluorescence in situ hybridization. The segregation of the ROB and sSMC in sperm and possible interchromosomal effects were examined by fluorescence in situ hybridization. Synapsis, meiotic recombination, and meiotic inactivation were investigated in ejaculate spermatocytes of the 47,XY,+mar and 45,XY,rob(13;21) carriers using immunostaining. RESULT(S): In the 47,XY,+mar and 46,XY,rob(13;21),+mar carriers, the sSMC was found in 13.5% and 11.5 % of sperm, respectively. Analysis of meiotic segregation of chromosome 13 and 21 showed that 91.2% of sperm were normal/balanced in the 46,XY,rob(13;21),+mar case, whereas 88.4% of sperm were normal/balanced in the 45,XY,rob(13;21) case. Interchromosomal effects involving the sex chromosomes were found in both sSMC carriers. Both 47,XY,+mar and 45,XY,rob(13;21) carriers showed decreased global recombination, impaired synapsis, and an association of abnormal chromosomes with the XY body. CONCLUSION(S): Carriers of marker chromosomes produce sperm with markers at frequencies lower than theoretically expected. Carriers of ROB and sSMC showed decreased recombination, impaired synapsis, and association of abnormal chromosomes with the XY body, which may contribute to an interchromosomal effect. Using immunofluorescence techniques to analyze ejaculate-derived spermatocytes from subfertile men provides a novel technique for examining meiosis without the need for a testicular biopsy.

Failure of homologous synapsis and sex-specific reproduction problems.

The prophase of meiosis I ensures the correct segregation of chromosomes to each daughter cell. This includes the pairing, synapsis, and recombination of homologous chromosomes. A subset of chromosomal abnormalities, including translocation and inversion, disturbs these processes, resulting in the failure to complete synapsis. This activates the meiotic pachytene checkpoint, and the gametes are fated to undergo cell cycle arrest and subsequent apoptosis. Spermatogenic cells appear to be more vulnerable to the pachytene checkpoint, and male carriers of chromosomal abnormalities are more susceptible to infertility. In contrast, oocytes tend to bypass the checkpoint and instead generate other problems, such as chromosome imbalance that often leads to recurrent pregnancy loss in female carriers. Recent advances in genetic manipulation technologies have increased our knowledge about the pachytene checkpoint and surveillance systems that detect chromosomal synapsis. This review focuses on the consequences of synapsis failure in humans and provides an overview of the mechanisms involved. We also discuss the sexual dimorphism of the involved pathways that leads to the differences in reproductive outcomes between males and females.