Subfertility in young male mice mutant for chromatin remodeler CECR2

Defects in spermatogenesis are an important cause of male infertility. Multiple aspects of spermatogenesis are controlled by chromatin remodelers, including regulating transcription. We previously described mutations in chromatin remodeling gene Cecr2 that resulted in the lethal neural tube defect exencephaly in most mutant mice, and subfertility in mice that were non-penetrant for exencephaly. Here, we show that the severity of male subfertility is dependent on age. Cecr2GT/Del males contain two mutant alleles, one of which is hypomorphic and therefore produces a small amount of protein. These males sire the fewest pups just after sexual maturity (88% fewer than Cecr2+/+ at P42-60) but improve with age (49% fewer than Cecr2+/+ at P81-100), although never completely recovering to Cecr2+/+ (wild type) levels. When young, they also have defects in testis histology, in vivo fertilization frequency, sperm number and motility, and testis weight that show similar improvement with age. Immunostaining of staged seminiferous tubules showed CECR2 in type A, In and B spermatogonia, and less in preleptotene and leptotene spermatocytes. Histological defects were first apparent in Cecr2GT/Del testes at P24, and RNA-seq analysis revealed 387 differentially expressed genes. This included 66 genes on the X chromosome (almost double the number on any other chromosome), all more highly expressed in Cecr2GT/Del testes. This inappropriate expression of X chromosome genes could be caused by a failure of effective meiotic sex chromosome inactivation. We identify several abnormally expressed genes that may contribute to defects in spermatogenesis at P24. Our results support a role for Cecr2 in juvenile spermatogenesis.

On the Origin and Evolution of Drosophila New Genes during Spermatogenesis

The origin of functional new genes is a basic biological process that has significant contribution to organismal diversity. Previous studies in both Drosophila and mammals showed that new genes tend to be expressed in testes and avoid the X chromosome, presumably because of meiotic sex chromosome inactivation (MSCI). Here, we analyze the published single-cell transcriptome data of Drosophila adult testis and find an enrichment of male germline mitotic genes, but an underrepresentation of meiotic genes on the X chromosome. This can be attributed to an excess of autosomal meiotic genes that were derived from their X-linked mitotic progenitors, which provides direct cell-level evidence for MSCI in Drosophila. We reveal that new genes, particularly those produced by retrotransposition, tend to exhibit an expression shift toward late spermatogenesis compared with their parental copies, probably due to the more intensive sperm competition or sexual conflict. Our results dissect the complex factors including age, the origination mechanisms and the chromosomal locations that influence the new gene origination and evolution in testes, and identify new gene cases that show divergent cell-level expression patterns from their progenitors for future functional studies.

The molecular evolution of spermatogenesis across mammals

The testis is a key male reproductive organ that produces gametes through the process of spermatogenesis. Testis morphologies and spermatogenesis evolve rapidly in mammals, presumably due to the evolutionary pressure on males to be reproductively successful. The rapid evolution of the testis was shown to be reflected at the molecular level based on bulk-tissue work, but the molecular evolution of individual spermatogenic cell types across mammalian lineages remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from eleven species that cover the three major mammalian lineages (eutherians, marsupials, egg-laying monotremes) and birds (the evolutionary outgroup), and include seven key primates. Our analyses reveal that the rapid evolution of the testis is driven by accelerated fixation rates of gene expression changes, amino acid altering substitutions, and newly emerged genes in late spermatogenic stages - likely facilitated by reduced pleiotropic constraints, haploid selection, and a transcriptionally permissive chromatin environment. We identify temporal expression changes of individual genes across species, which may have contributed to the emergence of species-specific phenotypes, but also conserved expression programs underlying ancestral spermatogenic processes. Sex chromosome analyses show that genes predominantly expressed in spermatogonia (i.e., germ cells fueling spermatogenesis) and Sertoli cells (i.e., somatic supporting cells) independently accumulated on X chromosomes across mammals during evolution, presumably due to male-beneficial selective forces. Further work uncovered that the process of meiotic sex chromosome inactivation (MSCI) also occurs in monotremes and hence is common to the different mammalian sex chromosome systems, contrary to previous inferences. Thus, the general mechanism of meiotic silencing of unsynapsed chromatin (MSUC), which underlies MSCI, represents an ancestral mammalian feature. Together, our study illuminates the cellular and molecular evolution of mammalian spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Transcriptional progression during meiotic prophase I reveals sex-specific features and X chromosome dynamics in human fetal female germline

During gametogenesis in mammals, meiosis ensures the production of haploid gametes. The timing and length of meiosis to produce female and male gametes differ considerably. In contrast to males, meiotic prophase I in females initiates during development. Hence, the knowledge regarding progression through meiotic prophase I is mainly focused on human male spermatogenesis and female oocyte maturation during adulthood. Therefore, it remains unclear how the different stages of meiotic prophase I between human oogenesis and spermatogenesis compare. Analysis of single-cell transcriptomics data from human fetal germ cells (FGC) allowed us to identify the molecular signatures of female meiotic prophase I stages leptotene, zygotene, pachytene and diplotene. We have compared those between male and female germ cells in similar stages of meiotic prophase I and revealed conserved and specific features between sexes. We identified not only key players involved in the process of meiosis, but also highlighted the molecular components that could be responsible for changes in cellular morphology that occur during this developmental period, when the female FGC acquire their typical (sex-specific) oocyte shape as well as sex-differences in the regulation of DNA methylation. Analysis of X-linked expression between sexes during meiotic prophase I suggested a transient X-linked enrichment during female pachytene, that contrasts with the meiotic sex chromosome inactivation in males. Our study of the events that take place during meiotic prophase I provide a better understanding not only of female meiosis during development, but also highlights biomarkers that can be used to study infertility and offers insights in germline sex dimorphism in humans.

Epigenetic Dysregulation of Mammalian Male Meiosis Caused by Interference of Recombination and Synapsis

Meiosis involves a series of specific chromosome events, namely homologous synapsis, recombination, and segregation. Disruption of either recombination or synapsis in mammals results in the interruption of meiosis progression during the first meiotic prophase. This is usually accompanied by a defective transcriptional inactivation of the X and Y chromosomes, which triggers a meiosis breakdown in many mutant models. However, epigenetic changes and transcriptional regulation are also expected to affect autosomes. In this work, we studied the dynamics of epigenetic markers related to chromatin silencing, transcriptional regulation, and meiotic sex chromosome inactivation throughout meiosis in knockout mice for genes encoding for recombination proteins SPO11, DMC1, HOP2 and MLH1, and the synaptonemal complex proteins SYCP1 and SYCP3. These models are defective in recombination and/or synapsis and promote apoptosis at different stages of progression. Our results indicate that impairment of recombination and synapsis alter the dynamics and localization pattern of epigenetic marks, as well as the transcriptional regulation of both autosomes and sex chromosomes throughout prophase-I progression. We also observed that the morphological progression of spermatocytes throughout meiosis and the dynamics of epigenetic marks are processes that can be desynchronized upon synapsis or recombination alteration. Moreover, we detected an overlap of early and late epigenetic signatures in most mutants, indicating that the normal epigenetic transitions are disrupted. This can alter the transcriptional shift that occurs in spermatocytes in mid prophase-I and suggest that the epigenetic regulation of sex chromosomes, but also of autosomes, is an important factor in the impairment of meiosis progression in mammals.

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.

Epigenetics drive the evolution of sex chromosomes in animals and plants

We review how epigenetics affect sex chromosome evolution in animals and plants. In a few species, sex is determined epigenetically through the action of Y-encoded small RNAs. Epigenetics is also responsible for changing the sex of individuals through time, even in species that carry sex chromosomes, and could favour species adaptation through breeding system plasticity. The Y chromosome accumulates repeats that become epigenetically silenced which leads to an epigenetic conflict with the expression of Y genes and could accelerate Y degeneration. Y heterochromatin can be lost through ageing, which activates transposable elements and lowers male longevity. Y chromosome degeneration has led to the evolution of meiotic sex chromosome inactivation in eutherians (placentals) and marsupials, and dosage compensation mechanisms in animals and plants. X-inactivation convergently evolved in eutherians and marsupials via two independently evolved non-coding RNAs. In Drosophila , male X upregulation by the male specific lethal (MSL) complex can spread to neo-X chromosomes through the transposition of transposable elements that carry an MSL-binding motif. We discuss similarities and possible differences between plants and animals and suggest future directions for this dynamic field of research. This article is part of the theme issue ‘How does epigenetics influence the course of evolution?’

Phosphoproteomics of ATR Signaling in Prophase I of Mouse Meiosis

During mammalian meiosis, the ATR kinase plays crucial roles in the coordination of DNA repair, meiotic sex chromosome inactivation and checkpoint signaling. Despite the importance of ATR in meiosis, the meiotic ATR signaling network remains largely unknown. Here we defined ATR signaling during prophase I in mice. Quantitative analysis of phosphoproteomes obtained after genetic ablation of the ATR-activating 9-1-1 complex or chemical inhibition of ATR revealed over 12,000 phosphorylation sites, of which 863 phosphorylation sites were dependent on both 9-1-1 and ATR. ATR and 9-1-1-dependent signaling was enriched for S/T-Q and S/T-X-X-K motifs and included proteins involved in DNA damage signaling, DNA repair, and piRNA and mRNA metabolism. We find that ATR targets the RNA processing factors SETX and RANBP3 and regulate their localization to the sex body. Overall, our analysis establishes a comprehensive map of ATR signaling in spermatocytes and highlights potential meiotic-specific actions of ATR during prophase I.