Approximately one in ten men suffers from infertility most often resulting from an abnormal spermatogenesis. The cause, whether genetic or environmental, remains often unknown, with ~50% of male infertility defined as ‘idiopathic’. Spermatogenesis, the process by which spermatozoa develop from immature germ cells in the seminiferous tubules of the testis, is associated with a highly dynamic genetic program and extensive chromatin changes.

Our group studies spermatogenesis at the gene and the chromatin levels to identify and characterize novel regulators/pathways required for sperm differentiation and male fertility. Our goal is to produce fundamental knowledge on the molecular mechanisms controlling spermatogenesis in order to better understand the genetic and epigenetic causes of male infertility.

Our project also aims at studying the impact of spermatozoa epigenetic program on reproductive efficiency, embryo development and progeny’s health. Indeed it is now well-known that, upon fertilization, the sperm cell contributes to the embryo with more than its DNA. Epigenetic information (chromatin, RNA molecules, DNA modifications) is also transmitted to the embryo and could affect embryo development and offspring health, in case of deregulation. A better characterization of the molecular mechanisms controlling spermatogenesis is needed to better understand their long-term impact.

Spermatogenesis is a very dynamic differentiation process in terms of gene regulation and chromatin remodeling, and an excellent model to study these essential biological processes. Our findings will therefore contribute to better understand how gene expression and chromatin are regulated, as a whole.

To address these questions, we combine in vivo and in vitro models and use molecular and cell biology, biochemistry and reproductive biology techniques. In particular we perform large-scale (‘omics) analyses such as transcriptomics, epigenetics and proteomics

Gene regulation and chromatin remodeling during spermiogenesis. Impact on male reproduction, progeny development and health

During the last stage of spermatogenesis (i.e. spermiogenesis) post-meiotic haploid cells called spermatids undergo profound morphological and functional changes to become spermatozoa (Blanco & Cocquet, 2019). They acquire their specific shape through the loss of most of their cytoplasm, the biogenesis of a flagellum and an acrosome, and the extreme compaction of their nucleus and chromatin. The high level of sperm chromatin compaction results from the replacement of most histones by protamines. It is essential for male fertility and to preserve paternal genome integrity until fertilization.

In the last decade, we have extensively studied two multicopy genes, named Slx/Slxl1 and Sly, which are respectively encoded by the mouse X and Y chromosomes. We have shown that absence/knock-down of Sly leads to a wide range of spermiogenesis defects, such as deformed spermheads, reduced motility, abnormal chromatin compaction and DNA damage, which cause male infertility. Those defects are associated with deregulation of hundreds of sex chromosome-encoded genes, as well as a smaller portion of autosomal genes (such as the genes of the Speer family) (Cocquet et al. PLos Biol 2009; Riel et al. J Cell Sci 2013; Moretti et al. Cell Death & Diff 2017 ). Interestingly, we have observed that Slx/Slxl1, the X-linked homologs of Sly, have a role opposite to that of Sly on gene expression (Cocquet et al. PLoS Genet 2012).

From Moretti et al. 2017. Localization of the histone methyl transferase DOT1L (green) in mouse testis section at different stages of sperm differentiation.
DAPI (in blue) stains nuclei and Lectin-PNA (in red) marks the acrosomes.

Using Slx/Slxl1 and Sly as entry points, we have identified novel actors of gene/chromatin regulation during spermiogenesis. By ChIP seq, we have shown that SLY protein is enriched at the promoter of thousands of genes involved in gene expression, chromatin regulation, and the ubiquitin pathway. Among those genes is Dot1l, which encodes the only known H3K79 methyltransferase; both Dot1l expression and H3K79 methylation level are downregulated Sly-deficient in spermatids. In parallel, we have used co-immunoprecipitation assays followed by mass spectrometry to identify SLY protein partners, and found that SLY interacts with SMRT/NcoR, a protein complex involved in transcriptional regulation, and which appears to be relevant to gene regulation during spermiogenesis (Moretti et al. Cell Death & Diff 2017).

From Moretti et al. 2017. Model presenting the mechanism by which SLY controls gene expression and chromatin remodeling during sperm differentiation. In WT round spermatids (left panel), SLY (in blue) interacts with the SMRT/N-CoR complex (which comprises TBL1XR1, TBL1X, NCOR1 and HDAC3) and is located at the start of genes involved in gene regulation, chromatin regulation and the ubiquitin pathway. In particular, SLY directly controls the expression of X-chromosome-encoded genes coding for H2.A variants (such as H2A.B3) and of the H3K79 methyltransferase DOT1L. In elongating spermatids, there is a wave of H3K79 dimethylation (orange circles) and of histone H4 acetylation (green circles); those modifications are expected to be a prerequisite to the efficient removal of nucleosomes (light pink oval) and replacement by protamines (purple oval), a process which is required to achieve optimal compaction of the spermatozoa nucleus. When SLY is knocked down (right panel), X-encoded H2.A variants are upregulated and more incorporated in the spermatid chromatin, while DOT1L is downregulated. DOT1L downregulation leads to a decrease in dimethylated H3K79 and acetylated histone H4 in elongating spermatids. Alterations in the spermatid chromatin structure affect the replacement of nucleosomes by protamines and lead to a higher proportion of nucleosomes and a decreased proportion of protamines. As a result, Sly-deficient spermatozoa are abnormally shaped, less compact and present a higher susceptibility to DNA damage than WT spermatozoa.

Schéma 2 projet Régulation (épi)génétique au cours de la spermatogenèse équipe Vaiman

Molecular mechanism of the genomic conflict between the mouse sex chromosomes - competition between Slx/Slxl1 and Sly.

While studying the molecular roles of Slx/Slxl1 and Sly, we discovered that they are involved in an intragenomic conflict that causes segregation distortion in addition to male infertility. Indeed, Slx/Slxl1 and Sly are "selfish" genes – each promoting its own transmission to the detriment of the other. Slx/Slxl1 and Sly genes are present dozens of times on the mouse X and Y chromosomes, respectively. Their number of copies is not the same depending on the species and has co-evolved during rodent evolution. The “conflict” for transmission in which Slx/Slxl1 and Sly are engaged led to a genetic arms race in which the amplification of the number of copies of one is counterbalanced by the amplification of the other. An imbalance in the number of copies of one versus the other (obtained by genetic mutants inducing a "knock-down" of Slx/Slxl1 or Sly) produces a skewed sex ratio of the progeny associated with hypofertility, or infertility, if copy number ratio is severely imbalanced (Cocquet et al. PLoS Genet 2012).

Our project aims at studying the competition at the molecular level and identifying other actors of the conflict.

We have recently shown that SLX/SLXL1 and SLY proteins are present at the promoter of thousands of spermatid genes, and that the knockdown of Sly increases the presence of SLX/SLXL1 at these promoters (Moretti et al. MBE 2020). Competition at the promoter level is mediated via SSTY protein, with which SLX/SLXL1 or SLY (but not the 2 together) interacts. Interestingly, SSTY belongs to a family of proteins, SPINDLIN, which recognizes the chromatin mark H3K4me3, characteristic of the promoter of active (expressed) genes. When SLY predominates, the genes with which it is associated are under-expressed, conversely when SLX/SLXL1 predominates these genes are over-expressed. To complete the elucidation of the molecular mechanism, we found that SLY, but not SLX/SLXL1, interacts with proteins of the SMRT/N-Cor complex that repress transcription; in case of Sly knockdown, this complex is less recruited at the promoters of SLX/SLY target genes resulting in upregulation. This result explains, at least in part, the fact that SLY acts as a repressor and SLX/SLXL1, as an activator.

From an evolutionary point of view, it is important to note that Ssty gene is itself multicopy and carried by the Y chromosome. Besides, many of SLX/SLXL1, SLY and SSTY target genes are also multicopy genes that were co-amplified during Muroid evolution, particularly Speer/Takusan genes amplified >150 times on chromosome 14.

Our work is an important step towards elucidating the molecular mechanism behind the competition between X and Y genes - a genomic conflict that has influenced genome organization and evolution of several rodents. This type of phenomenon is predicted to be quite widespread, and create hybrid incompatibility, lineage divergence and eventually speciation.

Figure: From Cocquet et al. PLoS Genet 2012. Localization of SLX and SLY proteins in spermatids. SLX and SLY proteins (in green, top and bottom panel respectively) co-localize with the X or Y chromosome (in yellow, X or Y paint) and with Speer gene cluster (in red, DNA FISH). DAPI (blue) was used to stain nuclei.