EPIGEFXY · Epigenetic regulation of the sex chromosomes and male infertility

Epigenetic regulation of the sex chromosomes and male infertility

Human infertility affects ~15% of couples, each partner being equally likely to be affected. Sperm anomalies are commonly found in infertile men and the majority are presumed to have a genetic origin; however, the causal genes remain largely unknown with ~75% of male infertility defined as ‘idiopathic’. In mice and men, sperm differentiation requires a high proportion of genes located on the sex chromosomes (i.e. the X and the Y). Expression of these genes is tightly controlled by epigenetic processes which remain to be studied. Alterations in these processes could significantly contribute to male infertility. The aim of our project was to better understand the epigenetic regulation of sex chromosomes during sperm differentiation and its impact on sperm development and male fertility. For this we studied mouse models which present male infertility associated with an epigenetic deregulation of XY gene expression in germ cells. The long-term goal is to determine if abnormal epigenetic regulation of XY genes is at the basis of unexplained cases of human infertility. For this purpose, mouse models which had been developed by the Marie Curie fellow (Julie Cocquet) while a postdoctoral fellow in Paul Burgoyne’s lab (NIMR, London, UK) were transferred to the host lab (Daniel Vaiman’s group at the Cochin Institute, Paris, France). The MC funding has allowed a young researcher (Julie Cocquet, the MC fellow) with expertise in the fields of mouse genetics, molecular and cell biology, to join a very dynamic and supportive environment (the host lab: Daniel Vaiman’s group at the Cochin Institute, Paris, France) which studies epigenetics and human reproductive biology. There, the MC fellow has developed her own research project, ‘the study of the epigenetic regulation of XY genes during sperm differentiation’ (see below for results) and has progressively become independent: she has now been recruited by Inserm, and leads her own team in the host group. The project results were as follow: 1) Study of Slx and Sly genes Slx and Sly are multicopy genes present on the X and Y chromosomes, respectively. We have demonstrated that Slx and Sly are both involved in the epigenetic regulation of XY gene expression during spermiogenesis, but with overall opposite effects: Slx is an activator while Sly is a repressor of XY gene expression. When absent, Sly leads to male infertility due to sperm defects caused by deregulation of XY genes; this deregulation can be compensated by absence of Slx. We have shown that this perturbation of XY gene expression is caused by changes in some of the epigenetic marks associated with the XY chromatin. Interestingly, our findings have also an impact in term of genome evolution: the conflict in which Slx and Sly are involved is probably responsible for the amplification of many X and Y genes and may have played an important role in speciation. These results have been published in PLoS Genetics in 2012. 2) SLY and sperm DNA damage. As part of our collaboration with Dr. Monika Ward s group (Institute for Biogenesis, Hawaii, USA) we have demonstrated that sperm from males deficient in Sly gene present DNA damage and abnormal chromatin compaction. These anomalies are likely caused by the deregulation of XY genes in germ cells induced by the absence of Sly. Many of the XY deregulated genes are good candidates (such as histone variants) to explain these anomalies. These results have been published in Journal of Cell Science in 2013. 3) Identification of novel actors of the regulation of XY gene expression While studying SLY and SLX proteins, we have identified one of their protein partner: SSTY. We have developed an antibody to study its pattern of expression and observed that SSTY is specifically express in differentiating germ cells where it colocalizes with the sex chromosomes. All these observations are in favour of a role of SSTY, together with SLY and SLX, in the epigenetic regulation of XY gene expression. These results are currently under evaluation for publication in a peer-reviewed journal. In parallel, we have also developed co-immunoprecipitation assays followed by mass spectrometry analyses to identify SLY protein partners. We have obtained a list of 21 putative partners which we now need to validate by a complementary approach. 4) In depth characterization of the epigenetic changes associated with sperm differentiation defects Since the beginning of the project, we have set up a technique to purify mouse germ cells via FACS. We have collected fractions of purified round spermatids from wild type and mutant mice with the aim to identify the epigenetic changes (DNA methylation, histones post translational modifications and variants) in abnormal versus normal germ cells. We are currently developing the protocols to study these changes by large-scale analyses. All in all, the funding has been used to identify and characterize novel factors involved in the regulation of XY gene expression during sperm differentiation; these results have been published in international peer-reviewed journals (or are currently in the peer-reviewing process). Importantly, this work is a first step to a better understanding of the epigenetic mechanisms controlling sex chromosome expression during sperm differentiation and their impact on male infertility. We are first studying this process using mouse models and will then apply our knowledge to the study of human pathology. Our long-term aim is to contribute to the identification of the causes of yet unexplained (‘idiopathic’) male infertilities. The European Union parliament has acknowledged that infertility is one of the causes of demographic decline throughout Europe. The growing number of infertility cases means that there is a progressive increase in the need for assisted reproductive technologies (ART). The number of live ART births is ~1% to 4.2% of total live births in the EU, depending on the country. With ART, the genetic and epigenetic causes of infertility may now be transmitted to subsequent generations, even in cases of severe spermiogenic failure. Though it is still controversial, a growing number of reports suggest that the use of ART could lead to a higher incidence of birth and developmental defects. In animals, the epigenetic status of the paternal germ cells has been shown to influence the phenotype of the offspring; alteration in the epigenetic program of the father’s germ cells, in case of spermiogenic defects or in case of exposure to particular environmental factors, would lead to unhealthy offspring. A better knowledge of the epigenetic events occurring during spermatogenesis is therefore of critical importance.

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