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Infertility is estimated to affect 10-15% of couples in the human population and in 50% of these the problem is attributed to the male partner. Numerous causes are already known, but for about half of cases, a reason behind the condition is still to be found. Familial studies have placed the genetic contribution to male infertility at about 60% in the cases studied. This has led to the intensification of the search for fertility genes. In this context, a candidate gene approach has been chosen to identify novel genes involved in spermatogenesis and perhaps in fertility. Expression profiling of testis transcripts during the first wave of spermatogenesis have been obtained by microarray chip technology in our laboratory. This relied on the comparison of gene expression levels not only between adult and juvenile wild-type mice, but also between juvenile wild-type mice and juvenile Dazl-/-
knockout mice, the latter being a model for infertility. The aim of this thesis was to characterise three novel genes which increased expression during normal spermatogenic development but which remained at basal levels in the infertility mouse model. These
genes were named Mouse Maelstrom (mMael), Central Element Synaptonemal Complex protein 1 (Cesc1) and SYnaptonemal complex Central Element protein 1 (Syce1).
Mouse Maelstrom is a gene predicted to contain twelve exons. Northern blotting and RT-PCR have shown mMael to be expressed in testis and very weakly in ovary, which indicates a role in gametogenesis. mMAEL protein sequence is predicted to contain an HMG box domain, typically found in non-histone components of chromatin and transcription factors, and one or two novel globular domains. The domains are conserved across species with orthologues found in vertebrates and invertebrates. The orthologue found in Drosophila melanogaster, Maelstrom, has been characterised and shown to be a component of the nuage, a cluster of perinuclear granules present in the germ cells. The
human orthologue is also expressed in the testis. In mouse testis, mMael transcripts are found in germ cells, mainly from meiotic cells where the gene is most expressed, to round spermatids. At the protein level, in agreement with the data obtained for the mRNA, mMAEL is highly expressed in the nucleus and cytoplasm of meiotic cells, but also round and early elongating spermatids. As meiosis progresses through the pachytene stage, the protein accumulates in what appears to be the XY body and in perinuclear granules in the round spermatids. Since the orthologue in Drosophila localises to the nuage, it is possible that the granules are chromatoid bodies, the equivalent structure found in mouse and which is composed of proteins and RNA. Also, yeast-two-hybrid analysis has identified a component of the chromatoid body, RanBPM, as an interactor of mMAEL. RanBPM in turn, viii
interacts with MVH, the mouse orthologue of Drosophila Vasa, which is required for Maelstrom localisation to the nuage. The same yeast-two-hybrid assay identified proteins involved in gene silencing mechanisms, mSin3B and SNF5, as mMAEL interactors.
Considering the localisation of mMAEL to what appears to be the XY body and the fact that Drosophila mutants for Maelstrom fail to form the karyosome, mMAEL could also be involved in sex chromatin repression. Taken together, these data indicate that MAEL may have a conserved function across species, which could be related not only to RNA metabolism but to heterochromatin formation as well.
Cesc1 and Syce1 are two genes highly expressed in and specific to testis respectively. Both genes encode proteins that lack known functional domains, but contain regions of coiled-coil and an alpha-hairpin fold, in the case of CESC1. CESC1 is conserved in vertebrates but SYCE1 orthologues are only found in mammals. In testis, both proteins are restricted to the nucleus of meiotic cells, specifically to the synaptonemal complex (SC). Immunofluorescent co-localisation of CESC1 or SYCE1 with a meiotic cohesin that marks the axial/lateral elements of the SC, STAG3, demonstrated that these proteins are only found in the synapsed regions of the SC. Furthermore, their localisation overlaps with SYCP1 distribution, a component of the transverse filaments of the SC. Ultrastructural localisation has shown that CESC1 and SYCE1 localise to the central element of the SC, which was never observed for any other protein. SYCP1, being a component of the transverse filaments localises to the entire central region, rather than the central element alone. Immunoprecipitation, in vitro pull-down assays and yeast-two-hybrid analysis demonstrated the interaction of CESC1 and SYCE1 with themselves, with each other and with SYCP1.
Moreover, co-expression studies in COS-7 cells suggested that SYCP1 could be recruiting both proteins to the central element of the SC. This was further supported by CESC1 and SYCE1 mimicking of SYCP1 distribution in Sycp3 mutant spermatocytes and oocytes. Finally, in Sycp1 mutant spermatocytes, SYCE1 is delocalised from the SC. Together, these results show that these two novel proteins are components of the central element of the SC and that they form a complex with SYCP1. Additionally, they suggest that SYCP1 recruits CESC1 and SYCE1 to the central element. It is hypothesised that CESC1 and SYCE1 could have a role in SC assembly, stability and recombination.
In summary, the data presented in this thesis reports on the characterisation of three novel genes that are involved in different processes of spermatogenesis. Future efforts should determine if mutations in the human orthologues of these genes could also be
responsible for human infertility.