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We identified DMXL2 from RNA-sequencing data as a gene preferentially expressed in differentiating-ovaries in the goat species. In order to determine its role during ovarian morphogenesis, we investigated functional studies in mice. Dmxl2 invalidation (KO) is lethal within the few hours following birth. The analysis of its expression in several tissues let us show that the heart, olfactory mucosa and brain expressed Dmxl2 during development. Our results further suggest that the lethality may be due to an alteration of the synaptic transmission.Besides, both male and female gonads express Dmxl2. Notably in the ovary, its expression increases few days before follicle formation. We show that DMXL2 protein is dynamically expressed in germ cells and granulosa cells; DMXL2 is strongly detected in the oocytes whereas its expression in the granulosa cells fluctuates depending on the stage of folliculogenesis. To study Dmxl2 implication during follicle formation, and to bypass the issue of lethality at birth, we performed allograft of KO ovaries on nude recipient mice. KO grafts show an alteration in follicle formation, followed by ovarian dysgenesis.To conclude, our work shows that Dmxl2 is involved in follicle formation in mice, and makes it a new candidate to explain some cases of premature ovarian failure. The conditional knock-out of Dmxl2 in germ cells or in granulosa cells is in progress in the laboratory (please refer to Clara Gobé’s poster) and will permit to precise its specific role in each cell types.

Maeva Elzaiat is the recipient of a Ph.D. fellowship from Université Paris Sud-11 and from the Fondation pour la Recherche Médicale (FDT20140930877)Aurelie Allais Bonnet is the recipient of a fellowship from ANR-09-GENM-009-03 GENIDOVà reprendre pour la pagination officielle; FOXL2 loss of function in goats leads to the early transdifferentiation of ovaries into testes, then to the full sex reversal of XX homozygous mutants. By contrast, Foxl2 loss of function in mice induces an arrest of follicle formation after birth, followed by complete female sterility. In order to understand the molecular role of FOXL2 during ovarian differentiation in the goat species, putative FOXL2 target genes were determined at the earliest stage of gonadal sex-specific differentiation by comparing the mRNA profiles of XX gonads expressing the FOXL2 protein or not. Of these 163 deregulated genes, around two-thirds corresponded to testicular genes that were up-regulated when FOXL2 was absent, and only 19 represented female-associated genes, down-regulated in the absence of FOXL2. FOXL2 should therefore be viewed as an antitestis gene rather than as a female-promoting gene. In particular, the key testis-determining gene DMRT1 was found to be up-regulated ahead of SOX9, thus suggesting in goats that SOX9 primary up-regulation may require DMRT1. Overall, our results equated to FOXL2 being an antitestis gene, allowing us to propose an alternative model for the sex-determination process in goats that differs slightly from that demonstrated in mice.

In goats, the PIS (Polled Intersex Syndrome) mutation results in hornlessness of heterozygous (PIS+/-) and homozygous (PIS-/-) animals of both sexes, and to a female-to-male sex-reversal in XX animals homozygous for the mutation (XX PIS-/-). The PIS mutation is a regulatory mutation which corresponds to a 11.7 kb-deletion on goat chromosome 1, inducing a long-range transcriptional effect on FOXL2 and various non-coding RNAs [3-5]. For several years, we have accumulated evidences that these ncRNAs are involved in FOXL2 expression regulation [5, 1; see Pannetier et al. abstract], and we consider now that the sole "acting" gene of the PIS locus is FOXL2. Thus, in goats, the transcriptional silencing of FOXL2 in the early-developing XX PIS-/- gonad is responsible for testis formation in a female genetic context. In order to highlight new crucial genes for ovarian differentiation that could be FOXL2 target genes, we performed a high-throughput sequencing of transcripts from male (XY PIS+/+), female (XX PIS+/+) and sex-reversed (XX PIS-/-) gonads at 36 days post-coïtum (dpc), the key stage of gonadic switch in goats. At this stage, we previously showed that in the sex-reversed gonad, FOXL2 was not detected as expected, but SOX9 and in theory other male genes were not up-regulated yet [4]. Consequently, mis-regulated genes between XX sex-reversed gonad and normal ovary at this stage may be FOXL2 target genes and crucial actors of ovarian differentiation. The RNA-sequencing results confirmed by RT-qPCR, showed that FOXL2 inhibits testicular differentiation on top of being an activator of ovarian development. This inhibition takes place at three levels; first, FOXL2 represses androgens production, then, it protects XX germ cells fate by inhibiting CYP26b1 expression, and finally it represses Sertoli cells differentiation, by inhibiting the DMRT1 gene that should lead to SOX9 up-regulation. Indeed, our data show that DMRT1 is up-regulated in 36 dpc XX sex-reversed gonads whereas SOX9 remains expressed as in normal ovaries. As DMRT1 is considered as the testis determining factor in at least three species of non-mammalian vertebrates where SRY is absent [2, 7, 8], and because DMRT1 haplo-insufficiency is involved in XY male-to-female sex reversal in humans [6], its up-regulation in XX sex-reversed gonads suggests its primary involvement in SOX9 up-regulation during the PIS sex-reversal process in goats.

DNA methylation of cytosines is a critical epigenetic modification in mammals that plays crucial roles in transcriptional regulation, chromatin remodelling and genomic imprinting. Dynamic erasure and reestablishment of DNA methylation marks are required for spermatogenesis and the normal function of mature sperm. DNA methylation is catalysed by DNA methyltransferase enzymes (DNMT) providing either maintenance (DNMT1) or de novo (DNMT3A/B/L) DNA methylation processes. The DNA methylation dynamics during spermatogenesis has been previously described in mice and humans but nothing is known in productive livestock. However, these investigations in ruminants could be helpful to determine epigenetic events likely to be crucial for male fertility. Interestingly, a recent study from our laboratory pointed DNA undermethylation of bull spermatozoa compared to other mammals such as humans, mice, sheep or goats [1]. This result raises the question of the dynamic of DNA methylation in bovine male germline. We thus propose to define and compare the methylation dynamics of bovine and caprine germ cells during spermatogenesis. Analyses of 5mC immunohistochemistry were performed on adult testis sections to illustrate the presence of DNA methylation marks in various spermatogenic cells. As purification of the different spermatogenic populations is required to characterize DNA methylation more precisely, we firstly developed a flow cytometry-based method (Hoechtst-FACS) for cell sorting from goat testis. Fraction enrichments were evaluated using stage-specific markers by real time qPCR analyses. In addition, de novo DNA methyltransferase expression was studied on the different fractions of purified spermatogenic cells, giving some clues on DNA methylation dynamics. In the future, spermatogenic cells will be purified from both caprine and bovine testes, and together with gene expression analyses (RNA-sequencing), DNA methylation will be determined for each cell types by Reduced-Representation Bisulfite Sequencing (RRBS)