Reproduction Abstracts

Context: DMRTA2 belongs to a family of genes coding for proteins containing a DM-domain that are conserved among vertebrates and widely involved in gonadal differentiation. We recently identified Dmrta2 gene expression through transcriptome analysis performed in murine female embryonic germ cells and retrieved its expression in human fetal ovaries. The role of DMRTA2 is poorly documented and we thus conducted this study to clarify its implication in human fetal ovaries.

Material and methods: Human fetal ovaries (8 – 11 weeks post fertilization) were harvested from material available following legally induced abortions and were grafted in two immunodeficient NMRI:Nu/Nu mice. Recipient mice were treated with siRNA targeting specifically the sequence of human DMRTA2.

Results and discussion: The xenograft model did recapitulate all the stages of female germ cell development with no overt change when compared to ungrafted ovaries. After 10 days of treatment with siRNA, we observed of robust inhibition of DMRTA2. This inhibition did not alter germ cell density or apoptosis. RT-qPCR and immunostaining analyses indicated that the expression of markers of undifferentiated germ cells was unchanged while the expression of markers of differentiating, pre-meiotic and meiotic germ cells were systematically decreased. This study reveals for the first time the requirement of DMRTA2 for the normal development of human female embryonic germ cells. DMRTA2 appears required to allow the differentiation of oogonia shortly prior their entry into meiosis. Additionally, we set up an original model of xenograft that should prove useful for future investigations of the human germline development.

The mammalian target of rapamycin (mTOR) signaling pathway functions as a central regulator of cell growth, proliferation, and survival. We previously reported that during meiotic maturation, the expression levels of mTOR in oocytes remain similar from the germinal vesicle (GV) stage to metaphase II (MII). To investigate the role played by mTOR during meiotic resumption, we cultured murine cumulus – oocyte complexes (COCs) in the presence of mTOR inhibitors. mTOR expression was detected in the cumulus cells. The COCs were cultured for 18 h in a medium containing dbcAMP with or without the mTOR inhibitor PI-103 or rapamycin. We observed cumulus expansion but the oocytes were arrested at the GV stage. These oocytes were then transferred to fresh maturation medium containing FSH with or without an mTOR inhibitor before culturing for 8 more hours. We found premature development of the first polar body in oocytes treated with the mTOR inhibitor. This result suggests that mTOR inhibition induces early progression of oocytes. Further, when GV-stage oocytes were cultured for 18 h in maturation medium lacking FSH but containing the mTOR inhibitor PI-103 or rapamycin, the cumulus cells expanded and the first polar body successfully developed. In addition, we found that the mRNA expression of hyaluronan synthase (HAS) in the cumulus cells increased after treatment with the mTOR inhibitor. In conclusion, our data suggest a role for mTOR signaling during cumulus expansion and meiotic maturation in mice. In the presence of an mTOR inhibitor, cumulus expansion occurred and meiotic maturation progressed without gonadotropin stimulation.