The oocyte regulates GDF9 and BMP15 expression in bovine cumulus cells

Unlike in the mouse, the presence of the oocyte is not required for expansion of the cumulus-oocyte complex (COC) in cattle. However, we have previously demonstrated that oocyte secreted factors, although not absolutely required, do enhance cumulus expansion in cattle. Major oocyte secreted factors regulating COC maturation are members of the TGFβ family, BMP15 and GDF9. Previous data suggest that BMP15 and GDF9 are expressed in bovine cumulus cells. Thus, the existence of an intra-cumulus loop mediated by TGFβ signalling in cattle constitutes an interesting hypothesis. In this study, we tested the hypothesis that BMP15 and GDF9 are expressed in cumulus cells during IVM in cattle and that mRNA abundance is regulated by the oocyte.COCs were aspirated from follicles 3-8 mm in diameter and COCs (grade 1 or 2) were divided in three groups: intact COCs (COC), oocytectomized COCs (OOX), and OOX plus denuded oocytes (DO). Oocytectomy was performed by aspiration of the ooplasm with a micromanipulator. COCs were matured in groups of 20 for 4 or 22 hours (n = 4/time). IVM was performed in TCM199 with Earl’s salts supplemented with either FSH (1µg/mL) or AREG (100 µg/mL), 0.4% BSA, 22 µg/Ml sodium pyruvate, 75µg/mL of amikacin at 38,5°C and 5.5%CO2 in humid atmosphere. Abundance of mRNA encoding BMP15 and GDF9 in cumulus cells was assessed by real-time RT-PCR using Power SybrGreen (LifeTech®) and CYC-A as the housekeeping gene. Effects of treatments were analysed by ANOVA and groups were compared with the Tukey test.Expression of BMP15 and GDF9 mRNA was detected in bovine cumulus cells. PCR cycle numbers were on average 29 and 28 for BMP15 and GDF9, respectively, at 4 hours of culture, and 29 and 31, at 22 hours of culture. Oocytectomy significantly reduced BMP15 and GDF9 mRNA expression in COCs cultured with AREG for 4 or 22 hours, but not with FSH. Co-culture of OOX with DO restored BMP15 and GDF9 expression . In conclusion, these data suggest that BMP15 and GDF9 mRNA expression in bovine cumulus cells is regulated by the oocyte. This effect is abolished by supra-physiological levesl of FSH used in IVM in cattle.