PARENT SESSION

(79) OOCYTE-SECRETED FACTORS PREVENT BOVINE CUMULUS APOPTOSIS: PARACRINE REGULATION BY BONE MORPHOGENETIC PROTEINS.

Hussein, Tamer¹, Froiland, David¹, Thompson, Jeremy¹, Gilchrist, Robert¹, ¹ University of Adelaide, Research Centre for Reproductive Health, Adelaide, SA, Australia

ABSTRACT- Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell (CC) functions. Characteristically, CC have a low incidence of apoptosis and we proposed that this is due to oocyte-secreted factors (OSF) acting in an anti-apoptotic manner. Bovine cumulus-oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytectomized (OOX) by microsurgical removal of the oocyte. OOX were treated with doses of either denuded oocytes (DO) or various growth factors for 24h (± rFSH; 0.1 IU/ml). Proportions of apoptotic CC were assessed using TUNEL and laser confocal scanning microscopy followed by image analysis. Quantification of Bcl-2 and Bax proteins in OOX was undertaken by Western analysis. Oocyte removal led to a significant increase in CC apoptosis compared to COC controls (35% vs. 9% TUNEL +ve, respectively; P<0.001). Levels of OOX apoptosis were significantly reversed (P<0.001) in a dose-dependent manner when co-cultured with DO. Furthermore, the anti-apoptotic effect of OSF followed a gradient from the site of the oocyte(s). Growth differentiation factor 9 (GDF9) had no significant effect on CC apoptosis. In contrast, CC apoptosis was significantly (P<0.001) reduced by bone morphogenetic proteins (BMP) 15, 6 or 7. Accordingly, levels of anti-apoptotic Bcl-2 were high in OOX+DO and OOX+BMP15, and low with OOX+GDF9 or OOX alone, whereas the reverse was observed for pro-apoptotic Bax. DO, BMP15 and BMP6 were also able to protect CC from undergoing apoptosis induced by staurosporine. FSH partially prevented apoptosis in all treatment groups (P<0.001). Follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined blocked 50% of the anti-apoptotic actions of DO. These results are the first to demonstrate that OSF, and particularly BMP15 and BMP6, maintain the low incidence of CC apoptosis by establishing a localized gradient of bone morphogenetic proteins.

KEY WORDS: oocyte-secreted factors, bone morphogenetic protein 15, cumulus cell, apoptosis