Gap junctional communication and connexin43 expression in relation to apoptotic cell death and survival of granulosa cells.

Ovarian follicular atresia in all vertebrates is mediated via apoptosis that is initiated in the granulosa cell layer. Here we investigated the relation between connexin expression, cell coupling, and apoptosis in avian granulosa cells. Results from qualitative and quantitative immunocytochemical analysis and Western blotting of connexin43 (Cx43) and electron microscopic observations of gap junctions were compared with functional data on gap junctional coupling obtained by fluorescence recovery after photobleaching in four experimental groups: a control group of freshly isolated granulosa cells, 24-hr serum-free cultures as the apoptosis-inducing condition, and two other groups in which apoptosis was inhibited by either hormone substitution or exposure to elevated extracellular calcium. Our work shows that apoptosis induction in granulosa cells is accompanied by an increased level of cell coupling and that decreasing cell coupling with the gap junction blocker alpha-glycyrrhetinic acid dose-dependently inhibits apoptosis. The level of Cx43 expression was inversely related to the apoptotic index, suggesting that Cx43 expression plays a role in granulosa cell survival. Our study supports the hypothesis that gap junctional coupling plays a role in propagating a cell death message and suggests a role for Cx43 expression per se in granulosa cell survival.

Expression of calcium-sensing receptor in quail granulosa explants: a key to survival during folliculogenesis.

This study investigated the potential role of the calcium-sensing receptor (CaR) in mediating survival of granulosa cells (GCs) in follicles of the Japanese quail (Coturnix coturnix japonica). Immunoreactivity of CaR was shown in GCs of quail preovulatory follicles as well as in the remnants of the GC layer after ovulation. Conversely, the CaR could not be detected by immunocytochemistry in the granulosa of smaller undifferentiated follicles. The presence of CaR in follicles destined to ovulate was confirmed by immunoblot and the receptor was identified as a protein of 115-125 kDa. Addition of different CaR agonists to granulosa explants in culture for 24 hr caused inhibition of apoptosis elicited by gonadotropin withdrawal on its own or in combination with C(8)-ceramide addition. Furthermore, R-568, a specific, positive allosteric modulator of CaR, not only inhibited apoptosis but also increased GC number per viewing field in cultured granulosa explants. This observation could be attributed not to a rise in GC proliferation but to a more compact tissue structure, including a distinct distribution pattern of connexin-43 gap junction proteins. Incubation in the presence of LY294002, a specific phosphatidylinositol-3 kinase inhibitor, increased GC apoptosis, indicating that this pathway is involved in GC survival signaling. However, LY294002-induced apoptosis was considerably attenuated by incubation with R-568, indicating that other pathways might be major contributors to the survival mediated by CaR agonists. We provide direct evidence for the presence of CaR in preovulatory granulosa explants and suggest a pivotal role for CaR in follicle selection.