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 C8‐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. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.