Faculty of Medicine and Health Sciences, Department of Basic Medical Sciences, Ghent University, Ghent, Belgium.
Anat Rec (Hoboken). 2010 May;293(5):890-9. doi: 10.1002/ar.21100.
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.
本研究探讨了钙敏感受体（CaR）在介导日本鹌鹑（Coturnix coturnix japonica）卵泡颗粒细胞（GC）存活中的潜在作用。CaR 的免疫反应性在鹌鹑排卵前卵泡的 GC 中以及排卵后 GC 层的残余物中均有显示。相反，免疫细胞化学在较小的未分化卵泡的颗粒细胞中无法检测到 CaR。免疫印迹证实了 CaR 存在于即将排卵的卵泡中，受体被鉴定为 115-125 kDa 的蛋白质。将不同的 CaR 激动剂添加到培养的颗粒细胞外植体中 24 小时，可抑制单独或与 C（8）-神经酰胺添加联合使用时促性腺激素撤退引起的细胞凋亡。此外，CaR 的特异性正变构调节剂 R-568 不仅抑制细胞凋亡，而且增加了培养的颗粒细胞外植体中每个观察视野的 GC 数量。这种观察结果可能归因于 GC 增殖的增加，而是由于组织结构更紧凑，包括缝隙连接蛋白-43 间隙连接蛋白的明显分布模式。在存在特定的磷脂酰肌醇-3 激酶抑制剂 LY294002 的情况下孵育会增加 GC 凋亡，表明该途径参与 GC 存活信号转导。然而，LY294002 诱导的凋亡在与 R-568 孵育时明显减弱，表明其他途径可能是 CaR 激动剂介导的存活的主要贡献者。我们提供了 CaR 存在于排卵前颗粒细胞外植体中的直接证据，并表明 CaR 在卵泡选择中起关键作用。
