PROGESTERONE REGULATION OF GRANULOSA CELL FUNCTION

这项提议的总体目标是检验这样一个假设 黄体酮通过激活颗粒细胞(GC)调节颗粒细胞功能 具有某些GABAA受体功能的膜受体。我们有 最近发现黄体酮具有抗细胞凋亡和抗有丝分裂的作用 这种作用可被GABA拮抗剂荷包牡丹碱减弱。此外， 黄体酮的作用可被针对黄体酮的抗体减弱 核黄体酮受体的配体结合域。什么时候 在蛋白质印迹分析中，该抗体识别60 kDa 卵巢和GC裂解物中的蛋白质。这个60 kDa的蛋白质也是 在卵巢裂解物的膜制剂中检测到。最后， 孕激素受体抗体检测到一个60 kDa的蛋白 用抗GABAA受体抗体进行免疫沉淀。这些 研究结果支持我们的假设，即黄体酮调节其作用 在卵巢内通过膜型受体，该受体具有分子 体重约60 kDa，并具有一些药理和 GABAA受体的免疫学特性。在这份提案中，我们 将通过一系列实验直接验证这一假设 使用1)药物来定义黄体酮的功能，2) 竞争性孕酮测定法和Scatchard曲线图分析 孕酮与GCs结合的特异性，3)蛋白质印迹和 免疫细胞化学检测该推定基因的表达模式 卵泡生长过程中的60 kDa膜孕酮受体 分化和闭锁。 有人认为黄体酮通过这个假定的60 kDa起作用。 膜孕酮受体对GC有丝分裂和活性的调节 在排卵前卵泡内。这一概念与 传统观点认为黄体酮的生物学作用几乎是 仅限于非卵巢组织。因此，建议的 研究将提供对细胞的新的重要见解 孕酮调节GC功能的机制。进一步 这种卵巢机制的特征最终可能导致 特异性修饰的拮抗剂和/或激动剂的研究进展 黄体酮在卵巢内的作用而不改变其基因组 在非卵巢靶细胞内的作用。因此，这些推定的 拮抗剂/激动剂在卵巢癌治疗中的应用 癌症、不孕不育和卵巢早衰。

The overall goal of this proposal is to test the hypothesis that progesterone regulates granulosa cell (GC) function by activating a membrane receptor that has some GABAA receptor features. We have recently shown that progesterone's anti-apoptotic and anti-mitotic actions are attenuated by the GABA antagonist, bicuculline. Further, progesterone's actions can be attenuated by an antibody directed against the ligand binding domain of the nuclear progesterone receptor. When used in a western blot analysis, this antibody recognizes a 60 kDa protein within ovarian and GC lysates. This 60 kDa protein is also detected within membrane preparations of ovarian lysates. Finally, the progesterone receptor antibody detects a 60 kDa protein after immunoprecipitation with an antibody against the GABAA receptor. These findings support our hypothesis that progesterone mediates its actions within the ovary through a membrane-type receptor that has a molecular weight of approximately 60 kDa and has some pharmacological and immunological characteristics of a GABAA-receptor. In this proposal we will directly test this hypothesis through a series of experiments which use 1) pharmacological agents to define progesterone's function, 2) competitive progesterone assays and scatchard plot analysis to assess the specificity of progesterone binding to GCs, 3) western blot and immunocytochemistry to detect the expression pattern of this putative 60 kDa membrane progesterone receptor during follicular growth, differentiation and atresia. It is proposed that progesterone acts through this putative 60 kDa membrane progesterone receptor to regulate both GC mitosis and viability within the preovulatory follicle. The concept is in marked contrast to the traditional view that progesterone's biological actions are almost exclusively restricted to non-ovarian tissues. Therefore, the proposed studies will provide new and important insights into the cellular mechanisms through which progesterone regulates GC function. Further characterization of this ovarian mechanism could ultimately lead to the development of antagonist and/or agonist which specifically modify progesterone's actions within the ovary without altering its genomic action within non-ovarian target cells. As such, these putative antagonists/agonist could find applications in the treatment of ovarian cancers, infertility and premature ovarian failure.