PROGESTERONE REGULATION OF GRANULOSA CELL FUNCTION

本提案的总体目标是检验以下假设： 孕酮通过激活一种受体来调节颗粒细胞（GC）的功能。 膜受体具有一些GABAA受体特征。 我们有 最近表明孕酮的抗凋亡和抗有丝分裂 作用被GABA拮抗剂荷包牡丹碱减弱。 此外，本发明还 孕酮的作用可以被一种针对 核孕酮受体的配体结合域。 当 用于蛋白质印迹分析，该抗体识别60 kDa的 卵巢和GC裂解物中的蛋白质。 这种60 kDa的蛋白质也是 在卵巢裂解物的膜制备物中检测到。 最后 孕酮受体抗体检测60 kDa的蛋白质后， 用针对GABAA受体的抗体进行免疫沉淀。这些 研究结果支持了我们的假设，孕酮介导了它的作用 通过一种膜型受体， 分子量约为60 kDa，具有一定的药理学和 GABAA受体的免疫学特征。 在本提案中，我们 将通过一系列实验直接验证这一假设， 使用1）药理学试剂来定义孕酮的功能，2） 竞争性孕酮测定和Scatchard图分析来评估 孕酮与GC结合的特异性，3）蛋白质印迹， 免疫细胞化学检测这种假定的表达模式 60 kDa膜孕酮受体在卵泡生长过程中， 分化和闭锁。 据推测，孕酮通过这一假定的60 kDa的 膜孕酮受体调节GC有丝分裂和活力 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 仅限于非卵巢组织。 因此，建议 研究将为细胞生物学提供新的重要见解。 孕酮调节GC功能的机制。 进一步 这种卵巢机制的特征可能最终导致 开发特异性地调节 孕酮在卵巢内的作用而不改变其基因组 作用于非卵巢靶细胞。 因此，这些假定的 拮抗剂/激动剂可用于治疗卵巢癌 癌症不孕症和卵巢早衰

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.