MOLECULAR BIOLOGY OF STEROID RECEPTORS IN XENOPUS OOCYTES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在两栖动物发育完全的卵泡中，促性腺激素诱导卵泡细胞产生一种诱导成熟的类固醇——孕酮，孕酮直接作用于卵母细胞启动成熟。成熟是卵母细胞在减数分裂前期恢复并完成第一次减数分裂的过程。完成卵母细胞成熟是正常受精所必需的。早期的研究表明，黄体酮在卵母细胞成熟中的作用涉及一个高亲和受体。然而，人们对黄体酮受体知之甚少。本研究的目的是分离和纯化该受体。收集非洲爪蟾卵母细胞，经处理得到质膜。将黄体酮受体从质膜中溶解，并应用于稳态聚丙烯酰胺凝胶管中测定其结合活性。在此过程中，在管凝胶中观察到黄体酮结合活性的单峰。然后将管凝胶置于SDS-PAGE板凝胶上，分离管凝胶中的蛋白质。在银色染色板中，发现了一个与管凝胶中黄体酮结合活性峰值相对应并完全共迁移的单点。该蛋白经电洗脱，分子量约为90kda。为了确定细胞内孕酮受体是否参与卵母细胞成熟的调节，我们研究了孕酮类似物米非司酮对非洲爪蟾卵母细胞成熟的影响。我们发现米非司酮对卵母细胞成熟有促进作用。Western blotting分析提示存在4种黄体酮受体同工异构体，卵母细胞切片免疫细胞化学显示核质阳性信号，细胞质弱信号。结果表明，不同类型的黄体酮受体可能共同作用于两栖动物卵母细胞成熟。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In full-grown ovarian follicles of amphibians gonadotropin induces the follicular cells to produce a maturation-inducing steroid, progesterone, that acts directly on the oocyte to initiate maturation. Maturation is the process by which oocytes arrested in prophase I of meiosis resume and complete their first meiotic division. Completion of oocyte maturation is required for normal fertilization. Early studies have shown that the action of progesterone in oocyte maturation involves a high-affinity receptor. However, little is known about the progesterone receptor. The objective of this study is to isolate and purify the receptor. Oocytes were collected from Xenopus laevis and processed to obtain plasma membrane. Progesterone receptor was solubilized from plasma membranes and applied to steady state polyacrylamide gel tubes to determine its binding activity. Following this procedure, a single peak of progesterone binding activity was observed in the tube gels. Proteins in the tube gels were then separated by placing the tube gels on SDS-PAGE slab gels. In silver-stained slabs, a single spot was identified that corresponded to, and exclusively co-migrated with, the peak of the progesterone binding activity in the tube gels. This protein was electro-eluted and its molecular weight was about 90 KDa. To determine if intracellular progesterone receptors are involved in the regulation of oocyte maturation, we examined the effects of a progesterone analog, mifepristone, on Xenopus oocyte maturation. We found that mifepristone had a stimulatory effect on oocyte maturation. Western blotting analysis suggested the existence of four progesterone receptor isoforms and immunocytochemistry of the oocyte sections yielded a positive signal in the nucleoplasm and a weak signal in the cytoplasm of the oocytes. The results suggest that different types of progesterone receptors may act together to regulate oocyte maturation in amphibians.