MOLECULAR BIOLOGY OF STEROID RECEPTORS IN XENOPUS OOCYTES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在两栖动物成熟的卵泡中，促性腺激素诱导卵泡细胞产生一种成熟诱导类固醇，孕酮，它直接作用于卵母细胞，启动成熟。成熟是处于减数分裂前期I期的卵母细胞恢复并完成第一次减数分裂的过程。正常受精需要完成卵母细胞成熟。早期的研究表明，黄体酮在卵母细胞成熟过程中的作用涉及一种高亲和力受体。然而，人们对孕激素受体知之甚少。本研究的目的是分离纯化该受体。从非洲爪哇采集卵母细胞，处理后得到质膜。从质膜上增溶孕酮受体，并将其应用于稳态聚丙烯酰胺凝胶管，以测定其结合活性。在这一过程中，在试管凝胶中观察到黄体酮结合活性的单峰。然后通过将管凝胶放置在SDS-PAGE平板凝胶上来分离管凝胶中的蛋白质。在银染切片中，发现了一个与管状凝胶中孕酮结合活性峰值相对应的单一斑点，并与之唯一地共同迁移。该蛋白经电洗脱后，相对分子质量约为90 KDa。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哇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分析表明，卵母细胞存在四种孕激素受体亚型，卵母细胞切片的免疫细胞化学结果显示，卵母细胞的核浆呈阳性信号，胞浆呈弱信号。这些结果表明，不同类型的孕激素受体可能共同作用于两栖动物的卵母细胞成熟。

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.