MOLECULAR BIOLOGY OF STEROID RECEPTORS IN XENOPUS OOCYTES

爪蟾卵母细胞中类固醇受体的分子生物学

• 批准号: 7381741
• 负责人: Zhiming Liu
• 金额: $ 6.3万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381741
• 关键词: MOLECULAR; BIOLOGY; STEROID; RECEPTORS; XENOPUS

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在两栖动物的成熟卵泡中，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，它直接作用于卵母细胞以启动成熟。成熟是停滞在减数分裂前期I的卵母细胞恢复并完成其第一次减数分裂的过程。卵母细胞成熟的完成是正常受精所必需的。早期的研究表明，孕酮在卵母细胞成熟中的作用涉及高亲和力受体。然而，对孕酮受体知之甚少。本研究的目的是分离和纯化受体。从非洲爪蟾中收集卵母细胞并加工以获得质膜。从质膜溶解孕酮受体，并施加到稳态聚丙烯酰胺凝胶管，以确定其结合活性。在此过程之后，在管凝胶中观察到孕酮结合活性的单峰。然后通过将管凝胶置于SDS-PAGE平板凝胶上来分离管凝胶中的蛋白质。在银染色的平板中，鉴定出一个单独的斑点，其对应于管凝胶中孕酮结合活性的峰，并且仅与管凝胶中孕酮结合活性的峰共迁移。该蛋白质经电洗脱，其分子量约为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印迹分析表明，存在四个孕激素受体亚型和免疫细胞化学的卵母细胞切片产生了一个积极的信号，在核质和弱信号的卵母细胞的细胞质。 结果表明，不同类型的孕激素受体可能共同作用，以调节卵母细胞成熟的两栖动物。