MOLECULAR BIOLOGY OF STEROID RECEPTORS IN XENOPUS OOCYTES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。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。列出的机构是 中心，不一定是研究者的机构。 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。 这一过程包括染色体凝聚、萌发囊泡破裂和第一极体的排出。 在成熟完成时，卵母细胞减数分裂再次在中期II停止。 卵母细胞成熟的完成是正常排卵和受精所必需的。 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和低容量的受体。 然而，对细胞表面孕酮受体的特性知之甚少。 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处理以获得质膜。 用毛地黄皂苷从质膜溶解孕酮受体，并将其应用于天然稳态聚丙烯酰胺凝胶管以分离其配体结合活性。 按照该方法，沿着管凝胶一致地观察到孕酮结合活性的单峰，其在过量未标记孕酮存在下是饱和的。 在不同丙烯酰胺浓度的凝胶中，峰的迁移距离不同。 天然分子量分析显示孕酮结合蛋白约为107 KDa。 然后通过将管凝胶置于十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）平板凝胶上来分离管凝胶中的蛋白质。 在银染板，一个单一的点被确定为对应于，并专门共同迁移，在不同的酰胺浓度的第一维管凝胶中的孕酮结合活性的峰值。 该蛋白质从凝胶中电洗脱，并通过梯度SDS-PAGE显示为均质的。 在还原或非还原条件下的蛋白质的分子量约为90 KDa。 针对该90 KDa蛋白的特异性免疫血清显著抑制卵母细胞膜制剂的孕酮结合活性。 总之，爪蟾卵母细胞的膜孕酮受体被溶解和分离，并纯化了分子量约为90 KDa的单体蛋白，其具有膜结合孕酮受体的电泳和免疫学特征。 （由NIH Grant P20 RR 016480支持）。

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. This process involves chromosome condensation, germinal vesicle breakdown, and extrusion of the first polar body. At the completion of maturation, oocyte meiosis once again becomes arrested at metaphase II. Completion of oocyte maturation is required for normal ovulation and fertilization. Early studies have shown that the action of progesterone in oocyte maturation involves a high-affinity and low-capacity receptor. However, little is known about the characteristics of the cell surface 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 with digitonin and applied to native steady state polyacrylamide gel tubes to isolate its ligand binding activity. Following this procedure, a single peak of progesterone binding activity which was saturable in the presence of excess unlabeled progesterone was consistently observed along the tube gels. The migration distance of the peak varied in gels of different acrylamide concentrations. Native molecular weight analysis showed the progesterone binding protein to be about 107 KDa. Proteins in the tube gels were then separated by placing the tube gels on sodium dodecyl sulfate polyacrylamide gel electrophoresis (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 first-dimension tube gels of different acylamide concenrations. This protein was electro-eluted from the gels and shown to be homogenous by gradient SDS-PAGE. The molecular weight of the protein under reducing or nonreducing conditions was approximately 90 KDa. A specific immune serum directed against this 90 KDa protein significantly inhibited the progesterone binding activity of oocyte membrane preparations. In conclusion, the membrane progesterone receptor of Xenopus oocytes was solubilized and isolated and a monomeric protein at the molecular weight of approximately 90 KDa with electrophoretic and immunologic characteristics of the membrane-bound progesterone receptor was purified. (Supported by NIH Grant P20 RR016480).