CLONING MATURATION-INDUCING STEROID RECEPTOR

该研究项目的长期目标是确定成熟诱导类固醇（MIS）受体在脊椎动物卵母细胞成熟调控中的分子机制。很少有人知道这个MIS受体的理化特性。最近，我们从非洲爪蟾卵母细胞质膜中分离纯化了MIS受体，并制备了针对该受体的多克隆抗血清。本研究的目的是克隆MIS受体并对其进行序列分析，并研究其在非洲爪蟾卵母细胞中的亚细胞定位。由于非洲爪蟾的MIS是孕酮，我们的研究目标是克隆和测序孕酮受体的cDNA编码。将对纯化的受体蛋白进行微测序以获得内部氨基酸序列以设计寡核苷酸探针，将从卵母细胞制备cDNA文库并筛选阳性克隆。的cDNA 将对受体进行测序和分析。我们的具体目标是确定孕激素受体的成熟诱导活性。孕酮受体mRNA将通过cDNA的体外转录产生，并显微注射到未成熟的卵母细胞中。培养卵母细胞以合成孕酮受体。将测定卵母细胞对孕酮刺激的成熟反应。我们的具体目标3是检查非洲爪蟾卵母细胞中孕激素受体mRNA和蛋白的亚细胞定位。标记的RNA探针将用于原位杂交，以确定 mRNA的分布和多克隆抗血清将用于受体蛋白的免疫细胞化学定位。这一发现将有助于更好地了解MIS受体在配子发育控制中的作用机制。这些新知识将在临床应用中具有巨大的潜力，例如诱导无排卵患者的卵母细胞成熟和开发用于抑制人和动物卵母细胞成熟和排卵的避孕药。

The long-term goal of the research project is to determine the molecular mechanism of the maturation-inducing steroid (MIS) receptor in the regulation of oocyte maturation in vertebrates. Little is known about the physiochemical characteristics of this MIS receptor. Recently, we have isolated and purified the MIS receptor from the plasma membrane of Xenopus laevis oocytes and also produced a polyclonal antiserum against the receptor. The objective of this application is to clone and sequence the MIS receptor and to examine its subcellular localization in Xenopus oocyte. Since the MIS in Xenopus is progesterone, our specific aim 1 of the research is to clone and sequence the cDNA encoding the progesterone receptor. Purified receptor protein will be microsequenced to obtain internal amino acid sequences to design the oligonucleotide probes, cDNA libraries will be prepared from oocytes and screened for positive clones. The cDNA of the receptor will be sequenced and analyzed. Our specific aim 2 is to determine the maturation-inducing activity of the progesterone receptor. The progesterone receptor mRNA will be generated by in vitro transcription of the cDNA and microinjected into immature oocytes. The oocytes will be cultured to allow for the synthesis of the progesterone receptor. Maturation of the oocytes in response to progesterone stimulation will be assayed. Our specific aim 3 is to examine the subcellular localization of the progesterone receptor mRNA and the protein in Xenopus oocytes. Labeled RNA probes will be used for in situ hybridization to determine the distribution of mRNA and the polyclonal antiserum will be used in immunocytochemical localization of the receptor protein. It is expected that the findings will contribute to a better understanding of the mechanism of the MIS receptor in the control of gamete development. The new knowledge will have a great potential for clinic applications such as induction of oocyte maturation in anovulatory patients and development of contraceptive agents for inhibition of oocyte maturation and ovulation in humans and animals.