CLONING MATURATION-INDUCING STEROID RECEPTOR

克隆成熟诱导类固醇受体

• 批准号: 6972145
• 负责人: Zhiming Liu
• 金额: $ 13.77万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-05 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6972145
• 关键词: Xenopus oocyte; cell membrane; complementary DNA; genetic library; hormone regulation /control mechanism; immunocytochemistry; in situ hybridization; messenger RNA; microinjections; molecular cloning; nucleic acid probes; nucleic acid sequence; oogenesis; organ culture; progesterone receptors; receptor expression

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.

该研究项目的长期目标是确定成熟诱导类固醇（MIS）受体在调节脊椎动物卵母细胞成熟中的分子机制。人们对这种 MIS 受体的理化特性知之甚少。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。本应用的目的是克隆 MIS 受体并对其进行测序，并检查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 是黄体酮，因此我们研究的具体目标 1 是对编码黄体酮受体的 cDNA 进行克隆和测序。纯化的受体蛋白将被微测序以获得内部氨基酸序列以设计寡核苷酸探针，将从卵母细胞中制备cDNA文库并筛选阳性克隆。的 cDNA 受体将被测序和分析。我们的具体目标 2 是确定黄体酮受体的成熟诱导活性。孕激素受体 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。将培养卵母细胞以合成黄体酮受体。将测定卵母细胞响应黄体酮刺激的成熟情况。我们的具体目标 3 是检查爪蟾卵母细胞中黄体酮受体 mRNA 和蛋白质的亚细胞定位。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。预计这些发现将有助于更好地理解 MIS 受体控制配子发育的机制。新知识将具有巨大的临床应用潜力，例如诱导无排卵患者的卵母细胞成熟以及开发抑制人类和动物卵母细胞成熟和排卵的避孕药。