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.
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