This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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).
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