Progesterone is a steroid hormone required both for implantation of the fertilized ovum, as well as for the maintenance of pregnancy. The goal of the proposed research is to elucidate the mechanisms whereby the synthesis of progesterone is regulated in the ovary and placenta, organs characterized by exceedingly high rates of secretion of progesterone. Previously we have shown that low-density lipoprotein (LDL)-cholesterol is a major source of precursor for progesterone biosynthesis in these tissues. Furthermore, we have shown in preliminary studies that LDL induces the synthesis of the enzymes involved in the conversion of cholesterol to pregnenolone, the first step in progesterone biosynthesis. Utilizing luteal cells and granulosa cells in monolayer culture, the effect of gonadotropins to induce the synthesis of the enzymes involved in the conversion of cholesterol to pregnenolone will be investigated. Utilizing luteal, granulosa, trophoblastic and choriocarcinoma cells in monolayer culture, the effect of LDL to induce the synthesis of these proteins will be examined. Cells will be radiolabeled with 35S methionine, or else RNA will be extracted from the cells and used to program a cell-free translation system in the presence of [35S]methionine. Enzymes involved in the conversion of cholesterol to pregnenolone will be subjected to immunoprecipitation, separated by SDS-polyacrylamide gel electrophoresis, and visualized by means of autoradiography. Species of cDNA specific for these proteins will be isolated from a cDNA library prepared from bovine adrenocortical poly A RNA. These cDNA probes will be utilized to determine whether treatment of cells with gonadotropins, as well as LDL, leads to an increase in the content of mRNA species specific for these enzymes. In addition to progesterone secretion, the process of in vitro luteinization will be investigated in order to ascertain whether LDL is a major factor regulating this process. The results of these studies will allow us to evaluate the concept that LDL is a major physiological factor regulating progesterone secretion in the ovary and placenta and the process of luteinization in the ovary, and will enable us to explore the cellular and molecular mechanisms of a hitherto unknown action of LDL, namely, to induce the synthesis of the enzymes catalyzing the conversion of cholesterol to progesterone.
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