REGULATION OF REPRODUCTION

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The long term objective of this project is a thorough understanding of the roles of steroid hormones in mammalian reproduction. To begin these studies we will use laboratory rodents and rodent cell systems derived from reproductive tissues. In general, reproductive and other tissues (ovary, testis, placenta, adrenal) chronically regulate steroid hormone synthesis by regulating the abundance of the mRNAs encoding the enzymes that synthesize steroids. It is still unclear how this occurs; the abundances of these mRNAs may be regulated both transcriptionally and post-transcriptionally. Furthermore, although the same small set of enzymes mediate steroidogenesis in different tissues, the genes encoding these enzymes are regulated differently in different tissues, and differently in the same tissue among different mammals. Our work will focus initially on P450c 17. This one enzyme has two distinct activities, 17alpha hydroxylase and 17,20 lyase, but is encoded by only one gene. In the absence of any P45Ocl7 activity, mineralocorticoids are synthesized. In the presence of only the hydroxylase activity, glucocorticoids are synthesized, and in the presence of both hydroxylase and lyase activities, sex steroids are synthesized. P45Ocl7 thus is a key branch in the synthesis of all steroid hormones. We are just beginning to understand what signals on the genes for some steroidogenic enzymes are necessary for regulation by some factors, but little is known about the regulatory sequences or factors necessary for tissue-specific regulation of these genes. The physiologic regulation of steroidogenesis in the rat is well characterized. We have already cloned rat P45Ocl7 cDNA, and now wish to clone the rat P450c 17 gene and study its regulation. By stably transfecting it into cultured adrenal and Leydig cell lines, we can study how this gene is regulated in a tissue-specific manner by factors influencing both transcriptional and post-transcriptional events. In addition, by transiently transfecting adrenal, Leydig and non-endocrine fibroblast cell lines, with putative regulatory sequences of the rat P450c 17 gene, we can determine how this gene is regulated by stimulatory/repressive- and tissue-specific factors. We will identify the DNA sequences necessary for hormonal stimulation/repression and tissue-specific expression of this gene, and will begin to characterize the nuclear proteins necessary for this regulation. Successful completion of these studies will give us a better understanding of how adrenals and testes, using the same genes, synthesize different classes of steroid hormones.