The long term objective of this project is to determine the influence of genetic control on steroidogenesis. The studies proposed in this application are designed to examine the relationships among the structural and regulatory gene(s) that influence the expression of the enzymes involved in testicular steroid hormone biosynthesis. Specifically, it is proposed to determine the chromosomal location of genes determining quantitative and structural variation of the following steroidogenic enzymes: 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD), cholesterol side-chain cleavage (P-450scc) and 17 alpha-hydroxylase/C17-20 lyase (P-450-17 alpha). Whole testicular preparations, purified Leydig cells or adrenal glands from numerous strains of inbred mice will be surveyed for quantitative and qualitative differences of each of the enzymes. Recombitant inbred (RI) lines, derived from progenitor strains which show the greatest interstrain differences, will be used to map the chromosomal location of the structural and regulatory gene(s) for each enzyme. Quantitative differences in 3 beta-HSD will be determined by measuring enzyme activity; structural variants of this enzyme will be detected by measuring rate of heat inactivation (thermostability). Quantitative differences in P-450scc and P-450-17 alpha will be determined in lysates of purified Leydig cells or of adrenal glands using specific rabbit antisera generated against these enzymes plus 125I-labeled second antibody and analysis by immunoblotting. Structural variants of P-450scc and P-450-17 alpha will be detected by analyzing for variations in restriction fragment length of genomic DNA, from different inbred strains of mice, through hybridization with cDNA probes specific for P-450scc and P-450-17 alpha. The availability of specific antisers to the P-450scc and P-450-17 alpha and the cDNA clones specific for each of these enzymes provide sensitive reagents for the detection of quantitative and qualitative variants, respectively. Information gained from the proposed studies will provide a better understanding of the normal physiologic function of the Leydig cell as well as explanations for pathologic states due to genetic defects in steroid biosynthesis enzymes in testes and adrenal glands.
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