The long term objective of this project is to characterize the mechanisms that determine the capcity of Leydig cells to produce testosterone. We have approached this problem by studying the relationship of activities of steroid biosynthetic enzymes to maximal testosterone production in two functionally distinct populations of Leydig cells from normal and gonadotropin-treated rats. The specific aims of the present study are designed to evaluate additional sites in the steroidogenic pathway that may be decisive in determining Leydig cell capacity for testosterone production as well as to elucidate the mechanisms by which luteinizing hormone (LH) and cAMP either increase or decrease maximal testosterone production depending on the mode of their administration. Specifically we will determine: 1) whether the activity of the cholesterol side chain cleavage enzyme (P-450scc) differs in the two populations of Leydig cells and whether chronic in vivo treatment with LH increases the activity of P-450scc' using homogenates or enriched mitochondrial fractions of Leydig cells separated into two populations by Metrizamide gradient centrifugation; 2) the role of prolactin in the regulation of 5Alpha-reductase activity in purified rat Leydig cells during sexual maturation; and 3) the mechanism by which cAMP brings about steroidogenic desensitization using a newly developed mouse Leydig cell culture system. This third specific aim will examine the mechanisms by which cAMP causes decreases in microsomal and mitochondrial P-450 enzyme activities and the relationship among decreases in microsomal and mitochondrial P-450 activities, substrate depletion and reductions in maximal testosterone production. Enzyme activities will be studied using [3H]-labeled steroid substrates and measuring [3H]-labeled steroid products after extraction and purification of steroids by thin-layer chromatography. The proposed studies using the combination of in vivo and in vitro approaches to study regulation of Leydig cell function will contribute to a better understanding of the processes that determine maximal testosterone production at different stages of life and under different pathological conditions.