This project addresses issues which are fundamental to our understanding of testicular function, i.e., the ontogenic development of Leydig cells and the regulation of this biological process. Research in this area will provide the opportunity to understand the role of multiple factors in the processes of precursor cell proliferation, differentiation and maturation. Specifically, experiments are proposed whereby the ethane dimethylsulfonate (EDS)-treated mature male rat, in which Leydig cell death and depletion is followed by regeneration of Leydig cells, will be used as the animal model. The objectives described in this proposal are to 1) determine the stimulatory role of gonadotropins, using human chorionic gonadotropin (hCG) and 2) elucidate the inhibitory control by estradiol (E2) on the processes of precursor, cell proliferation, differentiation and maturation into functional Leydig cells. Precursor cell identification will be based, in part, on the presence of the E2 receptor. Precursor cell proliferation will be assessed by means of 3H-thymidine incorporation (TCA precipitable radioactivity and autoradiography) and increased interstitial cell and Leydig cell numbers (quantified histologically by light microscopy). Differentiation will be ascertained through the appearance of specific Leydig cell functions, i.e., 125I-labeled hCG binding and hCG/dbcAMP-stimulated testosterone production, as well as the histochemical appearance of steroidogenic enzymes and morphological analysis. Maturation will be determined by the transient rise and fall in androstanediol production, the binding of specific monoclonal antibodies against mature Leydig cells and the disappearance of morphologically identifiable lipid droplets. These studies will reveal the endocrine nature of hCG/LH regulation of Leydig precursor cell DNA synthesis , subsequent cell division and the morphological changes that give rise to identifiable, functional Leydig cells. The inhibitory role of E2 in the regulation of Leydig cell ontogeny will be studied with respect to the cell-type which is estrogen sensitive and the mechanisms for estrogenic suppression. This system offers a unique model for investigating the role of E2 as an autocrine or paracrine factor.
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