A key to understanding the relationship between steroidogenesis and spermatogenesis is to define qualitatively and quantitatively the steroidal microenvironment characteristic of each germ cell stage and to identify not only the cells which are sites of steroid production but also those which are steroid targets. Such studies would also address the mechanism by which interstitial and tubular functions are coordinated. Because of the structural complexities of the testis of common laboratory animals and man, it is technically difficult to obtain tissues in successive stages of the spermatogenetic cycle. To circumvent this problem, we have chosen for study species in which 1) germ cells in different stages of maturation are segregated topographically within the testis; 2) Leydig cells and specific Sertoli/germ cell units may be obtained in high yield in isolation from other testicular elements; 3) morphological and physiological changes in the testis are a normal part of seasonal breeding cycles. These animals will include Squalus, Necturus, squirrel, and boar. Using light and electron microscopy; autoradiography, immunocytochemistry; enzyme tracer analysis; enzyme isolation and purification methods; radioimmunoassay; steroid receptor and binding assays; and cell culture techniques, we will 1) characterize stage-dependent changes in steroidogenic pathways leading from cholesterol to androgen, estrogen, and their conjugates; 2) determine whether enzymic 'potential' of cell subfractions is reflected in the products actually formed by whole cells from endogenous precursors; 3) identify the cell types (Leydig vs Sertoli) responsible for each reaction; 4) determine the physicochemical characteristics, intratesticular location, and stage-dependent activities of androgen and estrogen receptors; 5) relate the state of receptor occupancy to in situ steroid production and metabolism; 6) develop an assay system for testing tubular and neural factors controlling Leydig cell differentiation and function; 7) utilize morphological and physiological criteria to assess stage-dependent characteristics of Sertoli/germ cell units in culture; 8) isolate, characterize, and purify P-450 enzymes from Leydig cell-rich glandular tissues for use ultimately as immunochemical probes for enzyme localization at the cellular and ultrastructural levels. Studies in this laboratory show the feasibility of using animal models outside the range of common laboratory species to materially advance our understanding of testicular function.
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