A remaining problem in male reproduction is the exact relationship between steroidogenesis and spermatogenesis. In mammals it is difficult to obtain successive spermatogenic stage for biochemical analysis, thus 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, Sertoli cells or Sertoli/germ cell units (follicles) are readily separated from other testis components; (3) morphological and physiological changes in the testis are a normal part of seasonal reproductive cycles (dogfish, salamander, squirrel). To date, we have documented stage-dependent variations in steroidogenic enzymes; steroid receptor number, occupancy and acceptors; and a novel sex steroid binding protein. We have also described the morphological correlates of these functional changes. Preliminary data show a pathway of communication from more mature to less mature stages and a role for steroids as parahormones. Techniques developed for culturing staged Sertoli/germ cell units (follicles) and staged Sertoli cells open new possibilities for studying the spermatogenic progression, Sertoli- germ cell interactions, and direct effects of steroids. Using light and electron microscopy; autoradiography; immunocytochemistry; enzyme tracer analysis; radioimmunoassay; receptor analysis; and cell culture techniques, we will: (1) Determine whether Sertoli cells alone account for stage-dependent steroidogenesis; (2) Determine whether stage-dependent changes in microsomal enzymes accurately reflect endogenous steroid production; (3) Describe the basic morphological and functional characteristics of cultured Sertoli cells from early, mid- and late stages; observe effects of time, germ cells and a normal 3-D configuration; (4) Measure steroidogenesis in Sertoli cells of a seasonal rodent during the annual cycle and localize P-450 enzymes by immunocytochemistry; (5) Pinpoint cellular targets of steroid action in the zoned testis using autoradiography; (6) Characterize steroid receptors in a seasonal rodent; measure changes during spermatogenesis; (7) Quantify germ cell/Sertoli cell proliferation, survival and differentiation in response to steroids in vitro; (8) Evaluate spermatogenesis in vivo after discrete intratesticular steroid placement; (9) Advance collaborative efforts to isolate testicular aromatase and use antibodies to study intratesticular distribution and molecular relatedness of this enzyme through evolution. Studies in this laboratory show the feasibility of using animal models outside the range of common laboratory species to advance materially our understanding of testicular endocrinology.
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