The long term objective of this research is to develop a serum-free, hormone/growth factor-supplemented medium that will support the completion of spermatogenesis in co-cultures of rat spermatogenic cells with Sertoli cells. We will attempt to determine whether various components of this culture medium interact synergistically with follicle-stimulating hormone (FSH) and androgens to stimulate secretion of Sertoli cell regulatory proteins or have direct actions on the developing spermatogenic cells. Development of an in vitro system for the study of spermatogenic cell development is necessary to fully understand the molecular basis for the regulation of spermatogenesis and causes of male infertility. We will study the effects of FSH and growth hormone on the accumulation of somatomedin-C (SM-C), a growth factor secreted by Sertoli cells, in media of Sertoli cells alone and Sertoli-spermatogeneic cell co-cultures. SM-C will be measured by radioimmunoassay. Newly-synthesized SM-C will be examined by immunoprecipitation of [35S]methionine-labeled protein using a specific anti-SM-C serum. SM-C immunoreactivity in cells will be studied by indirect immunofluorescence. To determine dose-dependent effects of FSH, growth hormone, insulin, SM-C, epidermal growth factor, transferrin and retinol on spermatogonia proliferation and differentiation of pre-leptotene spermatocytes into advanced stages of spermatogenesis, we will use [3H] thymidine combined with autoradiography in short- and long-term co-culture experiments. Studies of the effect of alternating high/low androgen concentrations in the presence or absence of FSH will determine the role of androgens in facilitating completion of spermatogenesis in vitro. Finally, two-dimensional polyacrylamide gel electrophoresis will be used to study newly-synthesized [35S]methionine-labeled proteins resulting from the interaction of Sertoli cells with specific types of spermatogenic cells co-cultured with Sertoli cells and during Sertoli-spermatogenic cell reaggregation experiments.