Sertoli cells play an important role in the bioregulation of spermatogenesis by providing mechanical support and chemical signals to spermatogenic cells. Work of this laboratory has shown that the intermediate filament protein, vimentin is phosphorylated in a FSH-dependent manner in cultured Sertoli cells. We now want to determine the mechanisms regulating the phosphorylation of vimentin and other cytoskeletal proteins of Sertoli cells during different stages of rat spermatogenesis. Two-dimensional polyacrylamide gel electrophoresis will be used to resolve [32P]orthophosphate and [Gamma-32P]ATP-labeled proteins following activation of cAMP- and Ca2+/calmodulin-dependent protein kinases. Preliminary studies have shown that Sertoli cells secrete specific proteins that are also spermatogenic stage-dependent. We propose the identification of factors that control the synthesis of these proteins during rat spermatogenesis. Seminiferous tubular segments corresponding to distinct spermatogenic stages identified by their transillumination pattern and histology will be labeled with [35S]methionine and secretory proteins resolved by gel electrophoresis. Sertoli cell-specific secretory proteins designated S80, S45 and S35 will be purified on DEAE-cellulose column chromatography and characterized. Antibodies against these proteins will be produced. The effect of growth factors on the regulation of the mitogenic and non-mitogenic function of Sertoli and peritubular cells will be evaluated using the complementation test of Stiles et al. and gel electrophoresis to define autocrine or paracrine effects. Factors involved in the modulation of Sertoli cell function induced by peritubular cells will be studied using a coculture method that allows cell-cell functional interaction but prevents cell-cell contact. We will use gel electrophoresis to determine the effect of peritubular cell-specific proteins, designated P95, P40 and P38, on the synthesis and secretion of [35S]methionine-labeled Sertoli cell-specific proteins, S80, S45 and S35. These Sertoli cell-specific secretory proteins are of interest because they have been shown to be expressed in a spermatogenic stage-dependent manner and, therefore, they may play an important role in spermatogenesis. Knowledge of the regulatory mechanisms of spermatogenesis are relevant to the development of acceptable methods of contraception and treatment of infertility of the male.
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