Both FSH and germ cells interact with Sertoli cells by different pathways to cause a rapid activation of protein kinases and/or phosphatases. Because the mechanism of germ cell activation of Sertoli cell protein phosphorylation appears to involve increased levels of intracellular calcium, the effect of hormone or germ cell treatment on calcium levels in cultured Sertoli cells will be studied using the fluorescent calcium indicators, Fura2 and Indo1. Patterns of protein phosphorylation also suggest that the mechanism by which germ cells affect Sertoli cells involves the phosphatidylinositol (PI) pathway. Therefore, studies of Sertoli cell PI metabolism will be conducted. The factor from germ cells which stimulates Sertoli cell protein phosphorylation will be isolated, characterized and used to produce antibodies in order to learn by which germ cell stage it is produced. Two proteins whose phosphorylation is specifically affected by germ cells will be purified by conventional liquid chromatography, metal chelate chromatography, affinity, chromatography and HPLC. These phosphoproteins will be isolated in order to develop antibodies to the phosphoproteins. The antibodies will then be used to localize the phosphoproteins at the light and electron microscope levels with Sertoli cells and the testis and to study the correlation of each phosphoprotein with the cycle of the seminiferous epithelium. Antibodies will also be used to screen a Sertoli cell cDNA lambda gtll expression library in order to determine the sequences of the phosphoproteins. It is expected that localization of the phosphoproteins and determination of their amino acid sequences will lead to identification of the proteins and their functions. The studies should contribute to understanding Sertoli-germ cell interaction and hormone-mediated response mechanisms of Sertoli cells during spermatogenesis.
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