The long-term goal of this research is to define specific paracrine mechanisms that regulate spermatogenic cell development. Although FSH and testosterone are required to establish and maintain spermatogenesis, this endocrine regulation is indirect and mediated mainly through Sertoli cells, undoubtedly through multiple paracrine mechanisms. Our studies show that the insulin-like II growth factor/mannose 6-phosphate (IGF-II/M6P) receptor is abundant on the surface of spermatogonia, and that specific binding of Sertoli cell ligands to this receptor elevates germ cell c-fos mRNA and ribosomal RNA levels. This is t he first direct demonstration that insulin-like growth factor II (IGF-II) and mannose 6-phosphate-bearing glycoproteins (M6P-glycoproteins) secreted by Sertoli cells modulate the expression of a specific germ cell gene. We hypothesize that these ligands secreted by Sertoli cells activate receptor-mediated signal transduction pathways in spermatogonia, modulating gene expression and DNA synthesis required for differentiation.
The specific aims of this proposal are to: 1) Identify specific ligands secreted by Sertoli cells that stimulate gene expression when they bind to IGF-II/M6P receptors on the surface of spermatogonia. These studies will fractionate Sertoli M6P-glycoproteins and identify specific proteins in the active fractions by immunoblotting and microsequencing. In addition, the baseline level of IGF-II secreted by Sertoli cells in culture will be quantitated by radioimmunoassay. 2) Determine signal transduction pathways in spermatogonia that are activated by ligand binding to cell surface IGF-II/M6P receptors. These studies will determine if Sertoli cell ligands for the IGF-II/M6P receptor activate phospholipase C and stimulate an influx of calcium, as shown for this receptor in other cell types, and determine if proteins are subsequently phosphorylated via protein kinase C activation. 3) Determine the effects of IGF-II/M6P receptor activation on gene expression and DNA synthesis and the expression of other genes in spermatogonia. These studies will determine if Sertoli cell ligands for this receptor modulate DNA synthesis in spermatogonia or the expression of other genes identified by mRNA differential display. These studies will define the pathway and effects of a specific receptor- mediated mechanism for paracrine regulation of spermatogonia by Sertoli cells, providing fundamental knowledge about the regulation of spermatogenesis applicable to the clinical management of infertility and the development of new contraceptive strategies.
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