SERTOLI GERM CELL INTERACTIONS VIA IGF II/M6P RECEPTORS

这项研究的长期目标是确定特定的旁分泌 调节生精细胞发育的机制。 虽然FSH和 睾丸激素是建立和维持精子发生所必需的， 内分泌调节是间接的并且主要通过支持细胞介导， 无疑是通过多种旁分泌机制。 我们的研究表明， 胰岛素样生长因子II/甘露糖6-磷酸（IGF-II/M6 P）受体 在精原细胞表面丰富， 该受体的支持细胞配体升高生殖细胞c-fos mRNA， 核糖体RNA水平。 这是第一次直接证明， 胰岛素样生长因子II（IGF-II）和带有甘露糖6-磷酸 支持细胞分泌的糖蛋白（M6 P-糖蛋白）调节 特定生殖细胞基因的表达。 我们假设这些配体 Sertoli细胞分泌激活受体介导的信号转导 精原细胞中的途径，调节基因表达和DNA合成 需要区别对待。 这项建议的具体目标是： 1)鉴定由支持细胞分泌的刺激基因的特异性配体 当它们与细胞表面的IGF-II/M6 P受体结合时， 精原细胞 这些研究将使Sertoli M6 P糖蛋白 并通过免疫印迹鉴定活性组分中的特异性蛋白质 和微测序。 此外，IGF-II分泌的基线水平， 将通过放射免疫测定法对培养物中的支持细胞进行定量。 2)确定精原细胞的信号转导途径， 通过与细胞表面IGF-II/M6 P受体结合的配体激活。 这些 研究将确定IGF-II/M6 P受体的支持细胞配体是否 激活磷脂酶C并刺激钙的内流，如图所示。 这种受体在其他细胞类型，并确定蛋白质是否 随后通过蛋白激酶C激活磷酸化。 3)确定IGF-II/M6 P受体激活对基因表达的影响 表达和DNA合成以及其他基因的表达 精原细胞 这些研究将确定是否支持细胞配体， 该受体调节精原细胞的DNA合成或 通过mRNA差异显示鉴定的其他基因。 这些研究将确定特定受体的途径和作用- Sertoli介导的精原细胞旁分泌调节机制 细胞，提供有关调节的基本知识， 精子发生适用于不孕症的临床管理， 制定新的避孕策略。

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.