BIOREGULATION OF SPERMATOGENESIS

Sertoli细胞在支持细胞的生物调节中发挥重要作用 通过为精子发生提供机械支持和化学信号 生精细胞。这个实验室的工作表明， 中间丝蛋白，波形蛋白在一种 培养的Sertoli细胞对FSH的依赖。我们现在要确定的是 波形蛋白等蛋白磷酸化的调控机制 大鼠不同发育阶段支持细胞骨架蛋白的研究 精子发生。双向聚丙烯酰胺凝胶电泳会 用于拆分[~(32)P]正磷酸和[~(32)P]-ATP标记的蛋白质 CAMP和钙/钙调素依赖蛋白激活后 激活剂。初步研究表明，支持细胞分泌 也依赖于精子发生阶段的特定蛋白质。我们建议 控制这些蛋白质合成的因素的鉴定 在大鼠精子发生过程中。对应的生精小管段 通过透光模式识别不同的生精阶段 组织学将用[35S]蛋氨酸和分泌蛋白进行标记 通过凝胶电泳法进行分离。Sertoli细胞特异性分泌蛋白 指定的S80、S45和S35将在DEAE-纤维素柱上纯化 对其进行了层析和表征。针对这些蛋白质的抗体将 被制作出来。生长因子在细胞周期调控中的作用 支持细胞和肾小管周细胞的有丝分裂和非有丝分裂功能 采用Siles等人的互补性检验进行评价。和凝胶 电泳法确定自分泌或旁分泌效应。涉及的因素 肾小管周细胞对支持细胞功能的调节作用 将使用一种共培养方法进行研究，这种方法允许细胞-细胞功能 相互作用，但阻止细胞与细胞接触。我们将使用凝胶 电泳法测定肾小管周细胞特异性的影响 蛋白质，命名为P95，P40和P38，在合成和分泌 [35S]蛋氨酸标记的Sertoli细胞特异性蛋白S80、S45和S35。 这些Sertoli细胞特有的分泌蛋白之所以令人感兴趣，是因为它们 已被证明以精子发生阶段依赖的方式表达 因此，它们可能在精子发生中发挥重要作用。 有关精子发生的调节机制的知识与 可接受的避孕和治疗方法的发展 男性不育症。

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.