GLYCOPROTEIN METABOLISM IN MAMMALIAN SPERMATOGENESIS

哺乳动物精子发生中的糖蛋白代谢

• 批准号: 2197187
• 负责人: CLARKE F MILLETTE
• 金额: $ 13.95万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2197187
• 关键词: Sertoli cells; cell adhesion molecules; cell cell interaction; cellular polarity; confocal scanning microscopy; developmental genetics; flow cytometry; gene expression; glycoprotein structure; hormone regulation /control mechanism; immunofluorescence technique; in situ hybridization; laboratory mouse; laboratory rat; molecular cloning; northern blottings; paracrine; protein purification; protein sequence; protein structure function; retinoids; sperm; spermatogenesis; testis; tissue /cell culture

The overall goal of this application is to determine the molecular mechanisms regulating the cellular interactions between Sertoli cells and differentiating spermatogenic cells within the mammalian seminiferous epithelium. We are attempting to identify particular plasma membrane associated constituents of Sertoli cells which recognize specific sub- classes of germ cells, including spermatogonia, spermatocytes and spermatids. Such testis-specific cell adhesion components, we hypothesize, are important in establishing and modulating the spermatogenic cycle in mammals. Recently, we have identified the first Sertoli cell surface polypeptides directly involved in adhesive interactions with spermatogenic cells. SGAMs, for Sertoli Cell Germ Cell Adhesion Molecules, are not found on spermatogenic cells. Antibodies specific for these proteins inhibit the in vitro adhesion of isolated rodent spermatogenic cells to Sertoli cell monolayers. SGAMs are distinguished from other known testicular cell-cell adhesion molecules such as N-cadherin, E-cadherin, or spermatocyte cell surface polypeptides. This application is designed to characterize SGAMs biochemically and to investigate their physiological function. Three Specific Aims are proposed. Specific Aim A: To isolate and characterize SGAM biochemically and to clone SGAM. SGAM will be isolated using affinity chromatography with monoclonal antibodies already available. Chemical analysis of isolated SGAM will include carbohydrate determinations, in situ peptide mapping and microsequencing studies. SGAM cDNA will be cloned, sequenced and assessed for homology to other known CAMs. Specific Aim B: To test the hypothesis that the expression and/or activity of SGAMs is modulated (a) by systemic hormones and (b) by paracrine interactions between testicular cells. Hormones to be tested for possible effects include retinol. Paracrine cell interactions to be examined will include studies of secretory products derived from spermatogenic cells, particularly primary pachytene spermatocytes. Aim B will also test the hypothesis that direct germ cell-Sertoli cell contact modulates SGAM expression. Specific Aim C: To test the hypothesis that SGAM expression is temporally restricted during the spermatogenic cycle. Experiments will include (a) evaluation of SGAM appearance using both morphological and molecular techniques, (b) direct functional analysis of the possible involvement of SGAM in Sertoli cell adhesion to early spermatogenic cells, including spermatogonia, and (c) confocal immunomicroscopic studies of intact seminiferous tubules at defined stages of spermatogenesis.

该应用的总体目标是确定分子 调节Sertoli细胞与细胞相互作用的机制 区分哺乳动物生精中的精子细胞 上皮。我们正在尝试识别特定的质膜 Sertoli细胞的相关成分，这些成分识别特定的亚下 生殖细胞的类别，包括精子，精子细胞和 精子。这种睾丸特异性细胞粘附成分，我们假设 在建立和调节精子周期中很重要 哺乳动物。最近，我们确定了第一个Sertoli细胞表面 多肽直接参与与精子的粘合剂相互作用 细胞。 SGAM，用于Sertoli细胞生殖细胞粘附分子，找不到SGAMS 在生精细胞上。针对这些蛋白质特异的抗体抑制 分离的啮齿动物精子细胞对Sertoli细胞的体外粘附 单层。 SGAM与其他已知的睾丸细胞细胞区分开 粘附分子，例如N-钙黏着蛋白，E-钙粘蛋白或精子细胞细胞 表面多肽。该应用程序旨在表征SGAMS 生化并研究其生理功能。三 提出了具体目标。特定目的A：隔离和特征 SGAM生化和克隆SGAM。 SGAM将使用亲和力隔离 带有单克隆抗体的色谱学已经可用。化学 分析SGAM的分析将包括确定碳水化合物 原位肽映射和微骨测序研究。 SGAM cDNA将是 克隆，测序并评估与其他已知CAM的同源性。具体的 目标B：检验SGAM表达和/或活性的假设 由全身激素和（b）通过旁分泌相互作用调节（a） 在睾丸细胞之间。激素要测试可能影响 包括视黄醇。旁分细胞相互作用将包括 对源自精子细胞的分泌产物的研究， 特别是原发性粉状精子细胞。 AIM B也将测试 直接生殖细胞静脉细胞接触调节SGAM的假设 表达。特定目的C：检验SGAM表达的假设是 在精子周期中受到时间限制。实验会 包括（a）使用形态学和同时评估SGAM外观 分子技术，（b）可能的直接功能分析 SGAM参与Sertoli细胞粘附于早期的精子细胞， 包括精子症，以及（c） 精子发生的定义阶段的完整精神小管。