SERTOLI CELL-GERM CELL SURFACE MEMBRANE INTERACTIONS

本研究的长期目标是： 支持细胞调节 睾丸中的生殖细胞发育。 特别感兴趣的是 过程和特定的分子，在运作的水平， 支持细胞和生殖细胞之间以及支持细胞和生殖细胞之间 相邻的支持细胞。 为了更好地了解这些细胞 睾丸表面的相互作用，四个具体的目标将是 在这个项目中解决：（1）一个面板的生成 特异性针对以下表面组分的单克隆抗体： 支持细胞;（2）抗原的生化特性 （3）检测这些抗体的抗体水平; 支持细胞表面抗原时空表达 在发育和成熟的周期性曲细精管中;和（4） 支持细胞表面抗原的鉴定 参与生殖细胞与支持细胞的结合， 支持细胞紧密连接的形成。 单克隆 抗体将从用完整的 Sertoli细胞和从Sertoli分离的质膜 细胞 将使用间接免疫荧光法筛选杂交瘤上清液。 免疫荧光显微镜观察大鼠睾丸的冷冻切片。 选择的单克隆抗体所针对的抗原 将通过免疫探测支持细胞质膜来鉴定 通过凝胶电泳分离并转移到 硝酸纤维素（Western印迹法）。 每个单克隆抗体 产生的抗体将被用于研究 用低温恒温器检测睾丸支持细胞表面抗原的表达 来自1、5、10、20、30和65日龄大鼠的切片。 舞台- 支持细胞表面成分的依赖性表达将是 使用低温恒温器切片进行研究， 生精小管只包含14个阶段中的一个， 精子发生 从最初的抗体组中 对于支持细胞表面，将通过其 抑制生殖细胞与支持细胞结合的能力， 体外细胞-细胞粘附测定。 抗体的第二子集将 基于抑制形成的能力进行鉴定 支持细胞紧密连接。 这里描述的实验将做出重大贡献 支持细胞在精子发生中的作用 通过识别在时间上或空间上 在生精小管中表达，在生殖细胞中起作用 识别和粘附以及支持细胞紧密的形成 交叉点 这一信息应立即应用于 生精功能障碍的考虑， 能够安全调节男性生殖细胞发育的方案。

The long-term goal of the research proposed here is the identification of mechanisms used by the Sertoli cell to regulate germ cell development in the testis. Of particular interest are processes and specific molecules that operate at the level of the plasma membrane between Sertoli cells and germ cells and between adjacent Sertoli cells. In order to better understand these cell surface interactions in the testis, four specific aims will be addressed in this project: (1) the generation of a panel of monoclonal antibodies that are specific for surface components of the Sertoli cell; (2) the biochemical characterization of antigens corresponding to these antibodies; (3) an examination of the temporal and spatial expression of Sertoli cell surface antigens in developing and in mature, cycling seminiferous tubules; and (4) the identification of Sertoli cell surface antigens that are involved in either the binding of germ cells to Sertoli cells or the formation of Sertoli cell tight junctions. Monoclonal antibodies will be generated from mice immunized with intact Sertoli cells and with plasma membranes isolated from Sertoli cells. Hybridoma supernatants will be screened using indirect immunofluorescence microscopy on cryostat sections of rat testis. Antigens against which selected monoclonal antibodies are directed will be identified by immunoprobing Sertoli cell plasma membranes that have been fractionated by gel electrophoresis and transferred to nitrocellulose (Western blotting). Each of the monoclonal antibodies generated will then be used to study the developmental expression of Sertoli cell surface antigens by examining cryostat sections from 1, 5, 10, 20,30, and 65 day old rats. The stage- dependent expression of Sertoli cell surface components will be studied using cryostat sections prepared from lengths of seminiferous tubules containing only one of the fourteen stages of spermatogenesis. From the original panel of antibodies specific for the Sertoli cell surface, a subset will be identified by their ability to inhibit germ cell binding to Sertoli cells in an in vitro cell-cell adhesion assay. A second subset of antibodies will be identified based on the ability to inhibit the formation of Sertoli cell tight junctions. The experiments described here will make a significant contribution to the understanding of Sertoli cell function in spermatogenesis by identifying surface components that are temporally or spatially expressed in seminiferous tubules and that function in germ cell recognition and adhesion and in the formation of Sertoli cell tight junctions. This information should find immediate application in the consideration of spermatogenic dysfunction and in the design of protocols able to safely regulate male germ cell development.