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.
