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.
Showing the most recent 10 out of 17 publications
