The long term objective of this proposal is the identification and biochemical characterization of cell surface constituents present on developing spermatogenic cells of the mouse, correlated with functional assays of individual plasma membrane glycoprotein. Studies proposed herein represent biochemical studies utilizing highly purified preparations of plasma membranes obtained from isolated populations of pachytene spermatocytes and round spermatids. Work already completed suggests that one particular cell surface glycoprotein, p151/6.0, is involved in germ cell-Sertoli cell adhesion and, in fact, that p151/6.0 could be a structural component of testicular desmosomes.
Five specific aims are proposed for the functional study of p151/6.0 in the mammalian testis: (1) Direct biochemical comparisons of p151/6.0 with known desmosomal components isolated from somatic cells will include peptide mapping, amino acid analysis and immunochemical assays. These experiments will determine whether structural homology between these proteins exists. (2) In situ localization of p151/6.0 will be accomplished using immunohistochemistry at both the light and electron microscopic level. Monoclonal antibodies prepared against both p151/6.0 and Mr 150,000 desmosome components will be compared. (3) In vitro systems for the co-culture of mouse germ cells and Sertoli cells will be adapted from procedures recently described by others for use in functional assays of p151/6.0. (4) Direct functional assays of p151/6.0 in vitro will include serological inhibition of cell adhesion and desmosome formation to provide a dynamic analysis of the role of p151/6.0 within the seminiferous tubule. (5) Finally, attempts at the isolation and biochemical analysis of desmosomes from the testicular epithelium will complement the preceding experiments. Isolation protocols will include both cell fractionation and immunoaffinity methods. The proposal constitutes a concerted approach for the functional study of a mouse spermatogenic cell surface glycoprotein and represents perhaps the first biochemical analysis of cellular adhesion within the seminiferous tubule. Data obtained should be relevant for explanations of the highly coordinated nature of spermatogenesis in mammals.
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