The overall goal of this project is the biochemical and physiological analysis of cell-surface associated fucosyltransferase (FT) during mammalian spermatogenesis. We will test the hypothesis that a family of related FT enzymes, each with a different glycosidic specificity, is involved in Sertoli-germ cell interactions, sperm maturation and fertilization. Preliminary studies have identified the spermatogenic stage-specific expression of FT in the mouse and have implicated this enzyme in sperm-egg recognition. In contrast to most somatic cells, a substantial fraction of the spermatogenic cell FT is associated with the cell surface as an integral plasma membrane ecto-enzyme. It appears that a family of FT enzymes, each with differing glycosidic specificities is involved. This application proposes five Specific Aims designed to extend our understanding of the role FT has in regulating spermatogenesis.
These Aims are (1) To determine the glycosidic specificity of FTs during spermatogenesis and sperm maturation. Methods used here will include newly developed thin layer chromatographic micropartitioning procedures as well as the introduction of neoglycolipid analysis for the determination of FT specificity. (2) To purify and characterize germ cell surface FT. Isolated plasma membrane from spermatogenic cells and intact purified germ cell classes will be used as source materials for affinity chromatographic enzyme purifications, using HPLC techniques. Comparisons will be made with FTS isolated by previously published protocols from sources other than the testis. (3) To examine the role of cell surface FT in modulating germ cell-Sertoli cell interactions. An in vitro cell-cell binding assay already developed by this laboratory will be employed here. This assay permits quantification of cell-cell binding using purified populations of spermatogenic cells. (4) To examine the function of spermatozoon cell surface FT in sperm-egg interactions. These experiments will entail in vitro binding studies between sperm and the zona pellucida. (5) To identify and characterize the chemical structure of endogenous testicular acceptors for germ cell surface FT. This final Specific Aim will involve HPLC fractionation of germ cell plasma membranes as well as biochemical analysis of (a) cell surface glycoproteins, (b) fucosylated lipids and (c) novel disaccharide glucosyl-fucose, linked O-glycosidically to polypeptides. Results from these studies will related directly to the molecular mechanisms underlying cell differentiation and movement within the mammalian seminiferous epithelium and during initial binding of sperm to the egg and its protective vestments at fertilization. In addition, these studies should provide new data on the interaction of the maturing sperm plasma membrane with epithelial cell-derived macromolecules during sperm epididymal transit.
| Raychoudhury, S S; Millette, C F (1997) Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats. Biol Reprod 56:1268-73 |
| Walden, P D; Millette, C F (1996) Increased activity associated with the MAST205 protein kinase complex during mammalian spermiogenesis. Biol Reprod 55:1039-44 |
| Raychoudhury, S S; Millette, C F (1995) Glycosidic specificity of fucosyltransferases present in rat epididymal spermatozoa. J Androl 16:448-56 |
| Raychoudhury, S S; Millette, C F (1994) Presence of multiple fucosyltransferases in rat Sertoli cells and spermatogenic cells. Biol Reprod 51:1006-13 |
| Raychoudhury, S S; Millette, C F (1993) Surface-associated glycosyltransferase activities in rat Sertoli cells in vitro. Mol Reprod Dev 36:195-202 |
