Maturation associated changes in glycoproteins from rat epididymal fluid and sperm membranes

Abstract

A 32,000 dalton integral membrane glycoprotein can be labeled uniquely by galactose oxidase/ ({dollar}\sp3{dollar}H) sodium borohydride on the plasma of rat caudal, but not caput, epididymal sperm. Another glycoprotein of the same molecular weight can be labeled in rat caudal, but not caput, epididymal fluid. These studies explore the relationship between the membrane and fluid galactose oxidase-sensitive glycoproteins and, in addition, their relatedness to the 32,000 dalton major secretory glycoproteins of caudal epididymal fluid.;Immunoprecipitation experiments, one dimensional peptide mapping and the inability to label the purified major acidic secretory glycoproteins by galactose oxidase/ ({dollar}\sp3{dollar}H) sodium borohydride established that the galactose oxidase-sensitive glycoproteins and major acidic secretory glycoproteins were not related. The membrane and fluid tritium-labeled glycoproteins were shown, by a combination of chemical and enzymatic peptide mapping experiments and sugar analysis, to be closely related but not identical. It was also found that efficient labeling of the 32,000 Mr fluid glycoprotein was only possible if protease inhibitors were omitted in the isolation of the fluid and subsequent labeling procedures. This suggests that the fluid glycoprotein was acquired by degradation of the membrane glycoprotein.;Polyclonal antibodies raised against the electrophoretically resolved, and subsequently eluted, caudal sperm membrane glycoprotein immunoprecipitated the tritium-labeled glycoproteins from the caudal epididymal fluid and sperm membrane. Furthermore, both glycoproteins were immunoprecipitated by polyclonal antibodies raised against caput sperm plasma membranes, suggesting that a precursor form of the caudal galactose oxidase-sensitive glycoprotein may be present on caput sperm and is a subject to galactosylation during the sperm transit along the epididymis.;Bacterial phosphatidylinositol-specific phospholipases C were used to test a possibility that the galactose oxidase-sensitive glycoprotein was bound to the sperm plasma membrane via a glycosyl phosphatidylinositol anchor. Digestion of the tritium-labeled membrane glycoprotein with these enzymes resulted in the complete release of the radiolabel. More importantly, PI-PLC's also hydrolyzed the water soluble galactose oxidase-sensitive glycoprotein present in epididymal fluid. The action of PI-PLC's was concentration dependent and rapid. Neither the membrane nor the fluid glycoprotein was affected by nitrous acid. The released fragments also did not contain either a phosphodiester or phosphomonoester linkage. These findings suggest that the galactose oxidase-sensitive glycoprotein(s) possesses an unusual moiety which is different from the PI-PLC-sensitive structures previously described.