Identification, localization and involvement of glycosidases in sperm-zona interaction using frozen-thawed ejaculated pig spermatozoa

摘要

To identify sperm glycosidases specific to sugar residues found in the zona pellucida of pig oocytes, frozen-thawed ejaculated spermatozoa were treated experimentally and assayed for activities of alpha-L-fucosidase, alpha-D-mannosidase, beta-D-galactosidase and N-acetyl-beta-D-glucosaminidase (GlcNAc'ase). When spermatozoa were incubated with calcium ionophore A23187 for 1 h, about 70 percent were acrosome-reacted and about 72, 46, 56 and 35 percent of the activities of alpha-L-fucosidase, alpha-D-mannosidase, beta-D-galactosidase and GlcNAc'ase, respectively, were lost from spermatozoa. However, lower levels of alpha-L-fucosidase, alpha-D-mannosidase and beta-D-galactosidase activity, and a higher level of GlcNAc'ase activity were released fromspermatozoa treated with protease. Furthermore, treating spermatozoa with the detergent n-octylglucoside caused the release of similar amounts of alpha-L-fucosidase and beta-D-galactosidase activity and higher amounts of alpha-D-mannosidase and GlcNAc'aseactivity, compared with those treated with calcium ionophore. Protease and n-octylglucoside treatments released similar amounts GlcNAc'ase activity. These results suggest that GlcNAc'ase is present mainly in the plasma membrane of pig spermatozoa, butthat alpha-L-fucosidase, alpha-D-mannosidase and beta-D-galactosidase are mainly found in the acrosome matrix including the outer acrosomal membrane, and that alpha-D-mannosidase may also be located in the inner acrosomal membrane. Maximal activity of alpha-D-mannosidase was at around physiological pH, but the other three glycosidases showed maximal activity at pH 4-5. When cumulus-free pig oocytes matured in culture were inseminated in the presence of various glycosidase inhibitors, the number of spermatozoa bound to the zona pellucida and the proportion of oocytes penetrated were greatly reduced by the inhibitors of alpha-L-fucosidase, alpha-D-mannosidase and beta-D-galactosidase, but not by an inhibitor of GlcNAc'ase. These results indicate that alpha-L-fucosidase, alpha-D-mannosidase and beta-D-galactosidase are all important glycosidases involved in pig sperm-zona interaction.