Identification, Localization and Involvement of Glycosidases in Sperm-Zona Interaction Using Frozen-Thawed Ejaculated Pig Spermatozoa

抄録

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 α-L-fucosidase, α-D-mannosidase, β-D-galactosidase and N-acetyl-β-D-glucosaminidase (GlcNAc’ase). When spermatozoa were incubated with calcium ionophore A23187 for 1 h, about 70% were acrosome-reacted and about 72, 46, 56 and 35% of the activities of α-L-fucosidase, α-D-mannosidase, β-D-galactosidase and GlcNAc’ase, respectively, were lost from spermatozoa. However, lower levels of α-L-fucosidase, α-D-mannosidase and β-D-galactosidase activity, and a higher level of GlcNAc’ase activity were released from spermatozoa treated with protease. Furthermore, treating spermatozoa with the detergent n-octylglucoside caused the release of similar amounts of α-L-fucosidase and β-D-galactosidase activity and higher amounts of α-D-mannosidase and GlcNAc’ase activity, 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, but that α-L-fucosidase, α-D-mannosidase and β-D-galactosidase are mainly found in the acrosome matrix including the outer acrosomal membrane, and that α-D-mannosidase may also be located in the inner acrosomal membrane. Maximal activity of α-D-mannosidase was at around physiological pH (7-8), 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 α-L-fucosidase, α-D-mannosidase and β-D-galactosidase, but not by an inhibitor of GlcNAc’ase. These results indicate that α-L-fucosidase, α-D-mannosidase and β-D-galactosidase are all important glycosidases involved in pig sperm-zona interaction.