Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that α-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm α-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm α-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained Lfucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm α-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of α-L-fucose on sperm binding and penetrating into ZP caused sperm α-L-fucosidase inhibited by α-Lfucose.

Abstract. 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-Dmannosidase, 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 noctylglucoside 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-Dgalactosidase are all important glycosidases involved in pig sperm-zona interaction.