Fertilization in the mouse is initiated by sperm beta 1,4-galactosyltransferase (GalTase) binding to terminal
N-acetylglucosamine residues on the zona pellucida
glycoprotein ZP3. Binding of ZP3 induces exocytosis of the sperm acrosome, whose contents are believed to digest a penetration slit in the
zona matrix through which sperm reach the egg. As a consequence of acrosomal exocytosis, GalTase is redistributed to the lateral aspect of the sperm head, where its function remains unknown. In this location, GalTase could conceivably impede
zona penetration by binding to
N-acetylglucosamine residues exposed on
zona pellucida glycoproteins. Therefore, in this study we investigated the presence and function of acrosomal
glycosidases capable of removing the GalTase-binding site from
zona pellucida glycoproteins.
beta-N-acetylglucosaminidase was found at very high levels in sperm, being more than 20-fold higher than other
glycosidases assayed. The specific isozymic variant was identified as
beta-hexosaminidase B.
beta-N-acetylglucosaminidase was localized to sperm acrosomes by biochemical and indirect immunofluorescence studies and was released during the acrosome reaction, as expected for an
enzyme involved in
zona penetration. To determine if, in fact, acrosomal
beta-N-acetylglucosaminidase facilitated penetration through the
zona, an assay was developed using eggs that were rendered incapable of triggering the block to polyspermy. A specific competitive inhibitor of
beta-N-acetylglucosaminidase activity,
PUGNAC, inhibited sperm penetration of the
zona in a dose-dependent manner, whereas a closely related
beta-glucosidase inhibitor, PUGLU, had no effect on
zona penetration or on
beta-N-acetylglucosaminidase activity. Neither
glycosidase inhibitor affected sperm motility or induction of the acrosome reaction. These results demonstrate that
beta-N-acetylglucosaminidase is found in sperm acrosomes and is released during the acrosome reaction, at which time it facilitates sperm penetration through the
zona. These results also imply that sperm have developed mechanisms to prevent the formation of stable interactions between surface receptors and their zona pellucida
ligands during penetration.