Toshio Hirayama

Mannose-binding molecules of rat spermatozoa and sperm-egg interaction.

We have previously reported the occurrence and partial characterisation of an alpha-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25-30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 degrees C in 5% CO2 in air. The sperm-zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These result suggest that mannose-binding molecule(s) such as alpha-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.

The measurement of hyaluronidase activity in human spermatozoa by substrate slide assay and its clinical application

Acrosomal hyaluronidase activity of individual sperm can be detected by a halo formation around the sperm head on hyaluronic acid substrate slides. The following results were obtained by this method in clinical practice. A significant correlation was found between hyaluronidase activity and sperm concentration in male infertility. Hyaluronidase activity increased as the concentration of sperm increased, and the least hyaluronidase activity was determined at 6 /ml. The group with more than 40% motility had a higher hyaluronidase activity than other groups with poor motility. Although there was no significant correlation between hyaluronidase and cumulus dispersion and fertilization rates in immature oocytes, there was an excellent correlation in mature oocytes in human in vitro fertilization. These findings suggest that a newly developed assay will be useful for evaluating sperm fertilizing capacity.