Specific inhibition of sperm β‐<i>N</i>‐acetylglucosaminidase by the synthetic inhibitor <i>N</i>‐acetylglucosaminono‐1,5‐lactone <i>O</i>‐(phenylcarbamoyl)oxime inhibits fertilization in the ascidian, <i>Phallusia mammillata</i>

Abstract

Increasing evidence has evolved from studies in ascidians and mammals that sperm β‐ N ‐acetylglucosaminidase (GlcNAc'ase) plays a crucial role in fertilization. In the ascidian Phallusia mammillata , GlcNAc'ase is the predominant sperm‐bound glycosidase and N‐acetylglucosamine (GlcNAc) is the prevailing glycoside residue on the vitelline coat. We report here that the GlcNAc'ase inhibitor O ‐(2‐acetamido‐2‐deoxy‐D‐glucopyrano‐sylidene)‐amino‐ N ‐phenylcarbamate (PUGNAC) is a potent competitive inhibitor of sperm‐bound GlcNAc'ase in P. mammillata . The inhibitor constant K i for the isolated enzyme is 47 nmol/L. Fertilization of eggs is inhibited by PUGNAC in a dose dependent competitive manner with 50% inhibition at an inhibitor concentration of 85 μmol/L. Further experiments, in which intact eggs possessing an egg coat were mixed with eggs from which the coat had been removed, showed that only fertilization of intact eggs was inhibited by PUGNAC. This finding suggests that PUGNAC prevents the binding of the sperm‐associated GlcNAc'ase to terminal GlcNAc residues on the vitelline coat, thus inhibiting sperm binding and subsequently fertilization. Furthermore and most importantly, it shows that treatment with PUGNAC does not affect the viability of sperm and that the process of sperm‐egg fusion is not affected.