Molecular cloning and sequence analysis of an ascidian egg β‐N‐acetylhexosaminidase with a potential role in fertilization

β‐N‐Acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate‐based transient sperm–egg coat binding. In ascidians and mammals, β‐hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata , N‐acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of β‐hexosaminidase. In the present study, P. mammillata β‐hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known β‐hexosaminidases; however, P. mammillata β‐hexosaminidase had a unique potential N‐glycosylation site. A phylogenetic analysis suggested that P. mammillata β‐hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage‐specific expression of β‐hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg β‐hexosaminidase forms are specific for the oocyte, test cells and follicle cells.