Abstract
During spawning, female Sicyonia ingentis simultaneously release ova and stored nonmotile sperm and mix them externally to initiate gamete interaction. Sperm bind to a thin vitelline envelope (VE) via their anterior appendage and within seconds are induced to undergo acrosomal exocytosis. The sperm penetrate the VE and become secondarily bound to the surface coat (SC), a glycocalyx on the oocyte surface. In this study, both extracellular matrices were isolated from S. ingentis oocytes. Isolated VEs mediated only primary sperm binding (i.e., before the acrosome reaction), while the isolated SCs mediated only secondary sperm binding (i.e., after acrosomal exocytosis). Isolated S. ingentis VEs were used to characterize primary sperm binding activity. The two extracellular matrices differ morphologically and possess different polypeptide profiles. Soluble fractions of isolated VEs inhibited primary sperm binding in a concentration dependent manner, and immunolocalization of VE components demonstrated highly localized VE binding sites at the tip of the sperm anterior appendage by which sperm bind eggs. Extensive Pronase digestion of VE components did not affect sperm binding activity of solubilized VE components, while complete deglycosylation with trifluoromethanesulfonic acid destroyed sperm binding activity. However, neither alkaline treatment nor enzyme digestion using glycosidases specific for asparagine and serine/ threonine linked oligosaccharides affected sperm binding activity.