Specific Glycoconjugates Are Present at the Oolemma of the Fertilization Site in the Egg ofDiscoglossus pictus(Anurans) and Bind Spermatozoa in anin VitroAssay

Abstract

In the egg of the anuranDiscoglossus pictus,the site of fertilization is restricted to the central portion of an animal hemisphere indentation (thedimple). Previous studies showed that the acrosome reaction ofD. pictussperm is triggered in the jelly, and yet sperm arrive at the dimple surface with the plasma membrane at an early stage of vesiculation. Reactivity of the dimple surface with specific lectins suggests that fucose might be utilized as a marker of glycoproteins located at the dimple surface. In this paper, proteins of the egg surface were labeled with the membrane impermeable sulfo-NHS-biotin. Four main bands of 200, 230, 260, and 270 kDa labeled only at the dimple surface, although they were detected in the cortex of the whole egg. The 270-kDa band reacted withGalanthus nivalisagglutinin only in the cortex of the dimple, suggesting that this band is differently glycosylated according to its localization. The α-l-fucose-specific lectinUlex europaeusagglutinin I was utilized both in lectin blotting and in affinity chromatography and cross-reacted with the 200- and 270/260-kDa bands. Furthermore, two polypeptides were obtained by exposure of intact eggs to lysylendoproteinase C. They were also reactive toUlex europaeusagglutinin I. The 200- and 270/260-kDa bands were eluted from the acrylamide gels and adsorbed to polystyrene beads. An assay for sperm binding to 200-kDa glycoprotein-bound beads was developed. Sperm stuck to the beads before but not after Ca-ionophore treatment. When the beads were coated with the 270/260-kDa glycoproteins, binding occurred after ionophore treatment. In these assays, the 200- and 270/260-kDa glycoproteins competitively inhibited sperm binding to the beads coated with the corresponding glycoprotein. These results indicate that the assayed glycoproteins, located either in the glycocalyx or in the plasma membrane of the fertilization site, are involved in sperm binding.