Specific glycoconjugates are present at the oolemma of the fertilization site in the egg of Discoglossus pictus (Anurans) and bind spermatozoa in an in vitro assay

In the egg of the anuran Discoglossus pictus, the site of fertilization is restricted to the central portion of an animal hemisphere indentation (the dimple). Previous studies showed that the acrosome reaction of D. pictus sp erm is triggered in the jelly, and yet sperm arrive at the dimple surface w ith the plasma membrane at an early stage of vesiculation. Reactivity of th e dimple surface with specific lectins suggests that fucose might be utiliz ed as a marker of glycoproteins located at the dimple surface. In this pape r, proteins of the egg surface were labeled with the membrane impermeable s ulfo-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 wh ole egg. The 270-kDa band reacted with Galanthus nivalis agglutinin only in the cortex of the dimple, suggesting that this band is differently glycosy lated according to its localization. The alpha-L-fucose-specific lectin Ule x europaeus agglutinin I was utilized both in lectin blotting and in affini ty chromatography and cross-reacted with the 200- and 270/260-kDa bands. Fu rthermore, two polypeptides were obtained by exposure of intact eggs to lys ylendoproteinase C. They were also reactive to Ulex europaeus agglutinin I. The 200- and 270/260-kDa bands were eluted from the acrylamide gels and ad sorbed to polystyrene beads. An assay for sperm binding to 200-kDa glycopro tein-bound beads was developed. Sperm stuck to the beads before but not aft er 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 bindi ng to the beads coated with the corresponding glycoprotein. These results i ndicate that the assayed glycoproteins, located either in the glycocalyx or in the plasma membrane of the fertilization site, are involved in sperm bi nding. (C) 1998 Academic Press.