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 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 sperm 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 with Galanthus nivalis agglutinin only in the cortex of the dimple, suggesting that this band is differently glycosylated according to its localization. The alpha-l-fucose-specific lectin Ulex europaeus agglutinin 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 to Ulex europaeus agglutinin 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.

?-spectrin has a stage-specific asymmetrical localization duringXenopus oogenesis

“…In this regard, Cl Ϫ channels are more concentrated in the animal hemisphere than in the opposite egg half (reviewed by Kline, 1988). In addition, in Discoglossus eggs, a 254/239 kDa spectrin heterodimer is tenfold more concentrated at the site of sperm entrance at the animal half, than in the rest of the egg, suggesting that it may anchor to the plasma membrane glycoproteins involved in fertilization (Tatone et al, 1993;Carotenuto, unpublished observations;Maturi et al, 1998). In Xenopus, by immunochemistry, we found that ␣-spectrin is located in the cortex, as well as in the cytoplasm of the whole oocytes of stages 4-6.…”

“…Oocytes were fixed in 2.5% glutaraldehyde in 50 mM phosphate buffer, pH 7.4, post-fixed in 1% OsO 4 , dehydrated in alcohol, and embedded in Epon, as previously reported (Maturi et al, 1998). Following staining with uranyl acetate and lead citrate, ultrathin sections were observed at a Philips 410 electron microscope at the Center for Ultrastructure Research (CIRUB) of the University of Naples, Federico II.…”

“…Sixty micrograms of proteins from oocytes of all the oogenetic stages were loaded on each gel. The samples were transferred electrophoretically to nitrocellulose paper (see Maturi et al, 1998) overnight at 200 mA in a cooling chamber. The efficiency of the transfer was evaluated by staining the nitrocellulose paper with Ponceau S and the corresponding gel with Coomassie Brilliant Blue.…”

p27BBP/eIF6 acts as an anti-apoptotic factor upstream of Bcl-2 during Xenopus laevis development

“…After boiling in sample buffer with 1 mM DTT, aliquots of 40 mg of proteins were analyzed through SDS-polyacrylamide gel electrophoresis (PAGE) 8% or 15% polyacrylamide using standard Tris glycine buffer and using molecular mass standards (200, 116, 97, 45, 31, 21 or 14 kDa) (Bio-rad, Hercules, CA, USA). Western blotting on nitrocellulose membrane was carried out as in Maturi et al 35 Incubation with the antibodies was carried out using an anti-p27 BrdU and HUA experiments. Xp27 BBP /eIF6 overexpressing embryos were injected at st.28 with BrdU (Roche, Mannheim, Germany) in the gut, fixed 2 h later and cryostat-sectioned.…”

Carbohydrate-Mediated Sperm-Egg Interaction and Species Specificity: A Clue from the <i>Unio elongatulus </i>Model

“…Fucose, for example, and to a greater extent its sulfated polymer fucoidan, are reported to be determinant for sperm-egg binding in different groups such as algae [Callow et al, 1985], echinoderms [Okinaga et al, 1992;Hoshi et al, 1994], ascidians [Rosati, 1985;Baginski et al, 1999], amphibians [Denis-Donini and Campanella, 1977;Maturi et al, 1998] and mammals [Benoff, 1997] including man [Oehninger et al, 1992;Oehninger, 2001]. The Unio model can be also listed as an example.…”