Maturi G, Infante V, Carotenuto R, Focarelli R, Caputo M, Campanella C
Dipartimento di Biologia Evolutiva e Comparata, Universita' di Napoli, via Mezzocannone n.8, Napoli, 80134, Italy.
Dev Biol. 1998 Dec 1;204(1):210-23. doi: 10.1006/dbio.1998.9083.
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.
在无尾两栖类盘舌蟾的卵中，受精位点局限于动物半球凹陷（即凹痕）的中央部分。先前的研究表明，盘舌蟾精子的顶体反应在卵胶中被触发，然而精子在囊泡化早期时，其质膜就已到达凹痕表面。凹痕表面与特定凝集素的反应性表明，岩藻糖可能被用作位于凹痕表面的糖蛋白的标志物。在本文中，卵表面的蛋白质用膜不可渗透的磺基 - NHS - 生物素进行标记。200、230、260和270 kDa的四条主要条带仅在凹痕表面被标记，尽管它们在整个卵的皮质中也能被检测到。270 kDa的条带仅在凹痕皮质中与雪花莲凝集素发生反应，这表明该条带根据其定位不同而具有不同的糖基化。α - l - 岩藻糖特异性凝集素荆豆凝集素I被用于凝集素印迹和亲和色谱法，并与200 kDa以及270/260 kDa的条带发生交叉反应。此外，完整的卵经赖氨酸内切蛋白酶C处理后获得了两种多肽。它们也与荆豆凝集素I发生反应。200 kDa以及270/260 kDa的条带从丙烯酰胺凝胶上洗脱下来并吸附到聚苯乙烯珠上。开发了一种检测精子与结合有200 kDa糖蛋白的珠子结合的方法。精子在钙离子载体处理之前会黏附在珠子上，但处理之后则不会。当珠子包被有270/260 kDa糖蛋白时，在离子载体处理后会发生结合。在这些实验中，200 kDa以及270/260 kDa的糖蛋白竞争性抑制精子与包被有相应糖蛋白的珠子的结合。这些结果表明，所检测的位于受精位点糖萼或质膜中的糖蛋白参与了精子结合。
