Infante Vincenzo, Caputo Mariangela, Riccio Stefania, De Filippis Anna, Carotenuto Rosa, Vaccaro Maria Carmen, Campanella Chiara
Dipartimento di Biologia Evolutiva e Comparata, Universita' di Napoli Federico II, via Mezzocannone 8, 80134 Naples, Italy.
Mol Reprod Dev. 2004 Jun;68(2):213-22. doi: 10.1002/mrd.20063.
In Xenopus, conflicting data related to sperm-vitelline envelope (VE) binding suggest that further experiments should be performed to study the role of VE glycoproteins in sperm binding. In this article, we studied the VE of Discoglossus pictus, where gp63, the product of the Dp ZP2 gene, has high molecular identity to Xenopus gp69/64 and to mouse ZP2 and only A23187-treated sperm bind to VE. Sperm bind to VE all over the egg, yet a sperm tuft was found only in the animal half of the egg, where the dimple, the site of fertilization, is located and an intense immunostain was detected in VE by an antiserum directed against gp69/64. The same antiserum inhibited sperm binding to VE. Sperm binding to beads coated with gp63, gp40, or gp75 was in the range of 62-70% for gp63-beads, 67-75% for 75 beads, and about 20% for BSA beads and gp40-coated beads. Soluble purified gp63 and gp75 competitively inhibited binding of sperm to gp63-coated beads. Similarly, the same glycoproteins inhibited sperm binding to gp75-coated beads. SDS-polyacrylamide gels (PAGE) of FE and comparison of VE and FE peptide maps showed that gp63 undergoes a minor shift to about 62 kDa in FE. In sperm binding assays with beads coated with FEs gp62, there was no binding. Following fertilization, in the region of the dimple, an F-layer is formed as well as an alteration of the VE structure. Lectin blots of the FE showed that the FE and in particular gp62 acquires a stronger affinity to Maackia amurensis agglutinin (MAA) with respect to VEs gp63. These results indicate that gps 63 and 75 are the sperm binding glycoproteins of D. pictus VE, where major post-fertilization changes occur as in other anuran species.
在非洲爪蟾中，与精子 - 卵黄膜（VE）结合相关的数据相互矛盾，这表明应进行进一步实验来研究VE糖蛋白在精子结合中的作用。在本文中，我们研究了斑腿泛树蛙的VE，其中Dp ZP2基因的产物gp63与非洲爪蟾的gp69/64以及小鼠的ZP2具有高度的分子同源性，并且只有经A23187处理的精子才能与VE结合。精子在整个卵细胞上都能与VE结合，但仅在卵细胞动物极的一半发现有精子簇，受精位点酒窝就位于此处，并且用针对gp69/64的抗血清在VE中检测到强烈的免疫染色。相同的抗血清可抑制精子与VE的结合。精子与包被有gp63、gp40或gp75的珠子的结合率，对于gp63包被的珠子在62 - 70%之间，对于gp75包被的珠子在67 - 75%之间，而对于牛血清白蛋白（BSA）包被的珠子和gp40包被的珠子约为20%。可溶性纯化的gp63和gp75竞争性抑制精子与gp63包被珠子的结合。同样，相同的糖蛋白也抑制精子与gp75包被珠子的结合。VE的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）以及VE和受精提取物（FE）肽图的比较表明，gp63在FE中会轻微迁移至约62 kDa。在用包被有FE的gp62的珠子进行的精子结合试验中，未出现结合现象。受精后，在酒窝区域会形成一个F层以及VE结构的改变。FE的凝集素印迹显示，相对于VE的gp63，FE尤其是gp62对黑果越桔凝集素（MAA）具有更强的亲和力。这些结果表明，gp63和gp75是斑腿泛树蛙VE的精子结合糖蛋白，在受精后会发生与其他无尾两栖类物种类似的主要变化。
