Sperm-Egg binding in ascidian

精子与卵黄外壳的特异性结合是许多动物受精的必要步骤。在褐藻（Halocynthia roretzi）和肠草（Ciona ninteinalis）中，糖蛋白中酸性糖链的L-聚焦基残基似乎对卵磷脂外壳的精子受体活性至关重要。精子被合成底物（对硝基苯或4-甲基伞花基α-聚焦物）阻断与酶及其β-异头物的结合，后者作为竞争性抑制剂。精子在正常海水（pH值8.2）中与卵磷脂外壳结合，在pH值4时分离。由于聚焦胺亲和柱在pH为8.2的正常海水中对精子提取物有明显的吸附作用，但程度低于pH为4时，这表明该酶能够在正常海水中与底物形成复合物。因此，糖精链的L聚焦残基似乎对精子受体的活性至关重要。本文利用α-L-聚焦酶的保守区，采用RT-PCR方法分离了一段编码褐藻精子α-L-聚焦酶的cDNA （HrFuc）。制备了针对大肠杆菌lacZ-HrFuc融合蛋白的多克隆抗体。该抗体与精子47 kDa蛋白杂交，可抑制海水中海鞘的受精。该cDNA产物可能介导精子结合。

Specific binding of spermatozoa to the vitelline coat is an necessary step for fertilization in various animals. In Halocynthia roretzi and Ciona intestinalis, L-fucosyl residues of acidic saccharide chains in glycoproteins appears to be essential for sperm-receptor activity of the vitelline coat. Sperm binding was blocked by synthetic substrates (p-nitrophenyl or 4-methylumbelliferyl α-fucoside) toward the enzyme and their β-anomers that act as competitive inhibitors. The spermatozoa bound the vitelline coat in normal sea water (pH 8.2) detached at pH 4. Since the fucosylamine affinity column adsorbed the sperm extract appreciably in normal sea water at pH 8.2, though less extent than at pH 4, it was suggested that the enzyme was able to form a complex with substrates in normal sea water. Thus L-fucose residues of the sacchride chains appeared to be essential for the activity of sperm-receptor.In the present paper, we isolate a cDNA (HrFuc) encoding Halocynthia roretzi sperm α-L-fucosidase by RT-PCR method using the conservative region of α-L-fucosidase. The polyclonal antibody was prepared against a lacZ-HrFuc fusion protein made in E. coli. This antibody crossed to sperm 47 kDa protein, and inhibited ascidian fertilization in sea water. This cDNA product might be mediate the sperm binding.