Egg jelly molecules : The role in fertilization and the formation in growing oocytes

在Sepharose 2B柱上，用凝胶层析法从海胆（hemicentrrotus pulcherrimus）卵冻中分离到一种聚焦硫酸糖缀合物（FSG）。FSG在H. pulcherrimus精子中诱导顶体反应呈浓度依赖性，尽管其活性约为原始未分离果冻的一半。合成的精子激活肽I （SAP-I:Gly- phe - asp - leu - asn -Gly-Gly-Gly- Gly- val -Gly）增加了FSG诱导的肌酸反应速率；顶体与FSG和sap - 1反应的最大速率为未分馏果冻的最大速率。FSG的Pronase酶切导致顶体反应的诱导减少50%，同时精子细胞中cAMP的升高。从海胆H. pulcherrimus的卵冻中也纯化出一种唾液糖蛋白。由唾液酸（90%,w/w）和蛋白质（10%,w/w）组成的唾液糖蛋白没有诱导顶体反应和精子异凝集。含有1 mol硫酸盐/mol聚焦物的FSG具有2.0倍的重量聚焦蛋白。在2-巯基乙醇（2- me）存在下，SDS-聚丙烯酰胺凝胶电泳（SDS- page）分离得到2个主要蛋白（258 kDa和237 kDa）和1个次要蛋白（120 kDa），而在没有2- me的0.1% SDS或SDS- page存在下，HPLC无法分离蛋白质。FSG先用无放射性的碘乙酸羧基化，然后用2-ME还原，最后用碳-14包覆的碘乙酸羧基化，在120 kDa蛋白对应的蛋白质中检测到最多的放射性。羧基化FSG诱导顶体反应的能力弱于完整FSG。氟吡喃丹是一种聚焦硫酸盐聚合物，没有引起顶体反应。这些结果表明，sap - 1通过作为FSG的特异性辅助因子促进顶体反应的诱导，而FSG的蛋白部分在诱导顶体反应中起重要作用。使用小鼠抗FSG抗血清对不同阶段卵母细胞进行免疫组化研究表明，FSG在年轻卵母细胞的细胞质中形成，并在卵发生过程中分泌到细胞表面。少

A fucose sulfate glycoconjugate (FSG) was isolated from the egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel chromatogrqaphy on a Sepharose 2B columnn. FSG induced the acrosome reaction in H. pulcherrimus spermatozoa in a concentration-dependent manner, although it showed about half the activity of the original unfractionated jelly. Synthetic sperm-activating peptide I (SAP-I:Gly- Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) increased the rate of the acrosoine reaction induced by FSG; the maximal rate of the acrosome reaction with FSG and SAP-I being that of the unfractionated jelly. Pronase digestion of FSG resulted in an 50% decrease in induction of the acrosome reaction and also in the elevation of cAMP in sperm cells.A sialoglycoprotein was also purified from the egg jelly of the sea urchin H. pulcherrimus. Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination.FSG which con … More tained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2- mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. Wliell FSG was first carboxyniethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymetliylated with carbon-14 lapeled iodoacetic acid, the most of radioactivity was detected in proteins corresponding to a 120 kDa protein. Carboxymetiiylated-FSG was less potent than intact FSG in induction of the acrosome reaction. Flicoidan, a fucose sulfate polymer, did not induce the acrosome reaction.These results indicate that SAP-I promotes induction of the acrosome reaction by acting a specific co-factor of FSG and the protein moiety of FSG plays an important role in induction of the acrosome reaction in H. pulcherrimus spermatozoa.Immunohistochemical study with various stages of oocytes using a mouse anti-FSG anti-serum indicated that FSG is formed in the cytoplasm of a young oocyte and secreted to the cell surface during the oogenesis. Less