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

在Sepharose2B柱上用凝胶层析法从海胆卵胶中分离得到岩藻糖硫酸酯(FSG)。FSG以一种浓度依赖的方式诱导了普氏沼虾精子的顶体反应，尽管它的活性大约是原始未分离凝胶的一半。合成的精子激活肽I(SAP-I：Gly-Phe-Asp-Leu-Asn-Gly-Val-Gly)可增加FSG诱导的顶体反应的速率，FSG和SAP-I的顶体反应速率最大的是普通凝胶。FSG的Pronase消化使顶体反应的诱导和精子细胞内cAMP的升高减少了50%。从海胆的卵冻中也纯化了唾液糖蛋白。唾液酸糖蛋白由唾液酸(90%，w/w)和蛋白质(10%，w/w)组成，不能引起顶体反应和精子等凝。FSG与…结果表明，一种摩尔硫酸盐/摩尔岩藻糖的蛋白质含量是岩藻糖的2.0倍。在2-ME存在下，完整FSG的蛋白质经SDS-PAGE分离为两种主要蛋白质(258 kDa和237 kDa)和一种次要蛋白质(120 KDa)，而在0.1%的SDS或不存在2-ME的SDS-PAGE中均不能分离到蛋白质。Wliell FSG首先用非放射性碘乙酸羧乙基化，然后用2-ME还原，最后用碳-14标记的碘乙酸羧甲基化，在对应于120 kDa蛋白中检测到大部分放射性。羧甲基化的FSG在诱导顶体反应方面不如完整的FSG。岩藻糖硫酸酯聚合物不能诱导顶体反应。这些结果表明，SAP-I通过作用于FSG的一个特定辅助因子来促进顶体反应的诱导，而FSG的蛋白部分在诱导卵子顶体反应中起重要作用。用小鼠抗FSG抗血清对不同阶段的卵母细胞进行的免疫组织化学研究表明，FSG形成于年轻卵母细胞的细胞质中，并在卵子发生过程中分泌到细胞表面。较少

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