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Molecular Mechanisms of Sperm-Egg Interactions in the Ascidians

海鞘精卵相互作用的分子机制

基本信息

批准号：
02044053
负责人：
HOSHI Motonori
金额：
$ 6.59万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

The ascidians are hermaphrodite and many species are seif-sterile. Using two species of self-sterile solitary ascidiansas model systems, Ciona intestinalis (Enterogona) and Halocynthia roretzi (Pleurogona), molecular mechanisms underlying the sperm-egg interactions were studied.We postulated previously that a sperm glycosidase (e.g., alpha-L-fucosidase in Ciona) and corresponding sugar chains of the vitelling coat (VC) mediate the binding of ascidian sperm to the homologous VC. Several lines of experimental evidence has further supported this hypothesis in both Ciona and Halocynthia. In H. roretzi, we have purified the major VC glycoprotein of about 70 kDa. It has terminal fucose residues and presumably serves as a sperm-receptor. Its amino acid sequence was partially revealed.A chymotrypsin-like protease was purified from Ciona sperm as a VC-lysin and proved to digest the outermost thin layer of the VC. It was also shown that a Zn^<++>-dependent metalloprotease is involved in sperm-eg … More g fusion in Ciona.By using in vitro fertilization assay of oocytes from Ciona ovaries, we have found that self-sterility is controlled by products, presumably a glycoprotein, of the overlying follicle cells. When fully-grown ovarian oocytes with a germinal vesicle were incubated in sea water at room temperature, they underwent germinal vesicle breakdown within 20 minutes and became fertile, and eventually acquired self-sterility within a maximum of 3 hours of incubation. When they were deprived of follicle cells by gentle shaking before incubation, most of them remained self-fertile even after 3 hours. If follicle cell-free oocytes were incubated with the free follicle cells, they became self-sterile as well as follicle cell-intact oocytes.The acquisition of self-sterility was accompanied by an ultrastructural change in the VC fixed with tannic acid : an electron-dense, thin layer appeared on the outer surface of the VC after 3 hours of incubation. This was the layer digested by the chymotryptic lysin in the sperm. Therefore it is suggested that the induction of acrosome reaction is the site modulated by allo-recognition between sperm and VC. Less

海鞘是雌雄同体，许多种类是自不育的。使用两种自育的孤立海鞘模型系统，海鞘（Enterogona）和Halocynthia roretzi（Pleurogona），研究了精卵相互作用的分子机制。我们之前假设精子糖苷酶（例如海鞘中的α-L-岩藻糖苷酶）和卵黄衣（VC）的相应糖链介导海鞘精子与同源 VC 的结合。几条实验证据进一步支持了 Ciona 和 Halocynthia 中的这一假设。在 H. roretzi 中，我们纯化了大约 70 kDa 的主要 VC 糖蛋白。它具有末端岩藻糖残基，可能充当精子受体。它的氨基酸序列被部分揭示。从玻璃海鞘精液中纯化出一种胰凝乳蛋白酶样蛋白酶，作为 VC 溶素，并证明可以消化 VC 的最外层薄层。研究还表明，Zn^<++> 依赖性金属蛋白酶参与 Ciona 中的精子融合。通过对 Ciona 卵巢中的卵母细胞进行体外受精测定，我们发现自体不育是由覆盖卵泡细胞的产物（可能是糖蛋白）控制的。当具有生发囊泡的完全生长的卵巢卵母细胞在室温下的海水中孵育时，它们在20分钟内发生生发囊泡破裂并变得可育，并最终在最多3小时的孵育内获得自体不育。当它们在孵化前通过轻轻摇动去除卵泡细胞时，即使在3小时后，大多数它们仍然保持自体受精。如果将无卵泡细胞的卵母细胞与游离卵泡细胞一起孵育，它们就会成为自不育以及卵泡细胞完整的卵母细胞。自不育的获得伴随着单宁酸固定的VC的超微结构变化：孵育3小时后，VC的外表面上出现电子致密的薄层。这是精子中糜蛋白酶溶素消化的层。因此提示顶体反应的诱导是精子与VC同种异体识别的调节位点。较少的