Studies on Sperm Function in Fertilization

精子在受精过程中的功能研究

基本信息

批准号：
09460154
负责人：
BABA Tadashi
金额：
$ 8.32万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460154/
关键词：
fertilization sperm egg zona pellucida mouse acrosome acrosin protease 卵子

项目摘要

To elucidate the sperm function (sperm/egg interaction) in mammalian fertilization, we have carried out biochemical and biological experiments using genetically modified mutant mice, and have obtained the following results :1) To elucidate the role of acrosin in fertilization, we have examined the involvement of acrosin in the acrosome reaction of sperm, using the acrosin-deficient mutant mice. When the ability of sperm to adhere (attach) and bind to the zona pellicida of cumulus-free eggs was assessed in vitro, no significant difference was observed between the wild-type and acrosin-deficient mice. Immunocytochemical analysis demonstrated that the release of several acrosomal proteins from the acrosome of AcrィイD2-/-ィエD2 mouse sperm was significantly delayed during the calcium ionophore- and solubilized zona pellucida-induced acrosome reaction, in spite of normal membrane vesiculation. These data indicate that the delayed sperm penetration of the zona pellucida in the acrosin-deficient … More mouse results from the altered rate of protein dispersal from the acrosome, and provide the first evidence that the major role of acrosin is to accelerate the dispersal of acrosomal components during acrosome reaction.2) To identify a novel candidate(s) for acrosomal proteins that act on the sperm/egg interaction, a DNA fragment was PCR-amplified from a cDNA library of acrosin-deficient mouse testis, and then used as a probe to screen a mouse testis cDNA library. Complementary DNA clones encoding each of four similar but different serine proteases, TESP1, TESP2, TESP3, and TESP4, have been identified.3) We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome. The time required for the dispersal of acrosomal components was demonstrated to be approximately 3 seconds after the onset of acrosome reaction.4) A porcine homologue of the major secretory protein of human epididymis, HE1 was purified from cauda epididymal fluids. The HE1 homologue was found to be a major cholesterol-binding protein, suggesting that it is involved in the regulation of the lipid composition of the sperm membranes during sperm maturation in epididymis. Less

为了阐明哺乳动物受精的精子功能（精子/卵的相互作用），我们使用一般修饰的突变小鼠进行了生化和生物学实验，并获得了以下结果：1）阐明了Acrosin在生育中的作用，我们研究了Acrosin在Acrosient actrosien cocters中的参与。当精子在体外评估精子（附着）并结合与无云卵的Zona pellicida的能力时，在野生型和Acrosin缺陷小鼠之间未观察到无显着差异。免疫细胞化学分析表明，在正常的膜囊肿的症状中，在钙离子载体和可溶性Zona Pearlucida诱导的钙离子载体和可溶性Zona Pearlucida诱导的钙和可溶性Zona Pearlucida诱导的钙载体和可溶性Zona Pearlucida期间，释放了几种顶体蛋白在丙烯型D2 - / - D2小鼠精子中显着延迟。这些数据表明，Zona pellucida在Acrosin缺乏的延迟的精子渗透延迟。更多的小鼠是由于蛋白质分散率的变化而导致的，并且提供了第一个证据，并提供了首先证据，表明Acrosin的主要作用是加速反应过程中Acrosin成分的分散。 PCR从缺乏Acrosin的小鼠睾丸的cDNA库中扩增，然后用作筛选小鼠睾丸cDNA库的探针。已经鉴定出了四种相似但不同的丝氨酸蛋白（TESP2，TESP3和TTESP4）中的每个中的互补DNA克隆。首席反应发作后约3秒钟，分散质量分散所需的时间。4）一种人类附子性主要秘密蛋白质的猪同源物，HE1从Cauda附子流体中纯化。发现HE1同源物是一种主要的胆固醇结合蛋白，这表明它参与了在附睾精子成熟过程中精子膜的脂质组成的调节。