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中精子的功能（精子/卵子相互作用），我们利用转基因突变小鼠进行了生化和生物学实验，并获得了以下结果：1）为了阐明顶体素在受精中的作用，我们检查了顶体肽在受精过程中的作用。顶体素参与精子的顶体反应，利用顶体素缺陷的突变小鼠当精子粘附（附着）并结合到透明带的能力。体外评估无卵丘卵的透明质，在野生型和顶体蛋白缺陷小鼠之间没有观察到显着差异，免疫细胞化学分析表明，AcryD2-/-D2 小鼠精子顶体中的几种顶体蛋白的释放显着。尽管膜囊泡正常，但在钙离子载体和溶解的透明带诱导的顶体反应期间延迟。这些数据表明，在顶体蛋白缺陷小鼠中，精子穿透透明带的延迟是由于顶体蛋白质分散速率改变造成的，并提供了第一个证据，表明顶体蛋白的主要作用是加速顶体的分散。 2) 为了鉴定作用于精子/卵子相互作用的顶体蛋白的新候选蛋白，从 cDNA 文库中 PCR 扩增了 DNA 片段顶体蛋白缺陷的小鼠睾丸，然后用作筛选小鼠睾丸 cDNA 文库的探针，已鉴定出编码四种相似但不同的丝氨酸蛋白酶 TESP1、TESP2、TESP3 和 TESP4 的互补 DNA 克隆。3) 我们。产生的转基因小鼠品系在精子顶体中积累突变的绿色荧光蛋白（EGFP）。顶体成分分散所需的时间被证明是大约3秒后。 4) HE1是人类附睾主要分泌蛋白的猪同源物，是从附睾尾液中纯化出来的。HE1同源物被发现是一种主要的胆固醇结合蛋白，表明它参与了调节。精子在附睾成熟过程中精子膜的脂质组成较少。