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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过程中精子的功能(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，获得了以下结果：1)为了阐明顶体酶在受精过程中的作用，我们利用顶体酶缺陷突变小鼠研究了顶体酶在精子顶体反应中的作用。当在体外评估精子附着(附着)和结合无积云卵子的透明带的能力时，野生型和顶体酶缺陷小鼠之间没有明显的差异。免疫细胞化学分析表明，在钙离子载体和增溶透明带诱导的顶体反应中，ィイD2-/-ィエD2小鼠精子顶体中几种顶体蛋白的释放显著延迟，尽管膜囊泡正常。这些数据表明在顶体酶缺陷的…中，精子穿透透明带的延迟更多的小鼠结果是由于顶体蛋白质扩散速率的改变，并首次提供了证据表明顶体在顶体反应中的主要作用是加速顶体成分的扩散。2)为了鉴定参与精子/卵子相互作用的顶体蛋白的新候选者(S)，从顶体酶缺失的小鼠睾丸文库中扩增出一段DNA片段，并将其作为探针用于筛选小鼠睾丸cDNA库。已经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)我们获得了在精子顶体中积累突变绿色荧光蛋白(EGFP)的转基因小鼠。在顶体反应开始后，顶体成分分散所需的时间约为3秒。4)从人附睾尾液中纯化了人附睾液中主要分泌蛋白HE1的猪同源物。HE1同源物被发现是一种主要的胆固醇结合蛋白，表明它参与了附睾精子成熟过程中精子膜脂类成分的调节。较少