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Immunocytochemical and ultrastructural study on the condition of Fertilization.

受精条件的免疫细胞化学和超微结构研究。

基本信息

批准号：
62570005
负责人：
TOYOTA Fumie
金额：
$ 1.22万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570005/
关键词：
Sperm maturation Capacitation Freeze fracture Membrane sterol Intramembranous particle Monoclonal antibody Heat shock protein Immuno fluorescence Sperm maturation Capacitation Freeze fracture Membrane sterol Intramembranous particl

项目摘要

Changes in the intramembranous particles (Imps) of the sperm plasma membrane during epididymal transport Spermatozoa taken from the various parts of the epididymis were studied by freeze fracture in several mammalian species. In general, large and small IMPs could be distinguished in the peri-acrosomal plasma membrane (PAPM). In hamsters, rats rabbits and boars, hexagonal arrangement was temporally formed by the small IMPs, but it was not found in the human spermatozoa, whose IMP- density was low. From these observations, following conditions may be needed for the IMP-pattern formation; (1) the presence of similarly sized numerous IMPs, (2) the movement of the IMPs, (3) the presence of IMP gathering factors such as a barrier for their movement.Increase of membrane sterol density in the hamster spermatozoa during their epididymal passage The density of filipin-sterol complexes (FSCs),was 390/mum^2 in the PAPM of the hamster spermatozoa taken from the initial segment of the epididymis and it became to about 500/mum^2. when the spermatozoa passed the caput flexure. The increase of FSC density may contribute to stabilize the membrane.Changes of the sperm plasma membrane during in vitro capacitation in the golden hamster Spermatozoa were incubated in Tyrode solution, and they were examined by freeze fracture. At two hours after incubation, FSC density in the PAPM reduced by 70-80%. At three hours after incubation, when the spermatozoa were capacitated, the majority of small IMPs had disappeared, and wide IMP free- area were formed in the PAPM.Production of monoclonal antibody Monoclonal antibodies were produced against extracted proteins from the isolated spermatogenic cells in mice. One of such antibody, 13D3, recognized hsc7l, which belongs to 7OkDa heat shock protein family,. Indirect immunofluorescence showed that hsc7l was present throughout the cytoplasm in the young spermatogenic cells, but localized in the middle piece of the spermatozoa.

精子质膜膜内颗粒在附睾运输过程中的变化用冷冻断裂法研究了几种哺乳动物附睾不同部位的精子。一般而言，可以在顶体周围质膜（PAPM）中区分大小IMP。在仓鼠、大鼠、家兔和公猪中，小IMP暂时形成六边形排列，但在人精子中未发现，其IMP-密度低。根据这些观察，IMP图案形成可能需要以下条件：（1）大小相似的大量IMP的存在;（2）IMP的运动;（3）IMP聚集因子的存在，如IMP运动的障碍。仓鼠精子在附睾通过过程中膜固醇密度的增加在取自仓鼠附睾起始段的精子的PAPM中，PAPM为390个/mum ^2，而在取自仓鼠附睾起始段的精子的PAPM中，PAPM为500个/mum ^2。精子通过头部曲的时间。金仓鼠精子体外获能过程中质膜的变化将精子置于台氏液中孵育，用冷冻断裂法观察精子质膜的变化。在孵育后两小时，PAPM中的FSC密度降低了70- 80%。孵育后3小时，精子获能，大部分小IMP消失，PAPM中形成了较宽的IMP自由区。单克隆抗体的产生单克隆抗体产生于小鼠分离的生精细胞中，可对抗提取的蛋白质。其中一株抗体13 D3识别70 kDa热休克蛋白家族成员hsc 71。间接免疫荧光显示hsc 7 l在年轻的生精细胞胞质中广泛表达，但仅定位于精子中段。