Immunocytochemical and ultrastructural study on the condition of Fertilization.

用冷冻断裂法研究了几种哺乳动物精子在附睾运输过程中膜内颗粒(Imps)的变化。一般情况下，顶体周围质膜(PAPM)可区分大小IMP。在仓鼠、大白兔和公猪中，小IMP在时间上形成六角排列，而在IMP密度较低的人精子中未发现这种排列。根据这些观察，形成IMP-型式可能需要以下条件：(1)相似大小的大量IMP的存在，(2)IMP的运动，(3)IMP聚集因素的存在，如运动的障碍。在金黄地鼠精子附睾部的传代过程中，膜类固醇密度增加。取自附睾头的金黄地鼠精子的PAPM中，FSCS的密度为390个/微米^2，当精子通过精子头部时，其密度变为约500个/微米。金黄地鼠精子体外获能过程中精子质膜的变化在泰罗德液中孵育，冷冻断裂法观察其变化。孵育2小时后，PAPM内FSC密度下降70-80%。培养3h后，精子获能后，大部分小IMP消失，并在PAPM内形成较宽的IMP自由区，产生抗小鼠生精细胞蛋白的单抗。其中一种抗体13D3识别hsc71，它属于7OkDa热休克蛋白家族。间接免疫荧光显示，hsc7l在幼龄生精细胞胞浆内呈阳性表达，但定位于精子中段。

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.