Basic studies on X- and Y-bearing sperm

本项目旨在通过自由流动电泳等手段分离哺乳动物携带x染色体和y染色体的精子，并检测它们在生化、细胞生物学和免疫学等方面可能存在的差异。这一结果可以应用于人类致命的性相关疾病的预防和家畜的性别选择。出于上述目的，我们购买了赫希曼ACE710自由流电泳仪。作为材料，主要使用人类精子，因为y精子可以用奎纳克里芥末染色的f体来识别。作为第一步，进行了几次试验来纯化成熟的正常精子样本。Percoll连续密度梯度离心与游向法相结合，获得了最佳的分离效果。通过这种方法，自由流动电泳得到了两个分离峰。醌芥子气染色显示，阳极附近的峰主要由x精子组成，而第二个峰则由y精子组成。采用聚合酶链反应（PCR）扩增X染色体和y染色体中不同内含子的淀粉原蛋白基因，进一步分析分离样品的X染色体和y染色体。所得DNA片段用琼脂糖凝胶电泳分析。根据目前得到的结果，y精子部分确实只含有y染色体，但x精子部分仍然被y精子污染。需要进一步纯化x精子部分。zeta电位测量结果显示，x精子为-17 mV， y精子为-12 mV。发现一种针对人类精子细胞表面的单克隆抗体可使y型精子的ζ电位向负方向移动。在其他动物中，迄今为止在老鼠身上获得了两个分离的精子峰值。关于X-和y -精子的分离，也进行了一些关于精子成熟、精子活力等的基础研究。

This project aimed to separate mammalian X-chromosome and Y-chromosome bearing sperm by free-flow electrophoresis and other means, and to detect putative differences between them biochemically, cell biologically and immunologically, etc. The results could be applied to prevention of fatal sex-linked diseases in human and to sex selection in domestic animals. For the above purpose, we purchased a Hirschmann ACE710 fred-flow electrophoretic apparatus. As materials, human sperm were mainly used, because the Y-sperm can be identified with F-body stained with quinacrine mustard. As the first step, several trials were made to purify mature normal sperm samples. The best results were obtained by a combination of Percoll continuous density gradient centrifugation and swim down method. By this way, two separated peaks were obtained at a high rate by free-flow electrophoresis. The quinacrine mustard staining revealed that the peak near the anode mainly consisted of X-sperm, while the second peak contained Y-sperm. The separated samples were further analyzed for X- and Y-chromosome by amplifying amelogenin gene an intron of which is different between X- and Y-chromosome by PCR (polymerase chain reaction). The obtained DNA fragments were analyzed by agarose gel electrophoresis. According to the results so far obtained, the Y-sperm fraction really contained only Y-chromosome, but the X-sperm fraction was still contaminated with Y-sperm. Further purification of the X-sperm fraction is needed. Measurements of zeta-potential resulted in -17 mV for X-sperm and -12 mV for Y-sperm. A monoclonal antibody against human sperm cell surface was found to shift the zeta-potential of Y-sperm toward more negative side. Among other animals, two separated sperm peaks have so far been obtained with the mouse. In connection with the separation of X- and Y-sperm, several fundamental studies on sperm maturation, sperm motility, etc., have also been conducted.