Basic studies on X- and Y-bearing sperm

本项目旨在通过自由流动电泳法和其他方法分离哺乳动物的X染色体和Y染色体精子，并从生化、细胞生物学和免疫学等方面检测它们之间的可能差异，其结果可用于人类致死性连锁疾病的预防和家畜的性别选择。为此，我们购买了Hirschmann ACE710FRED-FLOW电泳仪。作为材料，主要使用人精子，因为用喹啉芥末染色的F-Body可以识别Y-精子。作为第一步，进行了几次试验来提纯成熟的正常精子样本。Percoll连续密度梯度离心法与游泳法相结合效果最好。通过这种方法，在自由流电泳法中获得了高速分离的两个峰。喹啉芥末染色显示，阳极峰主要为X-精子，第二峰为Y-精子。通过聚合酶链式反应扩增X-染色体和Y-染色体不同内含子的釉原蛋白基因，进一步分析X-和Y-染色体。用琼脂糖凝胶电泳法对获得的DNA片段进行分析。根据目前获得的结果，Y精子部分确实只含有Y染色体，但X精子部分仍被Y精子污染。还需要进一步提纯X精子组分。测量Zeta-电位，X-精子为-17 mV，Y-精子为-12 mV。一种抗人精子表面的单抗可使Y精子的Zeta电位向负值偏移。在其他动物中，到目前为止，老鼠已经获得了两个分离的精子峰。在X-精子和Y-精子的分离方面，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.