| Research Abstract |
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.
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