ONO Michio Yokohama City University, Dept. Anatomy, Assistant Professor, 医学部, 助手 (50264601)
YAZAMA Futoshi Yokohama City University, Dept. Anatomy, Assistant Professor, 医学部, 助手 (00254160)
|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1998 : ¥2,100,000 (Direct Cost : ¥2,100,000)
Three projects were performed.
One is to isolate spermatogoniae by an elutriator and a fluorescence-activated cell sorter, and we obtained the fraction of spermatogoniae and the fraction and pachytene spermatocytes. Then the cDNAs obtained from the former were subtracted by the cDNAs from the latter in order to obtain cDNAs specific for the former. We obtained approximately 20 cDNA clones with the method, and their nature is currently under investigation.
The second is to subtract the cDNAs obtained from the testes of 5-day mice by the cDNAs from the cDNAs from W/Wv mutant mice. In spermatogenesis is not fully differentiated in 5-day mice and the spermatogoniae are the only germ cell present, and in W/Wv mice no germ cell is present. Therefore, this subtraction yields the cDNA fraction which is expressed in spermatogoniae and not in non-germ cells. We obtained 22 cDNA clones whose identity have not been reported, and 30 clones corresponding to known genes. Among clones whose genes have not been known, 2 seemed to be identical to the sequences released by Riken as a database of full length mouse cDNA libraries. We are donated with their full length cDNA from Riken, and performed in situ hybridization. One of the clones hybridized with spermatogoniae, and other locations where stem cells have been reported to localize. At present we are investigating the effect of forced expression in cultured cells. We are also investigating some of the already known genes whose function in the testis seemed to be interesting.
The third project is to investigate the localization of genes which have been reported to have some relationships in stem cell function, such as telomerase, Oct-3/4, Notch-1,2,3,and Delta. We have obtained some interesting results, for example, telomerase was localized to spermatogonia by in situ hybridization