Isolation, characterization, and transplantation of testicular stem cell (spermatogonia)

执行了三个项目。一种方法是用洗脱器和荧光活化细胞分选器分离精原细胞，得到精原细胞和粗质精母细胞的部分。然后将前者获得的cdna与后者获得的cdna相减，以获得前者特异性的cdna。我们用这种方法获得了大约20个cDNA克隆，目前正在研究它们的性质。第二种方法是用W/Wv突变小鼠的cdna减去从5天小鼠睾丸中获得的cdna。在精子发生中，5天小鼠未完全分化，精原体是唯一存在的生殖细胞，而W/Wv小鼠不存在生殖细胞。因此，这种减法产生的cDNA片段是表达在精原细胞，而不是在非生殖细胞。我们获得了22个身份尚未报道的cDNA克隆，30个与已知基因对应的克隆。在基因未知的克隆中，2个似乎与Riken发布的全长小鼠cDNA文库的序列相同。我们从理化学研究所捐赠了他们的全长cDNA，并进行了原位杂交。其中一个克隆与精原细胞杂交，而其他干细胞已被报道定位。目前我们正在研究强迫表达对培养细胞的影响。我们也在研究一些已知的基因，它们在睾丸中的功能似乎很有趣。第三个项目是研究已报道的与干细胞功能有一定关系的基因，如端粒酶、Oct-3/4、Notch-1、2、3和Delta的定位。我们获得了一些有趣的结果，例如，通过原位杂交，端粒酶被定位在精原细胞上

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