Functionally distinctive testicular cell lines of zebrafish to support male germ cell development

Here we describe two testicular cell lines of zebrafish with distinct functions to support the development of male germ cells. Twelve cell lines were established by single-colony isolation from tumor-like testis-derived ZtA6 cells, and the features characteristic of Sertoli cells such as phagocytic activity and transcription both of their specific genes, sox9a and Wilms' tumor suppressor WT1, and of the vas gene of germ cells were analyzed in the lines. Six lines exhibited these three characteristics of Sertoli cells. Despite the single-colony isolation, low levels of vas expression were detected in three lines. Two lines, ZtA6-2 and ZtA6-12, showed almost the same characteristics as Sertoli cells, but exhibited distinctive features when male germ cells were cocultured with each line as feeders. The in vitro fertilization by the culture of germ cells with ZtA6-12 produced more embryos than that with ZtA6-2. In contrast, ZtA6-2 gave rise to large clumps of germ cells after a 12-day culture, while ZtA6-12 still produced only small clumps. Expression of vas, strongly expressed in spermatogonia and premeiotic spermatocytes, was prolonged in the culture with ZtA6-2, while it reduced in that with ZtA6-12. Compared with the previous results obtained on the original ZtA6 cells, these results suggested that the function of the ZtA6-2 cells was directed to stimulate the proliferation of spermatogonia, and ZtA6-12 to stimulate the differentiation into sperm. These cell lines will facilitate investigation of Sertoli cell molecules that contribute to the proliferation and differentiation of spermatogonia, and the longer culture time of male germ cells by using these lines successively.