Establishment and Characterization of a Spermatogonial Stem Cell Line from Tiger Puffer Fish (Takifugu rubripes)

SIMPLE SUMMARY: Tiger puffer fish (Takifugu rubripes) is an economically important marine fish, in which the development of selective breeding is limited due to the long time of sexual maturation. In order to promote the breeding research of this species, we established the spermatogenic stem cell line of the tiger puffer fish. In this study, we isolated spermatogonial stem cells from the testis of the 6-month-old tiger puffer fish and cultured them stably in vitro for more than 60 generations. The characteristics of the in vitro cultured spermatogonial stem cell line showed polygonal in shape. The germ cell marker genes dnd, ddx4, piwil, dazl, stem cell marker genes sox2, myca and nanog, and spermatogonial stem cell marker genes gfra1b and ly75 were highly expressed in this cell line. The establishment of this spermatogonial stem cell line provides a dependable material for the reproduction research of tiger puffer fish. ABSTRACT: Tiger puffer fish (Takifugu rubripes) has become the main fish species cultured in China since the last century because of its high economic value. Male and female tiger puffer fish need 2 and 3 years each to reach sexual maturity, which limits the development of breeding research for this species. In recent years, in vitro culture of fish spermatogonial stem cells (SSCs) have shown potential in aquaculture. In the present study, we established a spermatogenic stem cell line from T. rubripes (TrSSCs). TrSSCs were characterized by polygonal morphology, predominantly retained 44 chromosomes, and grew rapidly at 26 °C and in L-15. TrSSCs were still able to grow stably after more than one year of in vitro culture. TrSSCs showed positive alkaline phosphatase staining. TrSSCs expressed germ cell-associated genes, including dnd, ddx4, piwil, gfra1b, sox2, myca, nanog, ly75, and dazl, as determined by semiquantitative assays, and almost all cells were found to express the germ cell genes ddx4 and gfra1b in a fluorescence in situ hybridization assay. In vitro, induction experiments demonstrated the TrSSCs possessed the ability to differentiate into other types of cells. Our research has enriched the fish spermatogonial stem cell resource bank, which will provide an efficient research model for sex determination and sex control breeding in fish, establishing a foundation for subsequent breeding research.