Establishment and Characterization of a Spermatogonial Stem Cell-like Line with Potential for Meiotic Differentiation in the Endangered Fish Rhinogobio nasutus.

Rhinogobio nasutus, a freshwater fish belonging to the family Cyprinidae, is primarily distributed in the upper and middle reaches of the Yellow River. However, overfishing and habitat degradation have resulted in a marked decline in its wild populations, and the species was listed as a Class II nationally protected aquatic wildlife species in China in 2021. Spermatogonial stem cells (SSCs) provide a valuable resource for fish germplasm conservation and reproductive biotechnology. In this study, we successfully established a long-term cultured germ-cell population with spermatogonial stem cell-like characteristics derived from R. nasutus (RnSSCs). These cells exhibited a morphology consistent with an undifferentiated spermatogonial phenotype and showed a diploid chromosome number of 2n = 50. Alkaline phosphatase (AP) staining showed strong positive activity, while RT-PCR detected the expression of germ-cell- and stem-cell-associated markers, with only weak expression of the somatic-cell-associated marker. Immunofluorescence staining revealed strong signals for both proliferating cell nuclear antigen (PCNA) and the germ cell-specific protein Vasa. After more than four months of in vitro culture, the cells continued to proliferate and retained expression of germ-cell- and stem-cell-associated markers. Following transplantation into zebrafish blastulae, fluorescently labelled cells were detected in the gonadal region of a small number of recipient embryos. Following hormonal induction, some cells developed a sperm-like morphology, accompanied by the expression of the meiotic-associated markers rec8 and dmc1. The establishment of the RnSSC line provides a renewable cellular resource for studying spermatogenesis and germ cell transplantation in R. nasutus. These findings provide a useful cellular resource for studying the reproductive biology of R. nasutus.