Generation of a Normal Long-Term-Cultured Chinese Hook Snout Carp Spermatogonial Stem Cell Line Capable of Sperm Production In Vitro

SIMPLE SUMMARY: In vitro-induced differentiation of sperm cells is a key technology for genetic resource conservation. In the past ten years, Opsariichthys bidens has become a famous and excellent aquatic species in some areas in China. However, its genetic resources have reduced dramatically. To protect against the decline of O. bidens, a long-term-cultured spermatogonial stem cell line (ObSSC) of adult O. bidens was successfully established. The result of our study showed that ObSSC had a diploid karyotype and stable growth over more than 2 years, with SSC-typical gene expression patterns. Furthermore, our research demonstrates the potential and regulation mechanism of fish spermatogonial stem cell differentiation into different cells of three germ layers. Our findings will assist further research on the genetic resource conservation of germplasm in a commercially and ecologically valuable fish species. ABSTRACT: Opsariichthys bidens belongs to the family Cyprinidae and is a small freshwater economic fish widely distributed in China. In recent years, the natural resources of O. bidens have been drastically reduced due to overfishing and the destruction of the water environment. The in vitro culture and long-term preservation of germ stem cells are the key technologies to keep genetic resources from degeneration. However, except for the establishment of the first long-term cultured medaka spermatogonia cell line (SSC) capable of producing sperm in vitro in 2004, no other long-term cultured SSC line has been found in other fish species. In this study, we successfully established another long-term-cultured spermatogonial stem cell line from Opsariichthys bidens (ObSSC). After more than 2 years of culture, ObSSC had a diploid karyotype and stable growth, with the typical gene expression patterns of SSC. Under in vitro culture, ObSSC could be induced to differentiate into sperm and other different types of somatic cells. In vivo, ObSSC could differentiate into different cells of three germ layers upon being transplanted into zebrafish embryos. Our research helps to explore the potential and regulation mechanism of fish SSC differentiation and spermatogenesis in vitro, provides a new way for solving the problem of fish genetic resource degradation and lays a foundation for further research on fish germ cell transplantation.