Abstract
To address the growing consumer demands for improved fish meat quality, desirable morphological traits, and sustainable production practices, researchers have intensified efforts in the selective breeding and genetic improvement of carp (Cyprinus carpio) varieties. However, traditional breeding methods are often time-consuming and inefficient, which poses challenges to the sustainable development of the carp aquaculture industry. The establishment of germ stem cell lines offers a crucial tool for the study of germ cells, genetic improvement, and species conservation. In this study, we successfully established a spermatogonial stem cell line (YRSSCs) from Yellow River carp (Cyprinus carpio haematopterus) that can be cultured in vitro for the long term. We optimized the culture conditions to maintain their self-renewal and differentiation capabilities. The results demonstrated that YRSSCs have a diploid karyotype and can stably proliferate for over a year in L-15 medium supplemented with 5 mmol/L HEPES, 50 μmol/L β-mercaptoethanol, 15% FBS, 2 ng/mL bFGF, 2 ng/mL LIF, 1% carp serum, 800 IU/mL penicillin, 0.8 mg/mL streptomycin, 2 μg/mL amphotericin B, 1% zebrafish embryo extract, and 1% glutamine at 30 °C in the absence of CO(2). The cells exhibited a typical germ stem cell gene expression profile, with strong expression of the vasa, plzf-a, and Oct4-a genes. Additionally, this study found that YRSSCs possess the ability to differentiate in vitro and functionally colonize in vivo within recipient bodies. This research explored the establishment of YRSSCs and their differentiation potential both in vitro and in vivo, providing a novel strategy for the genetic improvement of aquaculture fish species through germ stem cell-based gene editing and transplantation technologies.