Establishment and characterization of the gonadal cell lines derived from large yellow croaker (Larimichthys crocea) for gene expression studies

Fish cell lines are important for the study in resource conservation, genetic breeding, diseases control, and environmental pollutants detection. The establishment of gonadal cell lines will provide a suitable in vitro model for studying the molecular regulation mechanisms underlying sex determination and sex-related gene expression and function. Large yellow croaker (Larimichthys crocea) is the unique marine economic fish with the highest production in China's coastal waters. In this study, the continuous gonadal cell lines, ovary cell line of large yellow croaker (LYCO) and testis cell line of large yellow croaker (LYCT), derived from the ovary and testis of juvenile fish were established with modified Leibovitz's L-15 medium at 27 °C. The medium was perfected to improve the growth rate and the increment ability of the LYCO and LYCT. The characteristics of the two cell lines were investigated, including the concentration of zeta-life fetal bovine serum (FBS), different passage ratios, cryopreservation, and recovery. The cell lines were successfully transfected with pEGFP-N1 and pNanog-N1 by electroporation, and the transfection efficiency of cells was high; these results showed that the cell lines could be used to study the expression of exogenous and endogenous genes. The LYCO expressed the marker gene of ovarian follicle cells, Foxl2, while the LYCT expressed the marker gene of testicular sertoli cells, Dmrt1. However, LYCO and LYCT did not express Vasa, a marker gene of germ cells. Therefore, LYCO and LYCT appeared to consist of ovarian follicle cells and testicular sertoli cells, respectively. After the knockdown of Dmrt1 and Foxl2 in LYCO and LYCT, respectively, the results showed that the cell lines could be effective for RNA-mediated interference (RNAi) experiments and the study of the interactions among genes. As a first fish gonadal cell lines of L. crocea, which can provide a new model for the interactions between somatic cells and germ cells, and a basis for the further study of their reproduction and breeding in L.crocea. The results lay a foundation for the study of the gonadal cell culture in marine fish in the future.