Establishment, identification, and transcriptome analysis of a Sertoli cell line from ovoviviparous black rockfish...

Black rockfish (Sebastes schlegelii) is an economically important species with a unique ovoviviparous reproductive mode, in which reproduction is limited by incomplete fertilization. In order to understand the mechanism of spermatogenesis of black rockfish, a cell line derived from the testis, named SSTC, was successfully established and cultured in L-15 medium at 25 °C, and it was passaged to the 50th generation. The SSTC exhibited fibroblast-like and epithelial-like morphology during in vitro culture, and 62% of the SSTC retained the diploid karyotype with 48 chromosomes by the 30th passage (P30). To evaluate the ability of SSTC to express exogenous genes, lipofection and electrotransfection were performed, achieving transfection efficiencies of 9% and 12%, respectively. Transcriptome analysis showed that SSTC at P15 and P30 scarcely expressed germ cell and Leydig cell marker genes, while only expressing the Sertoli cell marker genes sox9a, amh, krt18 and fasl, indicating that SSTC mainly consists of Sertoli cells. GO and KEGG enrichment analyses revealed that, compared to the primary cells, the MAPK, TGF-β, and Wnt signaling pathways, which are crucial for spermatogenesis in Sertoli cells, were significantly upregulated in SSTC after passaging. Additionally, cell cycle-related pathways were upregulated, while pathways associated with cell adhesion, extracellular matrix, cell communication and membrane signal transduction were significantly downregulated. This study demonstrated that SSTC can be used as a tool for exploring the molecular mechanisms of gonadal differentiation and development in black rockfish, providing an effective platform for research on reproduction and endocrinology in this species.