Establishment and identification of immortalized sheep ovarian granulosa cells

BACKGROUND: Follicular granulosa cells (GCs) are an important cellular resource for studying animal reproductive functions. RESULTS: This study aimed to investigate SV40T lentiviral vector-mediated gene transfection in sheep ovarian granulosa cells (GCs) and systematically validate their biological characteristics and functions after immortalization. By optimizing infection conditions (MOI = 10), efficient transfection of the GFP-tagged SV40T gene was successfully achieved (approximately 90% efficiency), leading to the establishment of an immortalized sheep granulosa cell line (GCs-SV40T-GFP). This cell line could be stably passaged for over 50 generations while maintaining consistent cellular morphology and stable fluorescence expression. Compared to primary cells, the immortalized cells exhibited significantly accelerated proliferation, with flow cytometry revealing a marked increase in the proportion of cells in S and G1 phases (P < 0.05). Functional assays confirmed that these cells continuously secreted estradiol (P < 0.05). Further experiments demonstrated that even at the 50th passage, the immortalized cells still highly expressed key granulosa cell functional markers, FSHR and CYP19A1. Karyotype analysis confirmed the maintenance of a normal chromosomal karyotype across different passages (primary, 10th, 25th, and 50th). Additionally, the cells exhibited a dose-dependent functional response to hormonal stimulation, with FSHR protein expression significantly upregulated in response to increasing concentrations of exogenous estradiol (P < 0.05). In vivo safety evaluation showed that GCs-SV40T-GFP cells did not induce tumor formation in mice. CONCLUSIONS: In this study, a functionally stable immortalized sheep granulosa cell model was successfully constructed. At the same time, it also provides an important tool for animal reproductive function research and hormone regulation mechanism exploration.