Establishment and characterization of an immortalized bovine luteal cell line

The bovine corpus luteum (CL) is critical for pregnancy establishment and maintenance via progesterone (P₄) secretion. Primary bovine luteal cells (PBLC), the core in vitro model for reproductive and steroidogenesis research, are severely limited by high isolation cost, cellular heterogeneity, and restricted proliferative capacity. In this study, we established an immortalized bovine luteal cell line with stable in vitro proliferative capacity, termed SV40 T-immortalized bovine luteal cells (SV40T-IBLC), via lentiviral transduction of the Simian virus 40 (SV40) T antigen gene. The immortalized cells displayed a typical epithelial-like morphology with abundant small cytoplasmic lipid droplets. They secreted multiple functional hormones including P₄ and oxytocin, expressed synaptophysin, a key marker protein of mature functional luteal cells, and maintained stable expression of core steroidogenic genes and proteins such as STAR, 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP11A1, and PTGFR, with mRNA levels of these key genes showing no significant difference from those of parallel-cultured PBLC. Functionally, SV40T-IBLC exhibited time-dependent P₄ secretion, with P₄ concentration in the culture supernatant reaching 1.89 ± 0.14 ng/mL at 60 h, which was fully consistent with the secretory level and dynamic pattern of PBLC. After continuous in vitro culture up to the 50th passage, the cells maintained a normal diploid karyotype (29 pairs of autosomes and 1 pair of XX sex chromosomes), and no evidence of malignant transformation was observed in the anchorage-independent growth assay and in vivo tumorigenicity assay in nude mice. Furthermore, Co-culture with SV40T-IBLC significantly enhanced the viability of bovine endometrial epithelial cells (BEEC) by 34.2% after 72 h of co-incubation (p < 0.01), increased intracellular lipid droplet abundance of BEEC, and upregulated the mRNA expression of endometrial receptivity-related genes including EGF and LIF, with no significant alteration in BEEC apoptosis rate. In conclusion, this immortalized cell line retains core functional characteristics of PBLC, providing a stable, homogeneous in vitro model for bovine reproductive research.