SV40 T-immortalized bovine luteal cells (SV40T-IBLC) established, maintaining steroidogenesis and enhancing endometrial cell viability.

The bovine corpus luteum (CL) is essential for pregnancy establishment and maintenance through progesterone (P4) secretion. Primary bovine luteal cells (PBLC) are the standard in vitro model for reproductive and steroidogenesis research, but their utility is hampered by high isolation costs, cellular heterogeneity, and limited proliferative capacity. This creates a significant gap for consistent, high-throughput research into luteal function and steroid hormone regulation, necessitating a stable, homogeneous cell model.

Researchers established an immortalized bovine luteal cell line, termed SV40T-IBLC, by transducing primary bovine luteal cells with the Simian virus 40 (SV40) T antigen gene via lentivirus. The immortalized cells were characterized for epithelial-like morphology, presence of lipid droplets, and secretion of hormones including P4 and oxytocin. Expression of the luteal marker synaptophysin and core steroidogenic genes (STAR, 3β-HSD, CYP11A1, PTGFR) was assessed via mRNA levels. P4 secretion dynamics were measured over 60 h. Cell stability was evaluated through karyotyping up to the 50th passage and tumorigenicity assays (anchorage-independent growth, in vivo nude mice). Co-culture experiments with bovine endometrial epithelial cells (BEEC) assessed viability and gene expression after 72 h.

The SV40T-IBLC line exhibited typical epithelial-like morphology with abundant small cytoplasmic lipid droplets. They secreted multiple functional hormones, including P4 and oxytocin, and expressed synaptophysin. Core steroidogenic genes (STAR, 3β-HSD, CYP11A1, PTGFR) maintained stable mRNA expression levels, showing no significant difference from parallel-cultured PBLC. Functionally, SV40T-IBLC demonstrated time-dependent P4 secretion.

P4 concentration in the culture supernatant reached 1.89 ± 0.14 ng/mL at 60 h, fully consistent with PBLC secretory levels and dynamic patterns. After continuous in vitro culture up to the 50th passage, cells maintained a normal diploid karyotype (29 pairs of autosomes and 1 pair of XX sex chromosomes), with no evidence of malignant transformation in anchorage-independent growth or in vivo tumorigenicity assays. Co-culture with SV40T-IBLC significantly enhanced BEEC viability by 34.2% after 72 h (p < 0.01), increased intracellular lipid droplet abundance in BEEC, and upregulated mRNA expression of endometrial receptivity-related genes, including EGF and LIF.