Intelectin-1 promotes granulosa cells proliferation and modulates apoptosis via ERK1/2, AKT, and insulin receptor signaling pathways in Large White and Meishan pigs

Abstract

Maintaining the proper balance between granulosa cells (Gc) proliferation and apoptosis is crucial for folliculogenesis and female fertility. Our previous study showed expression of omentin-1 (intelectin-1, ITLN1) in the porcine ovarian follicles; however, its impact on Gc functions remains unknown. Therefore, this study aimed to determine the in vitro effects of ITLN1 on Gc proliferation and apoptosis in Large White (LW) and Meishan (MS) pigs. These breeds were chosen due to their distinct reproductive characteristics: MS pigs are known for maintaining a higher number of follicles during the follicular phase and exhibiting greater estradiol synthesis compared to LW pigs. Porcine Gc were incubated with ITLN1 (10–100 ng/mL) for 24–72 h, and the viability/proliferation (alamarBlue/BrdU assays), cell cycle progression (flow cytometry) and the gene and protein expression of proliferation/apoptotic markers (PCNA, cyclins A1, B2, D1, E1, caspases-3, −9, BCL-2, BAX, FAS, FADD, XIAP) (real-time PCR, western blotting) were assessed. Next, the effect of ITLN1 on the phosphorylation of several kinases (AKT, AMPK, ERK1/2, STAT3, PKA) and the gene and protein expression of the insulin receptor (INSR) were studied (real-time PCR, western blotting). Then, using pharmacological inhibitors of ERK1/2 (PD98059, 5 μM), AKT (LY294002, 10 μM) and INSR (1 μM), treated alone or with ITLN1 (S961, 50 ng/mL), we analyzed its involvement in the effects of ITLN1 on Gc proliferation/apoptosis. We demonstrated that ITLN1 had a mitogenic effect on Gc by enhancing cell cycle progression and modulating the levels of PCNA, cyclins and apoptotic factors via ERK1/2, AKT, and INSR, suggesting that ITLN1 is a newly identified regulator in ovarian folliculogenesis, regardless of the fatness degree of pigs.