Steroidogenic acute regulatory protein promotes granulosa cell function and steroidogenesis in Duolang sheep ovarian follicles

Objective: Steroidogenic acute regulatory protein (STAR) is the principal regulator of steroid hormone biosynthesis by mediating the rate-limiting transport of cholesterol into mitochondria.However, its functional significance in ovarian granulosa cells of the native Xinjiang Duolang sheep breed with moderate reproductive potential remains inadequately characterized.This study aimed to perform bioinformatic analysis of the STAR gene and directly evaluate its functional effects in primary granulosa cells of Duolang sheep.Methods: An 858-bp coding sequence of the ovine STAR gene was cloned, and bioinformatics analyses were performed to evaluate protein conservation across mammalian species.In cellular experiments, STAR gene expression was induced or inhibited in granulosa cells.Cell proliferative activity was evaluated using the CCK-8 assay.Quantitative real-time PCR (qPCR) and western blotting (WB) were used to determine the mRNA and protein expression levels of proliferation-related markers (PCNA, MYC, and CCND2), apoptosis-related factors (Bcl-2, Bax, and Caspase-3), steroidogenic enzymes (CYP11A1, 3β-HSD, and CYP19A1), and the estrogen receptor ERβ.Apoptotic cells were evaluated using Hoechst/propidium iodide (PI) double staining followed by fluorescence microscopy.The secretion of estradiol (E2) and progesterone (P4) was determined by enzyme-linked immunosorbent assay (ELISA).Results: Bioinformatics analysis revealed high conservation of the STAR protein across A c c e p t e d A r t i c l e overexpression was accompanied by increased mRNA expression of steroidogenic-enzyme-related genes, including CYP11A1, 3β-HSD, and CYP19A1, as well as the estrogen receptor gene ERβ, together with enhanced secretion of E2 and P4.Conclusion: Collectively, these gain-and loss-of-function data provide direct functional evidence of STAR-regulated proliferative activity, apoptosis and steroidogenesis in Duolang sheep primary granulosa cells.