GnIH Modulates Proliferation, Apoptosis, and Steroid Hormone Secretion in Tibetan Sheep Granulosa Cells

Gonadotropin-inhibitory hormone (GnIH) is a key neuropeptide that negatively regulates animal reproduction. This study aims to elucidate its functional mechanism in Tibetan sheep, a characteristic plateau-adapted breed. A total of 12 Tibetan sheep were used in this study. For granulosa cell experiments, granulosa cells were isolated from the ovaries of three independent Tibetan sheep to generate three biological replicates, and three technical replicates were set for qPCR and Western blot detection within each biological replicate. The mRNA levels of GnIH in gonadal axis tissues, including the hypothalamus, pituitary, ovary, uterus and oviduct, were detected using RT-qPCR, and fluorescence in situ hybridization was performed to identify the subcellular localization of GnIH in ovarian granulosa cells. The overexpression plasmid pcDNA3.1 GnIH and GnIH-targeted siRNA fragments were constructed and transfected into Tibetan sheep ovarian granulosa cells. Cell proliferation, apoptosis, cell cycle distribution, and hormone secretion were examined with the aid of CCK 8 assay, flow cytometry, RT-qPCR and ELISA. The results showed that GnIH was widely expressed in gonadal axis tissues of Tibetan ewes. The highest mRNA abundance of GnIH was found in the hypothalamus, followed by the ovary. GnIH signals were mainly localized in the cytoplasm of ovarian granulosa cells. GnIH overexpression reduced granulosa cell viability, arrested cell cycle progression, upregulated proapoptotic genes BAX, Caspase 3 and Caspase 9, and downregulated the proliferation-related gene PCNA. Simultaneously, the secretion of estradiol (E2) and progesterone (P4) was simultaneously reduced. Endogenous GnIH knockdown enhanced the proliferative capacity of granulosa cells, inhibited cell apoptosis, uplifted the proportions of S phase and G2/M phase cells, and elevated estradiol secretion. These results confirm that GnIH acts as a negative regulator of granulosa cell function. It inhibits follicular development by suppressing cell proliferation, promoting cell apoptosis and reducing steroid hormone synthesis. These in vitro observations offer preliminary clues regarding the regulatory role of GnIH in granulosa cell proliferation and apoptosis.