RFRP-3 suppresses ovarian granulosa cell function in Tan sheep via dual signaling pathways: PI3K/AKT/mTOR and MAPK/ERK1/2.

The inhibitory role of RFRP-3 in the hypothalamic-pituitary axis is established, but its direct intra-ovarian actions remain poorly defined. This study reveals, for the first time, the expression of RFRP-3 and its receptor GPR147 in ovarian granulosa cells of Tan sheep. We demonstrate that RFRP-3 acts locally to suppress steroidogenesis by downregulating key steroidogenic enzymes (STAR, CYP19A1, HSD17B2). Furthermore, it inhibits proliferation and promotes apoptosis by modulating cell-cycle regulators (PCNA, CCND1, CDK4, CDK1, CCNB) and apoptosis-related proteins (Bcl-2, Bax, p53). A pivotal and novel finding, revealed by transcriptome sequencing and Western blot, is that RFRP-3 exerts these dual effects through the concurrent inhibition of two major signaling pathways: it impedes steroid hormone synthesis via the MAPK/ERK1/2 pathway and inhibits cell proliferation and promotes apoptosis via the PI3K/AKT/mTOR pathway. This study provides the first evidence of a local RFRP-3 system in the Tan sheep ovary and delineates a unique dual-pathway mechanism that precisely controls granulosa cell function. These findings provide theoretical insights into novel mechanisms of reproductive regulation in Tan sheep and establish a foundation for developing targeted strategies to enhance their reproductive capacity.