The effects of RFRP-3, the mammalian ortholog of GnIH, on the female pig reproductive axis in vitro

•RFRP-3 inhibit GnRH release and expression.•Inhibitory effect of RFRP-3 on gonadotropin secretion and the expression of gonadotropin gene subunits.•Inhibitory effect of RFRP-3 on the E2 and P4 secretion and granulosa cells proliferation. RFamide-related peptide-3 (RFRP-3) has been proposed as a key inhibitory regulator of mammalian reproduction. To further determine the potential mechanisms and sites of action of RFRP-3, we systematically investigated the direct effect of RFRP-3 on the female pig reproductive axis in vitro. Initially, we confirmed that G protein-coupled receptor 147 (GPR147) was distributed in isolated hypothalamic, anterior pituitary and ovarian granulosa cells, suggesting that RFRP-3 could act on these cells in vitro. Subsequently, the direct effects of RFRP-3 on hormone and steroid secretion, the synthesis of subunit genes and the expression of proteins related to proliferation in the hypothalamus, pituitary and ovary were evaluated. Our results demonstrate that different doses of RFRP-3 inhibited the release and synthesis of gonadotrophin releasing hormone, gonadotrophin and steroid hormones and impacted the relative gene expression of KISS1 and GnRHR and the protein expression of cyclin B1, PCNA and ERK 1/2.