Abstract
RFamide-related peptide-3 (RFRP-3) may be involved in the inhibition of kisspeptin, but there is no direct evidence that RFRP-3 can directly act on kisspeptin neurons. The present study aimed to investigate the role and mechanism of RFRP-3 and kisspeptin in the hypothalamic-pituitary reproductive axis. In order to detect the expression and localization of RFRP-3 and kisspeptin in dorsomedial hypothalamic nucleus, double immunofluorescence method combined with confocal microscopy were performed. RFRP-3 was injected into the lateral ventricle of ovariectomized estrogen primed rats. Blood and brain tissues were collected at 60-, 120-, 240- and 360-min. Serum levels of gonadotropin-releasing hormone, luteinizing hormone and follicle-stimulating hormone were detected by ELISA. Kisspeptin expression in hypothalamus was detected by western blotting. Finally, surface plasmon resonance was used to verify whether RFRP-3 can directly interact with kisspeptin. Confocal images indicated that RFRP-3 and kisspeptin were co-expressed in the same neurons in the hypothalamus of ovariectomized estrogen-primed rats. Serum concentrations of gonadotropin-releasing hormone, luteinizing hormone and follicle-stimulating hormone were demonstrated to be significantly reduced following microinjection of RFRP-3 into the lateral ventricle for 60, 120, 240 and 360 min compared with the corresponding saline groups. The expression levels of kisspeptin in hypothalamus were gradually decreased following microinjection of RFRP-3 into the lateral ventricle. In addition, the affinity constant (KD) of RFRP-3 binding to kisspeptin was 6.005x10-5 M, indicating that RFRP-3 bound directly to kisspeptin in the range of protein-protein binding strength (KD, 10-3-10-6 M). In conclusion, RFRP-3 may regulate the hypothalamic-pituitary reproductive axis by inhibiting the expression of hypothalamic kisspeptin and direct binding.