TY - JOUR AB - 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. AD - Department of Immunology, Chengde Medical University, Chengde, Hebei 067000, P.R. China Graduate School, Chengde Medical University, Chengde, Hebei 067000, P.R. China Department of Human Anatomy, Chengde Medical University, Chengde, Hebei 067000, P.R. China Hebei Provincial Key Laboratory of Reproductive Medicine, Family Planning Science and Technology Research Institute of Hebei Province, Shijiazhuang, Hebei 050000, P.R. China AU - Cheng,Luyang AU - Yang,Songhe AU - Si,Lina AU - Wei,Meng AU - Guo,Sen AU - Chen,Zhihong AU - Wang,Shusong AU - Qiao,Yuebing DA - 2022/01/01 DO - 10.3892/etm.2021.10946 IS - 1 JO - Exp Ther Med KW - gonadotropin inhibitory hormone/RFamide‑related peptide‑3 kisspeptin hypothalamic‑pituitary reproductive axis luteinizing hormone follicle‑stimulating hormone PY - 2022 SN - 1792-0981 1792-1015 SP - 24 ST - Direct effect of RFRP‑3 microinjection into the lateral ventricle on the hypothalamic kisspeptin neurons in ovariectomized estrogen‑primed rats T2 - Experimental and Therapeutic Medicine TI - Direct effect of RFRP‑3 microinjection into the lateral ventricle on the hypothalamic kisspeptin neurons in ovariectomized estrogen‑primed rats UR - https://doi.org/10.3892/etm.2021.10946 VL - 23 ER -