Direct effects of RFRP-1, a mammalian GnIH ortholog, on ovarian activities of the cyclic mouse

Arg(R)-Phe(F)-amide related peptide-1 (RFRP-1) and -3 (RFRP-3) are known as mammalian orthologs of gonadotropin-inhibitory hormone (GnIH). In mammals, these RFRPs are expressed not only in the hypothalamus and but also in gonads. Inhibitory roles of the hypothalamic and gonadal RFRP-3 in reproduction have been documented in mammals. However, functional roles of the hypothalamic and gonadal RFRP-1 in reproduction are still unclear in mammals. Therefore, in vitro studies were conducted to elucidate the direct effect of RFRP-1, a mammalian GnIH ortholog, on ovarian activities, such as steroidogenesis, apoptosis, cell proliferation and metabolism in the cyclic mouse. The ovaries collected from the proestrus mice were cultured in vitro with different doses (Control, 1 ng/ml, 10 ng/ml and 100 ng/ml) of RFRP-1 for 24 h at 37 °C. A significant dose-dependent increase in estradiol release from the ovary was detected after the treatment of RFRP-1. Therefore, changes in the ovarian activities, such as steroidogenic markers (luteinizing hormone receptors; LH-R and 3β-hydroxysteroid dehydrogenase; 3β-HSD), apoptotic markers [Poly(ADP-ribose) polymerase-1; PARP-1 and cysteine-aspartic protease; caspase-3], a cell proliferation marker (proliferating cell nuclear antigen; PCNA) and metabolic markers (GLUT-4; glucose uptake) were assessed by the treatment of RFRP-1 in the proestrus ovary. The densitometry analysis showed the treatment of RFRP-1 significantly increased the expressions of LH-R and 3β-HSD, steroidogenic markers. In contrast, the expressions of PCNA, a cell proliferation maker; PARP-1 and caspase-3, apoptotic markers were significantly decreased. Interestingly, RFRP-1 treatment further increases significantly glucose uptake and GLUT-4 receptor expression. These findings indicate that RFRP-1 possesses a stimulatory effect on ovarian steroidogenesis in the proestrus mouse. This is the first evidence showing the direct action of RFRP-1 on steroidogenesis in any vertebrate. In addition, RFRP-1 may also act directly on ovarian folliculogenesis as an inhibitory factor.