RFamide-Related Peptide Neurons Modulate Reproductive Function and Stress Responses

Abstract

RF-amide related peptide 3 (RFRP-3) is a neuropeptide thought to inhibit central regulation of fertility. We investigated whether alterations in RFRP neuronal activity led to changes in puberty onset, fertility, and stress responses, including stress and glucocorticoid-induced suppression of pulsatile luteinizing hormone secretion. We first validated a novel RFRP-Cre mouse line, which we then used in combination with Cre-dependent neuronal ablation and DREADD technology to selectively ablate, stimulate, and inhibit RFRP neurons to interrogate their physiological roles in the regulation of fertility and stress responses. Chronic RFRP neuronal activation delayed male puberty onset and female reproductive cycle progression, but RFRP-activated and ablated mice exhibited apparently normal fertility. When subjected to either restraint-or glucocorticoidinduced stress paradigms. However, we observed a critical sex-specific role for RFRP neurons in mediating acute and chronic stress-induced reproductive suppression. Female mice exhibiting RFRP neuron ablation or silencing did not exhibit the stress-induced suppression in pulsatile luteinizing hormone secretion observed in control mice. Furthermore, RFRP neuronal activation markedly stimulated glucocorticoid secretion, demonstrating a feedback loop whereby stressful stimuli activate RFRP neurons, which in turn further activate the stress axis. These data provide evidence for a neuronal link between the stress and reproductive axes.

Neuroendocrine mechanisms underlying estrogen positive feedback and the LH surge

“…While intriguing, a similar circadian decline in RFRP-3 also occurs in males ( Poling et al, 2017 ), despite their lack of a surge, suggesting this temporal change may be unrelated to the surge event. Moreover, chronically activating RFRP-3 neurons in transgenic female mice to, in theory, provide long-lasting inhibitory input to the reproductive axis does not impact normal fertility or litters ( Mamgain et al, 2021 ); it may be that RFRP-3 serves as a modulator that can blunt the surge under inhibitory physiological conditions, such as during stress or metabolic challenge, rather than being a requisite component of the normal surge process.…”

Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice

“…Given the inhibitory effect of RFRP-3 on LH secretion [24,35,52], our findings support that RFRP-3 neurons facilitate the gating of the LH surge at the right time of the day and stage of the estrous cycle. However, given that RFRP-3 is now reported to also regulate food intake [64] and stress response [42], we cannot exclude the possibility that RFRP-3 neurons may regulate female reproduction indirectly through metabolicand stress-regulated mechanisms.…”

“…Adult female C57BL/6J mice (Charles River, France) and heterozygous RFRP-Cre mice on a C57BL/6J background [42] were housed two or three per cage under a 12-hour light: 12-hour dark cycle (lights on at 7:00 am, given as zeitgeber 0 (ZT0)) with controlled temperature (22°C) and ad libitum access to food and water. All protocols were reviewed by the Regional Committee for Ethics in Animal Experimentation and approved by the French Ministry of Education and Research (authorization #8452-2017010613574177v2).…”

Regulation of the gonadotropin‐releasing hormone neuron during stress

“…119 Third, ablation of RFRP3 neurons prevented restraint stress-induced suppression of LH pulses in female mice. 120 Evidence for the direct action of RFRP-3 on GnRH neurons includes the findings that GnRH cells contain the receptor for RFRP-3 (NPFFR1) 115,121 and that RFRP-3 terminals are found in close contact with GnRH cell bodies. 115,122,123 Additionally, RFRP-3 was shown to inhibit GnRH cell firing, an effect maintained following GABA and glutamate receptor blockade, suggesting a direct effect on GnRH cells.…”