Role of RFamide-related peptide-3 in the neuroendocrine regulation of the reproductive and stress axes

Abstract

RFamide-related peptide-3 (RFRP-3) is part of a class of neuropeptides characterised by an arginine-phenylalanine-amide motif at their C-terminus. Direct pituitary actions have been proposed for RFRP-3, either via autocrine/paracrine or hypophysiotrophic mechanisms, due to the presence of their G-protein coupled receptor (GPR147) in the pituitary gland. This novel neuropeptide has been shown to inhibit gonadotrophin-releasing hormone (GnRH) neuronal firing rate. However, at present there is little compelling evidence to suggest that RFRP-3 acts as a hypophysiotropic hormone in mammals.

In this thesis, I first investigated whether RFRP-3 could act as a hypophysiotrophic hormone in rats and mice. Using immunohistochemistry, I found that RFRP-3 neurons project from cell bodies in the dorsomedial nucleus of the hypothalamus (DMH) to various brain regions, including to the preoptic area, anteroventral periventricular nucleus through to the rostral aspects of the paraventricular nucleus, but not to the external zone of the median eminence (the ‘neurosecretory zone’). In addition, retrograde tract tracing techniques did not result in the labelling of RFRP-1/3 neurons from the peripheral circulation, suggesting that these cells do not project outside the blood-brain barrier.

I next investigated whether RFRP-3 neurons may influence GnRH and kisspeptin neuronal activity, as intense fibre staining was observed in the rostral preoptic nucleus and rostral periventricular region of the third ventricle (RP3V), respectively. Single-cell RT-PCR and quantitative RT-PCR showed presence of GPR147 mRNA on GnRH and RP3V kisspeptin neurons, respectively. In addition, dual-label immunofluorescence showed numerous close RFRP-3 fibre contacts on both GnRH and kisspeptin neurons in the rostral preoptic nucleus and RP3V regions, respectively.

I further analysed changes in RFRP mRNA levels and RFRP-3 cell numbers in rats at 2, 4, 6, 8 and 14 weeks of age using quantitative RT-PCR and immunohistochemistry to determine developmental changes in this system across pubertal development. A 3-fold up-regulation of RFRP mRNA level and a 2-fold increase in the number of RFRP-3 neurons was seen in the DMH of female rats during sexual development, plateauing at 6 weeks of age (both P < 0.001). In male rats this gradual developmental increase was not apparent; if anything levels decreased with pubertal development. These findings suggest a role for RFRP-3 in the regulation of adult female reproduction rather than being a causative agent in prepubertal infertility, and are consistent with the idea that RFRP-3 plays a regulatory role in the surge of GnRH and LH that causes ovulation to occur. A further study was conducted to investigate the effects of lactation on RFRP mRNA levels and RFRP-3 cell numbers. A 2-fold down-regulation of RFRP mRNA and similar decrease in the number of immunoreactive RFRP-3 neurons was observed (both P < 0.05). Further studies showed that this reduction in RFRP-3 mRNA level and peptide in lactating dams was due to elevated prolactin concentrations.

Immunohistochemistry was used to detect prolactin-induced phosphorylation of signal transducer and activator of transcription 5 (pSTAT5) as a functional marker of prolactin receptor activation in RFRP-1/3 neurons. Dual-label immunohistochemistry for pSTAT5 and RFRP-3 showed that 95% of RFRP-3 neurons were able to respond to prolactin. Furthermore, it was hypothesised that RFRP-3 may act as a conduit between prolactin and the stress axis. Therefore, I first tested whether RFRP-3 can modulate anxiety-related behaviour and acute stress-induced glucocorticoid secretion. Behavioural analysis showed an increase in anxiety-related behaviour and also increased glucocorticoid secretion following acute RFRP-3 treatment (both P < 0.05). Co-treatment with RFRP-3 receptor antagonist RF9 completely blocked these effects. I next tested whether RFRP-3 can modulate the known anxiolytic effects of prolactin and lactation. Prolactin treatment as well as lactation decreased anxiety-related behaviour, and prolactin also decreased glucocorticoid secretion (all P < 0.05). Co-treatment with RFRP-3 prevented the prolactin-induced anxiolytic effects, but only partially overcame the anxiolytic effects of lactation. Collectively, the studies in this thesis have helped to define new roles for RFRP-3 in the neuroendocrine regulation of fertility and stress in mammals