Development, Sex Steroid Regulation, and Phenotypic Characterization of RFamide-Related Peptide (Rfrp) Gene Expression and RFamide Receptors in the Mouse Hypothalamus

Abstract

Arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3, encoded by the Rfrp gene) is the mammalian ortholog of gonadotropin-inhibiting hormone and can inhibit GnRH neuronal activity and LH release. However, the development and regulation of the RFRP-3 system in both sexes is poorly understood. Using in situ hybridization, we examined changes in Rfrp-expressing neurons in mice of both sexes during development and under different adulthood hormonal milieus. We found no sex differences in Rfrp expression or cell number in adult mice. Interestingly, we identified two interspersed subpopulations of Rfrp cells (high Rfrp-expressing, HE; low Rfrp-expressing, LE), which have unique developmental and steroidal regulation characteristics. The number of LE cells robustly decreases during postnatal development, whereas HE cell number increases significantly before puberty. Using Bax knockout mice, we determined that the dramatic developmental decrease in LE Rfrp cells is not due primarily to BAX-dependent apoptosis. In adults, we found that estradiol and testosterone moderately repress Rfrp expression in both HE and LE cells, whereas the nonaromatizable androgen dihydrotestosterone has no effect. Using double-label in situ hybridization, we determined that approximately 25% of Rfrp neurons coexpress estrogen receptor-α in each sex, whereas Rfrp cells do not readily express androgen receptor in either sex, regardless of hormonal milieu. Lastly, when we looked at RFRP-3 receptors, we detected some coexpression of Gpr147 but no coexpression of Gpr74 in GnRH neurons of both intact and gonadectomized males and females. Thus, RFRP-3 may exert its effects on reproduction either directly, via Gpr147 in a subset of GnRH neurons, and/or indirectly, via upstream regulators of GnRH.

Molecular, cellular, morphological, physiological and behavioral aspects of gonadotropin-inhibitory hormone

“…On the other hand, the inhibitory effects of RFRP-3 are independent of E2 status in adult female mice (Xiang et al, 2015) (Table 1). Poling et al (2012) examined changes in GnIH neurons in mice of both sexes during development and under different hormonal milieus in adults. They identified two interspersed subpopulations of GnIH cells (high GnIH-expressing, HE; low GnIH-expressing, LE).…”

RFamide-related peptide-3 (RFRP-3) suppresses sexual maturation in a eusocial mammal

“…In all vertebrates studied, a subset of GnRH neurons express RFRP-3's high-affinity receptor, GPR147 (11,12), and are apposed by RFRP-3 fibers originating in the dorsomedial nucleus of the hypothalamus (DMH) or paraventricular nucleus (PVN) (13)(14)(15). RFRP-3 suppresses gonadotropin secretion through central mechanisms (13,14,(16)(17)(18)(19)(20)(21)(22), but may also act locally in the gonads to drive changes in gonadal functioning (23)(24)(25).…”

Diurnal regulation of hypothalamic kisspeptin is disrupted during mouse pregnancy

“…Data also suggest that RFRP-3 exhibits reduced expression during proestrus, and thus lowers GnRH inhibition and positively drives the GnRH/LH surge and ovulation (Gibson et al 2008). Other studies showing that estrogen lowers RFRP3 mRNA levels also suggest that RFRP-3 may also play a role in estrogen positive feedback (Molnár et al 2011, Poling et al 2012.…”

“…However, it is also important to note that there is currently no consensus in the literature on the regulation of RFRP neurons by estradiol. Estradiol treatment has been shown to have markedly different effects on RFRP levels in female rodents: in some instances decreasing (Molnár et al 2011, Poling et al 2012, increasing (Iwasa et al 2012), or having no effect (Quennell et al 2010).…”