Hypothalamic action of phoenixin to control reproductive hormone secretion in females: importance of the orphan G protein-coupled receptor Gpr173

Abstract

Sexual maturation and maintenance of reproductive function are regulated by neurohormonal communication between the hypothalamus, pituitary, and gonads (referred to as the HPG axis). Phoenixin (PNX) is a newly identified, endogenous peptide abundantly produced in the hypothalamus and shown to be an important mediator of ovarian cyclicity. However, the underlying mechanisms by which phoenixin functions within the HPG axis are unknown. Previous in vitro studies demonstrated a direct action of PNX on gonadotrophs to potentiate gonadotrophin-releasing hormone (GnRH) induced luteinizing hormone (LH) secretion. Therefore, we hypothesized that centrally derived phoenixin regulates the preovulatory LH surge required for ovarian cyclicity. We observed a significant dose-related increase in the level of plasma LH in diestrous, female rats that were given an intracerebroventricular injection of PNX compared with vehicle-treated controls. While this suggests that even under low-estrogen conditions, PNX acts centrally to stimulate the HPG axis, further characterization is contingent on the elucidation of its cognate receptor. Using the "deductive ligand receptor matching strategy," we identified the orphan G protein-coupled receptor, Gpr173, as our top candidate. In cultured pituitary cells, siRNA-targeted compromise of Gpr173 abrogated PNX's action to potentiate GnRH-stimulated LH secretion. In addition, siRNA-mediated knockdown of endogenous Gpr173, which localized to several hypothalamic sites related to reproductive function, not only significantly extended the estrous cycle but also prevented the PNX-induced LH secretion in diestrous, female rats. These studies are the first to demonstrate a functional relationship between PNX and Gpr173 in reproductive physiology and identify a potential therapeutic target for ovulatory dysfunction.

Keywords: Gpr173; estrous cyclicity; luteinizing hormone; phoenixin.

Fig. 1.

Central administration of phoenixin stimulated luteinizing hormone (LH) secretion. Rats bearing a lateral ventricle cannula were injected with saline vehicle (n = 8), 1.0 nmol phoenixin (PNX)-20 amide (n = 9), or 3.0 nmol PNX-20 amide (n = 6) following a 10-min preinjection plasma collection for baseline determination by RIA. Plasma was collected at 5, 15, 30, and 60 min postinjection. At 5 and 15 min, administration of 3.0 nmol PNX resulted in a significant rise in circulating LH levels. The inset depicts significant dose-dependent increase in total LH secretion over the entire 60-min testing period. Data were analyzed by ANOVA with Scheffé's multiple-comparison test. *P < 0.05 and **P < 0.01 vs. vehicle treated. †P < 0.05 vs. 1.0 nmol PNX-treated.

Fig. 2.

Knockdown of Gpr173 abrogated the potentiative effect of phoenixin on gonadotrophin-releasing hormone (GnRH)-stimulated LH release. Anterior pituitary glands from random cycling, female rats were dispersed 24 h prior to Lipofectamine transfection with siRNA targeting either Gpr173 or eGFP. Cells were incubated overnight in either DMEM alone or containing 1 μM PNX-20 amide, followed by a 1-h exposure to GnRH. A: knockdown of Gpr173 prevented the ability of phoenixin to potentiate GnRH-stimulated LH secretion. (*P < 0.05, **P < 0.01, ***P < 0.001; One-way ANOVA with Scheffé's post hoc test). B: pretreatment with 100 nM Gpr173 siRNA resulted in ∼39% decrease in Gpr173 mRNA expression relative to eGFP siRNA pretreatment (***P < 0.001 vs. vehicle, †P < 0.05 vs. eGFP siRNA, using Mann-Whitney U-test).

Fig. 3.

Distribution of GPR173 mRNA in the rat forebrain. A–F: hybridization signal in distinct sections of the rat forebrain from rostral (A) to caudal (F) levels. Note high hybridization signal in the anteroventral periventricular nucleus (AVPV), in the medial preoptic nucleus (MPO), in the paraventricular and supraoptic nucleus of the hypothalamus (PVH and SON), in the ventromedial nucleus of the hypothalamus (VMH), in the compact formation of the dorsomedial nucleus of the hypothalamus (DMH), and in the medial nucleus of the amygdala (MeA). Moderate signal was also noticed in the lateral septum (LS), in the medial preoptic area (MPA) and in the ventral premammillary nucleus (PMV). Scale bar = 2 mm. Arc, arcuate nucleus.

Fig. 4.

Compromise of endogenous Gpr173-disrupted estrous cyclicity and inhibited PNX stimulation of LH release. A and B: cycling female rats implanted with a lateral ventricle cannulas were given two consecutive injections of 2 μl of saline containing 121 pmol (2 μg) of either Gpr173 siRNA or eGFP siRNA on the afternoons of estrus and diestrus day 1. Vaginal cytology was used to monitor progression through the estrous cycle. A: each bar indicates the phase of the estrous cycle for individual animals. Rats given Gpr173 siRNA (n = 6) exhibited a significant delay in the reappearance of estrus compared with eGFP siRNA-treated (n = 6). B: average estrous cycle duration was, therefore, significantly extended by the knockdown of Gpr173. (***P < 0.001 vs. eGFP siRNA-treated; one-way ANOVA) C and D: after consecutive intracerebroventricular injections (on estrus and diestrus day 1) of either Gpr173 siRNA (n = 9) or eGFP siRNA (n = 9), all rats were administered intracerebroventricularly 3.0 nmol PNX-20 amide. Plasmas were collected over 60 min for quantification of circulating LH levels by RIA and tissues [anterior pituitary, arcuate nucleus, medial basal hypothalamus (MBH), and anteroventral periventricular nucleus (AVPV)] collected for RNA isolation. C: animals pretreated intracerebroventricularly with eGFP siRNA exhibited a significant phoenixin-induced rise in plasma LH at 15 and 30 min postinjection. However, those pretreated with Gpr173 siRNA did not exhibit the stimulatory effect of phoenixin on LH release. (*P < 0.05 vs. eGFP siRNA-treated; one-way ANOVA). D: central administration of Gpr173 siRNA resulted in a significant reduction in the levels of Gpr173 in the arcuate nucleus (*P < 0.05 vs. eGFP siRNA-treated; Mann-Whitney U-test). E: relationship of the degree of GPR173 mRNA reduction and the LH response to intracerebroventricularly administered phoenixin. LH values plotted are those present 15 or 30 min following phoenixin administration.