Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge

Abstract

Kisspeptin and its receptor GPR54 have recently been identified as key signaling partners in the neural control of fertility in animal models and humans. The gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons of the neural network controlling fertility and are suspected to be the primary locus of kisspeptin-GPR54 signaling. Using mouse models, the present study addressed whether kisspeptin and GPR54 have a key role in the activation of GnRH neurons to generate the luteinizing hormone (LH) surge responsible for ovulation. Dual-label immunocytochemistry experiments showed that 40-60% of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) expressed estrogen receptor alpha and progesterone receptors. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, approximately 30% of RP3V kisspeptin neurons were found to express c-FOS in surging mice compared with 0% in nonsurging controls. A strong correlation was found between the percentage of c-FOS-positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 were essential for GnRH neuron activation and the LH surge, Gpr54- and Kiss1-null mice were examined. Whereas wild-type littermates all exhibited LH surges and c-FOS in approximately 50% of their GnRH neurons, none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin-GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. This broadens considerably the potential roles and therapeutic possibilities for kisspeptin and GPR54 in fertility regulation.

Figure 1.

RP3V kisspeptin neurons express ERα and PR. A, Dual-label immunocytochemistry showing AVPV kisspeptin neurons (brown) with ERα-immunoreactive nuclei (black). Arrowheads indicate dual-labeled cells. B, Dual-label immunocytochemistry showing PVpo kisspeptin neurons (brown) with PR-immunoreactive nuclei (black). C, Bar graph showing the mean + SEM number of kisspeptin-immunoreactive neurons counted per section at the three levels of the RP3V (AVPV, rPVpo, and cPVpo) in the ERα dual-labeling study. D, Bar graph showing the number of kisspeptin neurons counted per section in the AVPV, rPVpo, and cPVpo in the PR dual-labeling study. E, F, Percentage of kisspeptin neurons expressing ERα (E) and PR (F) throughout the RP3V. Scale bars, 10 μm.

Figure 2.

RP3V kisspeptin neurons express c-FOS at the time of the GnRH/LH surge. A, Dual-label immunocytochemistry showing GnRH neurons (brown) with c-FOS-immunoreactive nuclei (black). B, Dual-label immunocytochemistry showing AVPV kisspeptin neurons (brown) with c-FOS immunoreactive nuclei (black). C, Bar graph showing the mean + SEM number of GnRH neurons counted per section in the MS and rPOA of OVX+E- (E) and OVX+V- (V) treated wild-type mice. D, Percentage of rPOA GnRH neurons expressing c-FOS in OVX+E- and OVX+V-treated wild-type mice. E, Bar graph showing the number of kisspeptin neurons counted per section in the AVPV, rPVpo, and cPVpo in OVX+E- and OVX+V-treated wild-type mice. F, Bar graph showing the LH levels in OVX+E- and OVX+V-treated wild-type mice. G, Graph showing the positive correlation between percentage of GnRH neurons with c-FOS and percentage of RP3V kisspeptin neurons with c-FOS in the AVPV and the PVpo combined. H, Percentage of kisspeptin neurons at the three levels of the RP3V expressing c-FOS in OVX+E- and OVX+V-treated wild-type mice. *p < 0.05, ***p < 0.001, V versus E. Scale bars, 10 μm.

Figure 3.

Both the LH surge and GnRH neuron activation are absent in Gpr54- and Kiss1-null mice. A, Dual-label immunocytochemistry showing three GnRH neurons (brown) expressing c-FOS (black nuclei) in wild-type siblings. B, Dual-label immunocytochemistry showing absence of c-FOS staining in GnRH neurons of Gpr54-null mice. C, Dual-label immunocytochemistry showing absence of c-FOS staining in GnRH neurons of Kiss1-null mice. D, Bar graph showing the mean + SEM number of GnRH neurons counted per section in rPOA of wild-type siblings (wt) and Gpr54- (gpr54) and Kiss1- (Kiss-1) null mice. E, Mean + SEM LH levels in OVX+E+P mice of the three genotypes. F, Percentage of GnRH neurons expressing c-FOS in OVX+E+P mice of the three genotypes. G, Bar graph showing the mean + SEM number of kisspeptin neurons counted per section in the AVPV, rPVpo, and cPVpo of wild-type and Gpr54-null mice. H, Percentage of kisspeptin neurons expressing c-FOS in the AVPV, rPVpo, and cPVpo of OVX+E+P wild-type and Gpr54-null mice. *p < 0.05, **p < 0.01, ***p < 0.001, wt versus gpr54 or Kiss-1 or as indicated. Scale bars, 20 μm.