The role of kisspeptin-GPR54 signaling in the tonic regulation and surge release of gonadotropin-releasing hormone/luteinizing hormone

Abstract

The Kiss1 gene codes for kisspeptin, which binds to GPR54, a G-protein-coupled receptor. Kisspeptin and GPR54 are expressed in discrete regions of the forebrain, and they have been implicated in the neuroendocrine regulation of reproduction. Kiss1-expressing neurons are thought to regulate the secretion of gonadotropin-releasing hormone (GnRH) and thus coordinate the estrous cycle in rodents; however, the precise role of kisspeptin-GPR54 signaling in the regulation of gonadotropin secretion is unknown. In this study, we used female mice with deletions in the GPR54 gene [GPR54 knock-outs (KOs)] to test the hypothesis that kisspeptin-GPR54 signaling provides the drive necessary for tonic GnRH/luteinizing hormone (LH) release. We predicted that tonic GnRH/LH secretion would be disrupted in GPR54 KOs and that such animals would be incapable of showing a compensatory rise in LH secretion after ovariectomy. As predicted, we found that GPR54 KO mice do not exhibit a postovariectomy rise in LH, suggesting that tonic GnRH secretion is disrupted in the absence of kisspeptin-GPR54 signaling. We also postulated that kisspeptin-GPR54 signaling is critical for the generation of the estradiol (E)-induced GnRH/LH surge and thus E should be incapable of inducing an LH surge in the absence of GPR54. However, we found that E induced Fos expression in GnRH neurons and produced a GnRH-dependent LH surge in GPR54 KOs. Thus, in mice, kisspeptin-GPR54 signaling is required for the tonic stimulation of GnRH/LH secretion but is not required for generating the E-induced GnRH/LH surge.

Figure 1.

A, Photomicrographs of double-label ISH in WT (n = 5) and GPR54 KO (n = 5) brains. White arrows indicate GnRH neurons; black arrows with white outline indicate GnRH cells coexpressing GPR54 mRNA. Scale bars, 50 μm. B, Quantification of silver grains (GPR54 mRNA) on GnRH neurons. C, Serum LH levels in female WT and GPR54 KO mice after treatment with vehicle or kisspeptin (n = 4–5 per group). Double asterisks indicate significant difference at p < 0.01. Error bars indicate SEM.

Figure 2.

A, Representative photomicrographs of ISH for Kiss1 mRNA in the Arc and AVPV of OVX sham (OVX + Sham) and E-replaced (OVX + E) mice. Scale bars, 100 μm. B, Quantification of Kiss1-expressing cells in the Arc. Different letters represent significant difference (p < 0.05). C, Quantification of Kiss1-expressing cells in the AVPV. Different letters represent significant difference (p < 0.05; E WT, n = 5; E KO, n = 5; sham WT, n = 5; sham KO, n = 4). Error bars indicate SEM.

Figure 3.

Serum LH levels in OVX E and sham-replaced WT and GPR54 KO mice. Double asterisks indicate significant difference (p < 0.01; E WT, n = 5; E KO, n = 5; sham WT, n = 5; sham KO, n = 4). Error bars indicate SEM.

Figure 4.

Serum LH levels in WT and GPR54 KO mice in the A.M. and P.M.. Different letters indicate significant difference (p < 0.05; A.M. WT, n = 8; A.M. KO, n = 7; P.M. WT, n = 7; P.M. KO, n = 6). Error bars indicate SEM.

Figure 5.

Serum LH levels in WT and GPR54 KO mice in the A.M. and P.M. All mice in the P.M. groups were treated with either vehicle or acyline. Different letters indicate significant difference (p < 0.05; A.M. WT, n = 7; A.M. KO, n = 7; P.M. WT + Veh., n = 9; P.M. KO + Veh., n = 10; P.M. WT + acyline, n = 5; P.M. KO + acyline, n = 8). Error bars indicate SEM. Veh., Vehicle.

Figure 6.

A, Representative photomicrographs of GnRH and Fos double-label IHC in the rostral hypothalamus. Black arrows indicate Fos-stained nuclei. Red arrows indicate GnRH-stained cytoplasm. Red arrows with black outlines indicate cells stained for both GnRH and Fos. B, Percentage of GnRH neurons coexpressing Fos protein in the A.M. and P.M. Different letters indicate significant difference (p < 0.01; A.M. WT, n = 7; A.M. KO, n = 7; P.M. WT plus vehicle or acyline, n = 14; P.M. KO vehicle or acyline, n = 18). Scale bars, 50 μm. Error bars indicate SEM.