Kisspeptin Signaling Is Indispensable for Neurokinin B, but not Glutamate, Stimulation of Gonadotropin Secretion in Mice

Abstract

Kisspeptins (Kp), products of the Kiss1 gene that act via Gpr54 to potently stimulate GnRH secretion, operate as mediators of other regulatory signals of the gonadotropic axis. Mouse models of Gpr54 and/or Kiss1 inactivation have been used to address the contribution of Kp in the central control of gonadotropin secretion; yet, phenotypic and hormonal differences have been detected among the transgenic lines available. We report here a series of neuroendocrine analyses in male mice of a novel Gpr54 knockout (KO) model, generated by heterozygous crossing of a loxP-Gpr54/Protamine-Cre double mutant line. Gpr54-null males showed severe hypogonadotropic hypogonadism but retained robust responsiveness to GnRH. Gonadotropic responses to the agonist of ionotropic glutamate receptors, N-methyl-d-aspartate, were attenuated, but persisted, in Gpr54-null mice. In contrast, LH secretion after activation of metabotropic glutamate receptors was totally preserved in the absence of Gpr54 signaling. Detectable, albeit reduced, LH responses were also observed in Gpr54 KO mice after intracerebroventricular administration of galanin-like peptide or RF9, putative antagonist of neuropeptide FF receptors for the mammalian ortholog of gonadotropin-inhibiting hormone. In contrast, the stimulatory effect of senktide, agonist of neurokinin B (NKB; cotransmitter of Kiss1 neurons), was totally abrogated in Gpr54 KO males. Lack of Kp signaling also eliminated feedback LH responses to testosterone withdrawal. However, residual but sustained increases of FSH were detected in gonadectomized Gpr54 KO males, in which testosterone replacement failed to fully suppress circulating FSH levels. In sum, our study provides novel evidence for the relative importance of Kp-dependent vs. -independent actions of several key regulators of GnRH secretion, such as glutamate, galanin-like peptide, and testosterone. In addition, our data document for the first time the indispensable role of Kp signaling in mediating the stimulatory effects of NKB on LH secretion, thus supporting the hypothesis that NKB actions on GnRH neurons are indirectly mediated via its ability to regulate Kiss1 neuronal output.

ENDOCRINOLOGY AND ADOLESCENCE: Deciphering puberty: novel partners, novel mechanisms

“…Of note, the stimulatory effects of senktide on LH secretion were abrogated in Gpr54 null mice, thus suggesting that these require the integrity of kisspeptin signaling to manifest (35), a contention that is also supported by recent findings in the monkey (36). Notwithstanding these solid pharmacological data, evidence for null or even inhibitory responses to NKB agonists has also been presented (37,38).…”

“…In keeping with the hypogonadotropic phenotype of patients with inactivating mutations of the NKB system, a number of studies in ewes, monkeys, mice, and rats have documented stimulatory actions of the NKB agonist, senktide, on LH secretion (28,33,34,35). Of note, the stimulatory effects of senktide on LH secretion were abrogated in Gpr54 null mice, thus suggesting that these require the integrity of kisspeptin signaling to manifest (35), a contention that is also supported by recent findings in the monkey (36).…”

“…In turn, NKB may act on KNDy neurons to finely tune (predominantly, stimulate) kisspeptin release, therefore inducing GnRH secretion in an indirect manner. This possibility is, at least partially, supported by a solid body of experimental evidence, including the following observations: i) central senktide injection induces expression of C-fos in ARC KNDy neurons (34); ii) senktide elicits LH secretion in a GnRH-dependent manner (28); iii) the LH-releasing effects of senktide are not detected in the absence of proper Gpr54 signaling (35); iv) desensitization to the effects of continuous NKB stimulation takes place at a level upstream of GnRH neurons (28); v) substantial NK3R expression is detected in KNDy, but not GnRH neurons, in rodents and sheep (23); and vi) senktide induces the electrical activation of Kiss1 neurons, as revealed by electrophysiological recordings in Kiss1-CreGFP mice (40). To add further refinement to the system, the third partner of the KNDy trio, Dyn, has been long recognized as an inhibitor of gonadotropin secretion, likely via its ability to repress the release of kisspeptins on of GnRH neurons (12).…”

Neuroendocrine integration of nutritional signals on reproduction

“…Physiologically, GALP administration was shown to stimulate LH release in mice (Kauffman et al 2005), which occurs in a kisspeptin-independent manner (Garcia-Galiano et al 2012), and GALP infusion was sufficient to rescue the onset of puberty in food-restricted weanling male and female rats (Mohr et al 2012), demonstrating GALP is modulated by nutritional signals and also facilitates fertility in vivo. Accordingly, rats exhibiting insulin-dependent diabetes have reduced Galp mRNA compared to control animals, and brain insulin administration reversed this effect .…”

“…As reviewed by Iremonger and coworkers (Iremonger et al 2010), many studies have shown that glutamate agonists and antagonists injected into the brain can stimulate or inhibit LH secretion, respectively (Lopez et al 1990, Brann & Mahesh 1995, Ping et al 1997. Glutamate likely exerts its influence via direct effects on GNRH neurons, as TenaSempere's lab demonstrated that glutamate-stimulated LH secretion occurs in a kisspeptin-independent manner (Garcia-Galiano et al 2012). Furthermore, neurons in the ventral premamillary nucleus (PMV) of the hypothalamus, which mediate some of leptin's metabolic influences on GNRH function (Donato et al 2011), are almost exclusively glutamatergic (Kocsis et al 2003), supporting a role for glutamate-expressing neurons in the metabolic control of fertility.…”