Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that operates as sensor of cellular energy status and effector for its coupling to cell growth and proliferation. At the hypothalamic arcuate nucleus, mTOR signaling has been recently proposed as transducer for leptin effects on energy homeostasis and food intake. However, whether central mTOR also participates in metabolic regulation of fertility remains unexplored. We provide herein evidence for the involvement of mTOR in the control of puberty onset and LH secretion, likely via modulation of hypothalamic expression of Kiss1. Acute activation of mTOR by l-leucine stimulated LH secretion in pubertal female rats, whereas chronic l-leucine infusion partially rescued the state of hypogonadotropism induced by food restriction. Conversely, blockade of central mTOR signaling by rapamycin caused inhibition of the gonadotropic axis at puberty, with significantly delayed vaginal opening, decreased LH and estradiol levels, and ovarian and uterine atrophy. Inactivation of mTOR also blunted the positive effects of leptin on puberty onset in food-restricted females. Yet the GnRH/LH system retained their ability to respond to ovariectomy and kisspeptin-10 after sustained blockade of mTOR, ruling out the possibility of unspecific disruption of GnRH function by rapamycin. Finally, mTOR inactivation evoked a significant decrease of Kiss1 expression at the hypothalamus, with dramatic suppression of Kiss1 mRNA levels at the arcuate nucleus. Altogether our results unveil the role of central mTOR signaling in the control of puberty onset and gonadotropin secretion, a phenomenon that involves the regulation of Kiss1 and may contribute to the functional coupling between energy balance and gonadal activation and function.

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.