The Kiss1 gene encodes a family of neuropeptides called kisspeptins, which activate the receptor G protein-coupled receptor-54 and play a role in the neuroendocrine regulation of GnRH secretion. We examined whether estradiol (E2) regulates KiSS-1 in the forebrain of the female mouse by comparing KiSS-1 mRNA expression among groups of ovary-intact (diestrus), ovariectomized (OVX), and OVX plus E2-treated mice. In the arcuate nucleus (Arc), KiSS-1 expression increased after ovariectomy and decreased with E2 treatment. Conversely, in the anteroventral periventricular nucleus (AVPV), KiSS-1 expression was reduced after ovariectomy and increased with E2 treatment. To determine whether the effects of E2 on KiSS-1 are mediated through estrogen receptor (ER)alpha or ERbeta, we evaluated the effects of E2 in OVX mice that lacked functional ERalpha or ERbeta. In OVX mice that lacked functional ERalpha, KiSS-1 mRNA did not respond to E2 in either the Arc or AVPV, suggesting that ERalpha is essential for mediating the inhibitory and stimulatory effects of E2. In contrast, KiSS-1 mRNA in OVX mice that lacked functional ERbeta responded to E2 exactly as wild-type animals. Double-label in situ hybridization revealed that virtually all KiSS-1-expressing neurons in the Arc and AVPV coexpress ERalpha, suggesting that the effects of E2 are mediated directly through KiSS-1 neurons. We conclude that KiSS-1 neurons in the Arc, which are inhibited by E2, may play a role in the negative feedback regulation of GnRH secretion, whereas KiSS-1 neurons in the AVPV, which are stimulated by E2, may participate in the positive feedback regulation of GnRH secretion.

Kisspeptins are products of the KiSS-1 gene, which bind to a G protein-coupled receptor known as GPR54. Mutations or targeted disruptions in the GPR54 gene cause hypogonadotropic hypogonadism in humans and mice, suggesting that kisspeptin signaling may be important for the regulation of gonadotropin secretion. To examine the effects of kisspeptin-54 (metastin) and kisspeptin-10 (the biologically active C-terminal decapeptide) on gonadotropin secretion in the mouse, we administered the kisspeptins directly into the lateral cerebral ventricle of the brain and demonstrated that both peptides stimulate LH secretion. Further characterization of kisspeptin-54 demonstrated that it stimulated both LH and FSH secretion, at doses as low as 1 fmol; moreover, this effect was shown to be blocked by pretreatment with acyline, a potent GnRH antagonist. To learn more about the functional anatomy of kisspeptins, we mapped the distribution of KiSS-1 mRNA in the hypothalamus. We observed that KiSS-1 mRNA is expressed in areas of the hypothalamus implicated in the neuroendocrine regulation of gonadotropin secretion, including the anteroventral periventricular nucleus, the periventricular nucleus, and the arcuate nucleus. We conclude that kisspeptin-GPR54 signaling may be part of the hypothalamic circuitry that governs the hypothalamic secretion of GnRH.

We examined the role of kisspeptin and its receptor, the G-protein-coupled receptor GPR54, in governing the onset of puberty in the mouse. In the adult male and female mouse, kisspeptin (10 -100 nM) evoked a remarkably potent, long-lasting depolarization of Ͼ90% of gonadotropin-releasing hormone (GnRH)-green fluorescent protein neurons in situ. In contrast, in juvenile [postnatal day 8 (P8) to P19] and prepubertal (P26 -P33) male mice, kisspeptin activated only 27 and 44% of GnRH neurons, respectively. This developmental recruitment of GnRH neurons into a kisspeptin-responsive pool was paralleled by an increase in the ability of centrally administered kisspeptin to evoke luteinizing hormone secretion in vivo. To learn more about the mechanisms through which kisspeptin-GPR54 signaling at the GnRH neuron may change over postnatal development, we performed quantitative in situ hybridization for kisspeptin and GPR54 transcripts. Approximately 90% of GnRH neurons were found to express GPR54 mRNA in both juvenile and adult mice, without a detectable difference in the mRNA content between the age groups. In contrast, the expression of KiSS-1 mRNA increased dramatically across the transition from juvenile to adult life in the anteroventral periventricular nucleus (AVPV; p Ͻ 0.001). These results demonstrate that kisspeptin exerts a potent depolarizing effect on the excitability of almost all adult GnRH neurons and that the responsiveness of GnRH neurons to kisspeptin increases over postnatal development. Together, these observations suggest that activation of GnRH neurons by kisspeptin at puberty reflects a dual process involving an increase in kisspeptin input from the AVPV and a post-transcriptional change in GPR54 signaling within the GnRH neuron.

The KiSS-1 gene codes for a family of neuropeptides called kisspeptins which bind to the G-protein-coupled receptor GPR54. To assess the possible effects of kisspeptins on gonadotropin secretion, we injected kisspeptin-52 into the lateral cerebral ventricles of adult male rats and found that kisspeptin-52 increased the serum levels of luteinizing hormone (p < 0.05). To determine whether the kisspeptin-52-induced stimulation of luteinizing hormone secretion was mediated by gonadotropin-releasing hormone (GnRH), we pretreated adult male rats with a GnRH antagonist (acyline), then challenged the animals with intracerebroventricularly administered kisspeptin-52. The GnRH antagonist blocked the kisspeptin-52-induced increase in luteinizing hormone. To examine whether kisspeptins stimulate transcriptional activity in GnRH neurons, we administered kisspeptin-52 intracerebroventricularly and found by immunocytochemistry that 86% of the GnRH neurons coexpressed Fos 2 h after the kisspeptin-52 challenge, whereas fewer than 1% of the GnRH neurons expressed Fos following injection of the vehicle alone (p < 0.001). To assess whether kisspeptins can directly act on GnRH neurons, we used double-label in situ hybridization and found that 77% of the GnRH neurons coexpress GPR54 mRNA. Finally, to determine whether KiSS-1 gene expression is regulated by gonadal hormones, we measured KiSS-1 mRNA levels by single-label in situ hybridization in intact and castrated males and found significantly higher levels in the arcuate nucleus of castrates. These results demonstrate that GnRH neurons are direct targets for regulation by kisspeptins and that KiSS-1 mRNA is regulated by gonadal hormones, suggesting that KiSS-1 neurons play an important role in the feedback regulation of gonadotropin secretion.

Kisspeptins are products of the Kiss1 gene, which bind to GPR54, a G protein-coupled receptor. Kisspeptins and GPR54 have been implicated in the neuroendocrine regulation of GnRH secretion. To test the hypothesis that testosterone regulates Kiss1 gene expression, we compared the expression of KiSS-1 mRNA among groups of intact, castrated, and castrated/testosterone (T)-treated male mice. In the arcuate nucleus (Arc), castration resulted in a significant increase in KiSS-1 mRNA, which was completely reversed with T replacement, whereas in the anteroventral periventricular nucleus, the results were the opposite, i.e. castration decreased and T increased KiSS-1 mRNA expression. In the Arc, the effects of T on KiSS-1 mRNA were completely mimicked by estrogen but only partially mimicked by dihydrotestosterone, a nonaromatizable androgen, suggesting that both estrogen receptor (ER) and androgen receptor (AR) play a role in T-mediated regulation of KiSS-1. Studies of the effects of T on KiSS-1 expression in mice with either a deletion of the ERalpha or a hypomorphic allele to the AR revealed that the effects of T are mediated by both ERalpha and AR pathways, which was confirmed by the presence of either ERalpha or AR coexpression in most KiSS-1 neurons in the Arc. These observations suggest that KiSS-1 neurons in the Arc, whose transcriptional activity is inhibited by T, are targets for the negative feedback regulation of GnRH secretion, whereas KiSS-1 neurons in the anteroventral periventricular nucleus, whose activity is stimulated by T, may mediate other T-dependent processes.