Neurons that produce gonadotropin-releasing hormone (GnRH) are the final common pathway by which the brain regulates reproduction. GnRH neurons are regulated by an afferent network of kisspeptin-producing neurons. Kisspeptin binds to its cognate receptor on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion, thus gating down-stream events supporting reproduction. We have developed kisspeptin antagonists to facilitate the direct determination of the role of kisspeptin neurons in the neuroendocrine regulation of reproduction. In vitro and in vivo studies of analogues of kisspeptin-10 with amino substitutions have identified several potent and specific antagonists. A selected antagonist was shown to inhibit the firing of GnRH neurons in the brain of the mouse and to reduce pulsatile GnRH secretion in female pubertal monkeys; the later supporting a key role of kisspeptin in puberty onset. This analog also inhibited the kisspeptin-induced release of luteinizing hormone (LH) in rats and mice and blocked the postcastration rise in LH in sheep, rats, and mice, suggesting that kisspeptin neurons mediate the negative feedback effect of sex steroids on gonadotropin secretion in mammals. The development of kisspeptin antagonists provides a valuable tool for investigating the physiological and pathophysiological roles of kisspeptin in the regulation of reproduction and could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer.

Neurokinin B (NKB) and its cognate receptor neurokinin 3 (NK3R) play a critical role in reproduction. NKB and NK3R are coexpressed with dynorphin (Dyn) and kisspeptin (Kiss1) genes in neurons of the arcuate nucleus (Arc). However, the mechanisms of action of NKB as a cotransmitter with kisspeptin and dynorphin remain poorly understood. We explored the role of NKB in the control of LH secretion in the female rat as follows. 1) We examined the effect of an NKB agonist (senktide, 600 pmol, administered into the lateral cerebral ventricle) on luteinizing hormone (LH) secretion. In the presence of physiological levels of estradiol (E 2), senktide induced a profound increase in serum levels of LH and a 10-fold increase in the number of Kiss1 neurons expressing c-fos in the Arc (P Ͻ 0.01 for both). 2) We mapped the distribution of NKB and NK3R mRNAs in the central forebrain and found that both are widely expressed, with intense expression in several hypothalamic nuclei that control reproduction, including the Arc. 3) We studied the effect of E 2 on the expression of NKB and NK3R mRNAs in the Arc and found that E 2 inhibits the expression of both genes (P Ͻ 0.01) and that the expression of NKB and NK3R reaches its nadir on the afternoon of proestrus (when circulating levels of E 2 are high). These observations suggest that NKB/NK3R signaling in Kiss1/NKB/ Dyn-producing neurons in the Arc has a pivotal role in the control of gonadotropin-releasing hormone (GnRH)/LH secretion and its regulation by E 2-dependent negative feedback in the rat. estradiol; hypothalamus NEUROKININ B (NKB) is a member of the tachykinin family that has recently emerged as a key neuropeptide in the control of reproductive function. Humans bearing loss-of-function mutations of TAC3 or TAC3R, which are homologous to NKB and its cognate receptor neurokinin 3 (NK3R, aka Tac2 and Tac3r), respectively, in rodents, exhibit hypogonadotropic hypogonadism and infertility (34,38). Clues regarding NKB's specific role in the regulation of reproduction come from studies in animals. In rodents, the compensatory rise of luteinizing hormone (LH) following ovariectomy (OVX) is ablated by treatment with the NKB agonist senktide (24,30). In addition, the expression of NKB and its receptor NK3R in the hypothalamic arcuate nucleus (Arc) is inhibited by estradiol (E 2 ) via estrogen receptor-␣ (9, 24). Together, these observations suggest that NKB/NK3R signaling plays an important role in the E 2 -dependent negative-feedback control of gonadotropin-releasing hormone (GnRH) and LH in mammals.In the Arc of sheep and rodents, NKB is coexpressed with kisspeptin and dynorphin, which are encoded by the Kiss1 and preprodynorphin (Dyn) genes, respectively (4, 15, 24). Kisspeptin is a potent GnRH secretagogue (25), and GnRH neurons express the kisspeptin receptor (Kiss1r, aka GPR54) (17), which plays a critical role in the neuroendocrine regulation of GnRH and LH secretion (8,25,31). Kisspeptin neurons in the Arc express estrogen receptor-␣ and are thought to be direct targets f...

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