No abstract

The KiSS-1 gene encodes a family of peptides called kisspeptins, which are endogenous ligands for the G protein-coupled receptor GPR54. Kisspeptin function appears to be critical for GnRH secretion and the initiation of puberty. To test the hypothesis that steroid hormones regulate KiSS-1 mRNA expression in the ewe, we examined the brains of ovary-intact (luteal phase) and ovariectomized (OVX) ewes, as well as OVX ewes that received estradiol (E) or progesterone (P) replacement. KiSS-1 mRNA-expressing cells were predominantly located in the arcuate nucleus (ARC). Here, expression was increased after OVX but returned to the level of gonad-intact animals with E treatment. Treatment with P partially restored KiSS-1 expression toward gonad-intact levels. Double-label immunohistochemistry revealed that approximately 86% of kisspeptin-immunoreactive cells in the ARC are also P-receptor positive. Finally, we tested the hypothesis that KiSS-1 mRNA is lower during anestrus, due to a non-steroid-dependent seasonal effect. In OVX ewes, expression in the ARC was lower at the time of year corresponding to anestrus. We conclude that KiSS-1 expression in the ARC of the ewe brain is negatively regulated by chronic levels of E and P, suggesting that both steroids may exert negative feedback control on GnRH secretion through altered kisspeptin signaling. Furthermore, a seasonal alteration in KiSS-1 expression in the ARC of OVX ewes strongly suggests that kisspeptin is fundamentally involved in the control of seasonal changes in reproductive function.

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.

Kisspeptin stimulates reproduction, and kisspeptin cells in the arcuate nucleus (ARC) express Ob-Rb in the mouse. Herein we report studies in ewes to determine whether kisspeptin cells express Ob-Rb and respond to leptin and whether reciprocal connections exist between kisspeptin cells and proopiomelanocortin (POMC) or neuropeptide Y (NPY) cells to modulate reproduction and metabolic function. Kiss1 mRNA was measured by in situ hybridization in ovariectomized ewes that were normal body weight, lean, or lean with leptin treatment by intracerebroventricular (icv) infusion (4 μg/h, 3 d). Kiss1 expression in the ARC and the preoptic area was lower in hypogonadotropic lean animals than animals of normal weight, and icv infusion of leptin partially restored Kiss1 expression in lean animals. Single-cell laser capture microdissection coupled with real-time PCR showed that Kiss1 cells in the preoptic area and ARC express Ob-Rb. Double-label fluorescent immunohistochemistry showed that reciprocal connections exist between kisspeptin cells and NPY and POMC cells. Accordingly, we treated ovariectomized ewes with kisspeptin (5 μg/h, icv) or vehicle for 20 h and examined POMC and NPY gene expression by in situ hybridization. Kisspeptin treatment reduced POMC and increased NPY gene expression. Thus, kisspeptin neurons respond to leptin and expression of Kiss1 mRNA is affected by leptin status. Kisspeptin cells communicate with NPY and POMC cells, altering expression of the relevant genes in the target cells; reciprocal connections also exist. This network between the three cell types could coordinate brain control of reproduction and metabolic homeostatic systems.