Kisspeptin Signaling Research Articles

Gonadotrophin‐Releasing Hormone Pulse Generator Activity in the Hypothalamus of the Goat

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.

Does Kisspeptin signaling offer a new way to treat infertility?

Does Kisspeptin signaling offer a new way to treat infertility?

Kisspeptin Signaling in Rodents: Insights from Knock-Out Mice.William H. Colledge, Ph.D.

Initiation of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus at puberty stimulates luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from the anterior pituitary to galvanize maturation of the gonads. A key molecular event that initiates this activation of GnRH secretion has recently been identified with the discovery that kisspeptin neuropeptides are potent agonists of GnRH release. Kisspeptins are a family of overlapping amidated peptides encoded by the Kiss1 gene that signal through the G-protein coupled receptor GPR54 (KISS1R). Five transgenic mouse lines have been generated with mutations in the Gpr54 gene and two lines with mutations in the Kiss1 gene. The phenotypes of these mutant mice are broadly similar, suggesting no obvious functional compensation by other ligands or receptors. The mutant mice fail to undergo puberty and have poor gonadal development and low sex steroid and gonadotropic hormone levels (hypogonadotropic hypogonadism). Spermatogenesis and ovulation are severely impaired and mutant females do not show normal estrous cyclicity. The gonads and the anterior pituitary retain functional responses to hormonal stimulation, however, consistent with the primary defect being a failure to secrete GnRH from the hypothalamus to activate the hypothalamic-pituitary-gonadal axis. These mutant mice are being used to interrogate the function of GPR54 and kisspeptins in key aspects of kisspeptin signaling including the role of these proteins in regulating the neuroendocrine responses of GnRH neurons. Kisspeptin expression is regulated by gonadal steroids (testosterone or estrogen) and this provides a possible mechanism by which these steroid hormones control GnRH secretion. We have shown that kisspeptin-GPR54 signaling is essential for GnRH neuronal activation and the pre-ovulatory LH surge. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, 30% of kisspeptin neurons express c-FOS in surging mice compared with 0% in non-surging controls. A strong correlation was found between the percentage of c-FOS positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 are essential for GnRH neuron activation and the LH surge, Gpr54 and Kiss1 null mice were examined. Wild-type mice all exhibited LH surges and c-FOS in ~50% of their GnRH neurons, but none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin-GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. We have also shown that kisspeptin can stimulate GnRH secretion by a direct action at GnRH nerve terminals in addition to its likely action at the cell bodies. These mutant mice provide a useful resource to further understand the hypothalamic regulation of mammalian reproduction, its integration with the pituitary-gonadal axis and to study the potential function of Gpr54 and Kiss1 in peripheral tissues. This research was funded by Takeda Cambridge and BBSRC Grant BB/C003861/1.

Dissection of Kisspeptin's Role in the Neuroendocrine Regulation of Gonadotropin Using Antagonists.Robert P. Millar, Ph.D.

Kisspeptin and its cognate receptor (GPR54) are central regulators of reproduction and are key mediators of nutritional, photoperiod, stress and steroid hormone regulation of the GnRH neuron. Kisspeptin also regulates trophoblast invasion and stimulates vasoconstriction. Thus, specific antagonists at GPR54 would be useful as tools to interrogate physiological and patho-physiological roles of kisspeptin. Antagonists would also have potential as therapeutic agents for treating a wide range of clinical conditions. We have systematically substituted single and combinations of amino acids in kisspeptin-10 in a detailed structure-activity-relationship study using receptor binding and inhibition of kisspeptin-10 stimulation of inositol phosphate (IP) mobilisation as outputs. These studies revealed an essential binding pharmacophore and identified residues required for GPR54 activation allowing for the subsequent derivation of a series of antagonists and partial agonists. The most potent antagonists had IC</SUB50>s in the nM range as measured by the inhibition of 10nM kisspeptin stimulation of IP. A selected antagonist potently inhibited (1nM) kisspeptin-10 stimulation of GnRH neuronal firing in mouse brain slices and GnRH secretion in thesus monkeys. It also inhibited kisspeptin-induced LH secretion in male rats and mice. The antagonist inhibited the post-castration LH rise in mice and rats when administered intracerebroventricularly, confirming that inhibition of kisspeptin signalling mediates gonadal steroid negative feedback. The antagonist had no effect on basal LH secretion in male rats and mice possibly indicating a lower tone of kisspeptin input. LH pulse frequency and amplitude were inhibited by intracerebroventricular antagonist administration to ovariectomized ewes. Antagonist also delayed puberty in female rats and inhibited the ovulatory LH surge confirming a role in these processes. In summary, we have developed GPR54 antagonists that provide useful tools to investigate the neuroendocrine regulation of reproduction and for potential therapeutic applications.

Effects of ghrelin on Kisspeptin mRNA expression in the hypothalamic medial preoptic area and pulsatile luteinising hormone secretion in the female rat

The orexigenic gut peptide ghrelin negatively modulates the hypothalamic–pituitary–gonadal (HPG) axis. Hyperghrelinaemia results during negative energy balance, a state often associated with delayed puberty and disrupted fertility, whilst exogenous ghrelin suppresses pulsatile luteinising hormone (LH) secretion. The recent identification of kisspeptin (Kiss1) and its G protein-coupled receptor (GPR)54 (Kiss1r) as an essential component of the HPG axis controlling gonadotrophin secretion raises the possibility that kisspeptin-Kiss1r signalling may play a critical role in the transduction of ghrelin-induced suppression of LH. Ovariectomised oestrogen-replaced rats were implanted with intravenous catheters and blood samples collected for detection of LH pulses prior to and after intravenous administration of ghrelin (3 nM/250 μl) or saline (250 μl) during ad libitum feeding or after overnight fasting. Quantitative RT-PCR was used to determine Kiss1 and Kiss1r mRNA levels in brain punches of the key hypothalamic sites regulating gonadotrophin secretion, the medial preoptic area (mPOA) and arcuate nucleus (ARC), collected 6 h following administration of ghrelin. Ghrelin significantly lowered LH pulse frequency in fed rats, an effect significantly enhanced by food deprivation. Fasting, ghrelin or their combination down-regulated Kiss1, without affecting Kiss1r, expression in the mPOA, and affected the expression of neither in the ARC. Considering the pivotal role for kisspeptin signalling in the activation of the HPG axis, the ability of ghrelin to down-regulate Kiss1 expression in mPOA may be a contributing factor in ghrelin-related suppression of pulsatile LH secretion.

Impact of neonatal exposure to the ERα agonist PPT, bisphenol-A or phytoestrogens on hypothalamic kisspeptin fiber density in male and female rats

Neonatal exposure to endocrine disrupting compounds (EDCs) can impair reproductive physiology, but the specific mechanisms by which this occurs remain largely unknown. Growing evidence suggests that kisspeptin (KISS) neurons play a significant role in the regulation of pubertal onset and ovulation, therefore disruption of KISS signaling could be a mechanism by which EDCs impair reproductive maturation and function. We have previously demonstrated that neonatal exposure to phytoestrogens decreases KISS fiber density in the anterior hypothalamus of female rats, an effect which was associated with early persistent estrus and the impaired activation gonadotropin releasing hormone (GnRH) neurons. The goals of the present study were to (1) determine if an ERα selective agonist (PPT) or bisphenol-A (BPA) could produce similar effects on hypothalamic KISS content in female rats and (2) to determine if male KISS fiber density was also vulnerable to disruption by EDCs. We first examined the effects of neonatal exposure to PPT, a low (50 μg/kg bw) BPA dose, and a high (50 mg/kg bw) BPA dose on KISS immunoreactivity (-ir) in the anterior ventral periventricular (AVPV) and arcuate (ARC) nuclei of adult female rats, using estradiol benzoate (EB) and a sesame oil vehicle as controls. AVPV KISS-ir, following ovariectomy (OVX) and hormone priming, was significantly lower in the EB and PPT groups but not the BPA groups. ARC KISS-ir levels were significantly diminished in the EB and high dose BPA groups, and there was a nonsignificant trend for lower KISS-ir in the PPT group. We next examined effects of neonatal exposure to a low (50 μg/kg bw) dose of BPA and the phytoestrogens genistein (GEN) and equol (EQ) on KISS-ir in the AVPV and ARC of adult male rats, using OVX females as an additional control group. None of the compounds affected KISS-ir in the male hypothalamus. Our results suggest that the organization of hypothalamic KISS fibers may be vulnerable to disruption by EDC exposure and that females might be more sensitive than males.

Central and peripheral administration of kisspeptin activates gonadotropin but not somatotropin secretion in prepubertal gilts

It is well established that kisspeptin signaling is necessary for the onset of puberty in laboratory animals. However, the role that kisspeptin may have in regulating puberty in large domestic animals is unknown. We tested the hypothesis that either central or peripheral infusion of kisspeptin would stimulate gonadotropin and GH secretion in prepubertal gilts. In experiment 1, prepubertal gilts were fitted with i.c.v. cannula and indwelling jugular catheters. Animals were randomly assigned to receive 0, 10, or 100 microg kisspeptin in saline. In experiment 2, prepubertal gilts, fitted with indwelling jugular catheters, randomly received 0, 1, 2.5, or 5 mg kisspeptin in saline intravenously. Serial blood samples were collected every 15 min for 3 h before and 5 h after infusions, and serum concentrations of LH, FSH, and GH were determined. Mean concentrations of LH and FSH remained at basal levels for control animals but were increased (P<0.001) for animals receiving i.c.v. infusion of kisspeptin. Area under the LH and FSH curves following i.c.v. infusion of kisspeptin increased (P<0.001) in a dose-dependent manner. Concentrations of GH were unaffected by i.c.v. treatment. Peripheral administration of kisspeptin increased (P<0.05) serum concentrations of LH but not FSH or GH. Thus, kisspeptin can activate gonadotropic but not somatotropic hormone secretion in prepubertal gilts. The present data support the concept that kisspeptin plays a role in the mechanism involved in initiating puberty in swine.

Preanalytical Factors Affecting RIA Measurement of Plasma Kisspeptin

Kisspeptins are peptide products of the KiSS-1 metastasis-suppressor ( KISS1 ) gene and the natural ligands of the G-protein–coupled receptor GPR54. KISS1 was initially investigated as an antimetastasis gene. More recent studies have demonstrated that the kisspeptins are potent stimulators of the hypothalamo-pituitary-gonadal axis. Mice and humans with defective kisspeptin signaling show hypogonadotrophic hypogonadism and impaired sexual development (1)(2). Plasma kisspeptin concentrations are <2 pmol/L in men and nonpregnant women. KiSS-1 mRNA is highly expressed in the placenta, and plasma kisspeptin concentrations increase dramatically, to thousands of picomoles per liter, during pregnancy (3). In addition, plasma kisspeptin is increased in women with gestational trophoblastic tumors, thus raising the possibility of measuring plasma kisspeptin as a novel tumor marker (4). Previous studies that have measured plasma kisspeptin in women during pregnancy have found significantly different concentrations of circulating kisspeptin (3)(4) that may be attributable to differences in preanalytical variables, such as collection tube type, processing times, and storage conditions. Use of a standardized sample collection method …

Sexual Differentiation of Kiss1 Gene Expression in the Brain of the Rat

The Kiss1 gene codes for kisspeptins, which have been implicated in the neuroendocrine regulation of reproduction. In the brain, Kiss1 mRNA-expressing neurons are located in the arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. Kiss1 neurons in the AVPV appear to play a role in generating the preovulatory GnRH/LH surge, which occurs only in females and is organized perinatally by gonadal steroids. Because Kiss1 is involved in the sexually dimorphic GnRH/LH surge, we hypothesized that Kiss1 expression is sexually differentiated, with females having more Kiss1 neurons than either males or neonatally androgenized females. To test this, male and female rats were neonatally treated with androgen or vehicle; then, as adults, they were left intact or gonadectomized and implanted with capsules containing sex steroids or nothing. Kiss1 mRNA levels in the AVPV and ARC were determined by in situ hybridization. Normal females expressed significantly more Kiss1 mRNA in the AVPV than normal males, even under identical adult hormonal conditions. This Kiss1 sex difference was organized perinatally, as demonstrated by the observation that neonatally androgenized females displayed a male-like pattern of adulthood Kiss1 expression in the AVPV. In contrast, there was neither a sex difference nor an influence of neonatal treatment on Kiss1 expression in the ARC. Using double-labeling techniques, we determined that the sexually differentiated Kiss1 neurons in the AVPV are distinct from the sexually differentiated population of tyrosine hydroxylase (dopaminergic) neurons in this region. Our findings suggest that sex differences in kisspeptin signaling from the AVPV subserve the cellular mechanisms controlling the sexually differentiated GnRH/LH surge.

KiSS-1 Messenger Ribonucleic Acid Expression in the Hypothalamus of the Ewe Is Regulated by Sex Steroids and Season

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.

Kisspeptin Mediates the Photoperiodic Control of Reproduction in Hamsters

The KiSS-1 gene encodes kisspeptin, the endogenous ligand of the G-protein-coupled receptor GPR54. Recent data indicate that the KiSS-1/GPR54 system is critical for the regulation of reproduction and is required for puberty onset. In seasonal breeders, reproduction is tightly controlled by photoperiod (i.e., day length). The Syrian hamster is a seasonal model in which reproductive activity is promoted by long summer days (LD) and inhibited by short winter days (SD). Using in situ hybridization and immunohistochemistry, we show that KiSS-1 is expressed in the arcuate nucleus of LD hamsters. Importantly, the KiSS-1 mRNA level was lower in SD animals but not in SD-refractory animals, which spontaneously reactivated their sexual activity after several months in SD. These changes of expression are not secondary to the photoperiodic variations of gonadal steroids. In contrast, melatonin appears to be necessary for these seasonal changes because pineal-gland ablation prevented the SD-induced downregulation of KiSS-1 expression. Remarkably, a chronic administration of kisspeptin-10 restored the testicular activity of SD hamsters despite persisting photoinhibitory conditions. Overall, these findings are consistent with a role of KiSS-1/GPR54 in the seasonal control of reproduction. We propose that photoperiod, via melatonin, modulates KiSS-1 signaling to drive the reproductive axis.

Minireview: Kisspeptin Neurons as Central Processors in the Regulation of Gonadotropin-Releasing Hormone Secretion

The Kiss1 gene encodes a family of peptides called kisspeptins, which bind to the G protein-coupled receptor GPR54. Kisspeptin(s) and its receptor are expressed in the forebrain, and the discovery that mice and humans lacking a functional GPR54 fail to undergo puberty and exhibit hypogonadotropic hypogonadism implies that kisspeptin signaling plays an essential role in reproduction. Studies in several mammalian species have shown that kisspeptins stimulate the secretion of gonadotropins from the pituitary by stimulating the release of GnRH from the forebrain after the activation of GPR54, which is expressed by GnRH neurons. Kisspeptin is expressed abundantly in the arcuate nucleus (Arc) and the anteroventral periventricular nucleus (AVPV) of the forebrain. Both estradiol and testosterone regulate the expression of the Kiss1 gene in the Arc and AVPV; however, the response of the Kiss1 gene to these steroids is exactly opposite between these two nuclei. Estradiol and testosterone down-regulate Kiss1 mRNA in the Arc and up-regulate its expression in the AVPV. Thus, kisspeptin neurons in the Arc may participate in the negative feedback regulation of gonadotropin secretion, whereas kisspeptin neurons in the AVPV may contribute to generating the preovulatory gonadotropin surge in the female. Hypothalamic levels of Kiss1 and GPR54 mRNA increase dramatically at puberty, suggesting that kisspeptin signaling could mediate the neuroendocrine events that trigger the onset of puberty. Together, these observations demonstrate that kisspeptin-GPR54 signaling in the brain serves as an important conduit for controlling GnRH secretion in the developing and adult animal.

A role for kisspeptins in the regulation of gonadotropin secretion in the mouse.

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.