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Kisspeptin modulates GnRH secretion in mammals and peripheral administration of 10-amino acid fragment of kisspeptin (Kp10) induces LH release and ovulation in cattle. Experiments were done to determine if iv administration of kisspeptin will activate GnRH neurons (i.e., after crossing the blood-brain barrier) and if pre-treatment with a GnRH receptor blocker will alter kisspeptin-induced LH release (from gonadotrophs) and ovulation. In Experiment 1, cows (n = 3 per group) were given human-Kisspeptin10 (hKp10; 3 x 15 mg iv at 60-min intervals) or normal saline and euthanized 150 min after treatment was initiated. Every 20th free-floating section (50 μm thickness) from the preoptic area to hypothalamus was double immunostained to colocalize GnRH- (DAB) and activated neurons (cFOS; Nickel-DAB). Kisspeptin induced plasma LH release from 15 to 150 min (P = 0.01) but the proportion of activated GnRH neurons did not differ between groups (5.8% and 3.5%, respectively; P = 0.11). Immunogold electron microscopy detected close contacts between kisspeptin fibers and GnRH terminals in the median eminence. In Experiment 2, pubertal heifers (n = 5 per group) were treated with 1) hKp10 iv, 2) Cetrorelix (GnRH antagonist; im) + hKp10 iv or 3) saline on Day 6 of the follicular wave under low-progesterone condition. A rise in plasma LH concentration was detected from 15 to 240 min in the hKp10 group but not in cetrorelix or control group (P<0.001). Ovulations were detected only in the hKp10 group (4/5; P = 0.02). Cetrorelix treatment was associated with regression of the preovulatory dominant follicle and emergence of a new follicular wave 3.4±0.75 days after the treatment in all five heifers. Results support the hypothesis that the effect of peripheral kisspeptin is mediated downstream of GnRH synthesis and does not involve GnRH-independent LH release from gonadotrophs. Peripheral kisspeptin may release pre-synthesized GnRH from the nerve terminals in areas outside the blood-brain barrier., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Leonardi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In temperate regions goat's reproduction is seasonal. To obtain year-round breeding, hormonal treatments are currently applied. These treatments usually combine a progesterone analog with the pregnant mare serum gonadotropin (PMSG). However, their use has significant ethical and environmental drawbacks. Therefore, alternative methods to manage reproduction are needed. The discovery that in mammals the neuropeptide kisspeptin is a major positive regulator of hypothalamo-pituitary gonadal axis offered an attractive alternative strategy to control reproduction. We have previously designed a kisspeptin analog, called C6, which offers pharmacological advantages over endogenous kisspeptin. These include a longer lasting effect and enhanced activity following intramuscular injection. In the present work, we evaluated C6 effect on LH and FSH plasma concentrations in the Alpine goat breed and tested whether C6 could replace PMSG to trigger ovulation. An intramuscular injection of C6 (15 nmol/doe) given 24 hours after the end of progestogen treatment induced a surge-like peak of both LH and FSH. This was followed by an increase of progesterone, a hallmark of ovulation induction and corpus luteus formation. These results were obtained at three different time of the year: during the breeding season, the non-breeding season and at the onset of the breeding season. Furthermore, we compared the efficacy of C6 and PMSG to induce fertile ovulations when these treatments are given at the onset of the breeding season and are followed by artificial insemination. The results of this first attempt were extremely promising with gestation rates of 45% and 64% for C6 and PMSG respectively. Pending optimization of the treatment procedure in order to improve efficacy, kisspeptin analogs could be the long sought-after alternative to PMSG., Competing Interests: The authors have declared that no competing interests exist.

Kisspeptin is a peptide encoded by the Kiss 1 gene and is also called metastin. Previous studies have generally focused on several functions of this peptide, including metastasis, puberty, vasoconstriction and reproduction. However, few studies have focused on the cardiac functions of kisspeptin. In the present study, cardiac histomorphology was observed via TEM (transmission electron microscope) and HE and Masson staining to observe instinctive changes. Serum metabolites levels were also measured and analyzed using GC/TOF-MS after injection with kisspeptin-10. A gene chip was employed to screen the potential genes and pathways in the myocardium at the transcriptional leve, while RT-PCR and Western Blot were conducted to verify the relevant mRNA and protein expression, respectively. Histopathological findings demonstrated that there were many irregular wavy contractions through HE staining and increased fibrosis around the heart cells through Masson staining after treatment with kisspeptin-10. Additionally, the main changes in ultrastructure, including changes in mitochondrial and broken mitochondrial cristae, could be observed with TEM after treatment with kisspeptin-10. The PCA scores plot of the serum metabolites was in the apparent partition after injection of kisspeptin-10. Twenty-six obviously changed metabolites were detected and classified as amino acids, carbohydrate metabolites, organic acids and other metabolites. Furthermore, gene chip analysis showed 1112 differentially expressed genes after treatment with kisspeptin-10, including 330 up-regulated genes and 782 down-regulated genes. These genes were enriched in several signaling pathways related to heart diseases. The RT-PCR result for ITGB8, ITGA4, ITGB7, MYL7, HIF1-α and BNP corresponded with the gene chip assay. Moreover, the upregulated genes ITGB8, ITGA4 and BNP also displayed consistent protein levels in Western Blot results. In summary, these findings suggest that kisspeptin-10 could alter the morphology and structure of myocardial cells, serum metabolite levels, and expression of genes and proteins in heart tissues. Our work determined the profound effects of kisspeptin-10 on the heart, which could further lead to the development of therapeutics related to kisspeptin-10, including antagonists and analogs.

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

Kisspeptins regulate the mammalian reproductive axis by stimulating release of gonadotrophin releasing hormone (GnRH). Different length kisspeptins (KP) are found of 54, 14, 13 or 10 amino-acids which share a common C-terminal 10-amino acid sequence. KP-54 and KP-10 have been widely used to stimulate the reproductive axis but data suggest that KP-54 and KP-10 are not equally effective at eliciting reproductive hormone secretion after peripheral delivery. To confirm this, we analysed the effect of systemic administration of KP-54 or KP-10 on luteinizing hormone (LH) secretion into the bloodstream of male mice. Plasma LH measurements 10 min or 2 hours after kisspeptin injection showed that KP-54 can sustain LH release far longer than KP-10, suggesting a differential mode of action of the two peptides. To investigate the mechanism for this, we evaluated the pharmacokinetics of the two peptides in vivo and their potential to cross the blood brain barrier (BBB). We found that KP-54 has a half-life of ~32 min in the bloodstream, while KP-10 has a half-life of ~4 min. To compensate for this difference in half-life, we repeated injections of KP-10 every 10 min over 1 hr but failed to reproduce the sustained rise in LH observed after a single KP-54 injection, suggesting that the failure of KP-10 to sustain LH release may not just be related to peptide clearance. We tested the ability of peripherally administered KP-54 and KP-10 to activate c-FOS in GnRH neurons behind the blood brain barrier (BBB) and found that only KP-54 could do this. These data are consistent with KP-54 being able to cross the BBB and suggest that KP10 may be less able to do so.

β-cells in the pancreatic islet respond to elevated plasma glucose by secreting insulin to maintain glucose homeostasis. In addition to glucose stimulation, insulin secretion is modulated by numerous G-protein coupled receptors (GPCRs). The GPCR ligands Kisspeptin-10 (KP) and glucagon-like peptide-1 (GLP-1) potentiate insulin secretion through Gq and Gs-coupled receptors, respectively. Despite many studies, the signaling mechanisms by which KP and GLP-1 potentiate insulin release are not thoroughly understood. We investigated the downstream signaling pathways of these ligands and their affects on cellular redox potential, intracellular calcium activity ([Ca(2+)]i), and insulin secretion from β-cells within intact murine islets. In contrast to previous studies performed on single β-cells, neither KP nor GLP-1 affect [Ca(2+)]i upon stimulation with glucose. KP significantly increases the cellular redox potential, while no effect is observed with GLP-1, suggesting that KP and GLP-1 potentiate insulin secretion through different mechanisms. Co-treatment with KP and the Gβγ-subunit inhibitor gallein inhibits insulin secretion similar to that observed with gallein alone, while co-treatment with gallein and GLP-1 does not differ from GLP-1 alone. In contrast, co-treatment with the Gβγ activator mSIRK and either KP or GLP-1 stimulates insulin release similar to mSIRK alone. Neither gallein nor mSIRK alter [Ca(2+)]i activity in the presence of KP or GLP-1. These data suggest that KP likely alters insulin secretion through a Gβγ-dependent process that stimulates glucose metabolism without altering Ca(2+) activity, while GLP-1 does so, at least partly, through a Gα-dependent pathway that is independent of both metabolism and Ca(2+).

The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A.

Context: KISS1R mutations have been reported in few patients with normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110)., Objective: To describe in detail nCHH patients with biallelic KISS1R mutations belonging to 2 unrelated families, and to functionally characterize a novel KISS1R mutation., Results: An original mutant, p.Tyr313His, was found in the homozygous state in 3 affected kindred (2 females and 1 male) from a consanguineous Portuguese family. This mutation, located in the seventh transmembrane domain, affects a highly conserved amino acid, perturbs the conformation of the transmembrane segment, and impairs MAP kinase signaling and intracellular calcium release. In the second family, a French Caucasian male patient with nCHH was found to carry two recurrent mutations in the compound heterozygous state (p.Leu102Pro/Stop399Arg). In this man, pulsatile GnRH (Gonadotropin Releasing Hormone) administration restored pulsatile LH (Luteinizing Hormone) secretion and testicular hormone secretion. Later, long-term combined gonadotropin therapy induced spermatogenesis, enabling 3 successive pregnancies that resulted in 2 miscarriages and the birth of a healthy boy., Conclusion: We show that a novel loss-of-function mutation (p.Tyr313His) in the KISS1R gene can cause familial nCHH, revealing the crucial role of this amino acid in KISS1R function. The observed restoration of gonadotropin secretion by exogenous GnRH administration further supports, in humans, the hypothalamic origin of the gonadotropin deficiency in this genetic form of nCHH.

Kisspeptin (product of the kiss1 gene) is the most potent known activator of the hypothalamo-pituitary-gonadal axis. Both kiss1 and the kisspeptin receptor are highly expressed in the hypothalamus of vertebrates, and low doses of kisspeptin have a robust and long-lasting stimulatory effect on the rate of action potential firing of hypophysiotropic gonadotropin releasing hormone-1 (GnRH1) neurons in mice. Fish have multiple populations of GnRH neurons distinguished by their location in the brain and the GnRH gene that they express. GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb are neuromodulatory and do not play a direct role in regulating pituitary-gonadal function. In medaka fish, the electrical activity of TN-GnRH3 neurons is modulated by visual cues from conspecifics, and is thought to act as a transmitter of information from the external environment to the central nervous system. TN-GnRH3 neurons also play a role in sexual motivation and arousal states, making them an important population of neurons to study for understanding coordination of complex behaviors. We investigated the role of kisspeptin in regulating electrical activity of TN-GnRH3 neurons in adult medaka. Using electrophysiology in an intact brain preparation, we show that a relatively brief treatment with 100 nM of kisspeptin had a long-lasting stimulatory effect on the electrical activity of an extrahypothalamic population of GnRH neurons. Dose-response analysis suggests a relatively narrow activational range of this neuropeptide. Further, blocking action potential firing with tetrodotoxin and blocking synaptic transmission with a low Ca(2+)/high Mg(2+) solution inhibited the stimulatory action of kisspeptin on electrical activity, indicating that kisspeptin is acting indirectly through synaptic regulation to excite TN-GnRH3 neurons. Our findings provide a new perspective on kisspeptin's broader functions within the central nervous system, through its regulation of an extrahypothalamic population of GnRH neurons involved in multiple neuromodulatory functions.

Splice products of the Kiss1 protein (kisspeptins) have been shown to be involved in a diverse range of functions, including puberty, metastasis and vasoconstriction in large human arteries. Circulating Kisspeptin-10 (Kp-10) plasma levels are low in normal individuals but are elevated during various disease states as well as pregnancy. Here, we investigated the potential of Kp-10, the shortest biologically active kisspeptin, to influence microvascular effects, concentrating on the cutaneous vasculature. Kp-10 caused a dose-dependent increase in oedema formation (0.3-10 nmol/injection site), assessed by Evans Blue albumin dye extravasation, in the dorsal skin of CD1 mice. Oedema formation was shown to be inhibited by the histamine H(1) receptor antagonist mepyramine. The response was characterised by a ring of pallor at the injection site in keeping with vasoconstrictor activity. Therefore, changes in dorsal skin blood flow were assessed by clearance of intradermally injected (99m)technetium. Kp-10 was found to significantly reduce clearance, in keeping with decreased blood flow and providing further evidence for vasoconstrictor activity. The decreased clearance was partially inhibited by co-treatment with the cyclo-oxygenase inhibitor indomethacin. Finally evidence for the kisspeptin receptor gene (Kiss1R), but not the kisspeptin peptide gene (Kiss1), mRNA expression was observed in heart, aorta and kidney samples from normal and angiotensin II induced hypertensive mice, with similar mRNA levels observed in each. We have evidence for two peripheral vasoactive roles for kisspeptin-10. Firstly, plasma extravasation indicative of ability to induce oedema formation and secondly decreased peripheral blood flow, indicating microvascular constriction. Thus Kp-10 has vasoactive properties in the peripheral microvasculature.

Kisspeptins, the ligands of the kisspeptin receptor known for its roles in reproduction and cancer, are also vasoconstrictor peptides in atherosclerosis-prone human aorta and coronary artery. The aim of this study was to further investigate the cardiovascular localisation and function of the kisspeptins and their receptor in human compared to rat and mouse heart. Immunohistochemistry and radioligand binding techniques were employed to investigate kisspeptin receptor localisation, density and pharmacological characteristics in cardiac tissues from all three species. Radioimmunoassay was used to detect kisspeptin peptide levels in human normal heart and to identify any pathological changes in myocardium from patients transplanted for cardiomyopathy or ischaemic heart disease. The cardiac function of kisspeptin receptor was studied in isolated human, rat and mouse paced atria, with a role for the receptor confirmed using mice with targeted disruption of Kiss1r. The data demonstrated that kisspeptin receptor-like immunoreactivity localised to endothelial and smooth muscle cells of intramyocardial blood vessels and to myocytes in human and rodent tissue. [(125)I]KP-14 bound saturably, with subnanomolar affinity to human and rodent myocardium (K(D) = 0.12 nM, human; K(D) = 0.44 nM, rat). Positive inotropic effects of kisspeptin were observed in rat, human and mouse. No response was observed in mice with targeted disruption of Kiss1r. In human heart a decrease in cardiac kisspeptin level was detected in ischaemic heart disease. Kisspeptin and its receptor are expressed in the human, rat and mouse heart and kisspeptins possess potent positive inotropic activity. The cardiovascular actions of the kisspeptins may contribute to the role of these peptides in pregnancy but the consequences of receptor activation must be considered if kisspeptin receptor agonists are developed for use in the treatment of reproductive disorders or cancer.

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.