Kisspeptin treatment reverses high prolactin levels and improves gonadal function in hypothyroid male rats.

We evaluated whether the administration of kisspeptin-10 (Kp10) is capable of restoring gonadal function in hypothyroid male rats. Hypothyroidism was induced with 6-propyl-2-thiouracil (PTU) for three months. In the last month, half of the hypothyroid animals were treated with Kp10. Hypothyroidism reduced testicular and sex gland mass, decreased the proliferation of the seminiferous epithelium, and compromised sperm morphology, motility, and vigor. A decrease in plasma LH and testosterone levels and an increase in prolactin secretion were observed in the hypothyroid rats. Hypothyroidism reduced Kiss1 and Kiss1r protein and gene expression and Star and Cyp11a1 mRNA levels in the testis. Furthermore, it reduced Lhb, Prl, and Drd2 and increased Tshb and Gnrhr expression in the pituitary. In the hypothalamus, hypothyroidism increased Pdyn and Kiss1r while reducing Gnrh1. Kp10 treatment in hypothyroid rats restored testicular and seminal vesicle morphology, improved sperm morphology and motility, reversed high prolactin levels, and increased LH and testosterone levels. In addition, Kp10 increased testicular expression of Kiss1, Kiss1r, Fshr, and Nr5a1 and pituitary Kiss1 expression. Our findings describe the inhibitory effects of hypothyroidism on the male gonadal axis and sperm quality and demonstrate that Kp10 treatment reverses high prolactin levels and improves gonadal function and sperm quality in hypothyroid rats.

Maternal cafeteria diet influences kisspeptin (Kiss1), kisspeptin receptor(Gpr54), and sirtuin (Sirt1) genes, hormonal and metabolic profiles, and reproductive functions in rat offspring in a sex-specific manner†.

Kisspeptin (KP, encoded by Kiss1, binding to the Gpr54 receptor) is a neuropeptide conveying information on the metabolic status to the hypothalamic-pituitary-gonadal axis. KP acts together with dynorphin A (encoded by Pdyn) and neurokinin B (encoded by Tac2) to regulate reproduction. KP is crucial for the onset of puberty and is under the control of sirtuin (encoded by Sirt1). We hypothesize that the maternal cafeteria (CAF) diet has adverse effects on the offspring's hormonal, metabolic, and reproductive functions due to sex-specific alterations in the expression of Kiss1, Gpr54, Pdyn, Tac2, and Sirt1 in the hypothalamus, and Kiss1, Gpr54, and Sirt1 in the liver. Rats were fed a CAF diet before pregnancy, during pregnancy, and during lactation. The vaginal opening was monitored. Offspring were sacrificed in three age points: PND 30, PND 35, and PND 60 (females) and PND 40, PND 45, and PND 60 (males). Their metabolic and hormonal status was assessed. mRNA for Kiss1, Gpr54, Pdyn, Tac2, and Sirt1 were measured by real-time PCR in the hypothalamus and/or livers. We found that CAF offspring had lower weight and altered body composition; increased cholesterol and triglyceride levels, sex-specific changes in glucose and insulin levels; sex-dependent changes in Sirt1/Kiss1 mRNA ratio in the hypothalamus; sex-specific alterations in Kiss1 and Sirt1 mRNA in the liver with more diversity in males; and a delayed puberty onset in females. We concluded that the mother's CAF diet leads to sex-specific alterations in metabolic and reproductive outcomes via Kiss1/Gpr54 and Sirt1 systems in offspring.

Treadmill exercise has healing effects on obesity-induced sexual behavior disorder through kisspeptin and kiss1R expression in male rats.

The basic objective of this study was to examine the possible effects of treadmill exercise on obesity-related sexual behavior disorder in obese male rats and the role of kisspeptin in this effect. The rats were separated from their mothers at the age of 3 weeks, and classified into four groups as Control (C): normal diet-sedentary group, Exercise (E): normal diet-exercise group, Obese (O): high-fat diet-sedentary group, Obese + Exercise (O+E): high-fat diet-exercise grouSexual behavioral testing was conducted in the rats. At the end of the study, brain samples were taken from the animals for gene expression analyses. The treadmill exercise caused a significant increase in the O+E Group compared to the O Group in kisspeptin and kiss1R gene expression and in EF, ML, IL, MF, IF, III, EL, PEI, IR1, MFT, IFT, IRT sexual behavior parameters (p<0.05), and a significant decrease in ML, IL, III, EL sexual behavior parameters (p<0.05). Treadmill exercise caused a significant decrease in EF, ML, IL, MF, IF, III, EL, PEI, IR1, MFT, IFT, IRT sexual behavior parameters and kisspeptin and kiss1R gene expression in the hypothalamus, hippocampus, prefrontal cortex and corpus striatum in E Group compared to C Group (p<0.05), and a significant increase in ML, IL, III, EL sexual behavior parameters (p<0.05). Based on this effect, we believe that it is caused by an increase in kisspeptin and kiss1R expression in the hypothalamus, hippocampus, prefrontal cortex and corpus striatum. In conclusion, treadmill exercise-induced kisspeptin secretion may increase GnRH secretion and cause hypothalamo-pituitary gonadal axis activation and ameliorative effect on deteriorated sexual function.

Kisspeptin regulates airway hyperresponsiveness and remodeling in a mouse model of asthma.

Asthma is a multifactorial disease of origin characterized by airway hyperresponsiveness (AHR) and airway remodeling. Several pieces of evidence from other pathologies suggest that Kisspeptins (Kp) regulate cell proliferation, migration, and invasion, mechanisms that are highly relevant to asthma. Our recent in vitro studies show Kp-10 (active peptide of Kp), via its receptor, KISS1R, inhibits human airway smooth muscle cell proliferation. Here, we hypothesize a crucial role for Kp-10 in regulating AHR and airway remodeling in vivo. Utilizing C57BL/6J mice, we assessed the effect of chronic intranasal Kp-10 exposure on mixed allergen (MA)-induced mouse model of asthma. MA-challenged mice showed significant deterioration of lung function compared to those exposed to vehicle (DPBS); Kp-10 treatment significantly improved the MA-altered lung functions. Mice treated with Kp-10 alone did not show any notable changes in lung functions. MA-exposed mice showed a significant reduction in KISS1R expression as compared to vehicle alone. MA-challenged mice showed significant alterations in immune cell infiltration in the airways and remodeling changes. Proinflammatory cytokines were significantly increased upon MA exposure, an effect abrogated by Kp-10 treatment. Furthermore, biochemical and histological studies showed Kp-10 exposure significantly reduced MA-induced smooth muscle mass and soluble collagen in the lung. Overall, our findings highlight the effect of chronic Kp-10 exposure in regulating MA-induced AHR and remodeling. © 2023 The Pathological Society of Great Britain and Ireland.

Effects of electroacupuncture on the kisspeptin-gonadotropin-releasing hormone (GnRH) /luteinizing hormone (LH) neural circuit abnormalities and androgen receptor expression of kisspeptin/neurokinin B/dynorphin neurons in PCOS rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, anovulation, and polycystic ovaries. Electroacupuncture (EA) can effectively improve hyperandrogenism and increase ovulation frequency in patients with PCOS. Pieces of suggest that androgen activity in the brain is associated with impaired steroid negative feedback in such patients. Studies have shown that EA regulated androgen receptor (AR) expression and local factor levels (such as anti-Müllerian hormone and inhibin B) in the ovary of PCOS rats. However, few studies have explored the effect of EA on androgen activity in the brain. OBJECTIVE: This study investigated the effect of EA on the kisspeptin-gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) neural circuit and sex hormone receptor expression in the hypothalamus of PCOS rats. METHODS: PCOS signs were induced by letrozole administration, and the induced rats were treated with low-frequency EA at Guan Yuan acupoint (CV4). The effect of EA on PCOS-like signs was evaluated by observing changes in the body weight, ovarian quality, ovarian morphology, and serum sex hormone levels in rats. To explore the mechanism of the effect of EA on PCOS-like signs, the neuropeptide content of the kisspeptin-GnRH/LH neural circuit was assessed using enzyme-linked immunosorbent assay(ELISA); AR and estrogen receptor α (ERα) coexpression on kisspeptin/neurokinin B/dynorphin (KNDy) neurons was determined via triple-label immunofluorescence; and protein and mRNA expression of Kiss1, Ar, Esr1, and kisspeptin receptor (Kiss1r) was evaluated via western blotting and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). RESULTS: The results revealed that the estrous cycle of rats in the EA treatment group recovered, and their body and ovary weight reduced; ovarian morphology improved; serum testosterone and LH levels significantly decreased; and kisspeptin, GnRH, and dynorphin levels in hypothalamic arcuate nucleus significantly decreased. Compared with controls, the number of AR/Kiss1-positive cells increased, number of ERα/Kiss1-positive cells decreased, and protein and mRNA expression of Kiss1, Ar, and Kiss1r significantly increased in PCOS rats. However, EA treatment reversed these changes and reduced the expression of Kiss1, Ar, and Kiss1r significantly. CONCLUSION: Improvement in the reproductive hallmarks of PCOS rats via EA may be achieved by regulating the kisspeptin-GnRH/LH circuit via androgen activity attenuation. Thus, the results provide an experimental basis for acupuncture as an adjuvant medical therapy on PCOS.

Kisspeptin and kisspeptin receptor immunoreactivity in euploid and aneuploid choriodecidual tissues of recurrent pregnancy losses.

OBJECTIVE: To study choriodecidual immunoreactivity of kisspeptin (KISS1) and its receptor (KISS1R) in recurrent pregnancy loss (RPL) due to aneuploidy (AnE) and unexplained (UE) RPL in comparison to control elective abortions (EAbs). DESIGN: This is a case-control study. SETTING: Tertiary care facility and affiliated research institute. PATIENT(S): Patients with either UE RPL (n = 10) or RPL due to AnE (n = 10) vs. a control group of patients who underwent EAb (n = 10). INTERVENTION(S): Immunohistochemistry of archived choriodecidual tissue samples. MAIN OUTCOME MEASURE(S): Histoscores of KISS1 and KISS1R immunoreactivity in the syncytiotrophoblast (SyT), cytotrophoblast (CyT), decidual glands (DeGs), and decidual stroma (DeS) across the 3 study groups. RESULT(S): There was no difference in both maternal and gestational ages among the 3 groups. Kisspeptin immunoreactivity was similar in the SyT, CyT, DeGs, and DeS of all groups. Similarly, KISS1R expression was not different in the DeGs or DeS among all study groups. In addition, there was no difference in KISS1R immunoreactivity in the SyTs and CyTs between patients with RPL due to AnE and those with UE RPL. However, KISS1R was significantly lower in the SyT and CyT of patients with RPL due to AnE and UE RPL than in those who underwent EAb. CONCLUSION(S): The expression of KISS1R is lower in the chorionic tissues of euploid (unexplained) and aneuploid RPLs than in the control group. The current results broaden our understanding of the role played by KISS1 and KISS1R in early placentation. Further investigation is necessary to determine whether KISS1 activity is the cause or a sequel of defective placentation.

Effects of benzo[a]pyrene on the reproductive axis: Impairment of kisspeptin signaling in human gonadotropin-releasing hormone primary neurons.

The neuroendocrine control of reproduction is strictly coordinated at the central level by the pulsatile release of gonadotropin-releasing hormone (GnRH) by the hypothalamic GnRH neurons. Alterations of the GnRH-network, especially during development, lead to long-term reproductive and systemic consequences, also causing infertility. Recent evidence shows that benzo[a]pyrene (BaP), a diffuse pollutant that can play a role as an endocrine disruptor, affects gonadal function and gamete maturation, whereas data demonstrating its impact at hypothalamic level are very scarce. This study investigated the effects of BaP (10 µM) in a primary cell culture isolated from the human fetal hypothalamus (hfHypo) and exhibiting a clear GnRH neuron phenotype. BaP significantly decreased gene and protein expression of both GnRH and kisspeptin receptor (KISS1R), the master regulator of GnRH neuron function. Moreover, BaP exposure increased phospho-ERK1/2 signaling, a well-known mechanism associated with KISS1R activation. Interestingly, BaP altered the electrophysiological membrane properties leading to a significant depolarizing effect and it also significantly increased GnRH release, with both effects being not affected by kisspeptin addition. In conclusion, our findings demonstrate that BaP may alter GnRH neuron phenotype and function, mainly interfering with KISS1R signaling and GnRH secretion and therefore with crucial mechanisms implicated in the central neuroendocrine control of reproduction.

Inverse Correlation of KISS1 and KISS1R Expression in Triple-negative Breast Carcinomas from African American Women.

BACKGROUND/AIM: The kisspeptin 1 (KISS1) gene encodes a precursor polypeptide which after proteolysis forms the kisspeptin-10 (KISS1) protein. KISS1, retains maximum physiological activity when it binds to its receptor (KISS1R), allowing KISS1 to effectively function as a suppressor of metastasis in melanomas and other types of cancer. The goal of this study was to evaluate the expression of KISS1 and KISS1R in breast carcinomas from African American (AA) women and correlate their association with clinicopathological features, including breast cancer subtypes, and outcomes. MATERIALS AND METHODS: Tissue microarrays were constructed from formalin-fixed, paraffin-embedded surgical blocks from 216 AA patients. KISS1 and KISS1R expression was assessed using immunohistochemistry. Univariate analysis was used to determine the association between the expression of KISS1 and KISS1R, and clinicopathological characteristics. Pearson correlation was also determined between immunohistochemical H-scores, tumor size, and the number of positive lymph nodes. Kaplan-Meier estimates of overall and disease-free survival were plotted, and log-rank tests were performed to compare estimates among groups. RESULTS: KISS1 protein expression was found to be higher in receptor-negative and triple-negative breast cancer (TNBC) compared to other subtypes (p<0.001). However, KISS1R expression was higher in non-TNBC tumors compared to other subtypes (p<0.001). Higher KISS1R expression was marginally negatively correlated with tumor size (p=0.077), and positively correlated with lymph-node positivity (p=0.056), and disease-free survival (p=0.092). CONCLUSION: Our study showed a significant inverse correlation between KISS1 and KISS1R in TNBC. This investigation implicates a role for KISS1 and KISS1R in the pathogenesis of TNBCs in AA women.

DNA hypermethylation of Kiss1r promoter and reduction of hepatic Kiss1r in female rats with type 2 diabetes.

Kisspeptin, produced from the brain and peripheral tissues, may constitute an important link in metabolic regulation in response to external cues, such as diet. The kisspeptin system is well described in the brain. However, its function and regulation in the peripheral tissues, especially in relation to metabolic disease and sex differences, remain to be elucidated. As Kiss1 and Kiss1r, encoding for kisspeptin and kisspeptin receptors, respectively, are altered by overnutrition/fasting and regulated by DNA methylation during puberty and cancer, epigenetic mechanisms in metabolic disorders are highly probable. In the present study, we experimentally induced type 2 diabetes mellitus (DM2) in female Wistar rats using high-fat diet/streptozocin. We analysed expression and DNA methylation of Kiss1 and Kiss1r in the peripheral tissues, using quantitative-reverse-transcription PCR (qRT-PCR) and pyrosequencing. We discovered differential expression of Kiss1 and Kiss1r in peripheral organs in DM2 females, as compared with healthy controls, and the profile differed from patterns reported earlier in males. DM2 in females was linked to the increased Kiss1 mRNA in the liver and increased Kiss1r mRNA in the liver and adipose tissue. However, Kiss1r promoter was hypermethylated in the liver, suggesting gene silencing. Indeed, the increase in DNA methylation of Kiss1r promoter was accompanied by a reduction in Kiss1r protein, implying epigenetic or translational gene repression. Our results deliver novel evidence for tissue-specific differences in Kiss1 and Kiss1r expression in peripheral organs in DM2 females and suggest DNA methylation as a player in regulation of the hepatic kisspeptin system in DM2.

Effects of high-fat diet and treadmill running on the hypothalamic Kiss-1-GPR54 signaling pathway in male growing rats.

BACKGROUND: Kiss-1 neuron, one of the metabolic sensors in the hypothalamus, is necessary for puberty initiation. It acts through G protein-coupled receptor, known as GPR54. In this study, the mechanism of the hypothalamic Kiss-1-GPR54 signaling pathway in a high-fat diet and exercise was investigated in growing male rats. METHODS: A total of 135 3-week-old male weaned rats were kept on a high-fat diet (HFD) and exercise (60-70% [Formula: see text], 1 h/day, 5 days/week). They were randomly divided, as follows: control group (C); normal diet + exercise group (CE); HFD group (H); and HFD + exercise group (HE). Hypothalamus, testis, and serum samples of each group were collected on postnatal day (PND) 21 (early childhood), 43 (puberty), and 56 (maturity). Immunofluorescence, quantitative real-time PCR, hematoxylin and eosin staining, and chemiluminescent immunoassays were used in the study. ANOVA was used to analyze the effects of age (PNDs 21, 43, and 56), exercise (exercise and sedentariness), and diet (high-fat and normal) on the biological indices of rats. RESULTS: mRNA and protein expression of Kiss-1 and GPR54 in the hypothalamus gradually increased along with growth and peaked at PND 43, while those in serum testosterone increased and peaked at PND 56. The high-fat diet increased the expression of the Kiss-1-GPR54 system in the hypothalamus, whereas the serum testosterone decreased during different stages of growth. Exercise decreased the expression of Kiss-1 at PND 56 and increased it at PND 43. Meanwhile, it decreased testosterone and the deposition of lipid droplets in the testis at all ages of development. CONCLUSIONS: The expression of Kiss-1-GPR54 in male rats showed fluctuating changes during growth and development. The high-fat diet was able to upregulate the expression of the Kiss-1-GPR54 system in the hypothalamus. The exercise was able to correct the adverse effect of the high-fat diet on the Kiss-1-GPR54 signaling pathway in the hypothalamus and the function of the hypothalamic-pituitary-gonadal (HPG) axis, but had age-specific effects on the male rats' development.

The Kisspeptin and Kisspeptin receptor in follicular microenvironment: is that really necessary for oocyte maturation and fertilisation?

The aim of this study was to determine whether Kisspeptin and Kisspeptin receptor in the follicular microenvironment is necessary for human oocyte maturation and fertilisation. The cumulus cell (CC) and follicle fluids (FF) obtained from the first aspirated follicles (n = 52) from 32 patients were divided into three groups considering nuclear maturation and fertilisation results of oocytes: (1) Metaphase I or germinal vesicle stage oocytes (incomplete nuclear maturation, n = 10), (2) unfertilised metaphase II oocytes (incomplete cytoplasmic maturation, n = 16), and (3) fertilised metaphase II oocytes (completed nuclear-cytoplasmic maturation, n = 26). The gene expression levels were assessed by RT-PCR. The levels of Kisspeptin (KISS1) and Kisspeptin receptor (KISS1R) were measured by ELISA. There were no significant efficacy KISS1 and KISS1R gene expressions in cumulus cells in terms of oocyte nuclear maturation stage (Group 1, vs Group 2 + Group 3) (respectively p = .49; p = .45). In terms of the cytoplasmic maturation stage (Group 2, vs Group 3); KISS1 and KISS1R expressions in CCs were comparable (respectively p = .07; p = .08). In FFs, KISS1 and KISS1R concentrations were similar between all groups (respectively p = .86; p = .26). In conclusion, the relative KISS1 and KISS1R expressions in CC and also KISS1 and KISS1R level of FF were independent of oocytes nuclear and/or cytoplasmic maturation. Impact statementWhat is already known on this subject? It has been demonstrated that Kisspeptin is an essential regulator of reproductive function and plays a key role in the modulation of GnRH secretion and gonadotropin release. Still, no information is available about the link between gene expression or concentration in the follicular microenvironment and oocyte development.What do the results of this study add? The study has shown that the relative Kisspeptin (KISS1) and Kisspeptin receptor (KISS1R) and expressions in cumulus cell (CC) and also KISS1 and KISS1R levels of follicle fluids (FF) were independent of oocytes nuclear and/or cytoplasmic maturation.What are the implications of these findings for clinical practice and/or further research? Based on the findings, it is difficult to establish a concept that kisspeptin can directly induce oocyte maturation. Nevertheless, to confirm these findings, further studies with a larger sample size are needed.

The Role of Kisspeptin in the Control of the Hypothalamic-Pituitary-Gonadal Axis and Reproduction.

The discovery of kisspeptin as a critical central regulatory factor of GnRH release has given people a novel understanding of the neuroendocrine regulation in human reproduction. Kisspeptin activates the signaling pathway by binding to its receptor kisspeptin receptor (KISS1R) to promote GnRH secretion, thereby regulating the hypothalamic-pituitary-gonadal axis (HPG) axis. Recent studies have shown that kisspeptin neurons located in arcuate nucleus (ARC) co-express neurokinin B (NKB) and dynorphin (Dyn). Such neurons are called KNDy neurons. KNDy neurons participate in the positive and negative feedback of estrogen to GnRH secretion. In addition, kisspeptin is a key factor in the initiation of puberty, and also regulates the processes of female follicle development, oocyte maturation, and ovulation through the HPG axis. In male reproduction, kisspeptin also plays an important role, getting involved in the regulation of Leydig cells, spermatogenesis, sperm functions and reproductive behaviors. Mutations in the KISS1 gene or disorders of the kisspeptin/KISS1R system may lead to clinical symptoms such as idiopathic hypogonadotropic hypogonadism (iHH), central precocious puberty (CPP) and female infertility. Understanding the influence of kisspeptin on the reproductive axis and related mechanisms will help the future application of kisspeptin in disease diagnosis and treatment. In this review, we critically appraise the role of kisspeptin in the HPG axis, including its signaling pathways, negative and positive feedback mechanisms, and its control on female and male reproduction.

Kisspeptins inhibit human airway smooth muscle proliferation.

Sex and gender disparity in asthma is recognized and suggests a modulatory role for sex steroids, particularly estrogen. However, there is a dichotomous role for estrogen in airway remodeling, making it unclear whether sex hormones are protective or detrimental in asthma and suggesting a need to explore mechanisms upstream or independent of estrogen. We hypothesize that kisspeptin (Kp)/KISS1R signaling serves this role. Airway smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. We explored the role of Kp/KISS1R in regulating ASM proliferation. We report potentially novel data indicating that Kp and KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from women compared with men and lower in patients with asthma compared with people without asthma. Proliferation studies showed that cleaved forms of Kp, particularly Kp-10, mitigated PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R increased basal ASM proliferation, which was further amplified by PDGF. The antiproliferative effect of Kp-10 in ASM was mediated by inhibition of MAPK/ERK/Akt pathways, with altered expression of PCNA, C/EBP-α, Ki-67, cyclin D1, and cyclin E leading to cell cycle arrest at G0/G1 phase. Overall, we demonstrate the importance of Kp/KISS1R signaling in regulating ASM proliferation and a potential therapeutic avenue to blunt remodeling in asthma.

Targeting hepatic kisspeptin receptor ameliorates nonalcoholic fatty liver disease in a mouse model.

Nonalcoholic fatty liver disease (NAFLD), the most common liver disease, has become a silent worldwide pandemic. The incidence of NAFLD correlates with the rise in obesity, type 2 diabetes, and metabolic syndrome. A hallmark featureof NAFLD is excessive hepatic fat accumulation or steatosis, due to dysregulated hepatic fat metabolism, which can progress to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Currently, there are no approved pharmacotherapies to treat this disease. Here, we have found that activation of the kisspeptin 1 receptor (KISS1R) signaling pathway has therapeutic effects in NAFLD. Using high-fat diet-fed mice, we demonstrated that a deletion of hepatic Kiss1r exacerbated hepatic steatosis. In contrast, enhanced stimulation of KISS1R protected against steatosis in wild-type C57BL/6J mice and decreased fibrosis using a diet-induced mouse model of NASH. Mechanistically, we found that hepatic KISS1R signaling activates the master energy regulator, AMPK, to thereby decrease lipogenesis and progression to NASH. In patients with NAFLD and in high-fat diet-fed mice, hepatic KISS1/KISS1R expression and plasma kisspeptin levels were elevated, suggesting a compensatory mechanism to reduce triglyceride synthesis. These findings establish KISS1R as a therapeutic target to treat NASH.

Evaluation of KISS1 Receptor Gene Expression in Egyptian Female Patients with Breast Cancer.

OBJECTIVE: Breast cancer is the second most common cancer in the world. Many metastasis suppressor genes were identified, including the KISS1 gene which encodes for a 145 amino acid protein (kisspeptin-145), which undergoes proteolytic cleavage resulting in kisspeptin-14, -13 and -10. All of these proteins can activate KISS1 receptor (KISS1R). The role of KP/KISS1R signaling in breast cancer remains controversial. The present study aimed to measure mRNA gene expression of KISS1 receptor in healthy and cancerous breast tissue and to evaluate the association of its level with the available molecular subtypes and the traditional clinico-pathological variables. METHODS: The study was done on 41 operable primary breast cancer patients. Biopsies from both tumor tissue and surrounding healthy mammary tissue were taken from all patients. KISS1R mRNA expression level was measured using a quantitative real time PCR.   Results: KISS1R mRNA expression was significantly higher in stage III patients compared to stage II patients. At a cut-off value for KISS1R mRNA expression of 1.75, stage II was discriminated from stage III. A significant positive correlation was found between KISS1R mRNA expression and tumor size as well as lymph nodes metastasis. KISS1R mRNA was highly expressed in ER negative cases compared to ER positive ones, and in PR negative cases compared to PR positive ones. There was a statistically significant difference in KISS1R mRNA expression levels and different molecular subtypes being over-expressed in HER2 and triple negative cancer cases. CONCLUSION: This study supports other studies suggesting that KISS1/KISS1R may not be acting as a metastasis suppressor in breast cancer. KISS1R mRNA is over expressed in advanced stages of breast cancer and hence it can be used as a prognostic marker for aggressiveness of breast cancer. Also being over expressed in triple negative patients, KISS1R could represent a promising therapeutic target in triple negative cases.

Maternal hypothyroidism reduces the expression of the kisspeptin/Kiss1r system in the maternal-fetal interface of rats.

Alterations of circulating and placental levels of kisspeptin have been associated with gestational diseases. However, there are still no studies on the placental and decidual expression of Kiss1 and its receptor Kiss1r in maternal hypothyroidism, which is the aim of this work. We demonstrate that the fetoplacental restriction caused by hypothyroidism in rats is associated with a reduction in the Kiss1r expression and reduced Kiss1 and Kiss1r mRNA levels in the decidua and/or placenta. This demonstrate that fetoplacental restriction in hypothyroid rats is linked with a suppression of the kisspeptin/Kiss1r system at the maternal-fetal interface.

Differential Expression of Kisspeptin System and Kisspeptin Receptor Trafficking during Spermatozoa Transit in the Epididymis.

The hypothalamus-pituitary-testis axis controls the production of spermatozoa, and the kisspeptin system, comprising Kiss1 and Kiss1 receptor (Kiss1R), is the main central gatekeeper. The activity of the kisspeptin system also occurs in testis and spermatozoa, but currently the need of peripheral kisspeptin to produce gametes is not fully understood. Hence, we characterized kisspeptin system in rat spermatozoa and epididymis caput and cauda and analyzed the possible presence of Kiss1 in the epididymal fluid. The presence of Kiss1 and Kiss1R in spermatozoa collected from epididymis caput and cauda was evaluated by Western blot; significant high Kiss1 levels in the caput (p < 0.001 vs. cauda) and constant levels of Kiss1R proteins were observed. Immunofluorescence analysis revealed that the localization of Kiss1R in sperm head shifts from the posterior region in the epididymis caput to perforatorium in the epididymis cauda. In spermatozoa-free epididymis, Western blot revealed higher expression of Kiss1 and Kiss1R in caput (p < 0.05 vs. cauda). Moreover, immunohistochemistry revealed that Kiss1 and Kiss1R proteins were mainly localized in the secretory epithelial cell types and in contractile myoid cells, respectively. Finally, both dot blot and Elisa revealed the presence of Kiss1 in the epididymal fluid collected from epididymis cauda and caput, indicating that rat epididymis and spermatozoa possess a complete kisspeptin system. In conclusion, we reported for the first time in rodents Kiss1R trafficking in spermatozoa during the epididymis transit and Kiss1 measure in the epididymal fluid, thus suggesting a possible role for the system in spermatozoa maturation and storage within the epididymis.

Kisspeptins and the neuroendocrine control of reproduction: Recent progress and new frontiers in kisspeptin research.

In late 2003, a major breakthrough in our understanding of the mechanisms that govern reproduction occurred with the identification of the reproductive roles of kisspeptins, encoded by the Kiss1 gene, and their receptor, Gpr54 (aka, Kiss1R). The discovery of this unsuspected reproductive facet attracted an extraordinary interest and boosted an intense research activity, in human and model species, that, in a relatively short period, established a series of basic concepts on the physiological roles of kisspeptins. Such fundamental knowledge, gathered in these early years of kisspeptin research, set the scene for the more recent in-depth dissection of the intimacies of the neuronal networks involving Kiss1 neurons, their precise mechanisms of regulation and the molecular underpinnings of the function of kisspeptins as pivotal regulators of all key aspects of reproductive function, from puberty onset to pulsatile gonadotropin secretion and the metabolic control of fertility. While no clear temporal boundaries between these two periods can be defined, in this review we will summarize the most prominent advances in kisspeptin research occurred in the last ten years, as a means to provide an up-dated view of the state of the art and potential paths of future progress in this dynamic, and ever growing domain of Neuroendocrinology.

Connecting nutritional deprivation and pubertal inhibition via GRK2-mediated repression of kisspeptin actions in GnRH neurons.

BACKGROUND: Perturbations in the timing of puberty, with potential adverse consequences in later health, are increasingly common. The underlying neurohormonal mechanisms are unfolded, but nutritional alterations are key contributors. Efforts to unveil the basis of normal puberty and its metabolic control have focused on mechanisms controlling expression of Kiss1, the gene encoding the puberty-activating neuropeptide, kisspeptin. However, other regulatory phenomena remain ill-defined. Here, we address the putative role of the G protein-coupled-receptor kinase-2, GRK2, in GnRH neurons, as modulator of pubertal timing via repression of the actions of kisspeptin, in normal maturation and conditions of nutritional deficiency. METHODS: Hypothalamic RNA and protein expression analyses were conducted in maturing female rats. Pharmacological studies involved central administration of GRK2 inhibitor, ßARK1-I, and assessment of gonadotropin responses to kisspeptin or phenotypic and hormonal markers of puberty, under normal nutrition or early subnutrition in female rats. In addition, a mouse line with selective ablation of GRK2 in GnRH neurons, aka G-GRKO, was generated, in which hormonal responses to kisspeptin and puberty onset were monitored, in normal conditions and after nutritional deprivation. RESULTS: Hypothalamic GRK2 expression increased along postnatal maturation in female rats, especially in the preoptic area, where most GnRH neurons reside, but decreased during the juvenile-to-pubertal transition. Blockade of GRK2 activity enhanced Ca+2 responses to kisspeptin in vitro, while central inhibition of GRK2 in vivo augmented gonadotropin responses to kisspeptin and advanced puberty onset. Postnatal undernutrition increased hypothalamic GRK2 expression and delayed puberty onset, the latter being partially reversed by central GRK2 inhibition. Conditional ablation of GRK2 in GnRH neurons enhanced gonadotropin responses to kisspeptin, accelerated puberty onset, and increased LH pulse frequency, while partially prevented the negative impact of subnutrition on pubertal timing and LH pulsatility in mice. CONCLUSIONS: Our data disclose a novel pathway whereby GRK2 negatively regulates kisspeptin actions in GnRH neurons, as major regulatory mechanism for tuning pubertal timing in nutritionally-compromised conditions.

GABAB Receptor Antagonism from Birth to Weaning Permanently Modifies Kiss1 Expression in the Hypothalamus and Gonads in Mice.

INTRODUCTION: The kisspeptin gene Kiss1 is expressed in two hypothalamic areas: anteroventral periventricular nucleus/periventricular nucleus (AVPV/PeN) and arcuate nucleus (ARC), and also in gonads. Several pieces of evidence suggests that gamma-amino butyric acid B receptors (GABAB) signaling can regulate Kiss1 expression. Here, we inhibited GABAB signaling from PND2 to PND21 and evaluated the hypothalamic-pituitary-gonadal (HPG) axis. METHODS: BALB/c mice were treated on postnatal days 2-21 (PND2-PND21) with CGP55845 (GABAB antagonist) and evaluated in PND21 and adulthood: gene expression (qPCR) in the hypothalamus and gonads, hormones by radioimmunoassay, gonad histochemistry (H&E), puberty onset, and estrous cycles. RESULTS: At PND21, CGP inhibited Kiss1 and Tac2 and increased Pdyn and Gabbr1 in the ARC of both sexes and decreased Th only in female AVPV/PeN. Serum follicle-stimulating hormone (FSH) and testis weight were decreased in CGP-males, and puberty onset was delayed. In adults, Kiss1, Tac2, Pdyn, Pgr, Cyp19a1, and Gad1 were downregulated, while Gabbr1 was upregulated in the ARC of both sexes. In the AVPV/PeN, Kiss1, Th, Cyp19a1, and Pgr were decreased while Gad1 was increased in CGP-females, whereas Cyp19a1 was increased in CGP-males. Serum FSH was increased in CGP-males while prolactin was increased in CGP-females. Testosterone and progesterone were increased in ovaries from CGP-females, in which Kiss1, Cyp19a1, and Esr1 were downregulated while Hsd3b2 was upregulated, together with increased atretic and decreased ovulatory follicles. Testes from CGP-males showed decreased progesterone, increased Gabbr1, Kiss1, Kiss1r, and Esr2 and decreased Cyp19a1, and clear signs of seminiferous tubules atrophy. CONCLUSION: These results demonstrate that appropriate GABAB signaling during this critical prepubertal period is necessary for the normal development of the HPG axis.