Kisspeptin Research Articles

PMON54 Exosomes Isolated from Conditioned Media of Immortalized Kisspeptin Neurons Exert Diverse Effects on Central and Peripheral in Vitro Cell Models.

Abstract Previous work in our laboratory explored the proteomic contents of exosome-like extracellular vesicles (EVs) released into the media in vitro from immortalized Kisspeptin (KP) neuronal cell lines KTaV-3 (derived from female mouse AVPV) and KTaR-1 (derived from KNDy neurons in the ARC). EVs were isolated from conditioned media via ultracentrifugation or filtration kit and validated using a NanoCyte, and LCMS-MS analysis revealed that relative abundance of exosomal cargo varied dependent upon estrogen (E2) exposure in vitro, with ∼150-170 proteins up-regulated and ∼200-220 proteins downregulated by E2 in EVs of KTaR-1 and KTaV-3 KP neurons. Since E2-regulated KP exosomal proteins included candidates implicated in the regulation of synaptic plasticity and signaling (i.e. annexins, semaphorins, connexins), we investigated the effects of exposure to purified EVs on gene expression in immortalized GnRH neurons (GT1-7 cells). Notably, EVs from 24h E2-treated KTaV-3 neurons induced increased expression of kiss1r in GT1-7 cells, in contrast to KTaV-3 neurons not exposed to E2, suggesting that AVPV KP neurons may signal an increase in KP receptivity in GnRH neurons in vivo via non-neuronal communication. Additionally, increases in expression of the synaptic scaffolding protein PSD-95 (dlg4) were seen in GT1-7 cells treated with E2-treated KTaV-3 EVs, suggesting AVPV KP neurons may use extracellular vesicles to modulate GnRH neuronal synaptic plasticity over the estrous cycle. While EVs isolated from KTaR-1 conditioned media did not alter GT1-7 kiss1r or dlg4 levels, treatment (24h) resulted in induction of selective gap junction hemichannel expression in GT1-7 cells. KTaR-1 (but not KTaV-3) EVs increased expression of Cx26 (gjb2) irrespective of E2 exposure, while induction of Cx43 (gja1) expression in GT1-7 cells was only observed following treatment with E2-deprived KTaR-1 EVs. Further, we found that KTaR-1 media and EVs can affect osteoblast function in vitro, including increases in sp7 and runx2 expression, E2-dependent modulation of wnt10b, and formation of bone matrix (evaluated by Alizarin Red assay) in cultured osteoblast lines, supporting recent studies implicating ARC KP neurons in bone remodeling. Lastly, we found significant levels of immunomodulatory pentraxins (PTX3) in KP EVs, with abundance dependent upon prior E2 exposure in KTaR-1 KP neurons, as revealed by ELISA. Together, results from these studies suggest that EVs may represent additional intercellular communication pathways utilized by Kiss-1 neurons to elicit changes in nearby neuronal populations and potentially even in the periphery, affecting inflammation and bone remodeling. Future studies will address mechanisms involved in the E2 regulation of exosomal cargo in these critical neuronal populations. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

OR17-3 Immortalized AVPV Kisspeptin Neurons Transiently Express GnRH Receptors under Specific Sex Steroid Conditions in vitro

Abstract In female animals, ovarian estradiol (E2) acts as negative feedback inhibition of GnRH secretion and positive feedback stimulation at ovulation. Both E2-regulated mechanisms work via stimulation or repression of distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While E2 clearly stimulates AVPV KP neurons to increase kiss1 expression during the preovulatory surge, mechanisms required for optimal potentiation of maximal GnRH surge release remain incompletely characterized. We used two immortalized KP cell lines demonstrating increased kiss1 expression under high E2 (KTaV-3), or kiss1 suppression under low E2 (KTaR-1), to probe GnRH receptor (GnRHR) expression levels under E2 conditions of varying doses and times of exposure. Additionally, we have begun to investigate transcriptional mechanisms underlying gnrhr expression in KP neurons in vitro. AVPV-derived KTaV-3 cells were treated with either E2 alone (5-100pM) or E2 with progesterone (4-40nM) for varying durations (4-96h), either constitutively or under modulating levels approximating changes found during the murine estrous cycle. All treatments were undertaken in serum-free (SF) media (DMEM) following a 50% serum synchronization. After total RNA isolation, cDNAs were probed with mouse gnrhr primers. Results reveal that gnrhr expression, not observed in typical growth media conditions, is induced in KTaV-3 cells to varying degrees dependent on prior E2/P4 exposure. Following shorter treatment times (12-24h), proestrus levels (100pM E2; 4nM P4) of sex steroids noticeably induced gnrhr, while exposure of KTaV-3 cells to a 96h estrous cycle revealed maximal gnrhr expression at the diestrus II→proestrus transition, with exposure cycles concluding with declining E2/rising P4 exhibiting no to minimal gnrhr expression. These results suggest that maximal KP neuronal expression of gnrhr may occur prior to the E2 peak on proestrus, potentially preparing AVPV KP neurons for GnRH surge release 12-16h later. Similar steroid conditions also increased dlx3 expression, a transcription factor required for GnRHR in pituitary, only in KTaV-3 cells. In KTaR-1 cells, gnrhr expression was observed only following E2 decreases, while dlx3 was constitutively elevated. Since cFos/JunB complex formation on the gnrhr promoter has been implicated in gonadotrope GnRHR levels, we explored how transfection of these transcription factors might affect gnrhr expression in KTaV-3 cells. Preliminary results reveal that fos/jun co-transfection stimulates gnrhr expression maximally at intermediate (DII) E2 levels, while higher E2 levels are required with fos or jun expression alone. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these results suggest KP neurons may possess the capacity to respond to intrahypothalamic GnRH only in certain estrous cycle stages, by differentially modulating GnRH receptivity in the AVPV. These findings supplement the prevailing model of feed-forward KP regulation of GnRH with potential short-loop KP-GnRH positive feedback regulation, providing an increased understanding of feedback mechanisms required for normal neuroendocrine regulation of reproduction. Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.

Intrauterine LPS inhibited arcuate Kiss1 expression, LH pulses, and ovarian function in rats.

Uterine inflammatory diseases are a major cause of infertility in humans and domestic animals. The current findings that intrauterine lipopolysaccharide is absorbed in systemic circulation and attenuates ovarian cyclic activities could provide a basis for developing novel treatments to improve fertility. Uterine inflammatory diseases are a major cause of infertility in humans and domestic animals. Circulating lipopolysaccharide (LPS), a bacterial endotoxin causing uterine inflammation, reportedly downregulates the hypothalamic-pituitary-gonadal axis to mediate ovarian dysfunction. In contrast, the mechanism whereby intrauterine LPS affects ovarian function has not been fully clarified. This study aimed to elucidate whether uterine exposure to LPS downregulates hypothalamic kisspeptin gene (Kiss1) expression, gonadotropin release, and ovarian function. Uterine inflammation was induced by intrauterine LPS administration to ovary-intact and ovariectomized female rats. As a result, plasma LPS concentrations were substantially higher in control rats until 48 h post injection, and the estrous cyclicity was disrupted with a prolonged diestrous phase. Three days post injection, the number of Graafian follicles and plasma estradiol concentration were reduced in LPS-treated rats, while numbers of Kiss1-expressing cells in the anteroventral periventricular nucleus and arcuate nucleus (ARC) were comparable in ovary-intact rats. Four days post injection, ovulation rate and plasma progesterone levels reduced significantly while gene expression of interleukin1β and tumor necrosis factor α was upregulated in the ovaries of LPS-treated rats that failed to ovulate. Furthermore, the number of Kiss1-expressing cells in the ARC and pulsatile luteinizing hormone (LH) release were significantly reduced in ovariectomized rats 24 h post injection. In conclusion, these results indicate that intrauterine LPS is absorbed in systemic circulation and attenuates ovarian function. This detrimental effect might be caused, at least partly, by the inhibition of ARC Kiss1 expression and LH pulses along with an induction of ovarian inflammatory response.

Integrative Analysis of miRNA-mRNA in Ovarian Granulosa Cells Treated with Kisspeptin in Tan Sheep

Kisspeptin is a peptide hormone encoded by the kiss-1 gene that regulates animal reproduction. Our studies revealed that kisspeptin can regulate steroid hormone production and promote cell proliferation in ovarian granulosa cells of Tan sheep, but the mechanism has not yet been fully understood. We speculated that kisspeptin might promote steroid hormone production and cell proliferation by mediating the expression of specific miRNA and mRNA in granulosa cells. Accordingly, after granulosa cells were treated with kisspeptin, the RNA of cells was extracted to construct a cDNA library, and miRNA-mRNA sequencing was performed. Results showed that 1303 expressed genes and 605 expressed miRNAs were identified. Furthermore, eight differentially expressed miRNAs were found, and their target genes were significantly enriched in progesterone synthesis/metabolism, hormone biosynthesis, ovulation cycle, and steroid metabolism regulation. Meanwhile, mRNA was significantly enriched in steroid biosynthesis, IL-17 signaling pathway, and GnRH signaling pathway. Integrative analysis of miRNA-mRNA revealed that the significantly different oar-let-7b targets eight genes, of which EGR1 (early growth response-1) might play a significant role in regulating the function of granulosa cells, and miR-10a regulates lipid metabolism and steroid hormone synthesis by targeting HNRNPD. Additionally, PPI analysis revealed genes that are not miRNA targets but crucial to other biological processes in granulosa cells, implying that kisspeptin may also indirectly regulate granulosa cell function by these pathways. The findings of this work may help understand the molecular mechanism of kisspeptin regulating steroid hormone secretion, cell proliferation, and other physiological functions in ovarian granulosa cells of Tan sheep.

Serum kisspeptin and proopiomelanocortin in cystic fibrosis: a single study

The determination of hormonal biomarkers is of increasing interest in many diseases, including cystic fibrosis (CF). Hormones that have not been estimated and described so far in CF include kisspeptin (KISS) and proopiomelanocortin (POMC), which are involved in the regulation of many processes, including appetite and fertility. Therefore, the aim of our study was to estimate the level of KISS and POMC in sera from CF patients and to determine the correlation between these hormones and clinical parameters. For this purpose, we estimated the levels of KISS and POMC in 38 CF patients and 16 healthy participants with enzyme-linked immunosorbent assay. We found significantly reduced levels of KISS and POMC in people with CF compared to healthy subjects (1.76 ± 0.46 vs. 2.27 ± 0.56 ng/mL, p < 0.05 and 6.25 ± 4.36 vs. 14.74 ± 6.24 ng/mL, p < 0.001, respectively). Furthermore, the level of both hormones was negatively correlated with age. The hormones studied did not correlate with the results of spirometry and each other. Thus, decreased KISS and POMC levels may be associated with lower body weight and delayed puberty in patients with CF.

Association of KISS1 Gene SNPs (rs12998) With Endocrine Parameters in Iraqi women Polycystic Ovary Syndrome

background: (PCOS) is among the most common types of gynecological hormonal abnormalities and metabolic problems that have an impact females throughout adolescence, with a prevalence of 6-10%.Hyperandrogenism, unreliable menstrual periods, and polycystic ovaries are the hallmarks of PCOS. The (HPG) axis is controlled by kisspeptins, peptides made by the KISS1 gene.Objectives: This investigation's objective about used to be to analyze the affiliation of kiss1 Gene SNPs (rs 12998 ) with Endocrine Parameters in PCOS .Methodology: A case -control find out about enrolled one hundred sixty members of which eighty female have PCOS and eighty symbolize the manage crew who had-been interestingly healthful. Patients with PCOS had been chosen patients from our health center and the teaching hospital's gynecology and obstetrics department in Tikrit. Assessment of serum ( kisspeptin, estradiol ,LH, FSH and testosterone) used to be performed the use of ELISA approach and the maker company's provided methodology was followed to extract genomic DNA from whole blood. KISS1 Gene SNPs rs12998 T&gt;C gene polymorphisms was once detected by using (T-ARMS-PCR) method.Results: Findings indicate, the frequency of the kiss1 gene rs12998 CT and TT genotypes in PCOS-affected women increased 1.63-fold and 8.88-fold in comparison to the group of healthy women which have odds ratio (OR); 1.63 and 8.88, 95% [CI], 0.667-3.988 and 2.014-39.222; p-value 0.28 &amp; 0.01**), respectively. The levels of Kisspeptin, LH, and testosterone also differ.Conclusions: Our find out about suggests a greater awareness of Kisspeptin serum levels were observed in PCOS as in contrast with healthy as well, the frequency of homozygous allele (CC) used to be greater in patient 15 than in controls 3 whilst the frequency of heterozygous allele (CT) have been now not drastically distinct in PCOS fifty six much less than the controls sixty one The allelic frequency of SNP rs12998 C/T used to be detected extra regularly with significant affiliation in female with PCOS than in

Effects of kisspeptin analogues on the behavior of &lt;i&gt;Danio rerio&lt;/i&gt;

BACKGROUND: Previously has been shown that fish kisspeptin 1 (Kiss1) acts on the brains serotonin system to reduce anxiety-phobic reactions in Danio rerio. The kissspeptin gene (kiss1) of teleost fish is also a conservative orthologue of the kissspeptin gene (KISS1/Kiss1) of mammals.&#x0D; AIM: In this work we investigated the possible anxiolytic effect of mammalian kisspeptin analogs Kiss1 in Danio rerio in comparison with antidepressants of the serotonin-type of action.&#x0D; MATERIALS AND METHODS: A novelty test was used: the fish was first placed in a beaker with a dissolved pharmacological substance (or H2O), and then in a viewing tank for 6 min, where the trajectory of movement, the length of the path, the number of movements to the upper part of the tank, the time spent in the lower part of the tank, number and time of the freezing were automatically recorded.&#x0D; RESULTS: It is shown that, in response to the novelty of being placed in a viewing tank, fish react by moving to the bottom, increasing friezing, and reducing the number of movements to the upper half of the tank. Against the background of antidepressants clomipramine, paroxetine or trazodone (0.51 mg per 1 l of water), the fish were not only in the lower, but also in the upper part of the viewing tank. The average path length did not change significantly. The time in the lower part of the tank decreased by more than 2 times compared with the control group of animals and showed a dose-dependent effect. The number of movements to the upper part of the tank per experience increased significantly. Mammalian kisspeptin analogues Cloud Clone (USA) in a dose 0.011 mg per 1 l of water caused a similar patterns of behavior in fish in response to novelty. At the same time, the effects of kisspeptin analogs were lower than those of antidepressants. The most effective dose for the action of the studied kisspeptin analogs was 0.1 mg per 1 l of water.&#x0D; CONCLUSIONS: Thus, mammalian kisspeptin analogs Kiss1 reduce anxiety-phobic responses to novelty in Danio rerio. Data on the unidirectional effects of mammalian kisspeptin analogs and serotonin-type antidepressants support the potential role of Kiss1 in modulating serotonin-dependent behaviours in Danio rerio. The data obtained support the hypothesis that kisspeptin may be involved in the regulation of anxiety-phobic states, apparently to maintain the emotional aspects of reproductive behavior, such as sexual motivation and arousal.

The role of KiSS1 gene on the growth and migration of prostate cancer and the underlying molecular mechanisms

Metastatic prostate cancers have a high mortality rate. KiSS1 was originally identified as a metastasis suppressor gene in metastatic melanoma and breast cancer, but its role in prostate cancer has been contradictory. This study was therefore undertaken to investigate the effects of KiSS1 overexpression on the growth and migration of human metastatic prostate cancer cells. We first tested the effect of KiSS1 overexpression on the growth and migration of DU145 human metastatic prostate cancer cells in vitro. DU145 cells were infected with the culture medium of 293T cells, which produce lentivirus particles containing KiSS1. A 2.5-fold increase in proliferation of KiSS1-overexpressing cancer cells was observed, and these cells formed tumor spheroids about 3 times larger than the vector control group. qPCR and immunoblotting revealed the association between increased cell growth and regulation of the PI3K/Akt and cell cycle genes, and also that increases in β-catenin and CD133 contribute to tumor aggregation. KiSS1 overexpression resulted in upregulation of the β-arrestin1/2 and Raf-MEK-ERK-NF-κB pathways via KiSS1R. Moreover, the migration and invasion of KiSS1-overexpressing cells were determined to be faster than the control group, along with 1.6-fold increased metastatic colonization of the KiSS1-overexpressing cancer cells. These were associated to the regulation of EMT gene expressions, such as E-cadherin and N-cadherin, and the upregulation of MMP9. In a xenograft mouse model inoculated with DU145 cells infected GFP or KiSS1 via a lentiviral vector, KiSS1 statistically significantly increased the tumor growth, with upregulation of PCNA and Ki-67 in the tumor tissues. In addition, KiSS1 increased the angiogenic capacity by upregulating VEGF-A and CD31, both in vitro and in vivo. Taken together, our results indicate that KiSS1 not only induces prostate cancer proliferation, but also promotes metastasis by increasing the migration, invasion, and angiogenesis of malignant cells.

Abstract 104: Kisspeptin Signaling And Pregnancy Outcomes Are Ameliorated In The Preeclamptic-like BPH/5 Mouse After Synchronization Of Sex Steroid Hormones

Kisspeptins (KP), encoded by Kiss1 , are reported to play a role in early-onset preeclampsia (ePE), a life-threatening hypertensive disorder of pregnancy. KP is higher in term ePE placentae than in normal pregnancies. However, the role of KP in PE has not been confirmed. The Blood Pressure High Subline 5 (BPH/5) mouse model spontaneously develops the main features of ePE, including impaired placental expansion and maternal hypertension. We have shown that Kiss1 is higher at the BPH/5 maternal-fetal interface on embryonic days (e) 4.5 and 7.5. BPH/5 females have abnormal circulating sex steroid hormones (SSH) during early gestation (e2.5), with lower estradiol-17ß (E2) and higher progesterone (P4). Since E2 and P4 may modulate uterine Kiss1 , we aimed to investigate the effects of artificial synchronization of SSH (AS-SSH) in BPH/5 uteroplacental KP signaling and placental development. We hypothesized that AS-SSH would prevent BPH/5 uteroplacental Kiss1 upregulation and normalize KP downstream expression of tissue inhibitor of metalloproteinase (TIMP) 2 and placental defects. AS-SSH was performed after ovariectomy of pregnant BPH/5 and C57 females (n = 8/strain) at e2.5 with one dose of E2 (25 ng/mouse, subcutaneously), and daily subcutaneous injections of P4 (1ng/mouse) until embryonic implantation sites (eIS) were collected 3 days post-surgery (n = 8/group). Contrary to natural (Nat) BPH/5 pregnancies, eIS Kiss1 and TIMP2 expression was not different between AS-SSH BPH/5 and C57 (p &gt; 0.05). Nat and AS-SSH pregnant mice were anesthetized at e12.5 to assess umbilical cord blood flow via ultrasound and uteroplacental units were harvested for placental morphometry (n = 4-6/group). Placental expansion into the maternal decidua was measured and expressed as the ratio of the placenta depth in relation to the placenta + decidua. Nat BPH/5 had lower placental expansion and umbilical blood flow than Nat C57, whereas AS-SSH BPH/5 had higher placental expansion and umbilical blood flow than Nat BPH/5 (p &lt; 0.05). In conclusion, synchronization of the E2 and P4 profile in the BPH/5 mouse early gestation mitigated Kiss1 and TIMP2 upregulation at the maternal-fetal interface and ameliorated the placental defects previously reported in this model.

A Novel SNPs of KISS1 Gene Strongly Associated with Litter Size in Indonesian Goat Breeds

Kisspeptin is a protein encoded by the KISS1 gene which behaves as a key role by stimulating gonadotropin releasing hormone (GnRH) neuron activity directly in the reproductive axis. The objective of the present study was to determine the genetic diversity within intron 1 of KISS1 gene and to verify their association with fecundity traits which can be devoted as a marker assisted selection (MAS) for breeding selection. This study was established on three Indonesian native goat populations (Kacang, Kejobong, and Senduro). The PCR products were then sequenced in both directions. The DNA sequencing alignment resulted fifteen variants (one indel and fourteen SNPs), with SNP1, SNP2, SNP3, and SNP9 being novel single nucleotide polymorphisms (SNPs) of KISS1 gene intron 1. Genotype, haplotype, and parity were significantly associated with litter size. SNP8, SNP9, and SNP10 were correlated with overall means of litter size (LS) and LS at the first and the third parities (p<0.05). Additionally, novel SNP9 were in remarkably strong linkage disequilibrium with SNP8 and SNP10 (D’=1.00; r≥0.58; χ2≥13.38), and allele A had putative binding sites for the NFIC which plays an important role in activating the expression of KISS1 gene. The H2 haplotype (CATAGCGCAACGCT) was found to have the highest litter size (p<0.0001). CC genotype at SNP8, AA genotype at novel SNP9, GA genotype at SNP10, and H2 haplotype were the excellent genotypes and haplotype that associated with the superior LS (p<0.05). Therefore, this result led to presume that these three SNPs and H2 haplotype can be acknowledged as prominent genetic markers for goat- breeding selection.

Expression of Kisspeptin in the Adult Hamster Testis.

Kisspeptins, products of KISS1 gene, are ligands of the G-protein coupled receptor (GPR54), and the kisspeptin-GPR54 signaling has an important role as an upstream regulator of gonadotropin releasing hormone (GnRH) neurons. Interestingly, extrahypothalamic expressions of kisspeptin/GPR-54 in gonads have been found in primates and experimental rodents such as rats and mice. Hamsters, another potent experimental rodent, also have a kisspeptin-GPR54 system in their ovaries. The presence of testicular kisspeptin-GPR54 system, however, remains to be solved. The present study was undertaken to determine whether the kisspeptin is expressed in hamster testis. To do this, reverse transcription-polymerase chain reactions (RT-PCRs) and immunohistochemistry (IHC) were employed. After the nest PCR, two cDNA products (320 and 280 bp, respectively) were detected by 3% agarose gel electrophoresis, and sequencing analysis revealed that the 320 bp product was correctly amplified from hamster kisspeptin cDNA. Modest immunoreactive (IR) kisspeptins were detected in Leydig-interstitial cells, and the weak signals were detected in germ cells, mostly in round spermatids and residual bodies of elongated spermatids. In the present study, we found the kisspeptin expression in the testis of Syrian hamster. Further studies on the local role(s) of testicular kisspeptin are expected for a better understanding the physiology of hamster testis, including photoperiodic gonadal regression specifically occurred in hamster gonads.

Association of KISS1 Gene SNPs (Rs35431622) with Metabolic Parameters in Iraqi Women Polycystic Ovary Syndrome

Background: Polycystic ovarian syndrome (PCOS) is among the most common types of gynecological hormonal abnormalities and metabolic problems that have an impact females throughout adolescence , with a prevalence of 6-10%.Hyperandrogenism, unreliable menstrual periods, and polycystic ovaries are the hallmarks of PCOS. Objectives: The goal of this find out about used to be to analyze the affiliation of kiss1 Gene SNPs (rs35431622) with metabolic Parameters in PCOS . Methodology: a case –control find out about enrolled one hundred sixty members of which eighty female have PCOS and eighty symbolize the manage crew who had-been interestingly healthful. Patients with PCOS had been chosen patients from our health center and the teaching hospital's gynecology and obstetrics department in Tikrit. Assessment of serum (fasting blood glucose , cholesreol .HDL and triglyceride) was performed using smart 150 full automated clinical chemistry machine .and insulin by ELISA KIT ,HOMA-IR By calculation and the maker company's provided methodology was followed to extract genomic DNA from whole blood. KISS1 Gene SNPs rs35431622 T&gt;A gene polymorphisms was once detected by using (T-ARMS-PCR) method . Results: The findings indicate, the frequency of the kiss1 gene rs35431622 AT and TT genotypes in PCOS-affected women increased 3.524-fold and 9.633- in comparison to the group of healthy women which have odds ratio; 3.524and 9.633, 95% confidence interval [CI], 1.622-7.657 and 2.916-31.816; P-value ≤0.01**and ≤0.01**) respectively. and The levels of FBS ,Insulin ,HOMA-IR, cholesterol, HDL and triglyceride showed significant difference among PCOS against control group (P≤ 0.0001) . Conclusions: Our find out about suggests a greater awareness of serum (FBS ,Insulin , cholesterol, HDL and triglyceride) seemed in PCOS companies as in contrast with control and also significant difference in the value of HOMA-IR as well, Cases and controls had statistically different frequencies of the identified polymorphisms, and Control 34 had a higher frequency of the homozygous allele (CC) than Cases 12 ., while the frequency of heterozygous allele (AT) were significantly different in cases 51 higher than the controls 41 as well the frequency of mutant allele (tt) were significantly different in cases 17 higher than the controls 5 .The allelic frequency of SNP rs35431622.C/T used.to,be detected extra regularly with significant affiliation infemale with PCOS than in control. Keywords: PCOS, kiss1,(IR)

Structure and functions of spexin as a new neuroendocrine signal

Spexin (SPX ) is a recently discovered endogenous peptide consisting of 14 amino acids. It was found that SPX, kisspeptin (KISS), and galanin (GAL) peptides belong to the same gene family and are also endogenous ligands of GAL2 and GAL3 receptors. The amino acid sequence of the SPX peptide is relatively conserved in vertebrates and invertebrates. The mRNA and protein of SPX are highly expressed both in peripheral organs and in the peripheral/central nervous system of mammals, birds, and fishes. Many biological roles of SPX has been found in non- mammal/mammals, including food intake, energy metabolism, reproduction, nociception, gastrointestinal motility, stress, and endocrine functions. This review collectively mentions the peptide structure of SPX, its receptors and distribution in tissues, and the biological activities of SPX on various organs.

Estrogen differentially regulates transcriptional landscapes of preoptic and arcuate kisspeptin neuron populations

Kisspeptin neurons residing in the rostral periventricular area of the third ventricle (KPRP3V) and the arcuate nucleus (KPARC) mediate positive and negative estrogen feedback, respectively. Here, we aim to compare transcriptional responses of KPRP3V and KPARC neurons to estrogen. Transgenic mice were ovariectomized and supplemented with either 17β-estradiol (E2) or vehicle. Fluorescently tagged KPRP3V neurons collected by laser-capture microdissection were subjected to RNA-seq. Bioinformatics identified 222 E2-dependent genes. Four genes encoding neuropeptide precursors (Nmb, Kiss1, Nts, Penk) were robustly, and Cartpt was subsignificantly upregulated, suggesting putative contribution of multiple neuropeptides to estrogen feedback mechanisms. Using overrepresentation analysis, the most affected KEGG pathways were neuroactive ligand-receptor interaction and dopaminergic synapse. Next, we re-analyzed our previously obtained KPARC neuron RNA-seq data from the same animals using identical bioinformatic criteria. The identified 1583 E2-induced changes included suppression of many neuropeptide precursors, granins, protein processing enzymes, and other genes related to the secretory pathway. In addition to distinct regulatory responses, KPRP3V and KPARC neurons exhibited sixty-two common changes in genes encoding three hormone receptors (Ghsr, Pgr, Npr2), GAD-65 (Gad2), calmodulin and its regulator (Calm1, Pcp4), among others. Thirty-four oppositely regulated genes (Kiss1, Vgf, Chrna7, Tmem35a) were also identified. The strikingly different transcriptional responses in the two neuron populations prompted us to explore the transcriptional mechanism further. We identified ten E2-dependent transcription factors in KPRP3V and seventy in KPARC neurons. While none of the ten transcription factors interacted with estrogen receptor-α, eight of the seventy did. We propose that an intricate, multi-layered transcriptional mechanism exists in KPARC neurons and a less complex one in KPRP3V neurons. These results shed new light on the complexity of estrogen-dependent regulatory mechanisms acting in the two functionally distinct kisspeptin neuron populations and implicate additional neuropeptides and mechanisms in estrogen feedback.

The role of Kisspeptin signaling in Oocyte maturation.

Kisspeptins (KPs) secreted from the hypothalamic KP neurons act on KP receptors (KPRs) in gonadotropin (GPN) releasing hormone (GnRH) neurons to produce GnRH. GnRH acts on pituitary gonadotrophs to induce secretion of GPNs, namely follicle stimulating hormone (FSH) and luteinizing hormone (LH), which are essential for ovarian follicle development, oocyte maturation and ovulation. Thus, hypothalamic KPs regulate oocyte maturation indirectly through GPNs. KPs and KPRs are also expressed in the ovarian follicles across species. Recent studies demonstrated that intraovarian KPs also act directly on the KPRs expressed in oocytes to promote oocyte maturation and ovulation. In this review article, we have summarized published reports on the role of hypothalamic and ovarian KP-signaling in oocyte maturation. Gonadal steroid hormones regulate KP secretion from hypothalamic KP neurons, which in turn induces GPN secretion from the hypothalamic-pituitary (HP) axis. On the other hand, GPNs secreted from the HP axis act on the granulosa cells (GCs) and upregulate the expression of ovarian KPs. While KPs are expressed predominantly in the GCs, the KPRs are in the oocytes. Expression of KPs in the ovaries increases with the progression of the estrous cycle and peaks during the preovulatory GPN surge. Intrafollicular KP levels in the ovaries rise with the advancement of developmental stages. Moreover, loss of KPRs in oocytes in mice leads to failure of oocyte maturation and ovulation similar to that of premature ovarian insufficiency (POI). These findings suggest that GC-derived KPs may act on the KPRs in oocytes during their preovulatory maturation. In addition to the intraovarian role of KP-signaling in oocyte maturation, in vivo, a direct role of KP has been identified during in vitro maturation of sheep, porcine, and rat oocytes. KP-stimulation of rat oocytes, in vitro, resulted in Ca2+ release and activation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1 and 2. In vitro treatment of rat or porcine oocytes with KPs upregulated messenger RNA levels of the factors that favor oocyte maturation. In clinical trials, human KP-54 has also been administered successfully to patients undergoing assisted reproductive technologies (ARTs) for increasing oocyte maturation. Exogenous KPs can induce GPN secretion from hypothalamus; however, the possibility of direct KP action on the oocytes cannot be excluded. Understanding the direct in vivo and in vitro roles of KP-signaling in oocyte maturation will help in developing novel KP-based ARTs.

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 statement What 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.

How does apelin affect LH levels? An investigation at the level of GnRH and KNDy neurons

Hypothalamic control of reproduction relies on GnRH and kisspeptin (KP) secretions. KP neurons are sensitive to sex steroids and metabolic status and their distribution overlaps with neurons producing apelin, a metabolic hormone known to decrease LH secretion in rats. Here, we observed neuroanatomical contacts between apelin fibers and both KP and GnRH neurons in the hypothalamus of male rodents. Intracerebroventricular apelin infusion for 2 weeks in male mice did not decrease LH levels nor did it affect gene expression for KP, neurokinin B and dynorphin. Finally, increasing apelin concentrations did not modulate Ca2+ levels of cultured GnRH neurons, while 10 μM apelin infusion on forskolin pretreated GnRH neurons revoked a rhythmic activity in 18% of GnRH neurons. These results suggest that acute apelin effect on LH secretion does not involve modulation of gene expression in KP neurons but may affect the secretory activity of GnRH neurons.

Regulation of the kiss2 promoter in yellowtail clownfish (Amphiprion clarkii) by cortisol via GRE-dependent GR pathway.

Kisspeptin plays a vital role in mediating the stress-induced reproductive regulation. Cortisol, known as a stress-related hormone, is involved in gonadal development and sexual differentiation by binding with glucocorticoid receptor (GR) to regulate the expression of kiss gene. In the present study, cortisol treatment in yellowtail clownfish (Amphiprion clarkii) showed that the expression of kiss (kiss1 and kiss2) and gr (gr1 and gr2) genes were increased significantly. We demonstrated that the yellowtail clownfish Kiss neurons co-express the glucocorticoid receptors in the telencephalon, mesencephalon, cerebellum, and hypothalamus. We further cloned the promoter of kiss2 gene in yellowtail clownfish and identified the presence of putative binding sites for glucocorticoid receptors, estrogen receptors, androgen receptors, progesterone receptors, AP1, and C/EBP. Applying transient transfection in HEK293T cells of the yellowtail clownfish kiss2 promoter, cortisol (dexamethasone) treatment was shown to enhance the promoter activities of the yellowtail clownfish kiss2 gene in the presence of GRs. Deletion analysis of kiss2 promoter indicated that cortisol-induced promoter activities were located between position −660 and −433 with GR1, and −912 and −775 with GR2, respectively. Finally, point mutation studies on the kiss2 promoter showed that cortisol-stimulated promoter activity was mediated by one GRE site located at position −573 in the presence of GR1 and by each GRE site located at position −883, −860, −851, and −843 in the presence of GR2. Results of the present study provide novel evidence that cortisol could regulate the transcription of kiss2 gene in the yellowtail clownfish via GRE-dependent GR pathway.

Associations of the KiSS-1 and GPR54 genetic polymorphism with polycystic ovary syndrome in Yunnan, China

Bsckground Polycystic ovary syndrome (PCOS) is a heterogeneous condition that increases the risk for serious health complications among women. Thus, in this study, we aimed to identify polymorphisms in the KiSS-1 and GPR54 genes and investigate the associations between KiSS-1, BMI, FSH, LH, and estradiol in Chinese women with PCOS. Methods This study recruited women with PCOS (n = 60) and controls (n = 60). We collected blood samples and measured endocrine indicators (kisspeptin, FSH, LH–FSH ratio, estradiol, prolactin, and testosterone). PCR and Snapshots were performed to screen single nucleotide polymorphisms (SNPs) of the KiSS-1 and GPR54 genes. Results Compared with the controls, endocrine and metabolic disturbances in women with PCOS were reflected in significantly higher levels of BMI, LH, LH–FSH ratio, prolactin, and testosterone. Serum kisspeptin levels were significantly higher and positively correlated with LH and T levels (p < 0.05). No identified SNPs of KiSS-1 and GPR54 showed significant differences in frequency between patients and controls. The mutant G allele was highly predisposed to polymorphism in the PCOS GPR54 gene r10407968. We observed a significant difference in estradiol (p = 0.023, p = 0.012) after comparing the values of LH, FSH, estradiol, progesterone, and testosterone among the three genotypes of rs4889 and rs5780218. The KiSS-1 rs4889 CC genotype and rs5780218 AA genotype showed significantly higher estradiol levels compared with the other two genotypes. Conclusions These results suggest that kisspeptin plays a potential role in the etiology of PCOS.

Increased serum levels of KiSS1-derived peptides in non-small cell lung cancer patient liquid biopsies and biological relevance

BackgroundThe secreted products of the metastasis suppressor gene KiSS1 may represent useful biomarkers in non-small cell lung cancer (NSCLC) but their levels in patients have remained poorly investigated. We previously found that forced expression of KiSS1 decreased the invasive capability of NSCLC drug-resistant cells and a pro-apoptotic role for KiSS1 has been proposed in head and neck cancer. Thus, we designed a translational investigation including a pilot study to analyze KiSS1 levels in liquid biopsies, and in vitro experiments to explore the biological relevance of KiSS1 modulation.MethodsKiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays.Results We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0–4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models, KiSS1 mRNA levels were increased by short term exposure to azacytidine, enhanced KiSS1 release was induced by the combination of azacytidine and cisplatin and KiSS1-derived peptides enhanced cisplatin-induced apoptosis. KiSS1 increase was observed upon exposure neurons-enriched cultures to tumor cell conditioned medium.ConclusionsOur results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.