Kisspeptin Research Articles

Characterization of Kiss/Kissr system and expression profiling through developmental stages indicate kiss1 to be the active isotype in Clarias magur.

Kisspeptin (Kiss) and kisspeptin receptor (Kissr) system is a key regulator of GnRH expression in several vertebrates. The Indian catfish, Clarias magur, is popular in the Indian sub-continent, and a neo-type of the Asian catfish, C. batrachus. Catfish breeding is constrained as males do not release milt captivity with/without stimulation. Magur Kiss/Kissr system comprising of kiss1, kiss2, kissr1, and kissr2 genes was characterized for the first time. Full-length mRNA was sequenced using RACE PCR. Neighbor-joining tree of predicted proteins shows one clade of teleost orthologs. Magur whole genome (NCBI GenBank) has single copies of each gene, though yet unannotated/misannotated. Anomalies in the nomenclature of earlier sequences in GenBank were noted. Relative gene expression was profiled during various ontogenic stages, in six tissues including brain and gonads at maturity, and also in brains and gonads of premature and spent fish. Expression of gnrh1, gnrhr1, and gnrhr2 was estimated concomitantly. The kiss1 was the first to be twofold upregulated (P < 0.05) at 12h post fertilization. Kiss/Kissr genes expressed primarily in the brain, ovary, and testis. Though kiss2 was 10 times higher than kiss1, only kiss1 showed significant modulation across stages and appears to be the active isotype that regulates GnRH in magur.

Effects of glycerol monolaurate on estradiol and follicle-stimulating hormones, offspring quality, and mRNA expression of reproductive-related genes of zebrafish (Danio rerio) females.

This study aims to explore whether glycerol monolaurate (GML) can improve reproductive performance of female zebrafish (Danio rerio) and the survival percentage of their offspring. Three kinds of isonitrogenous and isolipid diets, including basal diet (control) and basal diet containing 0.75g/kg GML (L_GML) and 1.5g/kg GML (H_GML), were prepared for 4weeks feeding trial. The results show that GML increased the GSI of female zebrafish. GML also enhanced reproductive performance of female zebrafish. Specifically, GML increased spawning number and hatching rate of female zebrafish. Moreover, GML significantly increased the levels of triglycerides (TG), lauric acid, and estradiol (E2) in the ovary (P < 0.05). Follicle-stimulating hormone (FSH) levels in the ovary and brain also significantly increased in the L_GML group (P < 0.05). Besides, dietary GML regulated the hypothalamus-pituitary-gonad (HPG) axis evidenced by the changed expression levels of HPG axis-related genes in the brain and ovary of the L_GML and H_GML groups compared with the control group. Furthermore, compared with the control group, the expression levels of HPG axis-related genes (kiss2, kiss1r, kiss2r, gnrh3, gnrhr1, gnrhr3, lhβ, and esr2b) in the brain of the L_GML group were significantly increased (P < 0.05), and the expression levels of HPG axis-related genes (kiss1, kiss2, kiss2r, gnrh2, gnrh3, gnrhr4, fshβ, lhβ, esr1, esr2a, and esr2b) in the brain of the H_GML group were significantly increased (P < 0.05). These results suggest that GML may stimulate the expression of gnrh2 and gnrh3 by increasing the expression level of kiss1 and kiss2 genes in the hypothalamus, thus promoting the synthesis of FSH and E2. The expression levels of genes associated with gonadotropin receptors (fshr and lhr) and gonadal steroid hormone synthesis (cyp11a1, cyp17, and cyp19a) in the ovary were also significantly upregulated by dietary GML (P < 0.05). The increasing expression level of cyp19a also may promote the FSH synthesis. Particularly, GML enhanced the richness and diversity and regulated the species composition of intestinal microbiota in female zebrafish. Changes in certain intestinal microorganisms may be related to the expression of certain genes involved in the HPG axis. In addition, L_GML and H_GML both significantly decreased larvae mortality at 96h post fertilization and their mortality during the first-feeding period (P < 0.05), revealing the enhanced the starvation tolerance of zebrafish larvae. In summary, dietary GML regulated genes related to HPG axis to promote the synthesis of E2 and FSH and altered gut microbiota in female zebrafish, and improved the survival percentage of their offspring.

Genetic variants of KISS1 gene in association with polycystic ovary syndrome– A meta-analysis

Polycystic ovary syndrome (PCOS) is a widely diagnosed syndrome associated with clinical and metabolic manifestations and even many cases of infertility. Sexual hormone imbalance can be considered as PCOS causal factor. Thus, kisspeptin (KISS1) regulating gonadotropin-releasing hormone (GnRH) levels plays a critical role in this syndrome. This study aimed to investigate the association of KISS1 genetic polymorphisms with PCOS using meta-analysis protocols. In accordance with PRISMA guidelines, systematic search of scientific databases was conducted to find included articles and then meta-analysis was performed using Review Manager 5.4 in dominant, recessive, and allelic models of inheritance. This meta-analysis included 2165 PCOS patients and 2301 healthy reproductive-aged female genotyped for five different polymorphisms of KISS1. In recessive and allelic models, three genetic variations specifically showed significant association with PCOS. This concerns rs12998 (G → A) which was proved as associated with higher risk of PCOS in recessive (p = 0.0002) and allelic models (P = 0.02). In addition, rs372790354 despite the small number of studies was associated with this syndrome in allelic model (P = 0.03). In additive model, rs4889 was shown as associated with higher PCOS risk (P = 0.02). This meta-analysis suggests rs4889, rs12998 and rs372790354 genetic variations in KISS1 as risk factors of PCOS. Results will improve our knowledge about PCOS and form a basis for future trials trying to find new markers for its prognosis and therapy.

Kisspeptin a potential therapeutic target in treatment of both metabolic and reproductive dysfunction.

Kisspeptins (KPs) are proteins that were first recognized to have antimetastatic action. Later, the critical role of this peptide in the regulation of reproduction was proved. In recent years, evidence has been accumulated supporting a role for KPs in regulating metabolic processes in a sexual dimorphic manner. It has been proposed that KPs regulate metabolism both indirectly via gonadal hormones and/or directly via the kisspeptin receptor in the brain, brown adipose tissue, and pancreas. The aim of the review is to provide both experimental and clinical evidence indicating that KPs are peptides linking metabolism and reproduction. We propose that KPs could be used as a potential target to treat both metabolic and reproductive abnormalities. Thus, we focus on the consequences of disruptions in KPs and their receptors in metabolic conditions such as diabetes, undernutrition, obesity, and reproductive disorders (hypogonadotropic hypogonadism and polycystic ovary syndrome). Data from both animal models and human subjects indicate that alterations in KPs in the case of metabolic imbalance lead also to disruptions in reproductive functions. Changes both in the hypothalamic and peripheral KP systems in animal models of the aforementioned disorders are discussed. Finally, an overview of current clinical studies involving KP in fertility and metabolism show fewer studies on metabolism (15%) and only one to date on both. Presented data indicate a dynamic and emerging field of KP studies as possible therapeutic targets in treatments of both reproductive and metabolic dysfunctions.

Expression Patterns of Leptin, Leptin Receptor, Kiss1, and HOTAIR Genes in Blood and Semen of Infertile Males with Oligospermia

Expression Patterns of Leptin, Leptin Receptor, Kiss1, and HOTAIR Genes in Blood and Semen of Infertile Males with Oligospermia

Polymorphism rs5780218, rs12998 and rs10158616 in KISS1 gene among the Hubei province Chinese girls with the central precocious puberty.

Central precocious puberty (CPP) results from early activation of the hypothalamic-pituitary-gonadal (HPG) axis. To elucidate the molecular genetic basis of CPP, we here investigated the effects of polymorphism rs5780218, rs12998 and rs10158616 in KISS1 gene on CPP susceptibility. The three KISS1 gene polymorphisms were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Sanger sequencing in 422 healthy Hubei Chinese girls and 384 Hubei Chinese girls with CPP. Single-locus analysis demonstrated that rs5780218 and rs12998 were significantly associated with CPP susceptibility in Hubei Chinese girls. Haplotype analysis exhibited that the AGG carrying the risk allele A of rs5780218 and the -GG carrying the protective allele - of rs5780218 were associated with increased and decreased CPP susceptibility in Hubei Chinese girls, respectively. The following meta-analysis confirmed the contribution of rs5780218 and rs12998 on CPP susceptibility in Chinese girls. rs5780218 and rs12998 in the KISS1 gene may participate in genetic susceptibility to CPP in Chinese girls, and the KISS1 gene rs5780218 may serve as a genetic biomarker of CPP. However, the present findings should be validated in future studies with larger sample sizes in other ethnic populations.

Current Insights in Prolactin Signaling and Ovulatory Function.

Prolactin (PRL) is a pleiotropic hormone released from lactotrophic cells of the anterior pituitary gland that also originates from extrapituitary sources and plays an important role in regulating lactation in mammals, as well as other actions. Acting in an endocrine and paracrine/autocrine manner, PRL regulates the hypothalamic-pituitary-ovarian axis, thus influencing the maturation of ovarian follicles and ovulation. This review provides a detailed discussion of the current knowledge on the role of PRL in the context of ovulation and ovulatory disorders, particularly with regard to hyperprolactinemia, which is one of the most common causes of infertility in women. Much attention has been given to the PRL structure and the PRL receptor (PRLR), as well as the diverse functions of PRLR signaling under normal and pathological conditions. The hormonal regulation of the menstrual cycle in connection with folliculogenesis and ovulation, as well as the current classifications of ovulation disorders, are also described. Finally, the state of knowledge regarding the importance of TIDA (tuberoinfundibular dopamine), KNDγ (kisspeptin/neurokinin B/dynorphin), and GnRH (gonadotropin-releasing hormone) neurons in PRL- and kisspeptin (KP)-dependent regulation of the hypothalamic-pituitary-gonadal (HPG) axis in women is reviewed. Based on this review, a rationale for influencing PRL signaling pathways in therapeutic activities accompanying ovulation disorders is presented.

Kisspeptin Regulates Cell Invasion and Migration in Endometrial Cancer.

Kisspeptin (a product of the KISS1 gene and its receptor) plays an important role in obstetrics, gynecology, and cancer cell metastasis and behavior. In hypothalamic-pituitary-gonadal axis and placentation, Kisspeptin/Kisspeptin receptor affects hormone release and represses trophoblast invasion into maternal deciduae. Endometrial cancer is one of the common gynecological cancers and is usually accompanied by metastasis, the risk factor that causes death. Recently, research has demonstrated that Kisspeptin/Kisspeptin receptor expression in aggressive-stage endometrial cancer tissues. However, the detailed mechanism of Kisspeptin/Kisspeptin receptor in regulating the motility of endometrial cancers is not well understood. In this study, we use endometrial cancer cell lines RL95-2, Ishikawa, HEC-1-A, and HEC-1-B as models to explore the molecular mechanism of Kisspeptin on cell motility. First, we discovered that Kisspeptin/Kisspeptin receptor was expressed in endometrial cancer cells, and Kisspeptin significantly regulated the migration and invasion of endometrial cancer cells. Furthermore, we explored the epithelial-mesenchymal transition marker expression and the underlying signals were regulated on Kisspeptin treatment. In conclusion, we suggest that Kisspeptin regulates endometrial cancer cell motility via FAK and Src expression and the ERK1/2, N-Cadherin, E-Cadherin, beta-Catenin, Twist, and matrix metalloproteinase signaling pathways. We expect these molecules could be candidates for the development of new approaches and therapeutic targets.

KISS1 and Kisspeptins Detection in Cell Lines.

KISS1 belongs to the family of metastasis suppressor genes. However, its role is not limited to blocking cancer metastasis. KISS1 and its by-product kisspeptins (KP) are important players in regulating the reproductive axis in different species and have new roles in controlling physiological balance and social behaviors. These diverse functions point to KISS1 as a potential therapeutic molecule. Here we describe a methodology to detect KISS1 and KP from cell lysate and conditioned media in cell lines. This will serve as a critical tool to study KISS1 processing in KP.

Enhancement of Melanocortin-4 Receptor (MC4R) and Constancy of Kiss1 mRNAs Expression in the Hypothalamic Arcuate Nucleus in a Model of Polycystic Ovary Syndrome Rat

Background: Hypothalamic Melanocortin-4 Receptor (MC4R) and kiss1/kisspeptin systems play roles in reproductive processes. This study was conducted to evaluate changes in MC4R and kiss1 genes expression in the arcuate nucleus (ARC) of the hypothalamus and its relationship with polycystic ovary syndrome (PCOS) in rats. Materials and Methods: In the current experimental study, 24 female rats were randomly and equally allocated into nulliparous and primiparous groups and then were divided into two subgroups of PCOS and control. PCOS was induced by exposure to continuous light. Sex-related hormones were evaluated by radioimmunoassay or immunoradiometric assay. Expressions of MC4R and kiss1 gene in the ARC of the hypothalamus of the rats were evaluated by real-time PCR. Histomorphometric alterations of ovaries were compared between groups. Results: Number of tertiary follicles and their size and number of atretic follicles in the PCOS subgroups were more than those in the controls (P&lt;0.05) whereas the number of secondary follicles and corpus luteum in the PCOS subgroups were lower than those in the controls (P&lt;0.05). Antrum and total diameters of tertiary follicles in the PCOS subgroups were greater and granulosa layer diameter was lower than those in the controls (P&lt;0.05). The MC4R mRNA expression in the PCOS subgroups was 6.5-fold in nulliparous and 3.5-fold in primiparous groups more than their controls’ pairs (P&lt;0.05). However, parity did not affect the expression of MC4R gene (P&gt;0.05).The kiss1 mRNA expression in the PCOS and control subgroups was not significantly different (P&gt;0.05). Conclusion: Overexpression of MC4R gene after PCOS induction in the ARC of the hypothalamus may link to metabolic disorders of induced PCOS in the rats. However, alteration in the kiss1 mRNA expression after PCOS induction was not observed in the rats. [GMJ.2018;7:e1070]

Genetic and epigenetic aspects of the KISS1 and KISS1R genes in pubertal development and central precocious puberty: A review

The onset of puberty is a pivotal developmental milestone, and the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone is a key factor in the initiation of puberty. Both kisspeptin and its receptor (KISS1) and KISS1 receptor (KISS1R) play significant roles in regulating GnRH release, and consequently, the initiation of puberty. Central precocious puberty (CPP) is a condition in which the development of puberty is driven by the premature activation of the hypothalamic-pituitary-gonadal axis. In girls, CPP is primarily idiopathic, and genetic and epigenetic aspects of &lt;i&gt;KISS1&lt;/i&gt; and &lt;i&gt;KISS1R&lt;/i&gt; have been implicated in its etiology. This review aimed to provide an overview of the current knowledge regarding mutations and polymorphisms in &lt;i&gt;KISS1&lt;/i&gt; and &lt;i&gt;KISS1R&lt;/i&gt; associated with CPP. Additionally, this study provides a comprehensive review of the epigenetic regulation of the &lt;i&gt;KISS1&lt;/i&gt; gene in the context of puberty onset and CPP.

Kisspeptin stimulates sheep ovarian follicular development in vitro through homologous receptors.

The present study was conducted to elucidate (1) the influence of kisspeptin (KP) on the in vitro development of preantral follicles (PFs) and (2) evolution of KP receptor gene (KISS1R) expression during ovarian follicular development in sheep. Kisspeptin was supplemented (0-100 µg/ml) in the culture medium of PFs for 6 days. The cumulus-oocyte complexes (COCs) from cultured PFs were subsequently matured to metaphase II (MII) for an additional 24 h. The proportions of PFs exhibiting growth, antrum formation, average increase in diameter, and maturation of oocytes to MII stage were the indicators of follicular development in vitro. The expression of the kisspeptin receptor gene at each development stages of in vivo developed (preantral, early antral, antral, large antral and COCs from Graafian follicles) and in vitro cultured PFs supplemented with KP was assessed using a real-time polymerase chain reaction. The best development in all the parameters under study was elicited with 10 µg/ml of KP. Supplementation of KP (10 µg/ml) in a medium containing other growth factors (insulin-like growth factor-I) and hormones (growth hormone, thyroxine, follicle-stimulating hormone) resulted in better PF development. The KISS1R gene was expressed in follicular cells and oocytes at all the development stages of both in vivo developed and in vitro cultured follicles. Higher KISS1R gene expression was supported by culture medium containing KP along with other hormones and growth factors. Accordingly, it is suggested that one of the mechanisms through which KP and other growth factors and hormones influence the ovarian follicular development in mammals is through the upregulation of expression of the KP receptor gene.

#363 : Kisspeptins and Human Reproduction: New Challenges in the Management of Couple Infertility

Background and Aims: Human reproduction is strictly controlled by a complex neurohormonal system including the gonadotropin-releasing hormone (GnRH) production, which stimulates anterior pituitary gonadotropes secretion and thereby gonadal function. Now, it is well known that GnRH neurons are regulated by Kisspeptins, a family of neuropeptides encoded by the Kiss1 gene and produced mainly in the arcuate and periventricular nuclei of the third ventricle in the hypothalamus. This review aims to provide an updated view in the physiological effects of kisspeptins in male and female reproduction and to highlight future research challenges in therapeutic alternatives for the management of couple infertility. Method: A non-systematic review of the literature was performed by screening PubMed up to April 2023 using the search terms including “Kisspeptin”, “gonadotropin-releasing hormone”, “male infertility”, “female infertility”, “sperm”, “oocyte”, and “Assisted Reproductive Technology”. Results: Many reports suggested the importance of kisspeptins in various aspects of male and female reproduction, from sexual differentiation and puberty onset to adult regulation of gonadotropin secretion and the metabolic control of fertility. Kisspeptin is involved in oocyte development, quality of semen, steroidogenesis in both the ovaries and testes, sexual desires and pregnancy. Recently, a potential clinical relevance of kisspeptins was noted with emerging interests in applied medicine. Some investigations are being carried out to develop these neuromodulators as a diagnostic marker in male and female infertility. Kisspeptin analogs could constitute a new therapeutic strategy based on hormonal manipulation of the hypothalamic-pituitary-gonadal axis. Conclusion: Further clinical trials are needed to provide the required evidence for the use of kisspeptin to trigger reproductive axis, gonadic functions and gametes differentiation guiding future clinical practice in couple infertility.

Kisspeptin upregulates β-cell serotonin production during pregnancy.

During pregnancy the maternal pancreatic islets of Langerhans undergo adaptive changes to compensate for gestational insulin resistance. The lactogenic hormones are well established to play a key role in regulating the islet adaptation to pregnancy, and one of the mechanisms through which they act is through upregulating β-cell serotonin production. During pregnancy islet serotonin levels are significantly elevated, where it is released from the β-cells to drive the adaptive response through paracrine and autocrine effects. We have previously shown that placental kisspeptin (KP) also plays a role in promoting the elevated insulin secretion and β-cell proliferation observed during pregnancy, although the precise mechanisms involved are unclear. In the present study we investigated the effects of KP on expression of pro-proliferative genes and serotonin biosynthesis within rodent islets. Whilst KP had limited effect on pro-proliferative gene expression at the time points tested, KP did significantly stimulate expression of the serotonin biosynthesis enzyme Tph-1. Furthermore, the islets of pregnant β-cell-specific GPR54 knockdown mice were found to contain significantly fewer serotonin-positive β-cells when compared to pregnant controls. Our previous studies suggested that reduced placental kisspeptin production, with consequent impaired kisspeptin-dependent β-cell compensation, may be a factor in the development of GDM in humans. These current data suggest that, similar to the lactogenic hormones, KP may also contribute to serotonin biosynthesis and subsequent islet signalling during pregnancy. Furthermore, upregulation of serotonin biosynthesis may represent a common mechanism through which multiple signals might influence the islet adaptation to pregnancy.

Regulatory Effects of the Kiss1 Gene in the Testis on Puberty and Reproduction in Hezuo and Landrance Boars

Regulatory Effects of the Kiss1 Gene in the Testis on Puberty and Reproduction in Hezuo and Landrance Boars

Changes in pituitary gonadotropin subunits and hypothalamic Kiss-1 gene expression by administration of sex steroids in ovary-intact female rats.

We examined how the sex steroids influence the synthesis of gonadotropins. The effects of sex steroids estradiol (E2), progesterone (P4), and dihydrotestosterone (DHT) in pituitary gonadotroph cell model (LβT2 cells) in vitro and ovary-intact rats in vivo were examined. The effects of sex steroids on Kiss1 gene expression in the hypothalamus were also examined in ovary-intact rats. In LβT2 cells, E2 increased common glycoprotein alpha (Cga) and luteinizing hormone beta (Lhb) subunit promoter activity as well as their mRNA expression. Although gonadotropin subunit promoter activity was not modulated by P4, Cga and Lhb mRNA expression was increased by P4. DHT inhibited Cga and Lhb mRNA expression with a concomitant decrease in their promoter activity. During the 2-week administration of exogenous E2 to ovary-intact rats, the estrous cycle determined by vaginal smears was disrupted. P4 or DHT administration completely eliminated the estrous cycle. Protein expression of all three gonadotropin subunits within the pituitary gland was inhibited by E2 or P4 treatment in vivo; however, DHT reduced Cga expression but did not modulate Lhb or follicle-stimulating hormone beta subunit expression. E2 administration significantly repressed Kiss1 mRNA expression in a posterior hypothalamic region that included the arcuate nucleus. P4 and DHT did not modulate Kiss1 mRNA expression in this region. In contrast, P4 administration significantly inhibited Kiss1 mRNA expression in the anterior region of the hypothalamus that included the anteroventral periventricular nucleus. The expression of gonadotropin-releasing hormone (Gnrh) mRNA in the anterior hypothalamic region, where the preoptic area is located, appeared to be decreased by treatment with E2 and P4. Our findings suggest that sex steroids have different effects in the hypothalamus and pituitary gland.

294 KISS1 Knockout Boars Have Decreased Concentrations of Gonadotropins Leading to Smaller Testes and Reduced Skatole in Backfat

Abstract In swine production, boars are castrated to prevent boar taint from testicular androgens and skatole accumulation in fat. Kisspeptin (KISS1) regulates secretion of gonadotropins and gonadal steroids. We used boars from our line of KISS1-/-pigs (10.3389/fgene.2022.1078991) to understand how loss of KISS1 function affects compounds causing boar taint. Boars were fed ad libitum during development. Body weights and testicular volume (TV) were collected at weaning, 40, 70, 100, 130, 160, and 190 d of age using TV = 4/3*π *a*b2*2 and subtracting scrotal skin thickness measured by a Harpenden skinfold caliper. Average daily gain (ADG) was calculated by subtracting the beginning from ending body weight at each age range and dividing by the number of days. At each age, a blood serum sample was collected by jugular venipuncture to quantify changes in hormones during development. Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone were quantified with radioimmunoassay and analyzed as repeated measures with genotype (KISS1+/+; KISS1+/-; KISS1-/-; n = 8 – 20 animals per genotype) and age as the fixed effects. Concentrations of androstenone and skatole in backfat (quantified using ELISA and HPLC, respectively) were log transformed and analyzed with ANOVA. Body weight did not differ among KISS1 genotypes but increased normally with age (P &amp;lt; 0.0001). During development, KISS1+/+ and KISS1+/- boars grew at similar rates. Although the ADG of KISS1-/-boars was similar to other genotypes up to 160 d of age, ADG was reduced at 190 d of age (age x genotype; P &amp;lt; 0.05). Mean concentrations of LH and FSH in KISS1+/+ and KISS1+/- boars were similar throughout development (LH, 1.16 ±0.11 and 1.34 ± 0.17 ng/mL; FSH, 0.88 ± 0.06 and 0.89 ± 0.05 ng/mL, respectively), and were greater than KISS1-/- boars (LH, 0.38 ± 0.23 ng/mL; FSH, 0.56 ± 0.07 ng/mL; P &amp;lt; 0.05). TV of KISS1+/+ and KISS1+/- boars did not differ during development and was greater than KISS1-/- boars (P &amp;lt; 0.05), for which testes could not be palpated after 100 d of age. Testosterone concentrations in KISS1+/+ and KISS1+/- increased 40-fold during development but were below detectable limits in KISS1-/- boars (age x genotype; P = 0.06). Androstenone and skatole in KISS1-/- boars (0.30 ± 1.24 µg/g and 12.6 ± 1.6 ng/g, respectively) at 190 d of age was less (P &amp;lt; 0.0001) than in KISS1+/+ and KISS1+/- boars (androstenone, 4.33 ± 1.16 µg/g; skatole, 69.2 ± 1.2 ng/g), which did not differ from one another. Thus, a single functional allele of KISS1 is sufficient to confer normal secretion of reproductive hormones, testicular growth, and skatole in boars. Moreover, knocking out the KISS1 gene will prevent boar taint, but might slightly reduce growth in later stages of development.

A comprehensive meta-analysis to identify susceptibility genetic variants for precocious puberty.

Currently, several genetic variants in ERα gene (rs2234693 and rs9340799), ERβ gene (rs1256049 and rs4986938), KISS1 gene (rs4889, rs1132506 and rs5780218), LIN28B gene (rs314263, rs314276 and rs314280), and MKRN3 gene (rs2239669) have been repeatedly explored for their contribution to precocious puberty (PP) susceptibility. However, the results remain conflicting rather than conclusive. We here performed a meta-analysis to identify the real susceptibility genetic variants for PP. After screening by inclusion criteria, 20 related studies were finally included in this meta-analysis. The odds ratios and 95% confidence intervals were calculated to assess the strength of association. Sensitive analysis, publication bias, and trial sequential analysis (TSA) were performed to evaluate the stability and reliability of results. Rs2234693, rs9340799, and rs1256049 were significantly associated with PP susceptibility (p<0.0084). Stratified analysis according to ethnicity showed that rs2234693 and rs9340799 were significantly associated with PP susceptibility in Asian and Chinese populations. Stratified analysis according to PP subtype showed that rs2234693 and rs9340799 were significantly associated with idiopathic central PP susceptibility in Asian and Chinese populations (p<0.0084). The results of publication bias, sensitivity analysis, and TSA provided solid evidence for the association between these three variants and PP susceptibility. Rs2234693 and rs9340799 in ERα gene and rs1256049 in ERβ gene may serve as susceptive factors for PP development. The present finding should be confirmed in replication studies and reinforced in functional studies, which will ultimately improve the feasibility of the application of these three PP-susceptible loci in clinical practice.

FRI282 Sexually Dimorphic Responses Of Kisspeptin Expression To Neonatal Exposure To Estrogen In Rats

Abstract Disclosure: B. Marzbanabbasabadi: None. P. Lin: None. C. Park: None. C. Ko: None. Kisspeptin neurons (Kiss1 neurons) are located mainly in anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of hypothalamus and play a key role as regulators of GnRH secretion in all mammalian species. In both females and males, ARC contains more Kiss1 neurons than AVPV. Importantly, unlike ARC, AVPV displays sexual dimorphism; female AVPV contains significantly more Kiss1 neurons than males’. Recent studies showed that neonatal exposure to estrogen decreased Kiss1 expression in the hypothalamus, impacting fertility, in both sexes. However, whether such impact was driven by decreased number of Kiss1 neurons or expression of Kiss1 gene is not known. In this study, we tested our hypothesis that neonatal estrogen exposure decreases both the number of Kiss1 neurons and the Kiss1 gene expression in AVPV, in a gender-dependent manner. This hypothesis was tested in rats by treating them with estradiol benzoate (EB, a prodrug that is converted to estradiol) and measuring the effect by counting the number of Kiss1 neurons (Kiss1 mRNA expressing cells) and comparing Kiss1 mRNA expression levels between EB-exposed and control hypothalami. Male and female rat pups were injected, on postnatal day 1, subcutaneously with 0.3 mg EB via microsphere that was designed to release EB for 2 weeks; unexposed age matching pups were used as controls (n=6-7/group). Male rats were euthanized at 2.5 months of age and their entire brains were collected. Female brains were also sampled at 2.5 months of age, but after synchronizing their estrous cycle by injecting them with PMSG and hCG following an established superovulation protocol. The brains were serially sectioned and the sections from AVPV and caudal region of ARC where Kiss1 neurons most highly populated were identified and used for localizing Kiss1 neurons and quantitating Kiss1 mRNA expression level, using RNAscope. Results showed that in the AVPV, EB-exposed rats had less Kiss1 neurons both in males (8.7±1.9 vs. 42.2±7.9 Kiss1 neurons/section in control, p=0.02) and females (26.0±5.65 vs. 141.6±15.8 Kiss1 neurons/section in control, p=0.03) and showed lower Kiss1 mRNA expression in males (0.33 folds, p=0.03) and in females (0.44 folds, p=0.04) as well. In the ARC, EB-exposed rats had less Kiss1 neurons in females (27.5±4.3 vs. 56.0±2.4/section in control, p=0.00), but had same number in males (31.7±2.3 vs. 42.8±7.6 kiss1 neurons/section in control, p=0.22). EB exposure increased Kiss1 mRNA expression in both males (2.39 folds, p=0.00) and females (1.55 folds, p=0.05). In conclusion, this study showed that neonatal exposure to estrogen alters the number of Kiss1 neurons and Kiss1 mRNA expression in AVPV in a gender-dependent manner. In addition, neonatal estrogen exposure also alters Kiss1 neuron numbers and Kiss1 gene expression in the ARC as well. Presentation: Friday, June 16, 2023

FRI273 Generation And In Vitro Characterization Of A Novel Immortalized Ovine Neuronal KNDy Cell Line

Abstract Disclosure: R. Toyoizumi: None. C.Y. Markgraf: None. A. Starman: None. E. Rodriguez: None. B.L. Lee: None. R. Bahn-Bales: None. O. Stricklin: None. C. Estill: None. C.E. Roselli: None. P.E. Chappell: None. The multiphenotypic hypothalamic KNDy neurons play a critical role in the neuroendocrine regulation of reproduction, and via their stimulation of GnRH release, are requisite for fertility and pubertal progression. In vivo evidence has accumulated identifying this more caudal population of Kisspeptin neurons in mediating predominantly negative feedback effects of gonadal steroids, and their ability to synthesize and secrete kisspeptin (KP), neurokinin B (NKB/Tac3), and dynorphin (Dyn) implicates them as crucial regulators of GnRH pulse generation. We have previously generated murine immortalized arcuate KNDy neuronal cell lines to explore molecular mechanisms underlying the steroid hormone regulation of these cells, and in the current study, we characterize a novel immortalized ovine neuronal cell line model of KNDy (oKNDy) cells derived from fetal female sheep brain (∼133 days of fetal development). Clonal lines were derived from isolated mediobasal hypothalamic explants using lentiviral infection of neurons with plasmids encoding SV40 large T-antigen. These neurons express ovine kiss1, tac3, and pdyn, as well as steroid receptors esr1, esr2, and pgr. Similar to KNDy neurons in vivo, receptors for Tac3 (tacr3) and Dyn (oprk1) were also found to be expressed in our oKNDy cells. Further, low physiological concentrations (5-50pM) of 17β-estradiol (E2) were found to significantly repress kiss1 and tac3 expression, while having no effect on tacr3. Concentrations of E2 and progesterone (P4) approximating proestrous levels maximally repressed kiss1 and tac3 after 24h, with elevations in pdyn noted under the same conditions. While cells exposed to higher E2 levels exhibited greater kiss1/tac3 repression, P4 exposure alone revealed stimulation of expression with increasing P4. Since these neurons were derived from the short-day seasonally breeding sheep, they can also be used to explore how circannual timing cues derived from the pituitary pars tuberalis may exert gating signals on the reproductive axis as previously modeled in vivo. Consistent with this, we have found high levels of thyroid hormone receptor α (thra) expression in our oKNDy neurons, suggesting they can respond to locally-synthesized T3. We are continuing to characterize these oKNDy neuronal cells in culture in order to provide insight into GnRH pulse generation and construct molecular models of neuronal responsiveness to steroids and other regulatory inputs. Presentation: Friday, June 16, 2023