GnRH-driven FTO-mediated RNA m6A modification promotes gonadotropin synthesis and secretion.

BACKGROUND: Gonadotropin precisely controls mammalian reproductive activities. Systematic analysis of the mechanisms by which epigenetic modifications regulate the synthesis and secretion of gonadotropin can be useful for more precise regulation of the animal reproductive process. Previous studies have identified many differential m6A modifications in the GnRH-treated adenohypophysis. However, the molecular mechanism by which m6A modification regulates gonadotropin synthesis and secretion remains unclear. RESULTS: Herein, it was found that GnRH can promote gonadotropin synthesis and secretion by promoting the expression of FTO. Highly expressed FTO binds to Foxp2 mRNA in the nucleus, exerting a demethylation function and reducing m6A modification. After Foxp2 mRNA exits the nucleus, the lack of m6A modification prevents YTHDF3 from binding to it, resulting in increased stability and upregulation of Foxp2 mRNA expression, which activates the cAMP/PKA signaling pathway to promote gonadotropin synthesis and secretion. CONCLUSIONS: Overall, the study reveals the molecular mechanism of GnRH regulating the gonadotropin synthesis and secretion through FTO-mediated m6A modification. The results of this study allow systematic interpretation of the regulatory mechanism of gonadotropin synthesis and secretion in the pituitary at the epigenetic level and provide a theoretical basis for the application of reproductive hormones in the regulation of animal artificial reproduction.

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Central precocious puberty (CPP) is a developmental disorder caused by early activation of the hypothalamic-pituitary-gonadal axis. The incidence of CPP is rapidly increasing, but the underlying mechanisms are not fully understood. Previous studies have shown that gain-of-function mutations in the KISS1R and KISS1 genes and loss-of-function mutations in the MKRN3, LIN28, and DLK1 genes may lead to early initiation of pubertal development. Recent research has also revealed the significant role of epigenetic factors such as DNA methylation and microRNAs in the regulation of gonadotropin-releasing hormone neurons, as well as the modulating effect of gene networks involving multiple variant genes on pubertal initiation. This review summarizes the genetic etiology and pathogenic mechanisms underlying CPP.

The Involvement of the microRNAs miR-466c and miR-340 in the Palmitate-Mediated Dysregulation of Gonadotropin-Releasing Hormone Gene Expression.

Diets high in saturated fatty acids are associated with obesity and infertility. Palmitate, the most prevalent circulating saturated fatty acid, is sensed by hypothalamic neurons, contributing to homeostatic dysregulation. Notably, palmitate elevates the mRNA levels of gonadotropin-releasing hormone (Gnrh) mRNA and its activating transcription factor, GATA binding protein 4 (Gata4). GATA4 is essential for basal Gnrh expression by binding to its enhancer region, with Oct-1 (Oct1) and CEBP-ß (Cebpb) playing regulatory roles. The pre- and post-transcriptional control of Gnrh by palmitate have not been investigated. Given the ability of palmitate to alter microRNAs (miRNAs), we hypothesized that palmitate-mediated dysregulation of Gnrh mRNA involves specific miRNAs. In the mHypoA-GnRH/GFP neurons, palmitate significantly downregulated six miRNAs (miR-125a, miR-181b, miR-340, miR-351, miR-466c and miR-503), and the repression was attenuated by co-treatment with 100 µM of oleate. Subsequent mimic transfections revealed that miR-466c significantly downregulates Gnrh, Gata4, and Chop mRNA and increases Per2, whereas miR-340 upregulates Gnrh, Gata4, Oct1, Cebpb, and Per2 mRNA. Our findings suggest that palmitate may indirectly regulate Gnrh at both the pre- and post-transcriptional levels by altering miR-466c and miR-340, which in turn regulate transcription factor expression levels. In summary, palmitate-mediated dysregulation of Gnrh and, consequently, reproductive function involves parallel transcriptional mechanisms.

Circadian patterns and photoperiodic modulation of clock gene expression and neuroendocrine hormone secretion in the marine teleost Larimichthys crocea.

The light/dark cycle, known as the photoperiod, plays a crucial role in influencing various physiological activities in fish, such as growth, feeding and reproduction. However, the underlying mechanisms of this influence are not fully understood. This study focuses on exploring the impact of different light regimes (LD: 12 h of light and 12 h of darkness; LL: 24 h of light and 0 h of darkness; DD: 0 h of light and 24 h of darkness) on the expression of clock genes (LcClocka, LcClockb, LcBmal, LcPer1, LcPer2) and the secretion of hormones (melatonin, GnRH, NPY) in the large yellow croaker, Larimichthys crocea. Real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assays were utilized to assess how photoperiod variations affect clock gene expression and hormone secretion. The results indicate that changes in photoperiod can disrupt the rhythmic patterns of clock genes, leading to phase shifts and decreased expression. Particularly under LL conditions, the pineal LcClocka, LcBmal and LcPer1 genes lose their rhythmicity, while LcClockb and LcPer2 genes exhibit phase shifts, highlighting the importance of dark phase entrainment for maintaining rhythmicity. Additionally, altered photoperiod affects the neuroendocrine system of L. crocea. In comparison to the LD condition, LL and DD treatments showed a phase delay of GnRH secretion and an acceleration of NPY synthesis. These findings provide valuable insights into the regulatory patterns of circadian rhythms in fish and may contribute to optimizing the light environment in the L. crocea farming industry.

The Adipokinetic Hormone (AKH) and the Adipokinetic Hormone/Corazonin-Related Peptide (ACP) Signalling Systems of the Yellow Fever Mosquito Aedes aegypti: Chemical Models of Binding.

Neuropeptides are the main regulators of physiological, developmental, and behavioural processes in insects. Three insect neuropeptide systems, the adipokinetic hormone (AKH), corazonin (Crz), and adipokinetic hormone/corazonin-related peptide (ACP), and their cognate receptors, are related to the vertebrate gonadotropin (GnRH) system and form the GnRH superfamily of peptides. In the current study, the two signalling systems, AKH and ACP, of the yellow fever mosquito, Aedes aegypti, were comparatively investigated with respect to ligand binding to their respective receptors. To achieve this, the solution structure of the hormones was determined by nuclear magnetic resonance distance restraint methodology. Atomic-scale models of the two G protein-coupled receptors were constructed with the help of homology modelling. Thereafter, the binding sites of the receptors were identified by blind docking of the ligands to the receptors, and models were derived for each hormone system showing how the ligands are bound to their receptors. Lastly, the two models were validated by comparing the computational results with experimentally derived data available from the literature. This mostly resulted in an acceptable agreement, proving the models to be largely correct and usable. The identification of an antagonist versus a true agonist may, however, require additional testing. The computational data also explains the exclusivity of the two systems that bind only the cognate ligand. This study forms the basis for further drug discovery studies.

The effect of gonadal hormones on the gene expression of brain-pituitary in protandrous black porgy, Acanthopagrus schlegelii.

In black porgy (Acanthopagrus schlegelii), the brain-pituitary-testis (Gnrh-Gths-Dmrt1) axis plays a vital role in male fate determination and maintenance, and then inhibiting female development in further (puberty). However, the feedback of gonadal hormones on regulating brain signaling remains unclear. In this study, we conducted short-term sex steroid treatment and surgery of gonadectomy to evaluate the feedback regulation between the gonads and the brain. The qPCR results show that male phase had the highest gths transcripts; treatment with estradiol-17ß (E2) or 17α-methyltestosterone (MT) resulted in the increased pituitary lhb transcripts. After surgery, apart from gnrh1, there is no difference in brain signaling genes between gonadectomy and sham fish. In the diencephalon/mesencephalon transcriptome, de novo assembly generated 283,528 unigenes; however, only 443 (0.16%) genes showed differentially expressed between sham and gonadectomy fish. In the present study, we found that exogenous sex steroids affect the gths transcription; this feedback control is related to the gonadal stage. Furthermore, gonadectomy may not affect gene expression of brain signaling (Gnrh-Gths axis). Our results support the communication between ovotestis and brain signaling (Gnrh-Gths-testicular Dmrt1) for the male fate.

Grid1 regulates the onset of puberty in female rats.

The study aimed to investigate the effect of Grid1, encoding the glutamate ionotropic receptor delta type subunit 1 (GluD1), on puberty onset in female rats. Grid1 mRNA and protein expression was detected in the hypothalamus of female rats at prepuberty and puberty. The levels of Grid1 mRNA in the hypothalamus, the fluorescence intensity in the arcuate nucleus and paraventricular nucleus of the prepubertal rats was significantly lower than pubertal. Additionally, the expression of Grid1 was suppressed in primary hypothalamus cells and prepubertal rat. Finally, investigated the effect of Grid1 knockdown on puberty onset and reproductive performance. Treatment of hypothalamic neurons with LV-Grid1 decreased the level of Grid1 and Rfrp-3 (encoding RFamide-related peptide 3) mRNA expression, but increased the Gnrh (encoding gonadotropin-releasing hormone) mRNA levels. After an ICV injection, the time for the rat vaginal opening occurred earlier. Moreover, Gnrh mRNA expression was increased, whereas Rfrp-3 mRNA expression was decreased in the hypothalamus. The concentration of progesterone (P4) in the serum was significantly decreased compare with control group. Ovary hematoxylin-eosin staining revealed that the LV-Grid1 group mainly contained primary and secondary follicles. The reproductive performance of the rats was not affected by the Grid1 knockdown. Therefore, Grid1 may affect the onset of puberty in female rats by regulating the levels of Gnrh, and Rfrp-3 in the hypothalamus, as well as the concentrations of P4, but not reproduction performance.

Aspartame Intake Delayed Puberty Onset in Female Offspring Rats and Girls.

SCOPE: The disturbance of the hypothalamic-pituitary-gonadal (HPG) axis, gut microbiota (GM) community, and short-chain fatty acids (SCFAs) is a triggering factor for pubertal onset. The study investigates the effects of the long-term intake of aspartame on puberty and GM in animals and humans. METHODS AND RESULTS: Aspartame-fed female offspring rats result in vaginal opening time prolongation, serum estrogen reduction, and serum luteinizing hormone elevation. , 60 mg kg-1 aspartame treatment decreases the mRNA levels of gonadotropin-releasing hormone (GnRH), Kiss1, and G protein-coupled receptor 54 (GPR54), increases the mRNA level of RFamide-related peptide-3 (RFRP-3), and decreases the expression of GnRH neurons in the hypothalamus. Significant differences in relative bacterial abundance at the genus levels and decreased fecal SCFA levels are noted by 60 mg kg-1 aspartame treatment. Among which, Escherichia-Shigella is negatively correlated with several SCFAs. In girls, high-dose aspartame consumption decreases the risk of precocious puberty. CONCLUSIONS: Aspartame reduces the chance of puberty occurring earlier than usual in female offspring and girls. Particularly, 60 mg kg-1 aspartame-fed female offspring delays pubertal onset through the dysregulation of HPG axis and GM composition by inhibiting the Kiss1/GPR54 system and inducing the RFRP-3. An acceptable dose of aspartame should be recommended during childhood.

Molecular and structural analysis of Hdh-MIRP3 and its impact on reproductive regulation in female Pacific abalone, Haliotis discus hannai.

Molluscan insulin-related peptides (MIRP) play a crucial role in various biological processes, including reproduction and larval development in mollusk species. To investigate the involvement of MIRP in the ovarian development of Pacific abalone (Haliotis discus hannai), the Hdh-MIRP3 was cloned from cerebral ganglion (CG). Hdh-MIRP3 cDNA was 993 bp long, encoded a 13.22 kDa peptide, comprising 118 amino acids. Fluorescence in situ hybridization confirmed the localization of Hdh-MIRP3 in the CG and ovary. Molecular docking revealed that Hdh-MIRP3 binds to the N-terminal region of Hdh-IRP-R. Tissue expression analysis showed the highest Hdh-MIRP3 expression in the CG, followed by ovarian tissue. Hdh-MIRP3 expression was significantly upregulated in the CG and ovary during the ripe stage of seasonal ovarian development and in effective accumulative temperature conditioned abalone. Furthermore, siRNA silencing of Hdh-MIRP3 significantly downregulated the expression of four reproduction-related genes, including Hdh-GnRH, Hdh-GnRH-R, Hdh-IRP-R, and Hdh-VTG in both the CG and ovary, and Hdh-MIRP3 as well. These results indicate that Hdh-MIRP3 acts as a regulator of ovarian development in Pacific abalone. Additionally, expression analysis indicated that Hdh-MIRP3 plays a role in embryonic and larval development. Overall, the present findings elucidate the role of Hdh-MIRP3 in reproductive development in female Pacific abalone.

Effects of dopamine and melatonin treatment on the expression of the genes associated with artificially induced sexual maturation in Japanese eel, Anguilla japonica.

Japanese eel (Anguilla japonica) is a commercially important fish species in Asia. Understanding factors like photoperiod, temperature, and lunar cycles is crucial for successful aquaculture and managing its reproduction. Melatonin and dopamine (DA) are essential for regulating reproduction in vertebrates, including fish. This study investigated the effects of melatonin and DA on the reproductive system of mature male Japanese eels to better understand reproductive regulation in fish. To clarify the effects of these hormones on sexual maturation in eels, a critical stage in the reproductive process, sexual maturation was induced by injecting human chorionic gonadotropin, which stimulates the production of sex hormones. To check the effect of melatonin and DA on sexual maturation, DA, melatonin, and DA + domperidone were intraperitoneally injected into fish from each group (six per treatment) at a dose of 1 mg/kg body weight. The fish were then examined using quantitative RT-PCR by comparing the messenger RNA level of reproduction-related genes (gonadotropin releasing hormone 1; gnrh1, gonadotropin releasing hormone 2; gnrh2, follicle stimulating hormone; fshß, luteinizing hormone; lhß and DA receptor 2b; d2b), involved in the gonadotropic axis in eels, to those that received a control injection. The results indicate significant differences in the expression levels of gnrh1, gnrh2 and d2b in the brain and d2b, fshß, lhß in the pituitary at different stages of sexual maturation. Melatonin appears to enhance the production of sex gonadotropins, whereas DA inhibits them. These findings suggest an interaction between melatonin and DA in regulating reproduction in Japanese eels.

Expression and localization of HPG axis-related genes in Carassius auratus with different ploidy.

Introduction: In the Dongting water system, the Carassius auratus (Crucian carp) complex is characterized by the coexistence of diploid forms (2n=100, 2nCC) and polyploidy forms. The diploid (2nCC) and triploid C.auratus (3n=150, 3nCC) had the same fertility levels, reaching sexual maturity at one year. Methods: The nucleotide sequence, gene expression, methylation, and immunofluorescence of the gonadotropin releasing hormone 2(Gnrh2), Gonadotropin hormone beta(Gthß), and Gonadotropin-releasing hormone receptor(Gthr) genes pivotal genes of the hypothalamic-pituitary-gonadal (HPG) axis were analyzed. Results: The analysis results indicated that Gnrh2, follicle-stimulating hormone receptor(Fshr), and Lethal hybrid rescue(Lhr) genes increased the copy number and distinct structural differentiation in 3nCC compared to that in 2nCC. The transcript levels of HPG axis genes in 3nCC were higher than 2nCC (P<0.05), which could promote the production and secretion of sex steroid hormones conducive to the gonadal development of 3nCC. Meanwhile, the DNA methylation levels in the promoter regions of the HPG axis genes were lower in 3nCC than in 2nCC. These results suggested that methylation of the promoter region had a potential regulatory effect on gene expression after triploidization. Immunofluorescence showed that the localization of the Fshß, Lhß, and Fshr genes between 3nCC and 2nCC remained unchanged, ensuring the normal expression of these genes at the corresponding sites after triploidization. Discussion: Relevant research results provide cell and molecular biology evidence for normal reproductive activities such as gonad development and gamete maturation in triploid C. auratus, and contribute to further understanding of the genetic basis for fertility restoration in triploid C. auratus.

Characterization of reproductive hormones and related gene expression in the hypothalamus and pituitary gland in the egg-laying interval in White King pigeon.

The egg-laying interval (LI) directly reflects the laying performance of breeding pigeons, influenced by reproductive hormones. This study aimed to assess reproductive hormone levels in serum and the expression of related genes and their receptors in the hypothalamus and pituitary gland in 4 stages: first (LI1), third (LI3), fifth (LI5), and seventh (LI7) days. The results showed that serum gonadotropin-releasing hormone (GnRH) level decreased from LI1 to LI7 (P < 0.01) and peaked in LI1. The serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels stayed at high levels from LI1 to LI5. The FSH level decreased slightly from LI5 to LI7 (P > 0.05), but the LH level decreased rapidly (P < 0.01). The prolactin (PRL) levels significantly increased in LI5 (P < 0.01) compared with LI1 and then stayed at a high level. The GnRH1 expression in the hypothalamus had no significant change in LI (P > 0.05). However, the GnRHR first decreased from LI1 to LI3 (P < 0.05) and then increased. The FSH mRNA level in the pituitary gland decreased from LI1 to LI3 and slightly increased in LI5 (P > 0.05). The change pattern of FSHR was similar to that of FSH and peaked in LI5 (P < 0.05). The LH expression level was the highest in LI5 and significantly higher than that in LI3 and LI7 (P < 0.05). However, the LHR mRNA level decreased in LI (P < 0.05). The expression patterns of PRL and PRLR were similar; they were upregulated in LI and peaked in LI7 (P < 0.01). The expression pattern of GnRHR was similar to that of FSH, LH, and FSHR, suggesting the critical role of GnRHR in LI. Furthermore, the expression levels of these genes peaked in LI5, closely correlating with the maturation of the first largest follicle in pigeons. PRL-PRLR signaling inhibited GnRH activity to promote ovulation. This study provided a basis for further investigating the molecular mechanisms underlying the regulation of reproduction in pigeons.

Central regulation of reproduction in amphibians.

This review article includes a literature review of synteny analysis of the amphibian gonadotropin-releasing hormone (GnRH) genes, the distribution of GnRH 1 and GnRH2 neurons in the central nervous system of amphibians, the function and regulation of hypophysiotropic GnRH1, and the function of GnRH1 in amphibian reproductive behaviors. It is generally accepted that GnRH is the key regulator of the hypothalamic-pituitary-gonadal axis. Three independent GnRH genes, GnRH1, GnRH2, and GnRH3, have been identified in vertebrates. Previous genome synteny analyses suggest that there are likely just two genes, gnrh1 and gnrh2, in amphibians. In three groups of amphibians: Anura, Urodela, and Gymnophiona, the distributions of GnRH1 and GnRH2 neurons in the central nervous system have also been previously reported. Moreover, these neuronal networks were determined to be structurally independent in all species examined. The somata of GnRH1 neurons are located in the terminal nerve, medial septum (MS), and preoptic area (POA), and some GnRH1 neurons in the MS and POA project into the median eminence. In contrast, the somata of GnRH2 neurons are located in the midbrain tegmentum. In amphibians, GnRH1 neurons originate from the embryonic olfactory placode, while GnRH2 neurons originate from the midbrain. The characterization and feedback regulation mechanisms of hypophysiotropic GnRH1 neurons in amphibians, the involvement of GnRH1 in amphibian reproductive behavior, and its possible mechanism of action should be elucidated in future.

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

OBJECTIVE: We examined how the sex steroids influence the synthesis of gonadotropins. MATERIALS AND METHODS: The effects of sex steroids estradiol (E2), progesterone (P4), and dihydrotestosterone (DHT) in pituitary gonadotroph cell model (LßT2 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. RESULTS: In LßT2 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. CONCLUSION: Our findings suggest that sex steroids have different effects in the hypothalamus and pituitary gland.

A novel lncRNA LOC105613571 binding with BDNF in pituitary promotes gonadotropin secretion by AKT/ERK-mTOR pathway in sheep associated with prolificacy.

The pituitary is a vital endocrine organ for synthesis and secretion of gonadotropic hormones (FSH and LH), and the gonadotropin showed fluctuations in animals with different fecundity. Long non-coding RNAs (lncRNAs) have been identified as regulatory factors for the reproductive process. However, the profiles of lncRNAs and their roles involved in sheep fecundity remains unclear. In this study, we performed RNA-sequencing for the sheep pituitary gland associated with different fecundity, and identified a novel candidate lncRNA LOC105613571 targeting BDNF related to gonadotropin secretion. Our results showed that expression of lncRNA LOC105613571 and BDNF could be significantly upregulated by GnRH stimulation in sheep pituitary cells in vitro. Notably, either lncRNA LOC105613571 or BDNF silencing inhibited cell proliferation while promoted cell apoptosis. Moreover, lncRNA LOC105613571 knockdown could also downregulate gonadotropin secretion via inactivation AKT, ERK and mTOR pathway. In addition, co-treatment with GnRH stimulation and lncRNA LOC105613571 or BDNF knockdown showed the opposite effect on sheep pituitary cells in vitro. In summary, BDNF-binding lncRNA LOC105613571 in sheep regulates pituitary cell proliferation and gonadotropin secretion via the AKT/ERK-mTOR pathway, providing new ideas for the molecular mechanisms of pituitary functions.

The functional study of a novel MKRN3 missense mutation associated with familial central precocious puberty.

Central precocious puberty (CPP) refers to a syndrome of early puberty initiation with a characteristic increase in the release of gonadotropin-releasing hormone (GnRH); therefore, it is also called GnRH-related precocious puberty. About a quarter of idiopathic central precocious puberty (ICPP) may be familial. Studies suggest that mutations of makorin ring finger protein 3 (MKRN3) can cause familial central precocious puberty (FCPP). In this report, we describe a Chinese female patient carrying a novel MKRN3 variant (c.980G>A/p.Arg327His) and presenting the CPP phenotype. This novel variant attenuated its own ubiquitination, degradation, and inhibition on the transcriptional and translational activity of GNRH1, which was verified through functional tests. We can consider this variant as a loss-of-function mutation, which subsides the inhibition of GnRH1-related signaling and gives rise to GnRH-related precocious puberty.

Effects of trichlorobisphenol A on the expression of proteins and genes associated with puberty initiation in GT1-7 cells and the relevant molecular mechanism.

This study evaluated the effects of Cl3BPA on kisspeptin-G-protein coupled receptor 54 (GPR54)/gonadotropin-releasing hormone (GnRH) (KGG) signals and analyzed the roles of estrogen receptor alpha (ERɑ) and G-protein coupled estrogen receptor 1 (GPER1) in regulating KGG signals. The results showed that Cl3BPA at 50 µM increased the levels of intracellular reactive oxygen species (ROS) and GnRH, upregulated the protein levels of kisspeptin and the expression of fshr, lhr and gnrh1 genes related to KGG in GT1-7 cells. In addition, 50 µM Cl3BPA significantly upregulated the phosphorylation of extracellular regulated protein kinases 1/2 (Erk1/2), the protein levels of GPER1 and the expression of the gper1 as well as the most target genes associated with mitogen-activated protein kinase (MAPK)/Erk1/2 pathways. Specific signal inhibitor experiments found that Cl3BPA activated KGG signals by activating the GPER1-mediated MAPK/Erk1/2 signaling pathway at the mRNA level. A docking test further confirmed the interactions between Cl3BPA and GPER1. The findings suggest that Cl3BPA might induce precocious puberty by increasing GnRH secretion together with KGG signaling upregulation, which is driven by GPER1-mediated signaling pathway. By comparison, ClxBPAs with fewer chlorine atoms had more obvious effects on the expression of proteins and partial genes related to KGG signals in GT1-7 cells.

Therapeutic effects of melatonin in female mice with central precocious puberty by regulating the hypothalamic Kiss-1/Kiss1R system.

In recent years, central precocious puberty (CPP) in children is becoming more common, which seriously affects their physical and psychological health and requires finding a safe and effective treatment method. The aim of this study was to investigate the therapeutic effect of melatonin on CPP. A CPP model was established by subcutaneous injection of 300 micrograms of danazol into 5-day-old female mice, followed by treatment with melatonin and leuprolide. The vaginal opening was checked daily. Mice were weighed, gonads were weighed, gonadal index was calculated, and gonadal development was observed by hematoxylin and eosin (HE) staining. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) levels were measured by ELISA. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus Kiss-1, Kiss-1 receptor (Kiss1R), gonadotropin-releasing hormone (GnRH), and pituitary GnRH receptor (GnRHR) were identified. The results showed that melatonin delayed vaginal opening time and reduced body weight, gonadal weight and indices in female CPP mice. Melatonin treatment prevents uterine wall thickening and ovarian luteinization in female CPP mice. Melatonin treatment reduces serum concentrations of FSH, LH, and E2 in female CPP mice. Melatonin suppressed the expressions of Kiss-1, Kiss1R and GnRH in the hypothalamus, and the expression of GnRHR in the pituitary of the female CPP mice. Our results suggest that melatonin can inhibit the hypothalamic-pituitary-gonadal (HPG) axis by down-regulating the Kiss-1/Kiss1R system, thereby treating CPP in female mice.

Relevance of augmented kisspeptin signaling through H364 KISS1R in central precocious puberty.

Understanding the pathophysiology of idiopathic central precocious puberty (ICPP) is essential, in view of its consequences on reproductive health and metabolic disorders in later life. Towards this, estimation of circulating levels of the neuropeptides, viz; Kisspeptin (Kp-10), Neurokinin B (NKB) and Neuropeptide Y (NPY), acting upstream to Gonadotropin-Releasing Hormone (GnRH), has shown promise. Insights can also be gained from functional studies on genetic variations implicated in ICPP. This study investigated the pathophysiology of ICPP in a girl by exploring the therapeutic relevance of the circulating levels of Kp-10, NKB, NPY and characterizing the nonsynonymous KISS1R variant, L364H, that she harbours, in a homozygous condition. Plasma levels of Kp-10, NKB and NPY before and after GnRH analog (GnRHa) treatment, were determined by ELISA. It was observed that GnRHa treatment resulted in suppression of circulating levels of Kp-10, NKB and NPY. Further, the H364 variant in KISS1R was generated by site directed mutagenesis. Post transient transfection of either L364 or H364 KISS1R variant in CHO cells, receptor expression was ascertained by western blotting, indirect immunofluorescence and flow cytometry. Kp-10 stimulated signalling response was also determined by phospho-ERK and inositol phosphate production. Structure-function studies revealed that, although the receptor expression in H364 KISS1R was comparable to L364 KISS1R, there was an enhanced signalling response through this variant at high doses of Kp-10. Thus, elevated levels of Kp-10, acting through H364 KISS1R, contributed to the manifestation of ICPP, providing further evidence that dysregulation of Kp-10/KISS1R axis impacts the onset of puberty.

TAC3 regulates GnRH/gonadotropin synthesis in female chickens.

Neurokinin B (NKB), a peptide encoded by the tachykinin 3 (TAC3), is critical for reproduction in all studied species. However, its potential roles in birds are less clear. Using the female chicken (c-) as a model, we showed that cTAC3 is composed of five exons with a full-length cDNA of 787 bp, which was predicted to generate the mature NKB peptide containing 10 amino acids. Using cell-based luciferase reporter assays, we demonstrated that cNKB could effectively and specifically activate tachykinin receptor 3 (TACR3) in HEK293 cells, suggesting its physiological function is likely achieved via activating cTACR3 signaling. Notably, cTAC3 and cTACR3 were predominantly and abundantly expressed in the hypothalamus of hens and meanwhile the mRNA expression of cTAC3 was continuously increased during development, suggesting that NKB-TACR3 may emerge as important components of the neuroendocrine reproductive axis. In support, intraperitoneal injection of cNKB could significantly promote hypothalamic cGnRH-Ι, and pituitary cFSHß and cLHß expression in female chickens. Surprisingly, cTAC3 and cTACR3 were also expressed in the pituitary gland, and cNKB treatment significantly increased cLHß and cFSHß expression in cultured primary pituitary cells, suggesting cNKB can also act directly at the pituitary level to stimulate gonadotropin synthesis. Collectively, our results reveal that cNKB functionally regulate GnRH/gonadotropin synthesis in female chickens.