Gonadotropin-inhibitory Hormone mRNA Expression Research Articles

Variation in Hypothalamic GnIH Expression and Its Association with GnRH and Kiss1 during Pubertal Progression in Male Rhesus Monkeys (Macaca mulatta).

Modulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion across postnatal development in higher primates is not fully understood. While gonadotropin-inhibitory hormone (GnIH) is reported to suppress reproductive axis activity in birds and rodents, little is known about the developmental trajectory of GnIH expression in rhesus monkeys throughout the pubertal transition. This study was aimed at examining the variation in GnIH immunoreactivity (-ir) and associated changes among GnIH, GnRH, and Kiss1 mRNA expression in the hypothalamus of infant, juvenile, prepubertal, and adult male rhesus monkeys. The brains from rhesus macaques were collected from infancy until adulthood and were examined using immunofluorescence and RT-qPCR. The mean GnIH-ir was found to be significantly higher in prepubertal animals (p < 0.01) compared to infants, and significantly reduced in adults (p < 0.001). Significantly higher (p < 0.001) GnRH and Kiss1 mRNA expression was noted in adults while GnIH mRNA expression was the highest at the prepubertal stage (p < 0.001). Significant negative correlations were seen between GnIH-GnRH (p < 0.01) and GnIH-Kiss1 (p < 0.001) expression. Our findings suggest a role for GnIH in the prepubertal suppression of the reproductive axis, with disinhibition of the adult reproductive axis occurring through decreases in GnIH. This pattern of expression suggests that GnIH may be a viable target for the development of novel therapeutics and contraceptives for humans.

Hypothalamic-Pituitary-Thyroid Axis Crosstalk With the Hypothalamic-Pituitary-Gonadal Axis and Metabolic Regulation in the Eurasian Tree Sparrow During Mating and Non-mating Periods.

Reproduction is an energetically costly phenomenon. Therefore, to optimize reproductive success, male birds invest enough energetic resources for maintaining well-developed testes. The hypothalamic–pituitary–thyroid (HPT) axis in birds can crosstalk with the hypothalamic–pituitary–gonadal (HPG) axis, thus orchestrating both the reproduction and metabolism. However, until now, how the free-living birds timely optimize both the energy metabolism and reproduction via HPT-axis is not understood. To uncover this physiological mechanism, we investigated the relationships among body mass, testis size, plasma hormones including thyroid-stimulating hormone (TSH), thyroxine (T4), triiodothyronine (T3), metabolites including glucose (Glu), triglyceride (TG), total cholesterol (TC), uric acid (UA), diencephalon mRNA expressions of type 2 (Dio2) and 3 (Dio3) iodothyronine deiodinase enzymes, thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH), gonadotropin-releasing hormone I (GnRH-I), and gonadotropin-inhibitory hormone (GnIH) in a male Eurasian tree sparrow (ETS, Passer montanus). We found significantly larger testis size; elevated diencephalon Dio2 and TRH mRNA expressions, plasma T3, and UA levels; and significantly lowered Glu, TG, and TC levels during mating relative to the non-mating stages in male ETSs. However, Dio3, TSH, GnRH-I, and GnIH mRNA expression did not vary with the stage. Furthermore, life-history stage dependent variation in plasma T3 had both direct effects on the available energy substrates and indirect effects on body mass and testis size, indicating a complex regulation of metabolic pathways through the HPT- and HPG-axes. The identified differences and relationships in mRNA expression, plasma T3 and metabolites, and testis size in male ETSs contribute to our understanding how free-living birds adjust their molecular, endocrinal, and biochemical features to orchestrate their reproductive physiology and metabolism for the maintenance of well-developed testes.

Molecular identification of GnIH and its potential role in reproductive physiology and male pregnancy of the lined seahorse (Hippocampus erectus)

The gonadotropin-inhibitory hormone (GnIH) plays a negative role in the hypothalamic-pituitary-gonadal (HPG) axis by inhibiting gonadotropin secretion in vertebrates. Male pregnancy and ovoviviparous behavior are unique phenomena among vertebrates. To better understand the neuroendocrine regulatory mechanisms in ovoviviparous fish with male pregnancy, we identified the orthologous GnIH gene in the lined seahorse (Hippocampus erectus). The full-length cDNA of the GnIH precursor was 658 base pairs with an open reading frame of 528 base pairs that encoded a 175-amino acid prepro-GnIH peptide. The seahorse GnIH precursor contained two putative LPXRFamide peptides. Both seahorse LPXRFa-1 and LPXRFa-2 were found to be unique among vertebrates. The synteny blocks of GnIH gene loci were conserved in mammals and teleosts. Tissue distribution analysis revealed that seahorse GnIH mRNA was mainly expressed in the hypothalamus, with relatively high levels observed in the brood pouch. The expression patterns of seahorse GnIH during different reproductive stages and pregnancy stages were also detected, and GnIH mRNA expression was significantly reduced during the early puberty stage. In addition, GnIH mRNA expression was significantly increased during the pregnancy stage compared to non-pregnancy stages. In summary, our results reveal the existence of GnIH in ovoviviparous fish and suggest its involvement in regulation of reproductive behavior and male pregnancy in the male seahorse.

Molecular characterization of gonadotropin-inhibitory hormone (GnIH) gene and effect of intramuscular injection of GnIH peptide on the reproductive axis in Catla catla

Gonadotropin-inhibitory hormone (GnIH) plays an important role in reproduction by inhibiting the expression of gonadotropins in birds and mammals, but in fishes, it is ambiguous. In this study, we cloned 606 bp long cDNA of GnIH from Catla catla brain (cGnIH). The encoded preproGnIH peptide generated three putative peptides (cGnIH-I, -II, -III) of different size. Phylogenetic analysis of GnIH showed clustering of different peptide sequence with its orthologs in separate clades. The real-time PCR analysis showed the expression of cGnIH in brain, gonads, intestine, stomach, heart, gill and liver with the highest expression in the brain and gonads of both sexes. The basal GnIH mRNA expression was higher in spawning and spent phase of the male brain and spawning phase of the female brain. In testis, the expression was highest in spent phase, while in ovary the expression did not change significantly during reproductive phases. The in vivo experiment of cGnIH-III peptide exhibited the higher expression of HPG axis genes, lhb, fshb, cgnrh, kiss2 and kiss1r and serum hormone level of LH and FSH as soon as 3 h after the intramuscular delivery. These results suggest that the GnIH is positively involved in regulation of reproduction in HPG axis of C. catla.

Reduction in voluntary food intake, but not fasting, stimulates hypothalamic gonadotropin-inhibitory hormone precursor mRNA expression in chicks under heat stress

Heat stress is an issue of rising concern across the globe. Recently, we found that mRNA expression of gonadotropin-inhibitory hormone (GnIH), an orexigenic neuropeptide, was increased in the heat-exposed chick brain when food intake was reduced. The aim of the current study was to examine mRNA expression of GnIH and of the glucocorticoid receptors (GRs) in the hypothalamus as well as the plasma corticosterone (CORT) and metabolites in 14-d-old chicks exposed to a high ambient temperature (HT; 40 ± 1 °C for 1 or 5 h) or a control thermoneutral temperature (CT; 30 ± 1 °C), either with free access to food or fasted. Heat stress caused a voluntary reduction of food intake and reduced plasma triacylglycerol concentration, but increased rectal temperature and plasma CORT and glucose concentrations (P < 0.05). Heat stress also increased (P < 0.05) the expression of diencephalic GnIH mRNA in chicks when they reduced food intake voluntarily, but did not do so under fasting conditions. Although the expression of GR mRNA was not altered as a result of heat stress, its expression was decreased (P < 0.05) in fasted chicks at 5 h in comparison with fed chicks. In addition, the rectal temperature of fasted chicks was lower than that of fed chicks under both CT and HT. In conclusion, voluntary reduction of food intake caused an increase in brain GnIH mRNA expression, plasma CORT, and body temperature in chicks under heat stress. Interestingly, brain GnIH mRNA expression was not induced by heat stress in fasted chicks and was not accompanied by a decrease in rectal temperature. These results suggest that the increased expression of brain GnIH mRNA in chicks under heat stress could be a consequence of a mechanism mediated by the voluntary reduction of food intake, but that it is not a consequence of fasting.

Gonadotropin-Inhibitory Hormone, the Piscine Ortholog of LPXRFa, Participates in 17β-Estradiol Feedback in Female Goldfish Reproduction

Gonadotropin-inhibitory hormone (GnIH) plays a critical role in regulating gonadotropin-releasing hormone, gonadotropin hormone, and steroidogenesis in teleosts. In the present study, we sought to determine whether 17β-estradiol (E2) acts directly on GnIH neurons to regulate reproduction in goldfish, a seasonal breeder, and we investigated the role of estrogen receptors (ERs) in mediating this process. We found that GnIH neurons coexpress three types of ERs. Ovariectomy and letrozole implantation into female goldfish at the vitellogenic stage elicited a substantial decrease in the expression of GnIH messenger RNA (mRNA), and E2 supplementation abolished this effect. In primary cultured hypothalamus cells, E2 increased GnIH mRNA levels; surprisingly, selective ERα and ERβ agonists showed opposite effects in regulating GnIH mRNA levels. Using genome walking, we isolated a 2329-bp section of the GnIH promoter sequence, and 7 half-estrogen response elements (EREs) were found in the promoter region. Luciferase assays and electrophoretic mobility shift assay results show that the half-ERE element at -2203 is the key site for competitive binding between ERα and ERβ. Ovariectomy and letrozole implantation into female goldfish in the maturating stage did not change the GnIH mRNA expression levels. Taken together, these findings suggest that E2 binds to multiple types of ERs, which competitively bind to the same half-ERE binding site of the GnIH promoter to achieve both positive and negative feedback in response to estrogen to regulate goldfish reproduction at different stages of ovarian development.

Time-related effects of various LED light spectra on reproductive hormones in the brain of the goldfish Carassius auratus

In this study, we examined differences among the gonadotropin-inhibitory hormone (GnIH), kisspeptin 1 (Kiss1), Kiss 1 receptor (G-protein-coupled receptor 54; GPR54), melatonin receptor 1 (MT1), and melatonin levels in brain cells of goldfish Carassius auratus exposed to white fluorescent light and three light-emitting diode (LED) wavelength and melatonin treatments in the culture medium. In the green and blue LED treatment groups, GnIH and MT1 mRNA expression levels were significantly lower than in the other groups; conversely, levels significantly increased in the melatonin treatment groups. Additionally, expression levels of Kiss1 and its receptor, GPR54, in the white fluorescent and red LED light groups were significantly lower than the other groups, but levels also significantly decreased in the melatonin treatment groups. These results suggest that white fluorescent and red wavelengths downregulate the production of neurohormones in the brains of C. auratus and thus may inhibit sexual maturation in goldfish.

Molecular Basis for the Activation of Gonadotropin-Inhibitory Hormone Gene Transcription by Corticosterone

The inhibitory effect of stress on reproductive function is potentially mediated by high concentrations of circulating glucocorticoids (GCs) acting via the GC receptor (GR). Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion. GnIH may mediate stress-induced reproductive dysfunction. However, it is not yet known whether GC-bound GR is directly involved in GnIH transcription. Here, we demonstrated the localization of GR mRNA in GnIH neurons in the paraventricular nucleus of quail, suggesting that GC can directly regulate GnIH transcription. We next showed that 24 hours of treatment with corticosterone (CORT) increase GnIH mRNA expression in the quail diencephalon. We further investigated the mechanism of activation of GnIH transcription by CORT using a GnIH-expressing neuronal cell line, rHypoE-23, derived from rat hypothalamus. We found the expression of GR mRNA in rHypoE-23 cells and increased GnIH mRNA expression by 24 hours of CORT treatment. We finally characterized the promoter activity of rat GnIH gene stimulated by CORT. Through DNA deletion analysis, we identified a CORT-responsive region at 2000-1501 bp upstream of GnIH precursor coding region. This region included 2 GC response elements (GREs) at -1665 and -1530 bp. Mutation of -1530 GRE abolished CORT responsiveness. We also found CORT-stimulated GR recruitment at the GnIH promoter region containing the -1530 GRE. These results provide a putative molecular basis for transcriptional activation of GnIH under stress by demonstrating that CORT directly induces GnIH transcription by recruitment of GR to its promoter.

Review: Melatonin stimulates the synthesis and release of gonadotropin-inhibitory hormone in birds

Gonadotropin-inhibitory hormone (GnIH), a neuropeptide that inhibits gonadotropin synthesis and release, was first identified in the quail hypothalamus. To understand the physiological role of GnIH, this review will demonstrate the mechanisms that regulate GnIH synthesis and release. Pinealectomy (Px) combined with orbital enucleation (Ex) decreased the synthesis of GnIH precursor mRNA and content of mature GnIH peptide in the diencephalon. Melatonin administration to Px plus Ex birds caused a dose-dependent increase in the synthesis of GnIH precursor mRNA and production of mature peptide. A melatonin receptor subtype, Mel1c, was expressed in GnIH-immunoreactive neurons, suggesting direct action of melatonin on GnIH neurons. Melatonin administration further increased GnIH release in a dose-dependent manner from hypothalamic explants in vitro. GnIH mRNA expression and GnIH release during the dark period were greater than those during the light period in explants from quail exposed to long-day photoperiods. Conversely, plasma luteinizing hormone (LH) concentration decreased during the dark period. This review summarizes that melatonin appears to act on GnIH neurons in stimulating not only GnIH synthesis but also its release, thus inhibiting plasma LH concentration in birds.

Neonatal dexamethasone exposure down-regulates GnRH expression through the GnIH pathway in female mice

Synthetic glucocorticoid (dexamethasone; DEX) treatment during the neonatal stage is known to affect reproductive activity. However, it is still unknown whether neonatal stress activates gonadotropin-inhibitory hormone (GnIH) synthesizing cells in the dorsomedial hypothalamus (DMH), which could have pronounced suppressive action on gonadotropin-releasing hormone (GnRH) neurons, leading to delayed pubertal onset. This study was designed to determine the effect of neonatal DEX (1.0mg/kg) exposure on reproductive maturation. Therefore, GnRH, GnIH and GnIH receptors, G-protein coupled receptors (GPR) 147 and GPR74 mRNA levels were measured using quantitative real-time PCR in female mice at postnatal (P) days 21, 30 and in estrus stage mice, aged between P45–50. DEX-treated females of P45–50 had delayed vaginal opening, and irregular estrus cycles and lower GnRH expression in the preoptic area (POA) when compared with age-matched controls. The expression levels of GPR147 and GPR74 mRNA in the POA increased significantly in DEX-treated female mice of P21 and P45–50 compared to controls. In addition, GPR147 and GPR74 mRNA expression was observed in laser captured single GnRH neurons in the POA. Although there was no difference in GnIH mRNA expression in the DMH, immunostained GnIH cell numbers in the DMH increased in DEX-treated females of P45–50 compared to controls. Taken together, the results show that the delayed pubertal onset could be due to the inhibition of GnRH gene expression after neonatal DEX treatment, which may be accounted for in part by the inhibitory signals from the up-regulated GnIH–GnIH receptor pathway to the POA.

RNA Interference of Gonadotropin-Inhibitory Hormone Gene Induces Arousal in Songbirds

Gonadotropin-inhibitory hormone (GnIH) was originally identified in quail as a hypothalamic neuropeptide inhibitor of pituitary gonadotropin synthesis and release. However, GnIH neuronal fibers do not only terminate in the median eminence to control anterior pituitary function but also extend widely in the brain, suggesting it has multiple roles in the regulation of behavior. To identify the role of GnIH neurons in the regulation of behavior, we investigated the effect of RNA interference (RNAi) of the GnIH gene on the behavior of white-crowned sparrows, a highly social songbird species. Administration of small interfering RNA against GnIH precursor mRNA into the third ventricle of male and female birds reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations. GnIH RNAi further enhanced song production of short duration in male birds when they were challenged by playbacks of novel male songs. These behaviors resembled those of breeding birds during territorial defense. The overall results suggest that GnIH gene silencing induces arousal. In addition, the activities of male and female birds were negatively correlated with GnIH mRNA expression in the paraventricular nucleus. Density of GnIH neuronal fibers in the ventral tegmental area was decreased by GnIH RNAi treatment in female birds, and the number of gonadotropin-releasing hormone neurons that received close appositions of GnIH neuronal fiber terminals was negatively correlated with the activity of male birds. In summary, GnIH may decrease arousal level resulting in the inhibition of specific motivated behavior such as in reproductive contexts.

Identification, Expression, and Physiological Functions of Siberian Hamster Gonadotropin-Inhibitory Hormone

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. To further understand its physiological roles in mammalian reproduction, we identified its precursor cDNA and endogenous mature peptides in the Siberian hamster brain. The Siberian hamster GnIH precursor cDNA encoded two RFamide-related peptide (RFRP) sequences. SPAPANKVPHSAANLPLRF-NH(2) (Siberian hamster RFRP-1) and TLSRVPSLPQRF-NH(2) (Siberian hamster RFRP-3) were confirmed as mature endogenous peptides by mass spectrometry from brain samples purified by immunoaffinity chromatography. GnIH mRNA expression was higher in long days (LD) compared with short days (SD). GnIH mRNA was also highly expressed in SD plus pinealectomized animals, whereas expression was suppressed by melatonin, a nocturnal pineal hormone, administration. GnIH-immunoreactive (-ir) neurons were localized to the dorsomedial region of the hypothalamus, and GnIH-ir fibers projected to hypothalamic and limbic structures. The density of GnIH-ir perikarya and fibers were higher in LD and SD plus pinealectomized hamsters than in LD plus melatonin or SD animals. The percentage of GnRH neurons receiving close appositions from GnIH-ir fiber terminals was also higher in LD than SD, and GnIH receptor was expressed in GnRH-ir neurons. Finally, central administration of hamster RFRP-1 or RFRP-3 inhibited LH release 5 and 30 min after administration in LD. In sharp contrast, both peptides stimulated LH release 30 min after administration in SD. These results suggest that GnIH peptides fine tune LH levels via its receptor expressed in GnRH-ir neurons in an opposing fashion across the seasons in Siberian hamsters.