The effect of gonadotropin-inhibitory hormone on steroidogenesis and spermatogenesis by acting through the hypothalamic-pituitary-testis axis in mice.

Gonadotropin inhibitory hormone (GnIH) is essential for regulating the reproduction of mammals and inhibiting testicular activities in mice. This study aimed to explore the mechanism of GnIH on spermatogenesis and steroidogenesis by acting through the hypothalamus-pituitary-testis axis of mice. Mice were subcutaneously injected with different doses of GnIH (1 µg/150 µL, 3 µg/150 µL, 6 µg/150 µL, 150 µL saline, twice daily) for 11 days. Subsequently, luteinizing hormone (LH), testosterone (T), and inhibin B (INH B) levels of peripheral blood were determined, and the expression of GnRH synthesis-related genes (GnRH-1, Kiss-1, NPY) and gonadotropin synthesis-related genes (FSH ß, LH ß, GnRH receptor) in the hypothalamus and pituitary gland were respectively detected. Additionally, the expression of steroidogenesis-related genes/proteins (P450scc, StAR and 3ß-HSD) and spermatogenesis-related proteins/genes including LH receptor (LHR), androgen receptor (AR), heat shock factor-2 (HSF-2) and INH B were analyzed using western blot and q-PCR. Results showed that GnIH treatment significantly reduced the concentration of LH in the peripheral blood. Further analysis revealed that GnIH treatment markedly reduced the expression of GnRHImRNA and Kiss-1 mRNA in the hypothalamus, and mRNA levels of FSH ß, LH ß, and GnRHR genes in the pituitary. We also observed that GnIH treatment significantly decreased T levels and expression of the P450scc, StAR, and 3ß-HSD proteins in the testis. Furthermore, GnIH treatment down-regulated LHR, AR proteins, and HSF-2 gene in the testis. Importantly, the INH B concentration of and INH ßb mRNA levels significantly declined following GnIH treatment. Additionally, GnIH treatment may induce germ cell apoptosis in the testis of mice. In conclusion, GnIH may suppress spermatogenesis and steroidogenesis by acting through the hypothalamus-pituitary-testis axis in mice.

Possible Role of GnIH as a Novel Link between Hyperphagia-Induced Obesity-Related Metabolic Derangements and Hypogonadism in Male Mice.

Gonadotropin-inhibitory hormone (GnIH) is a reproductive inhibitor and an endogenous orexigenic neuropeptide that may be involved in energy homeostasis and reproduction. However, whether GnIH is a molecular signal link of metabolism and the reproductive system, and thus, regulates reproductive activity as a function of the energy state, is still unknown. In the present study, we investigated the involvement of GnIH in glycolipid metabolism and reproduction in vivo, and in the coupling between these two processes in the testis level. Our results showed that chronic intraperitoneal injection of GnIH into male mice not only increased food intake and altered meal microstructure but also significantly elevated body mass due to the increased mass of liver and epididymal white adipose tissue (eWAT), despite the loss of testicular weight. Furthermore, chronic intraperitoneal administration of GnIH to male mice resulted in obesity-related glycolipid metabolic derangements, showing hyperlipidemia, hyperglycemia, glucose intolerance, and insulin resistance through changes in the expression of glucose and lipid metabolism-related genes in the pancreas and eWAT, respectively. Interestingly, the expression of GnIH and GPR147 was markedly increased in the testis of mice under conditions of energy imbalance, such as fasting, acute hypoglycemia, and hyperglycemia. In addition, chronic GnIH injection markedly inhibited glucose and lipid metabolism of mice testis while significantly decreasing testosterone synthesis and sperm quality, inducing hypogonadism. These observations indicated that orexigenic GnIH triggers hyperphagia-induced obesity-related metabolic derangements and hypogonadism in male mice, suggesting that GnIH is an emerging candidate for coupling metabolism and fertility by involvement in obesity and metabolic disorder-induced reproductive dysfunction of the testes.

Organization of the gonadotropin-inhibitory hormone (Lpxrfa) system in the brain of zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. However, the role of GnIH (Lpxrfa) in teleosts is unknown. In this study, a transgenic zebrafish (Danio rerio) line Tg(gnih:mCherry) was developed to determine the organization of GnIH neurons in the brain. Another transgenic line, Tg(gnih:mCherry; gnrh3:eGFP), was established to determine the positional relationships between GnIH and GnRH3 neurons. In these transgenic lines, the mCherry protein was specifically expressed in GnIH neurons, and eGFP was expressed exclusively in GnRH3 neurons. We found that GnIH cell somata were restricted to the posterior periventricular nucleus (NPPv). Most GnIH neuronal processes projected to the hypothalamus, but a few extended to the posterior tuberculum, telencephalon, and olfactory bulb. GnIH neuronal processes were in close apposition with GnRH3 cell somata and processes in the preoptic-hypothalamic area but were seldom in direct contact. However, in the olfactory bulb, GnIH neuronal processes were in proximity to the terminal nerve GnRH3 cell somata. Neither GnIH cell soma nor neuronal processes were detected in the pituitary, although GnIH receptor mRNAs (npffr1l1, npffr1l2, and npffr1l3) were detected. Intraperitoneal administration of GnIH-3 peptides promoted the transcription of brain gnrh3 as well as pituitary fshß but not lhß. Thus, GnIH cell somata were specifically distributed in the NPPv, and their fibers extended to the hypothalamus and advanced to the telencephalon and olfactory bulb. We conclude that GnIH may directly stimulate terminal nerve GnRH3 neurons in the zebrafish brain.

Genetic deciphering of the antagonistic activities of the melanin-concentrating hormone and melanocortin pathways in skin pigmentation.

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers.

Circadian rhythm in photoperiodic expressions of GnRH-I and GnIH regulating seasonal reproduction in the Eurasian tree sparrow, Passer montanus.

The present study investigates the involvement of circadian rhythm in photoperiodic expressions of GnRH-I and GnIH in the hypothalamus controlling seasonal reproduction in the Eurasian tree sparrow (Passer montanus). Groups of photosensitive birds were exposed for four weeks to resonance light dark cycles comprising of a light phase of 6 h (L) combined with dark phase of different durations (D) such that the period of LD cycles varied by 12 h increments viz. 12- (6 L/6D), 24- (6 L/18D), 36- (6 L/30D), 48- (6 L/42D), 60- (6 L/54D) and 72- (6 L/66D)h. In addition, a control group (C) was maintained under long day length (14 L/10D). Observations, recorded at the beginning and end of experiment, revealed significant testicular growth with corresponding increase in the hypothalamic expression of GnRH-I peptide but low levels of GnIH mRNA and peptide in the birds exposed to resonance cycles of 12, 36 and 60 h which were read as long days. On the other hand, birds experiencing resonance cycles of 24, 48 and 72 h read them as short days wherein they maintained their quiescent gonads and low levels of GnRH-I peptide but exhibited significant increase in GnIH mRNA and peptide expressions. Thus, sparrows responded to resonance light dark cycles differently despite the fact that each of them contained only 6 h of light. These findings suggest that an endogenous circadian rhythm is involved in photoperiodic expressions of above molecules and indicate a shift in their expressions depending upon whether the light falls in the photoinducible or non-photoinducible phase of an endogenous circadian rhythm.

Regulation of the hypothalamic GnRH-GnIH system by putrescine in adult female rats and GT1-7 neuronal cell line.

The gonadotropin-releasing hormone-gonadotropin inhibitor (GnRH-GnIH) system in the hypothalamus of mammals is the key factor that controls the entire reproductive system. The aim of this study was to immunolocalize GnIH (RFRP-3) in the hypothalamus during the estrous cycle and to study the effect of putrescine on the expression of GnRH-I and GnIH through both in vivo and in vitro (GT1-7 cells) approach and the circulatory levels of GnRH-I, GnIH, and gonadotropins were also investigated. The study also aims in analyzing all the immunofluorescence images by measuring the relative pixel count of an image. This study showed the effect of putrescine on the morphology of ovary, uterus, and the expression of the steroidogenic acute regulatory protein in the ovary. This study showed GnIH expression was intense during the diestrus and moderate during proestrus and estrus, whereas mild staining during the metestrus. The study further showed that putrescine supplementation to adult female rats increased both GnRH-I expression in the hypothalamus as well as the GnRH-I levels in circulation. The study, for the first time, also showed that putrescine supplementation decreased the expression and release of GnIH. These effects of upregulating GnRH-I expression and downregulating GnIH expression were confirmed by in vitro experiments using GT1-7 cells. Putrescine supplementation also increased the gonadotropin levels in the serum. To summarize, putrescine can regulate the hypothalamic-pituitary-gonadal axis by increasing the GnRH-I, luteinizing hormone, and follicle-stimulating hormone levels and suppressing GnIH levels. This is the first report showing the simultaneous effects of putrescine on the regulation of both GnRH-I and GnIH in the hypothalamus.

Dynamic Changes of RFRP3/GPR147 in the Precocious Puberty Model Female Rats.

BACKGROUND: Pubertal development is a complex physiological process regulated by the neuroendocrine system and hypothalamic-pituitary-gonadal axis. Sexual precocity is a common childhood endocrine disease.The pathogenesis of sexual precocity has not been fully elucidated. RFRP3/GPRl47 signal pathway is able to inhibit the reproductive capability in avians and mammals, probably by acting on the GnRH neuron and pituitary to regulate gonadotrophin synthesis and release. However, little is known about the role of RFRP3 in puberty development and sexual precocity. OBJECTIVE: To observe the dynamic changes of RFamide related peptide 3/G proteincoupled receptor 147 (RFRP3/GPR147) in hypothalamic during puberty development and explore their role in precocious puberty based on a female rat model. METHODS: The Sprague-Dawley female rats were randomly divided into three groups, normal, vehicle, and precocious puberty model. At 5 days old, the rat model with precocious puberty was prepared by subcutaneously injecting a mixture of danazoldissolved ethanol and glycol. At different day-age (15, 25, 30, 35, and 40 days), the levels of estradiol(E2), follicle-stimulating hormone(FSH), and luteinizing hormone (LH) in the peripheral blood were detected by the enzyme-linked immunosorbent assay, the messenger ribonucleic acid (mRNA) expressions of RFRP3, gonadotropin releasing hormone and GPR147 were examined by real-time polymerase chain reaction(R-T PCR). RFRP3 positive cells were observed using Immunofluorescence confocal microscopy. RESULTS: At 25 and 30 days, the levels of sex hormones and the uterus coefficients were significantly higher in the precocious puberty model group than those in the normal and vehicle groups. The ovarian morphological development in the precocious puberty model rats was significantly earlier than those in the normal and vehicle groups. The mRNA expressions of RFRP3/GPR147 and GnRH in the precocious puberty model group gradually increased and peaked at 25 days. The different day-age and the interaction have significant statistical significance on the expression of RFRP3 mRNA, while the levels of RFRP3 mRNA in the model group and vehicle groups have no significant statistical significance. There was statistical significance between the model group and vehicle groups in different day-age on the expression of GPR147 mRNA.The expression of hypothalamic RFRP3/GPR147 mRNA and RFRP3 positive cells gradually decreased with puberty onset. At 35 days, the levels of RFRP3 mRNA and GPR147 mRNA were significantly lower in the precocious puberty model group than those in the vehicle groups. Meanwhile, the levels of LH in the precocious puberty model rats reached its peak at this age. In the vehicle group, the levels of RFRP3 mRNA and serum LH were gradually increased and LH nearly peaked at 35 day-age. Subsequently, it gradually decreased and reached the lowest level at 35 day-age. The expression of RFRP3 mRNA and LH were positively correlated. CONCLUSION: The findings suggested that RFRP3/GPR147 signaling pathway may be involved in the pathogenesis of sexual precocity by regulating puberty development and sexual maturity in rats.

Specific neural phase relation of serotonin and dopamine modulate the testicular activity in Japanese quail.

Specific phase relation of serotonin and dopamine modulate the hypothalamo-hypophyseal-gonadal axis as well as photosexual responses in Japanese quail, but the effect of these specific phase relations on testicular activity and steroidogenesis is not yet been investigated. We hypothesized that temporal phase relation induced alteration in local testicular gonadotropin-releasing hormone (GnRH)-Gonadotropin-inhibitory hormone (GnIH) and their receptor system may modulate the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we have checked the alterations in the expression of gonadotropin-releasing hormone receptor (GnRH-R), gonadotropin-inhibitory hormone receptor (GnIH-R) messenger RNA (mRNA), growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), cell communication and gap junctional proteins (14-3-3 and connexin-43 [Cnx-43]), steroidogenic factor-1 (SF-1), steroidogenic acute regulatory (StAR) protein, steroidogenic enzyme (3ß-hydroxysteroid dehydrogenase [3ß-HSD]) in testis as well as androgen receptor (AR) in testis and epididymis of control, 8-, and 12-hr quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3ß-HSD in testis as well as AR in testis and epididymis in 8-hr quail, while 12-hr quail exhibited the opposite results that is significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3ß-HSD in testis as well as AR in testis and epididymis. The significantly increased intratesticular testosterone has been observed in the 12-hr quail while, 8-hr quail showed opposite result. Hence, it can be concluded that 12-hr quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in 8-hr quail.

Prenatal diagnosis of a familial 5p14.3-p14.1 deletion encompassing CDH18, CDH12, PMCHL1, PRDM9 and CDH10 in a fetus with congenital heart disease on prenatal ultrasound.

OBJECTIVE: We present prenatal diagnosis of a familial 5p14.3-p14.1 deletion in a fetus with congenital heart disease on prenatal ultrasound. CASE REPORT: A 33-year-old woman underwent amniocentesis at 18 weeks of gestation because of fetal ventricular septal defect (VSD) and echogenic bowel on prenatal ultrasound. Amniocentesis revealed a karyotype of 46,XX,del (5) (p14p14). Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed a 5.589-Mb 5p14.3-p14.1 deletion or arr 5p14.3p14.1 (19, 497, 649-25,086,268) × 1.0 [GRCh37 (hg19)] encompassing CDH18, CDH12, PMCHL1, PRDM9 and CDH10. Cytogenetic and aCGH analyses of the parents showed that the phenotypically normal mother carried the 5p14.3-p14.1 deletion. The father did not have such a deletion. The parents elected to continue the pregnancy, and a 3426-g female baby was delivered at 38 weeks of gestation with no gross abnormalities. The infant postnatally manifested VSD, atrial septal defect and patent ductus areriosus, and underwent cardiac surgery to treat the congenital heart disease. When follow-up at age 1 year and 4 months, she had a body weight of 8.8 Kg (50th-75th centile), a body height of 75.6 cm (85th-95th centile) and normal psychomotor development. CONCLUSION: Fetuses with a 5p14.3-p14.1 deletion may present congenital heart disease on prenatal ultrasound, and aCGH is helpful for prenatal diagnosis under such a circumstance.

Phoenixin participated in regulation of food intake and growth in spotted scat, Scatophagus argus.

Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2 d and 7 d fasting, while reduced significantly after re-feeding (P < 0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10 nM and 100 nM Pnx-20 and ghr1 incubated with 10 nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.

Melanin-concentrating hormone neurons contribute to dysregulation of rapid eye movement sleep in narcolepsy.

The lateral hypothalamus contains neurons producing orexins that promote wakefulness and suppress REM sleep as well as neurons producing melanin-concentrating hormone (MCH) that likely promote REM sleep. Narcolepsy with cataplexy is caused by selective loss of the orexin neurons, and the MCH neurons appear unaffected. As the orexin and MCH systems exert opposing effects on REM sleep, we hypothesized that imbalance in this REM sleep-regulating system due to activity in the MCH neurons may contribute to the striking REM sleep dysfunction characteristic of narcolepsy. To test this hypothesis, we chemogenetically activated the MCH neurons and pharmacologically blocked MCH signaling in a murine model of narcolepsy and studied the effects on sleep-wake behavior and cataplexy. To chemoactivate MCH neurons, we injected an adeno-associated viral vector containing the hM3Dq stimulatory DREADD into the lateral hypothalamus of orexin null mice that also express Cre recombinase in the MCH neurons (MCH-Cre::OX-KO mice) and into control MCH-Cre mice with normal orexin expression. In both lines of mice, activation of MCH neurons by clozapine-N-oxide (CNO) increased rapid eye movement (REM) sleep without altering other states. In mice lacking orexins, activation of the MCH neurons also increased abnormal intrusions of REM sleep manifest as cataplexy and short latency transitions into REM sleep (SLREM). Conversely, a MCH receptor 1 antagonist, SNAP 94847, almost completely eliminated SLREM and cataplexy in OX-KO mice. These findings affirm that MCH neurons promote REM sleep under normal circumstances, and their activity in mice lacking orexins likely triggers abnormal intrusions of REM sleep into non-REM sleep and wake, resulting in the SLREM and cataplexy characteristic of narcolepsy.

A Review of Single-Nucleotide Polymorphisms in Orexigenic Neuropeptides Targeting G Protein-Coupled Receptors.

Many physiological pathways are involved in appetite, food intake, and the maintenance of energy homeostasis. In particular, neuropeptides within the central nervous system have been demonstrated to be critical signaling molecules for modulating appetite. Both anorexigenic (appetite-decreasing) and orexigenic (appetite-stimulating) neuropeptides have been described. The biological effects of these neuropeptides can be observed following central administration in animal models. This review focuses on single nucleotide polymorphisms (SNPs) in six orexigenic neuropeptides: agouti-related protein (AGRP), galanin, melanin concentrating hormone (MCH), neuropeptide Y (NPY), orexin A, and orexin B. Following a brief summary of the neuropeptides and their orexigenic activities, reports associating SNPs within the orexigenic neuropeptides to energy homeostasis, food intake, obesity, and BMI in humans are reviewed. Additionally, the NIH tool Variation Viewer was utilized to identify missense SNPs within the mature, biologically active neuropeptide sequences. For SNPs found through Variation Viewer, a concise discussion on relevant pharmacological structure-activity relationship studies for select SNPs is included. This review is meant to update reported orexigenic neuropeptide SNPs and demonstrate the potential utility of genomic sequence databases for finding SNPs that may result in altered receptor signaling for neuropeptide pathways associated with appetite.

Characterization of LPXRFa receptor in the half-smooth tongue sole (Cynoglossus semilaevis): Molecular cloning, expression profiles, and differential activation of signaling pathways by LPXRFa peptides.

Gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide, serves as a key player in the regulation of reproduction across vertebrates, acting on the brain and pituitary to modulate reproductive physiology and behavior. However, little information is available in teleosts regarding the intracellular signal transduction pathway in response to GnIH. To this end, we first cloned the gene of LPXRFa (the piscine ortholog of GnIH) receptor in the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. The full-length cDNA of LPXRFa receptor was 2201 bp in size with an open reading frame (ORF) of 1365 bp that encoded 454 amino acids. Tissue distribution showed that LPXRFa receptor transcripts could be detected at high levels in the brain, to a lesser extent in the pituitary, and at low levels in the ovary and other peripheral tissues. In vitro functional analysis revealed that putative tongue sole LPXRFa-1 and LPXRFa-2 peptides significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity in COS-7 cells transfected with the novel receptor, and these stimulatory effects were evidently reduced by two inhibitors of the PLC/PKC pathway. In addition, neither LPXRFa-1 nor LPXRFa-2 altered the cAMP-responsive element (CRE)-luc activity, but only LPXRFa-2 could markedly decrease forskolin-induced CRE-luc activity in COS-7 cells expressing its cognate receptor. Taken together, our results encompass the first study reporting the existence of LPXRFa receptor in the order Pleuronectiformes and provide novel evidence of differential activation of signaling pathways by LPXRFa peptides in fish.

Identifying artificial selection signals in the chicken genome.

Identifying the signals of artificial selection can contribute to further shaping economically important traits. Here, a chicken 600k SNP-array was employed to detect the signals of artificial selection using 331 individuals from 9 breeds, including Jingfen (JF), Jinghong (JH), Araucanas (AR), White Leghorn (WL), Pekin-Bantam (PB), Shamo (SH), Gallus-Gallus-Spadiceus (GA), Rheinlander (RH) and Vorwerkhuhn (VO). Per the population genetic structure, 9 breeds were combined into 5 breed-pools, and a 'two-step' strategy was used to reveal the signals of artificial selection. GA, which has little artificial selection, was defined as the reference population, and a total of 204, 155, 305 and 323 potential artificial selection signals were identified in AR_VO, PB, RH_WL and JH_JF, respectively. We also found signals derived from standing and de-novo genetic variations have contributed to adaptive evolution during artificial selection. Further enrichment analysis suggests that the genomic regions of artificial selection signals harbour genes, including THSR, PTHLH and PMCH, responsible for economic traits, such as fertility, growth and immunization. Overall, this study found a series of genes that contribute to the improvement of chicken breeds and revealed the genetic mechanisms of adaptive evolution, which can be used as fundamental information in future chicken functional genomics study.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Age dependent variations in the deep brain photoreceptors (DBPs), GnRH-GnIH system and testicular steroidogenesis in Japanese quail, Coturnix coturnix japonica.

The complex physiology of aging involves a number of molecular and biochemical events, manifested as signs of senescence. Japanese quail is a very unique and advantageous model to study the signs and symptoms of senescence in the central and peripheral modules of HPG axis. In the present study, we have investigated the age dependent variations in hypothalamic deep brain photoreceptors (DBPs), central GnRH-I/II-GnIH-Mel1cR system, testicular GnRH-GnIH system, testicular steroidogenic genes and proteins, androgen receptor (AR) and serum testosterone level in quail of different age groups [3-wk (sexually immature), 6-wk (sexually mature and crossed the puberty), 16-wk (adult, sexually active and showing full breeding phase) and 144-wk (aged)]. Findings of our present study showed the differential expression of these genes/proteins in quail of different age groups. The low levels of the DBPs, GnRH-I, GnIH, Mel1cR in hypothalamus and GnRH-II in midbrain, significantly decreased testicular GnRH/GnRH-R-GnIH, steroidogenic genes/proteins and serum testosterone were observed in immature quail. The significantly increased expression of opsins in the DBPs, GnRH-I, GnIH, Mel1cR in hypothalamus and GnRH-II in midbrain influences the testicular GnRH-GnIH and stimulate the testicular steroidogenesis in mature and adult quail. In aged quail, the significantly decreased levels of hypothalamic DBPs, GnRH-I, GnIH, Mel1cR and midbrain GnRH-II modulates the testicular GnRH-GnIH and further suppresses the genes/proteins involved in steroidogenesis and results in reduced serum testosterone. Hence, it can be concluded from our findings that the testicular steroidogenesis and its neuroendocrine regulation varies with age, in Japanese quail.

Feeding of phytosterols reduced testosterone production by modulating GnRH and GnIH expression in the brain and testes of male Japanese quail (Coturnix coturnix japonica).

Phytosterols (PS), or plant sterols used as cholesterol-lowering agents, have been shown to act as endocrine-disrupting chemicals in some laboratory animals. Moreover, dietary PS efficiently pass through the blood-brain barrier and accumulate in brain cell membranes. We asked whether the accumulation of PS affects reproduction through the hypothalamic-pituitary-gonadal axis. Thirty male quail chicks were randomly divided into 3 groups (control, 80 mg/kg BW, and 800 mg/kg BW), and daily single doses of PS or vehicle were gavaged into the crop sac from 15 to 100 d of age. At the end of the entire period, half of each group was injected intramuscularly with either 10 µg of chicken gonadotropin-releasing hormone 1 (cGnRH-1) or phosphate-buffered saline solution (PBS) as the vehicle. Blood was collected before and 30 min after cGnRH-1 challenge by jugular venipuncture and decapitation, respectively. The results indicated that testosterone concentrations were low (P < 0.05) before (800 mg/kg BW) and after GnRH challenge in PS-treated quails compared with controls (P < 0.001). However, luteinizing hormone (LH) levels were not different among the groups before cGnRH-1 challenge. In addition, PS-gavaged animals failed to manifest increased LH levels after cGnRH-1 injection (P < 0.01). The same trends were observed in pituitary LH levels at 800 mg/kg BW PS after cGnRH-1 injection (P < 0.05). Real-time PCR results revealed that PS (800 mg/kg BW) feeding reduced expression of GnRH-1 in the brain and testes compared to controls. However, gonadotropin-inhibitory hormone (GnIH) expression was significantly elevated before and after GnRH-1 challenges in the brain and testes. Collectively, these results suggest that brain-mediated effects of PS on gonadal function occurs via the induction of GnIH gene expression, and these indirect effects are less potent than direct effects.

Accumbal D2 cells orchestrate innate risk-avoidance according to orexin signals.

Excitation of accumbal D2 cells governs vital actions, including avoidance of learned risks, but the origins of this excitation and roles of D2 cells in innate risk-avoidance are unclear. Hypothalamic neurons producing orexins (also called hypocretins) enhance innate risk-avoidance via poorly understood neurocircuits. We describe a direct orexin→D2 excitatory circuit and show that D2 cell activity is necessary for orexin-dependent innate risk-avoidance in mice, thus revealing an unsuspected hypothalamus-accumbens interplay in action selection.

Reproductive effects of synthetic progestin norgestrel in zebrafish (Danio rerio).

The aim of this study was to assess the adverse effects of synthetic progestin norgestrel (NGT) on the reproduction of zebrafish by measuring the egg production, histology and transcriptional expression profiles along the hypothalamic-pituitary-gonadal (HPG) axis in adult zebrafish. After a pre-exposure period of 7 days, adult zebrafish were exposed to 6, 29 and 69 ng L-1 NGT for 21 days. The results showed that exposure to 69 ng L-1 NGT led to a significant up-regulation of follicle stimulating hormone, beta polypeptide (fshb), luteinizing hormone, beta polypeptide (lhb), progesterone receptor (pgr), estrogen receptor 1 (esr1) and androgen receptor (ar) genes in the brains, as well as significant up-regulation of hydroxysteroid 20-beta dehydrogenase (hsd20b) and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2) genes and down-regulation of 11-beta-hydroxylase (cyp11b) gene in the ovaries of females. In the testes of males, an overall down-regulation of steroidogenic acute regulatory protein (star), cytochrome P450-mediated side-chain cleavage enzyme (cyp11a1), cyp11b, hsd20b, hydroxysteroid 17-beta dehydrogenase type 3 (hsd17b3), hsd11b2 and ar genes were observed following exposure to different treatments of NGT. These transcriptional alterations imply that NGT could exhibit the potent progestogenic and androgenic activities in zebrafish. Egg production as well as histology in the ovaries and testes was not affected by NGT. Taken together, the overall results demonstrated that NGT could significantly affect transcriptional expression levels of genes related to HPG axis in zebrafish, and whether that change translates to additional physiological effects is needed further research.

Testicular atrophy and reproductive quiescence in photorefractory and scotosensitive quail: Involvement of hypothalamic deep brain photoreceptors and GnRH-GnIH system.

Birds time their daily and seasonal activities in synchronization with circadian and annual periodicities in the environment, which is mainly provided by changes in photoperiod/day length conditions. Photoperiod appears to act at the level of eye, pineal and encephalic/deep brain photoperception and thus entrain the hypothalamic clock as well as reproductive circuitry in different avian species. In this article our focus of study is to elucidate out the underlying molecular mechanism of modulation of the hypothalamic reproductive circuitry following the photoperception through the hypothalamic photoreceptor cells and the subsequent alteration in the reproductive responses in quail, kept under different simulated photoperiodic conditions. Present study investigated the different simulated photoperiodic conditions induced hypothalamic DBP-GnRH-GnIH system mediated translation of photoperiodic information and subsequent exhibition of differential photosexual responses (scoto-/photo-sensitivity and refractoriness) in Japanese quail, Coturnix coturnix japonica. Paired testes weight and paired testicular volume increased 15.9 and 22.6-fold respectively in scotorefractory quail compare to that of scotosensitive phase and 12.8 and 24.3-fold in photosensitive quail compare to that of photorefractory phase. The pineal/eye melatonin (through melatonin receptor subtype Mel1cR) and hypothalamic deep brain photoreceptor (DBPs) cells directly modulate the hypothalamic GnRH-I/II and GnIH system and thus exhibit testicular stimulation or regression in response to different photoperiodic conditions (PS, PR, SS and SR). The hypothalamic alteration of DBP(s) and GnRH-GnIH system thus may induce the testicular stimulation in PS and SR quail and testicular regression in SS and PR quail.