Gonadotropin-inhibitory Hormone Research Articles

The saccus vasculosus of the neotropical cichlid fish Cichlasoma dimerus: characterization, developmental studies and its response to photoperiod.

The saccus vasculosus is an organ present in gnathostome fishes, located ventral to the hypothalamus and posterior to the pituitary gland, whose structure is highly variable among species. In some fishes, this organ is well-developed; however, its physiological function is still under debate. Recently, it has been proposed that this organ is a seasonal regulator of reproduction. In the present work, we examined the histology, ultrastructure, and development of the saccus vasculosus in Cichlasoma dimerus. In addition, immunohistochemical studies of proteins related to reproductive function were performed. Finally, the potential response of this organ to different photoperiods was explored. C. dimerus presented a well-developed saccus vasculosus consisting of a highly folded epithelium, composed of coronet and supporting cells, closely associated with blood vessels, and a highly branched lumen connected to the third ventricle. Coronet cells showed all the major characteristics described in other fish species. In addition, some of the vesicles of the globules were positive for thyrotropin beta subunit, while luteinizing hormone beta subunit immunostaining was observed at the edge of the apical processes of some coronet cells. Furthermore, neuropeptide Y and gonadotropin inhibitory hormone innervation in the saccus vasculosus of C. dimerus were shown. Finally, animals exposed to the long photoperiod showed lower levels of thyrotropin beta and common alpha subunits expression in the saccus compared to those of animals exposed to short photoperiod. All these results support the hypothesis that the saccus vasculosus is involved in the regulation of reproductive function in fish.

Peripheral 5-HT mediates GnIH-induced feeding behavior and energy metabolism disorder in chickens via the 5-HT2C receptor.

Gonadotropin-inhibitory hormone (GnIH) plays a critical role of reproduction in vertebrate since its discovery. Recently, a regulatory role of GnIH in appetite and the energy metabolism has emerged, despite its precise physiological mechanisms remain unknown. Thus, the present study evaluated the effects of a single or long-term GnIH treatments (administered via intraperitoneal injection) on the food intake, weight and glucolipid metabolism of chickens, while investigated the possible neuroendocrinology factors and its mechanism that involved in GnIH-induced obesity and glucolipid metabolism disorder. Our results showed that the intraperitoneal administration of GnIH to chickens resulted in marked body mass increased, hyperlipidemia, hyperglycemia and glucose intolerance. Subsequently, the results of metabolomics and pharmacological inhibition of 5-HT2C receptor studies revealed that blocked 5-HT2C receptor reinforced the effects of GnIH on food intake, body weight and the levels of blood glucose and lipid, resulted in GnIH-induced hyperglycaemia, hyperlipidemia and hepatic lipid deposition even worse, suggesting that peripheral 5-HT via 5-HT2C receptor may act as a negative feedback regulator to interplay with GnIH and jointly homeostatic control of energy balance in chickens. Our present study provide evidence of the cross-talk between GnIH and 5-HT in food intake and energy metabolism at the in vivo pharmacological level and to propose a molecular basis for these interactions, suggesting that functional interaction between GnIH and 5-HT may open new avenues to understand the mechanism of neuroendocrine network involved in appetite and energy metabolism as well as provide a new therapeutic strategy to prevent obesity, diabetes and metabolic disorders.

The possibly role of GnIH in stress and gut dysfunction in chicken

Stress is known to disrupt the intestinal barrier and induce intestinal dysfunction. A critical role for gonadotropin inhibitory hormone (GnIH) in stress has emerged. However, whether GnIH mediates stress-induced intestinal dysfunction remains unknown. The present study explored this question through in vivo and in vitro experiments in hens. Our in vivo experiments showed that continuous intraperitoneal injection of GnIH not only significantly increased the concentration of stress hormones in serum, but also significantly elevated the mRNA expression of glucocorticoid receptor (GR) in the duodenum and jejunum. Moreover, morphological and molecular analyses revealed that GnIH disrupted the physical and chemical barriers of the intestine and dramatically increased inflammatory factor levels in the intestine and serum of hens. Interestingly, the microbiomics results showed that GnIH altered the structure and composition of the gut flora in the cecum, revealing an increased abundance of harmful intestinal bacteria such as Desulfovibrionaceae. Similar results were found in in vitro studies in which the GnIH-induced intestinal mucosal barrier was disrupted, and inflammation increased in jejunal explants, although no significant difference was found in the expression of GR between the control and GnIH groups. Our results demonstrated that GnIH not only directly damaged intestinal barriers and elevated intestinal inflammation but also mediated stress and microflora imbalance-induced intestinal function disorder, suggesting that GnIH is a potential therapeutic target for gut dysfunction, stress-induced intestinal function disorder, and inflammatory bowel disease in animals and humans.

Gonadotropin-inhibitory hormone and its receptors in teleosts: Physiological roles and mechanisms of actions

Gonadotropin-inhibitory hormone (GnIH) was the first reported hypothalamic neuropeptide inhibiting reproduction in vertebrates. Since its discovery in the quail brain, its orthologs have been identified in a variety of vertebrate species and even protochordates. Depending on the species, the GnIH precursor polypeptides comprise two, three or four mature peptides of the RFamide family. It has been well documented that GnIH inhibits reproduction at the brain-pituitary–gonadal levels and participates in metabolism, stress response, and social behaviors in birds and mammals. However, most studies in fish have mainly been focused on the physiological roles of GnIH in the control of reproduction and results obtained are in some cases conflicting, leaving aside its potential roles in the regulation of other functions. In this manuscript we summarize the information available in fish with respect to the structural diversity of GnIH peptides and functional roles of GnIH in reproduction and other physiological processes. We also highlight the molecular mechanisms of GnIH actions on target cells and possible interactions with other neuroendocrine factors.

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 β, LH β, GnRH receptor) in the hypothalamus and pituitary gland were respectively detected. Additionally, the expression of steroidogenesis-related genes/proteins (P450scc, StAR and 3β-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 β, LH β, and GnRHR genes in the pituitary. We also observed that GnIH treatment significantly decreased T levels and expression of the P450scc, StAR, and 3β-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 βb 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.

Dual effects of gonadotropin-inhibitory hormone on testicular development in prepubertal Minxinan Black rabbits (Oryctolagus cuniculus).

Gonadotropin-inhibitory hormone (GnIH) is a neurohormone that not only suppresses reproduction at the brain level but also regulates steroidogenesis and gametogenesis at the gonad level. However, its function in gonadal physiology has received little attention in rabbits. The main objective of this study was to evaluate the effects of GnIH on testicular development and function in prepubertal Minxinan Black rabbits (Oryctolagus cuniculus). In the present study, we investigated the serum reproductive hormone concentration, testicular parameters, morphology of seminiferous tubules, apoptosis of testicular cells, and expression of reproductive-related genes in male prepubertal Minxinan Black rabbits intraperitoneally administered with 0, 0.5, 5, or 50 μg quail GnIH-related peptides (qGnIH) for 10 days. Compared with the vehicle, administration with 5 μg of qGnIH downregulated the serum testosterone concentration and mRNA levels of spermatogenic genes (PCNA, FSHR, INHβA, HSF1, and AR) and upregulated the apoptosis rate of testicular cells; administration with 50 μg of qGnIH decreased the serum testosterone concentration and hypothalamic GnIH gene mRNA level and increased the serum LH concentration, pituitary LHβ gene mRNA level, testicular weight, gonadosomatic index (GSI), and spermatogenic cell layer thickness. It is concluded that GnIH could exert dual actions on testicular development depending on the male prepubertal rabbits receiving different intraperitoneal doses.

Impact of stress on male fertility: role of gonadotropin inhibitory hormone.

Studies have implicated oxidative stress-sensitive signaling in the pathogenesis of stress-induced male infertility. However, apart from oxidative stress, gonadotropin inhibitory hormone (GnIH) plays a major role. The present study provides a detailed review of the role of GnIH in stress-induced male infertility. Available evidence-based data revealed that GnIH enhances the release of corticosteroids by activating the hypothalamic-pituitary-adrenal axis. GnIH also mediates the inhibition of the conversion of thyroxine (T4) to triiodothyronine (T3) by suppressing the hypothalamic-pituitary-thyroidal axis. In addition, GnIH inhibits gonadotropin-releasing hormone (GnRH), thus suppressing the hypothalamic-pituitary-testicular axis, and by extension testosterone biosynthesis. More so, GnIH inhibits kisspeptin release. These events distort testicular histoarchitecture, impair testicular and adrenal steroidogenesis, lower spermatogenesis, and deteriorate sperm quality and function. In conclusion, GnIH, via multiple mechanisms, plays a key role in stress-induced male infertility. Suppression of GnIH under stressful conditions may thus be a beneficial prophylactic and/or therapeutic strategy.

LPXRFa and LPXRFa-R in the female swamp eel (Monopterus albus): Molecular cloning, tissue distribution and gonadal function

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide known for inhibiting gonadotropin release. In teleosts, it is also referred to as LPXRFa. While the effects of LPXRFa on gonadotropin synthesis and release, and its impact on pituitary function in teleosts have been widely studied, its specific mechanism in fish reproduction remains relatively unexplored. In this paper, LPXRFa and LPXRFa-R genes were isolated from the swamp eel brain to assess the roles of LPXRFa in the swamp eel. Their ORFs were 612 bp and 1284 bp, encoding proteins of 203 and 427 amino acids, respectively. The LPXRFa gene encodes a polypeptide possibly cleaved into three mature peptides (LPXRFa-1, −2, and −3). The LPXRFa-R receptor protein comprises seven conserved transmembrane domains. Tissue distribution analysis revealed that LPXRFa-R expression was the highest in the hypothalamus, while LPXRFa showed the highest expression in the rest of brain, with little to no expression in other tissues. The role of LPXRFa in the reproduction of the swamp eel was investigated by studying the effect of intraperitoneal injection with each of the three peptides. The intraperitoneal injection of LPXRFa-2 down-regulated serum FSH and LH levels, while that of LPXRFa-3 down-regulated serum FSH but up-regulated LH levels. Additionally, both LPXRFa-2 and LPXRFa-3 increased the expression levels of fshr, cbx3, dmrt1, gsdf, sox9al, amh, foxl2, and rab37 in the ovaries. Moreover, LPXRFa-2 also increased the ovarian expression of lhr, while LPXRFa-3 had no effect on it. Except upregulating cbx3 and foxl2 expression, LPXRFa-1 neither impacted the serum FSH and LH levels, nor did it alter the expression of other gonadal and reproductive-related genes. In summary, our findings provide the first evidence of a LPXRFa/LPXRFa-R system in the order Synbranchiformes and suggest its involvement in sex differentiation, gonadal development, and sexual reversal in the swamp eel.

Global cerebral ischemia in adult female rats interrupts estrous cyclicity and induces lasting changes in hypothalamic-pituitary-gonadal axis signaling peptides

Persistent post-ischemic alterations to the hypothalamic–pituitary–adrenal (HPA) axis occur following global cerebral ischemia (GCI) in rodents. However, similar effects on hypothalamic-pituitary–gonadal (HPG) axis activation remain to be determined. Therefore, this study evaluated the effects of GCI in adult female rats (via four-vessel occlusion) on the regularity of the estrous cycle for 24-days post ischemia. A second objective aimed to assess persistent alterations of HPG axis activation through determination of the expression of estrogen receptor alpha (ERα), kisspeptin (Kiss1), and gonadotropin-inhibitory hormone (GnIH/RFamide-related peptide; RFRP3) in the medial preoptic area (POA), arcuate nucleus (ARC), dorsomedial nucleus (DMH) of the hypothalamus, and CA1 of the hippocampus 25 days post ischemia. Expression of glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN) and CA1 served as a proxy of altered HPA axis activation. Our findings demonstrated interruption of the estrous cycle in 87.5 % of ischemic rats, marked by persistent diestrus, lasting on average 11.86 days. Moreover, compared to sham-operated controls, ischemic female rats showed reduced Kiss1 expression in the hypothalamic ARC and POA, concomitant with elevated ERα in the ARC and increased GnIH in the DMH and CA1. Reduced GR expression in the CA1 was associated with increased GR-immunoreactivity in the PVN, indicative of lasting dysregulation of HPA axis activation. Together, these findings demonstrate GCI disruption of female rats’ estrous cycle over multiple days, with a lasting impact on HPG axis regulators within the reproductive axis.

Lunar Age-Dependent Oscillations in Expression of the Genes for Kisspeptin, GnIH, and Their Receptors in the Grass Puffer during the Spawning Season.

Grass puffer is a semilunar-synchronized spawner: spawning occurs on beaches only for several days of spring tide around new moon (lunar age 0) and full moon (lunar age 15) every 2 weeks from spring to early summer. To investigate the role of kisspeptin and gonadotropin-inhibitory hormone (GnIH) in the semilunar-synchronized spawning, lunar age-dependent expression of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kissr2), GnIH (gnih), GnIH receptor (gnihr), gonadotropin-releasing hormone 1 (GnRH1) (gnrh1), and three gonadotropin (GTH) subunits (gpa, fshb, lhb) was examined in the male grass puffer, which was kept in an aquarium under natural light condition in a lunar month during the spawning period. In the brain, both kiss2 and kissr2 showed lunar variations with a peak at lunar age 10, while both gnih and gnihr showed semilunar variations with two peaks at lunar age 0 and 20. On the other hand, gnrh1 showed semilunar variation with two peaks at lunar age 0 and 15. In the pituitary, kiss2, kissr2, gnih, and gnihr showed similar variations to those shown in the brain. The fshb and lhb mRNA levels showed semilunar variations with two peaks at lunar age 0 and 15. The present study shows lunar and semilunar oscillations of kiss2/kissr2 and gnih/gnihr expressions, respectively, with their peaks around spring tide in the brain and pituitary along with the semilunar expressions of gnrh1 and the pituitary GTH subunit genes. These results suggest that the lunar age-dependent expressions of the kisspeptin, GnIH, and their receptor genes may be primarily important in the control of the precisely timed semilunar spawning of the grass puffer.

Effects of gonadotropin-inhibitory hormone on testicular development and reproduction-related gene expression in roosters

Gonadotropin-inhibitory hormone (GnIH) plays a crucial role in regulating reproduction in the hypothalamus of poultry and has been intensely investigated since its discovery. This study aimed to assess the effects of GnIH on testicular development, as well as on reproduction-related hormone release and gene expression levels in roosters. The administration of exogenous GnIH resulted in a significant reduction in testis weight, testis volume and semen quality (p < 0.05). Additionally, exogenous GnIH significantly up-regulates the expression of GnIH, and down-regulates the expression of PRL (p < 0.05). GnIH application also decreased the GnRH, vasoactive intestinal peptide (VIP) and luteinizing hormone β subunit(LHβ)gene expression levels. Meanwhile, by neutralizing the effects of endogenous GnIH through immunization, testicular development on day 150 in roosters was significantly promoted. Compared to the control condition, GnIH immunization significantly down-regulated the expression of the VIP and PRL genes (p < 0.05). In conclusion, we found that exogenous GnIH treatment inhibited testicular development, reduces PRL gene expression, and suppressed reproductive performance in roosters. Conversely, GnIH immunization down-regulated VIP and PRL genes, activates the reproductive system, and promotes the reproductive activity and testicular development of roosters.

Molecular regulation and genetic basis of gonadotropin-releasing hormone genes: A review

This review systematically introduces basic information on the hypothalamic pituitary-gonad axis and provides knowledge on the regulation, location, function, reproduction, gene mutations, disorders, sexual behavior, life cycle, and effect of environmental factors on the gonadotropin-releasing hormone gene. On the other hand, this review focused on the GnRH gene, regulations, receptor structures, and their signaling pathways, in addition to its related genes and its effect on crucial hormones such as follicle-stimulating hormone, luteinizing hormone, testosterone, estradiol, and progesterone. Additionally, gonadotropin-inhibiting hormone and its related peptides, such as R-Famide peptides, were found to decrease hormone secretion by working on the hypothalamic pituitary gonads axis to inhibit the biosynthesis process of gonadotropin alpha and beta subunits. Additionally, the roles of crucial hormones in reproduction and fertility, as well as the disruption, resulted from mutations. Special characteristics of several hormones and pulsatile secretion of gonadotropin-releasing hormone were also summarized.

Physiological role of dietary energy in the sexual maturity: clues of body size, gonad development, and serum biochemical parameters of Chinese indigenous chicken

Sexual maturity is a crucial factor in the formation and development of poultry reproductive capacity. The nutritional status has been confirmed to play an important role in the regulation of sexual maturity. To investigate the effect of dietary energy levels on sexual maturity in chicken, diets with 3 energy levels (group L: 2,573 kcal/kg, group C: 2,836 kcal/kg, group H: 3,122 kcal/kg) were implemented to feed Guangyuan Gray chickens. During this trial, body weight, body size, organ development, sexual maturity, reproductive performance and blood biochemical parameters were monitored. The earlier sexual maturity was observed in group H, as well as a heavier first egg weight, larger interpubic distance and higher total cholesterol (T-CHO) content at sexual maturity. The dietary energy levels had no significant effect on body weight at first egg and egg production at 300 d of age. Although dietary energy levels had a significant effect on body weight, comb length, tibia length and girth, abdominal fat weight, oviduct weight and length, T-CHO, triglyceride (TG) content and estradiol (E2) level during the rearing period. No significant difference of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) level among 3 groups was observed during the trial. The dietary energy levels had effects on mRNA expression of GnRH, estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2) in hypothalamus, gonadotropin inhibitory hormone receptor (GnIHR) in pituitary and luteinizing hormone receptor (LHR), ESR2 in ovary. The GnIHR/GnRHR ratio in pituitary was higher before sexual maturity and decreased at sexual maturity. The results of correlations analysis found that all the body size, carcass traits, serum biochemical parameters negatively correlated with age at first egg except for interpubic distance and serum blood glucose content. Collectively, dietary energy levels had effects on sexual maturity of chicken, which may be achieved by affecting body weight, gonad development, endocrine and the mRNA expression of genes related to hypothalamus-pituitary-gonad axis. These results further set our understanding of how dietary energy regulates sexual maturity.

Differential activation of neuropeptide FF receptors by gonadotropin-inhibitory hormone peptides in the European sea bass

Neuropeptide FF (NPFF) and gonadotropin-inhibitory hormone (GnIH) are thought to be paralogous, and a recent study has revealed that both NPFF and GnIH peptides can activate the GnIH receptor (GnIHR, also called NPFFR1) in the European sea bass (Dicentrarchus labrax). However, whether GnIH can bind to the NPFF receptor (NPFFR2) is still yet unknown in this species. Accordingly, we further investigated the potential interactions between GnIH and NPFFR2 (two NPFFR2 forms present in sea bass, namely NPFFR2-1 and NPFFR2-2) on the intracellular signaling pathways. Neither GnIH1 nor GnIH2 had any effect on basal CRE-luc activity, while forskolin-stimulated CRE-luc activity was significantly reduced when COS-7 cells expressing sea bass NPFFR2-1 and NPFFR2-2 were challenged with these two GnIH peptides. NPFF and NPAF also inhibited forskolin-induced CRE-luc activity via their cognate receptors. An evident stimulation of SRE-luc activity was observed when COS-7 cells transfected with NPFFR2-1 and NPFFR2-2 were treated with NPFF and NPAF, whereas GnIH peptides had no effect, except a slight but significant increase elicited by 1000 nM of GnIH1 in COS-7 cells expressing NPFFR2-2. Moreover, only GnIH2 exerted an inhibitory action on NFAT-RE-luc activity in COS-7 cells expressing NPFFR2-1. None of GnIH or NPFF peptides altered ERK phosphorylation levels via NPFFR2 receptors. Our results provide new evidence that sea bass GnIH peptides may exert their functions partially via NPFFR2, and PKA, PKC and Ca2+ routes are potential mediators.

Molecular cloning and bioinformatic characterization of Gonadotropin Inhibitory Hormone (GnIH) and its receptors in the freshwater murrel, Channa punctatus (Bloch, 1793).

Gonadotropin inhibitory hormone belonging to the RFamide peptide family, a hypothalamic neuropeptide, regulates Hypothalamus-pituitary-gonadal (HPG) axis and inhibits gonadal development. GnIH polypeptide precursor has an Arg-Phe-NH2 (RFamide) motif at the C-terminal, which has LPXRF (X = Q or L) domain. The actions of GnIH are mediated through G-protein coupled receptors and upto threereceptors have been characterized in many teleosts. GnIH exerts its inhibitory effect on the HPG axis through direct interaction with GnRH and Kisspeptin neurons in the brain and acts directly on the pituitary gonadotrophs. To decipher the role of GnIH in Indian freshwater murrel, Channa punctatus, we sequenced the cDNA encoding GnIH and its two receptors. The identified GnIH mRNA encodes three RFamide peptides having -MPMRF, -MPQRF, and -LPQRFamide motifs. In silico analysis of the amino acid sequence of GnIH exhibits its molecular and functional properties and the protein-protein interaction with significant factors regulating the HPG axis. The 3-D structure of GnIH and its receptors, provides more relevant information about the active residues of these proteins which might be involved in their functioning and interaction with other proteins. Molecular dynamic simulation of GnIH protein has provided more insight into its dynamic behavior. The expression of GnIH and its receptors, shows an inverse correlation with gonadal development during the annual reproductive cycle.

Gonadotropin inhibitory-hormone modulates neurosteroids-synthesizing enzymes expression and aggressive behavior in male sea bass, Dicentrarchus labrax

Neurosteroids are involved in the regulation of multiple behavioral and physiological processes and metabolic activities in the vertebrate brain. However, central mechanisms of how neurosteroid synthesis is regulated is far to be understood. Gonadotropin-inhibitory hormone (GNIH) is a hypothalamic neuropeptide that negatively regulates gonadotropin secretion but also inhibits sexual and aggressive behaviors in birds and mammals by modulating aromatase enzyme and neuroestrogen synthesis. In a previous study performed in male sea bass, we reported that Gnih inhibited the reproductive axis by acting at the three levels of the brain-pituitary-gonad axis. Moreover, the presence of Gnih cells and fibers in the telencephalon, mesencephalon and rhombencephalon suggests a role of Gnih in regulating other important brain functions in sea bass, including behavior. In this study, we have analyzed the effects of the intracerebroventricular (icv) injection of sbGnih-2 on the brain and pituitary expression of the main neurosteroids-synthesizing enzymes (stAR, cyp17, 3β-hsd, 17β-hsd, cyp19b, cyp7b), as well as on estrogen and androgen receptors (erα, erβ1, erβ2, ar). A combination of immunohistochemistry and in situ hybridization was also used to identify putative interaction of Gnih- and aromatase-positive cells. We also performed a mirror test study as a proxy to measure aggression levels and agonistic behavior after icv injection of sbGnih-2. Central administration of sbGnih-2 at different doses reduced the transcript levels of 3β-hsd and 17β-hsd, and increased the expression of cyp19b (brain aromatase) in the sea bass brain. Neuroanatomical results suggest that paracrine and neuroendocrine actions could mediate Gnih effects on aromatase expression. Central administration of sbGnih-2 also decreased the pituitary expression of 17β-hsd and estrogen receptors (erβ2). The mirror test analysis showed that sbGnih-2 affected the agonistic/aggressive behavior of sea bass as revealed by the decreased interaction with the mirror, lower time spent in the mirror zone, increased latency to establish contact with the mirror and higher mean distance to the mirror zone. In contrast, locomotor activity parameters measured were not affected by sbGnih-2 injection. Taken together, our results showed for the first time in fish that Gnih inhibits social-aggressive behavior and affects the gene expression of neurosteroid-synthesizing enzymes giving rise to neuroandrogens and neuroestrogens in the sea bass brain.

RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence.

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10-6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E2 and P4 were significantly lower with 10-6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10-6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3β-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

Food deprivation differentially modulates gene expression of LPXRFa and kisspeptin systems in the brain-pituitary axis of half-smooth tongue sole (Cynoglossus semilaevis).

LPXRFa, also known as gonadotropin-inhibitory hormone (GnIH), and kisspeptin (Kiss) are two major hypothalamic peptides that modulate the reproductive axis of vertebrates, including teleosts. However, little information is available regarding the actions of nutritional status on the regulation of these two neuroendocrine systems in fish. Herein, we assessed the effects of starvation and refeeding on the expression of lpxrfa, kiss2 and their receptors (lpxrfa-r and kiss2r respectively) at the brain-pituitary level of half-smooth tongue sole (Cynoglossus semilaevis). Food deprivation for 4 weeks induced a rise in brain lpxrfa as well as brain and pituitary lpxrfa-r mRNA levels, and refeeding restored brain lpxrfa and lpxrfa-r expression back to normal. However, pituitary lpxrfa-r mRNA levels still remained high after 1 week of refeeding. Neither lpxrfa nor kiss2 transcripts in the pituitary were altered by fasting, but their mRNA levels increased significantly after 1 week of refeeding, and declined back to the control levels after 2 weeks of refeeding. None of brain kiss2 and kiss2r along with pituitary kiss2r transcripts were modified by the nutritional status. In summary, our results revealed an interaction between energy status and the elements of LPXRFa and Kiss systems in the brain-pituitary axis of half-smooth tongue sole. Food deprivation and refeeding differentially regulated the two systems, which provided additional evidence for the involvement of the LPXRFa and Kiss systems in the regulation of reproduction by energy balance in non-mammalian species.

Synthetic microfiber exposure negatively affects reproductive parameters in male medaka (Oryzias latipes)

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E2) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process.

Effects of RFRP‑3 on an ovariectomized estrogen‑primed rat model and HEC‑1A human endometrial carcinoma cells.

The hypothalamic peptide gonadotropin inhibitory hormone (GnIH) is a relatively novel hypothalamic neuropeptide, identified in 2000. It can influence the hypothalamic-pituitary-gonadal axis and reproductive function through various neuroendocrine systems. The present study aimed to explore the effects and potential underlying molecular mechanism of RFamide-related peptide-3 (RFRP-3) injection on the uterine fluid protein profile of ovariectomized estrogen-primed (OEP) rats using proteomics. In addition, the possible effects of RFRP-3 on the viability and apoptosis of the human endometrial cancer cell line HEC-1A and associated molecular mechanism were investigated. The OEP rat model was established through injection with GnIH/RFRP-3 through the lateral ventricle. At 6 h after injection, the protein components of uterine fluid of rats in the experimental and control groups were analyzed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Differentially expressed proteins (DEPs) were analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interactions (PPI) were investigated using the STRING database. PPI networks were then established before hub proteins were selected using OmicsBean software. The expression of one of the hub proteins, Kras, was then detected using western blot analysis. Cell Counting Kit-8, Annexin V-FITC/PI, reverse transcription-quantitative PCR and western blotting were also performed to analyze cell viability and apoptosis. In total, 417 DEPs were obtained using LC-MS/MS, including 279 upregulated and 138 downregulated proteins. GO analysis revealed that the majority of the DEPs were secretory proteins. According to KEGG enrichment analysis, the DEPs found were generally involved in tumor-associated pathways. In particular, five hub proteins, namely G protein subunit α (Gna)13, Gnaq, Gnai3, Kras and MMP9, were obtained following PPI network analysis. Western blot analysis showed that expression of the hub protein Kras was downregulated following treatment with 10,000 ng/ml RFRP-3. RFRP-3 treatment (10,000 ng/ml) also suppressed HEC-1A cell viability, induced apoptosis, downregulated Bcl-2 and upregulated Bax protein expression, compared with those in the control group. In addition, compared with those in the control group, RFRP-3 significantly reduced the mRNA expression levels of PI3K, AKT and mTOR, while upregulating those of LC3-II. Compared with those in the control group, RFRP-3 significantly decreased the protein expression levels of PI3K, AKT, mTOR and p62, in addition to decreasing AKT phosphorylation. By contrast, RFRP-3 significantly increased the LC3-II/I ratio and G protein-coupled receptor 147 (GPR147) protein expression. In conclusion, the present data suggest that RFRP-3 can alter the protein expression profile of the uterine fluid of OEP rats by upregulating MMP9 expression whilst downregulating that of key hub proteins Gna13, GnaQ, Gnai3 and Kras. Furthermore, RFRP-3 can inhibit HEC-1A cell viability while promoting apoptosis. The underlying molecular mechanism may involve activation of GPR147 receptor by the direct binding of RFRP-3, which further downregulates the hub protein Kras to switch on the PI3K/AKT/mTOR pathway. This subsequently reduces the Bcl-2 expression and promotes Bax expression to induce autophagy.