GnRH Gene Expression Research Articles

Decoding the effect of photoperiodic cues in transducing kisspeptin-melatonin circuit during the pubertal onset in common carp.

This study unravels the intricate interplay between photoperiod, melatonin, and kisspeptin to orchestrate the pubertal onset of Common carp. Female fingerlings exposed to long days (LD) exhibited a hormonal crescendo, with upregulated hypothalamic-pituitary-ovarian (HPO) axis genes (kiss1, kiss1r, kiss2, gnrh2, gnrh3) and their downstream targets (lhr, fshr, ar1, esr1). However, the expression of the melatonin receptor (mtnr1a) diminished in LD, suggesting a potential inhibitory role. This hormonal symphony was further amplified by increased activity of key transcriptional regulators (gata1, gata2, cdx1, sp1, n-myc, hoxc8, plc, tac3, tacr3) and decreased expression of delayed puberty genes (mkrn1, dlk1). In contrast, short days (SD) muted this hormonal chorus, with decreased gnrh gene and regulator expression, elevated mtnr1a, and suppressed gonadal development. In in-vitro, estradiol mimicked the LD effect, boosting gnrh and regulator genes while dampening mtnr1a and melatonin-responsive genes. Conversely, melatonin acted as a conductor, downregulating gnrh and regulator genes and amplifying mtnr1a. Our findings illuminate the crucial roles of melatonin and kisspeptin as opposing forces in regulating pubertal timing. LD-induced melatonin suppression allows the kisspeptin symphony to flourish, triggering GnRH release and, ultimately, gonadal maturation. This delicate dance between photoperiod, melatonin, and kisspeptin orchestrates common carp's transition from juvenile to reproductive life.

Status of Expression of GnRH gene in Fed, Starved, and Refed Clarias Gariepinus (African Catfish)

Inadequate nutritional status impairs gonadal activities, which may induce reproductive difficulties in Clarias gariepinus. This research was aimed at evaluating the influence of starvation and subsequent refeeding on the expression of GnRH gene in Clarias gariepinus. One hundred and eighty (180) healthy juvenile Clarias gariepinus (African catfish) used in this study were randomly placed into two groups in triplicates. Fish in the control group were fed to satiation while fish in the treatment group were starved and refed and the fish in the control group were continuously fed. Brain tissue samples were collected after 7 days and 14 days of starvation, histopathology, and gene expression studies were done. The histological sections of brain tissue of week one starved and fed, C. gariepinus show neuronal cells on a background of neuropil while week two starved reveal badly distorted morphological architecture and degenerated neuronal cells. However, the week two re-fed, C. gariepinus brain tissue shows vacuolation of neuropil while the week two continuous fed shows no abnormalities. No mortality was recorded during starvation and the entire experimental period. In the second week of starvation, a decrease in the fold change (1.7) was observed when compared to week one while the fold change (1.2) observed in week three (week one of re-fed) is lower than the fold change observed in week one and two of starvation. The fold change (1.7) observed in week four (week two re-fed) is higher than the fold change observed in week three. The highest fold change (2.13) was observed in week one of starvation while the lowest fold change (1.24) was observed in week one of re-feeding. This study reveals that exposure of C. gariepinus to prolonged food deprivation alters the expression of GnRH, which might lead to impairment in the reproductive success of Clarias gariepinus.

Astaxanthin ameliorates the impairment consequence of prenatal bacterial lipopolysaccharide exposure in adult male offspring NMRI mice

In this study, the potential effects of astaxanthin (AST) were investigated on preventing the prenatal LPS-induced injures in mothers and adult male offspring of NMRI mice.Pregnant mice were randomly divided into four groups: 1. Saline + vehicle; 2. Saline + AST: received astaxanthin (4 mg/kg for 3 days, ip) on 11–13 gestation days; 3. LPS + vehicle (LPS-treated group): injected with LPS (20 µg/kg, sc) on gestation day 11; 4. LPS + AST: administrated LPS and astaxanthin on gestation days 11 and 11–13, respectively. In each group, maternal care behaviors and TNF-α serum levels were examined until weaning of male offspring at 23 days. At 60 days old, male pups underwent analysis of body weight and length, serum gonadotropins and testosterone hormone levels, sperm quality, gonadal and brain tissues morphologies, and the expression of SOX9 and GnRH genes by real-time PCR.Serum TNF-α level increased significantly in mothers treated with LPS, while AST reduced it. In adult male offspring, serum hormone levels, sperm quality, and the number of spermatocytes and Leydig cells in the testes improved when AST was administrated. According to histological studies of the brain, neurons in the LPS-treated group were smaller and less active, whereas neurons in the LPS + AST group were larger, more numerous, and more active. LPS significantly reduced GnRH expression, while AST induction improved its expression.AST administration during pregnancy prevented the adverse effects of prenatal exposure to LPS, presumably through its genomic and non-genomic effects, in adult male offspring.

Effective accumulative temperature affects gonadal maturation by controlling expression of GnRH, GnRH receptor, serotonin receptor and APGWamide gene in Pacific abalone, Haliotis discus hannai during broodstock conditioning in hatcheries.

Water temperature is a crucial environmental factor that influences reproductive function of abalone. Broodstock conditioning exposed to effective accumulative temperature (EAT) is a common practice in abalone hatcheries. To understand the molecular mechanism underlying the regulation of gonadal maturation and reproduction of Haliotis discus hannai exposed to EAT and induced spawning period, changes in expression of neuroendocrine genes encoding two gonadotropin releasing hormone (Hdh-GnRH, GnRH-like peptide), GnRH receptor (HdhGnRH-R), serotonin receptor (5-HTHdh) and Hdh-APGWamide in neural ganglia and gonadal tissues were examined. Gonadosomatic index (GSI) was significantly increased with increasing EAT °C-days. Expression levels of Hdh-GnRH, GnRH-like peptide, HdhGnRH-R, 5-HTHdh and Hdh-APGWamide mRNA were significantly increased with increasing EAT °C-days in ganglion (where the gene synthesized) and gonadal tissues. The significant increase in mRNA expression of each examined gene started from EAT 500 to 750°C-days, reached an initial peak at 1000°C-days, suggesting gonadal maturation started from the onset of EAT and slowly continued until 750°C-days, then at 1000°C-days reached to initial peak developmental period. The maturation reached to spawning state at 1000°C-days and peaked at 1500°C-days. Hdh-GnRH showed significantly higher mRNA expression in pleuropedal ganglion and branchial ganglion, whereas GnRH like peptide showed higher expression in cerebral ganglion, and HdhGnRH-R, 5-HTHdh and Hdh-APGWamide showed higher expression in pleuropedal ganglion. All genes were expressed higher at higher EAT °C-days. During induced spawning period, higher mRNA expression of examined genes was observed at the time of spawning; however, a sharp decrease occurred after spawning, suggesting that these genes are involved in spawning activities. Taken together, these results indicate that an increase of EAT °C-days can increase expression of neuroendocrine genes and enhance gonadal maturation. Besides all these genes are involved in the process of spawning induction, and increase of GSI has a positive correlation with the increase of gene expression.

Short-term regular moderate exercise improved male hypothalamic-pituitary-gonadal axis function via the reduction of hypothalamic neurokinin B expression in adult rats

Introduction: In the arcuate nucleus, kisspeptin, neurokinin-B and pro-dynorphin (KNDy) neurons control the function of gonadotropin-releasing hormone (GnRH) neurons. Early investigations indicated that exercise with various intensities affects luteinizing hormone (LH) and testosterone (T) in different ways. Meanwhile the molecular mechanisms underlying its function not yet been fully understood. Accordingly, the present study evaluated the role of alterations in the levels of KNDy mRNA upstream of GnRH neurons in conveying the effects of various short-term exercise intensities on the male hypothermic-pituitary-gonadal (HPG) axis. Methods: Twenty-one adult Wistar rats were randomly divided into 3 groups: control, one-month regular moderate exercise (ME) and one-month regular intensive exercise (IE). In ME (22m/min) and IE (35m/min) groups, the rats were treated 5 days a week for 60min each day. Finally, we assessed serum levels of LH and T using the ELIZA technique and KNDy and Gnrh mRNA expression by the real-time PCR method. Results: The results revealed that in ME group the expression of Nkb was reduced and the expression of Gnrh mRNA and the LH and T serum levels were increased. However, intensive exercise did not change the serum levels of LH and T or the relative expression of kiss1, Nkb, Pdyn and Gnrh genes. Conclusion: The results suggested that monthly moderate exercise improved male reproductive axis function, while intensive exercise did not have an adverse effect on the reproductive axis. These various effects on the male HPG axis may be propagated by the change in hypothalamic Nkb gene expression.

In-Vitro Exposure to Endocrine Disruptors Alters Inflammatory Markers in Whole Hypothalami and Immortalized GnRH Neurons

Bisphenol A (BPA), a monomer of polycarbonate plastics, and Benzophenones (BPs), used as UV-filters, are endocrine disrupting chemicals (EDC) found in everyday products. Previously, we showed that the in-vitro exposure to BPA decreased Kiss-induced GnRH expression in GN11 cells (donated by Dr. Susan Wray, NIH), immature GnRH neurons, and that exposure to all the EDC decreased Kiss-induced GnRH gene expression in GT1-7 cells (donated by Dr. Pamela Mellon, UCSD), mature GnRH neurons. In this study, we analyzed the effect of in-vitro exposure to the same EDC (BPA, BP2 or BP3, Sigma, 1x10-9 M) or medium as control (C) in mature GnRH neurons (GT1-7 cells), and isolated hypothalami from adult Balb/c males on Glial fibrillary acidic protein (GFAP) and cytokine gene expression. Cells were exposed to the compounds for 12 or 24 h, in DMEM (high glucose) with charcoal-stripped FBS, and the hypothalami for 6 h to the EDC, in Krebs-Ringer buffer. After the incubations, RNA was extracted using Tri-Reagent (Molecular Research Center, OH, USA), 1-2 µg RNA was reverse transcribed and Real-Time PCR performed using specific primers. Results were expressed as Mean±SE and analyzed by T-test or ANOVA using Statistica v12 (StatSoft Inc, USA) Twenty-four hour BPA exposure increased il18 in GT1-7 cells (C=1.0±0.04, BPA=1.2±0.1, T-test p<0.05, n=7), whereas neither BP2 nor BP3 had an effect on il18 expression (ANOVA: ns, n=7). When il6 was analyzed, 24-hour BPA decreased its expression relative to C and to 12-hour BPA, whereas BP3 had a dual effect depending on the time-point analyzed, increasing the expression at 12-hour stimulation and decreasing it after 24-hour stimulation (DMSO-12h=0.82±0.08, DMSO-24h= 1.01±0.13, BPA-12h=1.00±0.11, BPA-24h=0.68±0.06, BP2-12h=0.75±0.12, BP2-24h=0.82±0.18, BP3-12h=1.20±0.10, BP3-24h=0.83±0.05; Repeated Measures Two-way ANOVA: BPA-24h different from DMSO-24h and BPA-12h p<0.05, BP3-12h different from DMSO-12h and from BP3-24h p<0.05, n=4). In the hypothalami, 6-hour BPA exposure increased gfap gene expression (C=0.8±0.2, BPA=1.7±0.4; T-test p<0.05, n=9), whereas neither BP2 nor BP3 had any significant effect (C=0.8±0.2, BP2=1.4±0.3, BP3=1.0±0.3, ANOVA ns, n=9). There was no significant change in il18, il6 or il1b gene expression in whole hypothalami with any of the EDC tested (ANOVA ns). Our results show that the EDC herein studied have the potential to alter the inflammatory state of mature GnRH neurons and to activate astrocytes in the hypothalamus. The pattern for cytokine expression in the whole tissue could be different from the one observed in GnRH neurons, as the hypothalamus contain multiple cell types, and effects can be different in the different cells. More experiments are needed to dissect the mechanisms involved in the effects observed. Funding: CONICET, ANPCyT, UBA, International Society for Neurochemistry, Asoc. ORT Arg., Fund. R. Barón, Fund. Williams.

Maternal Exposure to Dibutyl Phthalate (DBP) or Diethylstilbestrol (DES) Leads to Long-Term Changes in Hypothalamic Gene Expression and Sexual Behavior.

Xenobiotic exposure during pregnancy and lactation has been linked to perinatal changes in male reproductive outcomes and other endocrine parameters. This pilot study wished to assess whether brief maternal exposure of rats to xenobiotics dibutyl phthalate (DBP) or diethylstilbestrol (DES) might also cause long-term changes in hypothalamic gene expression or in reproductive behavior of the resulting offspring. Time-mated female Sprague Dawley rats were given either DBP (500 mg/kg body weight, every second day from GD14.5 to PND6), DES (125 µg/kg body weight at GD14.5 and GD16.5 only), or vehicle (n = 8–12 per group) and mild endocrine disruption was confirmed by monitoring postnatal anogenital distance. Hypothalamic RNA from male and female offspring at PND10, PND24 and PND90 was analyzed by qRT-PCR for expression of aromatase, oxytocin, vasopressin, ER-alpha, ER-beta, kisspeptin, and GnRH genes. Reproductive behavior was monitored in male and female offspring from PND60 to PND90. Particularly, DES treatment led to significant changes in hypothalamic gene expression, which for the oxytocin gene was still evident at PND90, as well as in sexual behavior. In conclusion, maternal xenobiotic exposure may not only alter endocrine systems in offspring but, by impacting on brain development at a critical time, can have long-term effects on male or female sexual behavior.

Brain-derived neurotrophic factor (BDNF) affects the activity of the gonadotrophic axis in sheep

This study aimed to examine the hypothesis that BDNF modulates the activity of the gonadotrophic axis in sheep. Central infusions of BDNF were administered to sexually mature Polish Merino sheep. The sheep were randomly divided into three groups: the control group received intracerebroventricular (ICV) infusions of the vehicle, the BDNF 10 group received ICV infusions of BDNF at 10 μg/480 μL/day, and the BDNF 60 group was infused with BDNF at 60 μg/480 μL/day. A series of four infusions on three consecutive days was performed. Blood samples were collected on days 0 and 3 of the infusions. Immediately after the experiment, all the sheep were slaughtered, and selected structures of the hypothalamus and pituitaries were collected for Real Time RT-qPCR analysis. The collected plasma samples, as well as parts of pituitaries were stored for radioimmunoassay analysis of LH and FSH.Central treatment with exogenous BDNF stimulated GnRH mRNA expression in the preoptic area, as well as GnRH-R mRNA in the pituitary. Furthermore, substantial changes in the KNDy mRNA expression in the mediobasal hypothalamus were observed after the ICV BDNF administration. Additionally, central BDNF infusion caused a decrease in LH concentration and a simultaneous increase in FSH concentration in peripheral blood. Neither pulse amplitude nor pulse frequency for any gonadotrophin was affected in both groups of sheep that received BDNF infusion.Our results revealed that exogenous BDNF affects GnRH and KNDy gene expression and changes in the LH and FSH pituitary cell secretory activities. These findings suggest that BDNF may participate in the mechanism modulating the activity of the gonadotrophic axis at the central level in female sheep.

The Effect of Vitex Agnuse Castus Fruit Powder on Hypothalamic GnRH Gene Expression in Laying Hens

The Effect of Vitex Agnuse Castus Fruit Powder on Hypothalamic GnRH Gene Expression in Laying Hens

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Effects of High-Glucose and High-Fat Condition on Estrogen Receptor- and Sexual Precocity-Related Genes in GT1-7 Cells.

BackgroundThis study was designed to investigate the effect of high-glucose and high-fat condition on estrogen receptor- and sexual precocity-related genes in GT1-7 cells.Material/MethodsIn this study, CCK8 was used to detect cell viability, and TUNEL assay was used to detect apoptosis levels of GT1-7 cells after treatment with glucosamine and palmitate. The expression level of GnRH was measured by ELISA and RT-qPCR. RT-qPCR and Western blot were used to detect the expression of ERβ, CD36, and GPR54 in GT1-7 cells, and the expression of ERβ was detected using immunohistochemistry analysis. Finally, after adding the intervening drug tamoxifen to GT1-7 cells, the expression level of GnRH was measured by ELISA and Western blot analysis was used to detect the expression of GPR54 and GnRH.ResultsGnRH secretion in the high-fat and high-glucose group increased continuously over time and peaked at 18 h, and GnRH gene expression peaked at 12 h. High-fat and high-glucose conditions also significantly increased the levels of estrogen receptors β (ERβ), fatty acid translocase protein (CD36), and G Protein-Coupled Receptors 54 (GPR54) in GT1-7 cells. After estrogen receptors β (ER) was inhibited, GnRH secretion and GPR54 expression were decreased at 12 h and 18 h.ConclusionsOur study demonstrates that high-glucose and high-fat conditions promote the secretion of GnRH and ER and the expression of genes related to sexual precocity in GT1-7 cells.

Deletion of the Homeodomain Protein Six6 From GnRH Neurons Decreases GnRH Gene Expression, Resulting in Infertility.

Hypothalamic GnRH (luteinizing hormone-releasing hormone) neurons are crucial for the hypothalamic-pituitary-gonadal (HPG) axis, which regulates mammalian fertility. Insufficient GnRH disrupts the HPG axis and is often associated with the genetic condition idiopathic hypogonadotropic hypogonadism (IHH). The homeodomain protein sine oculis-related homeobox 6 (Six6) is required for the development of GnRH neurons. Although it is known that Six6 is specifically expressed within a more mature GnRH neuronal cell line and that overexpression of Six6 induces GnRH transcription in these cells, the direct role of Six6 within the GnRH neuron in vivo is unknown. Here we find that global Six6 knockout (KO) embryos show apoptosis of GnRH neurons beginning at embryonic day 14.5 with 90% loss of GnRH neurons by postnatal day 1. We sought to determine whether the hypogonadism and infertility reported in the Six6KO mice are generated via actions within the GnRH neuron in vivo by creating a Six6-flox mouse and crossing it with the LHRHcre mouse. Loss of Six6 specifically within the GnRH neuron abolished GnRH expression in ∼0% of GnRH neurons. We further demonstrated that deletion of Six6 only within the GnRH neuron leads to infertility, hypogonadism, hypogonadotropism, and delayed puberty. We conclude that Six6 plays distinct roles in maintaining fertility in the GnRH neuron vs in the migratory environment of the GnRH neuron by maintaining expression of GnRH and survival of GnRH neurons, respectively. These results increase knowledge of the role of Six6 in the brain and may offer insight into the mechanism of IHH.

Gonadoliberin – Synthesis, Secretion, Molecular Mechanisms and Targets of Action

Decapeptide gonadoliberin (GnRH) is the most important regulator of the hypothalamic-pituitary-gonadal (HPG) axis that controls the synthesis and secretion of the luteinizing and follicle-stimulating hormones by gonadotrophs in the adenohypophysis. GnRH is produced by the specialized hypothalamic neurons using the site-specific proteolysis of the precursor protein and is secreted into the portal pituitary system, where it binds to the specific receptors. These receptors belong to the family of G protein-coupled receptors, and they are located on the surface of gonadotrophs and mediate the regulatory effects of GnRH on the gonadotropins production. The result of GnRH binding to them is the activation of phospholipase C and the calcium-dependent pathways, the stimulation of different forms of mitogen-activated protein kinases, as well as the activation of the enzyme adenylyl cyclase and the triggering of cAMP-dependent signaling pathways in the gonadotrophs. The gonadotropins, kisspeptin, sex steroid hormones, insulin, melatonin and a number of transcription factors have an important role in the regulation of GnRH1 gene expression, which encodes the GnRH precursor, as well as the synthesis and secretion of GnRH. The functional activity of GnRH-producing neurons depends on their migration to the hypothalamic region at the early stages of ontogenesis, which is controlled by anosmin, ephrins, and lactosamine-rich surface glycoconjugate. Dysregulation of the migration of GnRH-producing neurons and the impaired production and secretion of GnRH, lead to hypogonadotropic hypogonadism and other dysfunctions of the reproductive system. This review is devoted to the current state of the problem of regulating the synthesis and secretion of GnRH, the mechanisms of migration of hypothalamic GnRH-producing neurons at the early stages of brain development, the functional activity of the GnRH-producing neurons in the adult hypothalamus and the molecular mechanisms of GnRH action on the pituitary gonadotrophs. New experimental data are analyzed, which significantly change the current understanding of the functioning of GnRH-producing neurons and the secretion of GnRH, which is very important for the development of effective approaches for correcting the functions of the HPG axis.

Cyanidin-3-O-glucoside protects against cadmium-induced dysfunction of sex hormone secretion via the regulation of hypothalamus-pituitary-gonadal axis in male pubertal mice

Cadmium (Cd) has been generally recognized as an endocrine-disrupting chemical for its toxic effects on the hypothalamus-pituitary-gonadal (HPG) axis accompanied by dysfunction in sex hormone secretion. Particularly, exposure to Cd during puberty versus post-puberty exhibits differing age-dependent effects that require further examination. This study sought to determine if cyanidin-3-O-glucoside (C3G), a typical anthocyanin with neuroprotective bioactivity, could protect against Cd-induced sex hormone-disorder in Pubertal male mice. C3G treatment reversed the disruption of hormone levels and increased Gnrh1 gene expression in the hypothalamus. In addition, the levels of gonadotropins, including luteinizing hormone (LH) and follicle stimulating hormone (FSH), were reversed by C3G. Interestingly, C3G improved the expression of LH and FSH receptor in the testis in mice exposed to Cd. Furthermore, C3G activated the signaling pathway related to the synthesis of testosterone processing. In conclusion, C3G protected against Cd-induced dysfunction of sex hormone secretion through the regulation of the HPG axis in male mice during puberty. The results of this study suggest that consumption of anthocyanins can be protective against metal-induced male reproductive dysfunction.

SUN-468 Sex Differences in Obesity-Mediated Impairment of Reproductive Function

Increasing prevalence in obesity has become a significant public concern. C57BL/6J mice are prone to diet-induced obesity (DIO) when fed high-fat diet (HFD), and develop chronic inflammation and metabolic syndrome, making them a good model to analyze mechanisms whereby obesity elicits pathologies. DIO mice demonstrated profound sex differences in response to HFD with respect to inflammation and hypothalamic function. First, we determined that males are prone to DIO, while females are resistant. Ovariectomized females, on the other hand, are susceptible to DIO, implying protection by ovarian hormones. Males, but not females, exhibit changes in gonadotropin hormone concentration and hypothalamic neuropeptide expression. Surprisingly, ovariectomized females remain resistant to neuroendocrine changes, showing that ovarian hormones are not necessary for protection. Second, obese mice exhibit sex differences in DIO-induced inflammation. Proinflammatory cytokines are increased specifically in males, while anti-inflammatory cytokines are increased in the hypothalami of females but not males, which may provide protection to female mice. Again, females are protected from the increase in inflammatory cytokines regardless of the presence of ovarian hormones. Third, males and females deposit fat differentially in subcutaneous and visceral depots. Obese males exhibit reduced synapses in GnRH neurons and diminished GnRH gene expression, which may stem from obesity-mediated activation of immune cells or from direct effect of proinflammatory cytokines on GnRH gene expression, respectively. Proinflammatory cytokines directly repress GnRH gene expression in GT1-7 cells model, while in vivo, obese males show activation of a cytokine-induced transcription factor in GnRH neurons. Our studies suggest that inflammation-induced hypothalamic changes are potentially responsible for diminished levels of gonadotropin hormones, testosterone, and sperm numbers, which corresponds to the observations in obese humans. Taken together, we propose a potential mechanism of obesity-induced impairment of hypothalamic function, and our data implicate neuro-immune mechanisms underlying sex-specific differences in obesity-induced impairment of the hypothalamic function with potential consequences for reproduction and fertility.

MON-031 In Vitro Exposure to Bisphenol A, Benzophenones 2 or 3, Have Different Effects on GnRH Gene Expression and Secretion in Mature and Immature GnRH Neurons

Bisphenol A, (BPA), a component of polycarbonate plastics, and Benzophenones (BPs), UV filters, are endocrine disrupting chemicals (EDC). Previously we showed that the in vitro exposure to BPA decreased Kiss-induced GnRH expression in GN11 cells (Dr. Susan Wray, NIH), immature GnRH neurons (1). In this study, we analyzed the in vitro effects of BPA, BP2 and BP3 (1x10-9M), or medium as control (C) in mature GnRH neurons (GT1-7 cells, donated by Dr Pamela Mellon, UCSD, USA, and isolated hypothalami from adult Balb/c males). Effects of BP2 and BP3 (1x10-9M) were also analyzed in GN11 cells. We exposed the cell lines for 24 h and the hypothalami for 6 h to the EDC. GT1-7 and GN11 cells were further incubated with Kiss (1x10-9M, Phoenix Pharmaceuticals, Inc, CA, USA) for 4 h to analyze gene expression (qPCR) and for 1 h to analyze GnRH secretion (RIA). Hypothalami exposed to BPA showed increased GnRH expression compared to C (Relative expression: C:1.0±0.2; BPA:2.2±0.5; Student T-test: BPA vs C p<0.05, n=9), whereas neither BP2 nor BP3 had any effect (ANOVA: ns). There was no effect on GnRH secretion (ANOVA: ns). GT1-7 cells exposed to C showed Kiss-induced GnRH gene expression, while the exposure to the EDC abolished this response (Relative expression: C-Basal:1.0±0.1, C-Kiss:1.6±0.3, BPA-Basal:1.3±0.2, BPA-Kiss:1.1±0.2, BP2-Basal:1.2±0.1, BP2-Kiss:1.4±0.2, BP3-Basal:1.3±0.3, BP3-Kiss:1.1±0.2; Repeated measures Two-way ANOVA: Interaction p<0.05, n=6). There was no difference in Kiss-stimulated GnRH release in EDC-exposed cells vs C-exposed cells (Repeated measures Two-way ANOVA: Interaction ns; Main Effect: Time of Kiss exposure: p<0.01, n=7). In GN11 cells, exposure to BP2 inhibited Kiss-induced GnRH gene expression, whereas there was no such effect in cells exposed to BP3 (Relative Expression: C-Basal:1.1±0.02, C-Kiss:1.3±0.1, BP2-Basal:1.2±0.1, BP2-Kiss:1.0±0.1, BP3-Basal:1.0±0.1, BP3-Kiss:1.3±0.1; Repeated measures Two-way ANOVA: Interaction p<0.01, n=8). Exposure to BP3 significantly increased GnRH secretion and abolished the response to Kiss, whereas BP2 significantly decreased Kiss-stimulated release [GnRH (pg/ml): C-Basal: 8.5±1.3, C-Kiss: 32.9±8.8, BP2-Basal: 16.5±4.9, BP2-Kiss: 16.9±5.1, BP3-Basal: 18.0±3.2, BP3-Kiss: 20.8±5.6, Repeated measures Two-way ANOVA: Interaction p<0.05; C-Kiss vs C-Basal, BP3-Basal vs C-Basal, BP2-Kiss vs C-Kiss p<0.05, n=9]. Our results show that EDC exposure alters the physiology of mature and immature GnRH neurons, although effects are different in the two cell lines, and they also differ in mature GnRH neurons vs hypothalamus. Further studies are needed to dissect the mechanisms involved. 1- Endoc. Rev. 38(3S), SAT-256, 2017. Nothing to disclose. Funding: CONICET, ANPCyT, UBA, International Society for Neurochemistry, Asoc. ORT Arg., Fund. R. Barón, Fund. Williams.

Central Injection of Substance P Antagonizes the RF Amide-Related Peptide-3 Impacts on Hypothalamic KISS-1 and GnRH Gene Expressions in Male Wistar Rats

Background: Gonadotropin-inhibitory hormone and its mammalian orthologues (RFRP: RF amid related peptide) are known to inhibit the secretion of gonadotropins. In addition, substance P (SP) a member of tachykinin’s family is demonstrated that can increase the firing rate of kisspeptin/neurokinin B/dynorphin (KNDy) neurons and provokes the of secretion gonadotropins. In this experimental study we investigated the effects of co-administration of RFRP-3 and SP on the expression of KISS-1 and GnRH genes in male rats. Methods: forty-two mature Wistar rats were randomly allocated in 7 groups (n=6 in each group). Animals in each group intracerebroventricularly received either saline+DMSO, 1nmol SP, 5 nmol RFRP-3, 1nmol SP plus 5 nmol RFRP-3, 1 nmol SP plus10 nmol RF9 (RFRP-3 receptor antagonist), 1 nmol SP plus 1 nmol P234 (kisspeptin receptor antagonist) plus 5 nmol RFRP-3 or 1 nmol SP plus 5 nmol CP-96,345 (SP receptor antagonist) plus 5 nmol RFRP-3 in final volume of 3 µl. After two hours following injections hypothalamic samples were collected for evaluating the expression of target genes by real-time PCR technique. Results: Injections in SP and SP plus RF9 groups increase the expression of the both GnRH and KISS-1 genes (P&lt;0.05). Injections in RFRP-3 group and SP plus RFRP-3 plus SP antagonist group significantly decrease the expression of both GnRH and KISS-1 genes (P&lt;0.05). Injections of SP plus RFRP-3 group and SP plus RFRP-3 plus P234 group did not significantly change the expression of GnRH and KISS-1 genes. Conclusion: The results indicate that SP antagonizes the effects of RFRP-3 on the expression of Hypothalamic KISS-1 and GnRH genes.

Effects of glyphosate on the ovarian function of pregnant mice, the secretion of hormones and the sex ratio of their fetuses

Glyphosate is the active ingredient of the commercial formulation Roundup®, which is used worldwide. This study aimed to investigate the toxic effects of pure glyphosate or Roundup® on pregnant mice and their fetuses during pregnancy. From gestation days (GDs) 1–19, ICR mice were orally administered distilled water, 0.5% glyphosate solution or 0.5%-glyphosate Roundup® solution. The ovaries and serum were collected at GD19. The results showed decreases in body weight gain and, ovary and liver weight in glyphosate-treated mice. Additionally, histopathological alterations in the ovary including increased atretic follicles, interstitial fibrosis and decreased mature follicles were observed in the groups treated with glyphosate. The serum concentrations of both progesterone and estrogen were markedly altered after glyphosate exposure, and there were also changes in the expression of GnRH, LHR, FSHR, 3β-HSD and Cyp19a1 genes at the hypothalamic-pituitary-ovarian axis. Furthermore, oxidative stress was observed in the treated mice, increasing the activity of T-AOC, CAT and GSH-Px, as well as the MDA content in both the serum and ovary. With regard to litters, the sex ratio was significantly altered by pure glyphosate. These results show that glyphosate is able to cause several effects on pregnant mice, such as ovarian failure, interference with hormone secretion by affecting the steroidogenesis-related gene expression, and oxidative stress. The sex ratio of litters was also influenced by prenatal exposure to pure glyphosate.

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Obesity-related cellular stressors regulate gonadotropin releasing hormone gene expression via c-Fos/AP-1.

Obesity is a risk factor for infertility, but mechanisms underlying this risk are unclear. Fertility is regulated by hypothalamic gonadotropin-releasing hormone, encoded by the Gnrh1 gene. Because obesity promotes endoplasmic reticulum (ER) stress, we sought to determine how tunicamycin-induced ER stress affected Gnrh1 gene expression in the mouse hypothalamic cell line GT1-7. Tunicamycin repressed expression of Gnrh1 in a PKC- and JNK-dependent manner, while upregulating expression of a known Gnrh1 repressor, Fos. Obesity is associated with increased circulating free fatty acids, and exposure to palmitate promoted ER stress and inflammation. Fos expression increased with palmitate dose, but Gnrh1 expression was upregulated with low-dose palmitate and repressed with high-dose palmitate. Using a small molecule inhibitor, we determined that AP-1 was required for Gnrh1 repression by high-dose palmitate or tunicamycin-induced ER stress. These findings suggest that hypogonadism driven by decreased hypothalamic GnRH may be a component of obesity-related infertility.