Brain and testicular metabonomics revealed the protective effects of Guilingji on senile sexual dysfunction rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Guilingji (GLJ), which has been used to treat male diseases in China for centuries, contains 28 Chinese herbs and was previously established as an effective treatment for male sexual dysfunction. However, its mechanism of action remains unclear. AIM OF THE STUDY: To explore the efficacy and mechanism of action of GLJ in improving senile sexual dysfunction (SSD) in aging rats. MATERIALS AND METHODS: An aging rat model of SSD was induced by the subcutaneous injection of d-galactose (300 mg⋅kg-1) and used to analyse the effects of GLJ (different concentrations of 37.5, 75, and 150 mg⋅kg-1) on the mating of aging rats. At the end of the 8th week, histopathological analysis of testicular tissues, assessment of the hypothalamic-pituitary-gonadal (HPG) axis hormone levels in serum or brain, and metabonomics analysis of the brain and testicular tissue with liquid chromatography-mass spectrometry was performed to explore the mechanism of action of GLJ. RESULT: After treatment with GLJ, the mount and ejaculation latency levels were increased in the treatment group than those in model group (P < 0.05), moreover, the testicular morphology was improved. Gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) levels in rats were also improved significant (P < 0.05) compared with those in the model group. Furthermore, the metabonomics results in the testicular and brain tissue showed that GLJ improved SSD by adjusting amino acid and lipid metabolism. CONCLUSION: This study integrated the complementary metabolic profiles of the target tissues. GLJ might affect SSD rats by regulating amino acid and lipid metabolism and may modulate sensitivity to the signaling pathway in the HPG axis. This study provides an essential basis for the broad clinical application of GLJ.

Decreased angiotensin receptor 1 expression in ± AT1 Knockout mice testis results in male infertility and GnRH reduction.

BACKGROUND: This study aimed to detect the effect of angiotensin receptor 1 (AT1) knock out (KO) on spermatogenesis and hypothalamic-pituitary-gonadal (HPG) axis hormone expression. METHODS: Normal C57BL/6 male mice were used as control group or treated with angiotensin receptor blocker, in addition heterozygous ± AT1KO mice were generated. After caged at a ratio of 2 to 1 with females, pregnancy rates of female mice were determined by detection of vaginal plugs. Deformity rate of spermatozoa was evaluated by eosin staining and morphology evaluation. The AT1 mRNA expression in the testes of male ± AT1KO mice was detected by quantitative real-time polymerase chain reaction (QRT-PCR). Serum GnRH level was determined by ELISA. RESULTS: Compared to control, ± AT1KO mice showed reduced expression of AT1 in testes, pituitary and hypothalamus. In addition, decreased level of GnRH, but not follicle stimulating hormone (FSH) or luteinizing hormone (LH), in ± AT1KO mice was detected. Treatment with angiotensin receptor blocker (ARB) did not have significant effects on HPG hormones. ± AT1KO mice exhibited male infertility and significant abnormality of sperm morphology. CONCLUSION: Reduced AT1 knockout resulted in male infertility, potentially by inducing abnormal spermatogenesis. Both testis and HPG axis signaling may be involved.

Cellular and molecular features of EDC exposure: consequences for the GnRH network.

The onset of puberty and the female ovulatory cycle are important developmental milestones of the reproductive system. These processes are controlled by a tightly organized network of neurotransmitters and neuropeptides, as well as genetic, epigenetic and hormonal factors, which ultimately drive the pulsatile secretion of gonadotropin-releasing hormone. They also strongly depend on organizational processes that take place during fetal and early postnatal life. Therefore, exposure to environmental pollutants such as endocrine-disrupting chemicals (EDCs) during critical periods of development can result in altered brain development, delayed or advanced puberty and long-term reproductive consequences, such as impaired fertility. The gonads and peripheral organs are targets of EDCs, and research from the past few years suggests that the organization of the neuroendocrine control of reproduction is also sensitive to environmental cues and disruption. Among other mechanisms, EDCs interfere with the action of steroidal and non-steroidal receptors, and alter enzymatic, metabolic and epigenetic pathways during development. In this Review, we discuss the cellular and molecular consequences of perinatal exposure (mostly in rodents) to representative EDCs with a focus on the neuroendocrine control of reproduction, pubertal timing and the female ovulatory cycle.

Erythropoietin-producing hepatocellular receptor A7 restrains estrogen negative feedback of luteinizing hormone via ephrin A5 in the hypothalamus of female rats.

We have previously shown that systemic injection of erythropoietin-producing hepatocellular receptor A7 (EPHA7)-Fc raises serum luteinizing hormone (LH) levels before ovulation in female rats, indicating the induction of EPHA7 in ovulation. In this study, we aimed to identify the mechanism and hypothalamus-pituitary-ovary (HPO) axis level underlying the promotion of LH secretion by EPHA7. Using an ovariectomized (OVX) rat model, in conjunction with low-dose 17ß-estradiol (E2) treatment, we investigated the association between EPHA7-ephrin (EFN)A5 signaling and E2 negative feedback. Various rat models (OVX, E2-treated OVX, and abarelix treated) were injected with the recombinant EPHA7-Fc protein through the caudal vein to investigate the molecular mechanism underlying the promotion of LH secretion by EPHA7. Efna5 was observed strongly expressed in the arcuate nucleus of the female rat by using RNAscope in situ hybridization. Our results indicated that E2, combined with estrogen receptor (ER)α, but not ERß, inhibited Efna5 and gonadotropin-releasing hormone 1 (Gnrh1) expressions in the hypothalamus. In addition, the systemic administration of EPHA7-Fc restrained the inhibition of Efna5 and Gnrh1 by E2, resulting in increased Efna5 and Gnrh1 expressions in the hypothalamus as well as increased serum LH levels. Collectively, our findings demonstrated the involvement of EPHA7-EFNA5 signaling in the regulation of LH and the E2 negative feedback pathway in the hypothalamus, highlighting the functional role of EPHA7 in female reproduction.

CSF1R inhibitor PLX5622 and environmental enrichment additively improve metabolic outcomes in middle-aged female mice.

As the elderly population grows, chronic metabolic dysfunction including obesity and diabetes are becoming increasingly common comorbidities. Hypothalamic inflammation through CNS resident microglia serves as a common pathway between developing obesity and developing systemic aging pathologies. Despite understanding aging as a life-long process involving interactions between individuals and their environment, limited studies address the dynamics of environment interactions with aging or aging therapeutics. We previously demonstrated environmental enrichment (EE) is an effective model for studying improved metabolic health and overall healthspan in mice, which acts through a brain-fat axis. Here we investigated the CSF1R inhibitor PLX5622 (PLX), which depletes microglia, and its effects on metabolic decline in aging in interaction with EE. PLX in combination with EE substantially improved metabolic outcomes in middle-aged female mice over PLX or EE alone. Chronic PLX treatment depleted 75% of microglia from the hypothalamus and reduced markers of inflammation without affecting brain-derived neurotrophic factor levels induced by EE. Adipose tissue remodeling and adipose tissue macrophage modulation were observed in response to CSF1R inhibition, which may contribute to the combined benefits seen in EE with PLX. Our study suggests benefits exist from combined drug and lifestyle interventions in aged animals.

Vasopressin regulates hypothalamic GnRH synthesis: Histomorphological evidence in hypothalamus and biological effects in GT1-7 cells.

AIMS: To investigate the direct histomorphological clues and observe the biological effects of VP acting on gonadotropin-releasing hormone (GnRH) secretion. MAIN METHODS: Immunofluorescence was conducted to investigate the expressions of GnRH and VP in experimental left varicocele (ELV) rats and ELV repair rats. The colocalization of GnRH and VP was observed by electron microscopy immunohistochemistry. The protein-protein interaction between GnRH and VP was tested by co-immunoprecipitation (co-IP) and the proximity ligation assay (PLA). The effects of intracellular and extracellular VP on GnRH and relative transcription factors (Oct-1, Otx2, Pbx1b and DREAM) were respectively evaluated in VP overexpressed and VP treated GT1-7 cells. KEY FINDINGS: Both hypothalamic GnRH and VP decreased in ELV rats and recovered by ELV repair. The overlapped immunolocalizations of GnRH and VP mainly distributed in the lateral part of the arcuate nucleus (ArcL) and median eminence (ME) with a Manders' overlap coefficient of 0.743 ±â€¯0.117. Immunoreactive substances of GnRH and VP existed in the same and adjacent terminals. VP overexpression did not cause any significant effects on the expressions of GnRH and Oct-1, as well as GnRH promoter activity. While 50-200 pg/ml VP treatments increased GnRH mRNA levels in a dose- and time-dependent manner in GT1-7 cells. Additionally, 200 pg/ml VP triggered a marked promotion of expressions of GnRH, Oct-1, Oxt2 Pbx1b and DREAM, as well as GnRH promoter activity (P < 0.05). SIGNIFICANCE: The results reveal the colocalization and interaction of VP and GnRH, which will be conducive to explain the effects and mechanisms of VP acting on reproduction.

Obestatin may affect the GnRH/KNDy gene network in sheep hypothalamus.

The effects of obestatin on gonadotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the effect of intracerebroventricular infusions of obestatin on the gonadotrophin-releasing hormone (GnRH) mRNA and protein expressions as well as on KNDy mRNA and kisspeptin (Kiss) peptide expressions in peripubertal female sheep. Animals were randomly divided into two groups: the control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h-1). The series of four 1-h infusions per day during three consecutive days were performed. After the end of the experiment parts of sheep brains were fixed in situ for immunohistochemical analysis, while the remaining brains were frozen for Real Time qPCR analysis. Substantial changes in the activity of the GnRH and KNDy gene network were observed in obestatin-infused sheep. In those animals an increase of GnRH mRNA expression in the preoptic area, a decrease of GnRH mRNA expression in the median eminence and an increase of GnRH immunoreactivity in the median eminence were found. Moreover, changes in the KNDy mRNA expression in mediobasal hypothalamus as well as decrease Kiss expression in arcuate nucleus and median eminence were observed. It was revealed that obestatin affects the GnRH and KNDy gene network as well as Kiss at the level of mRNA and protein expression. Thereby, it can be concluded that obestatin participates in the mechanism modulating gonadotrophic axis activity at the central level in peripubertal female sheep.

Progesterone-induced amplification and advancement of GnRH/LH surges are associated with changes in kisspeptin system in preoptic area of estradiol-primed female rats.

The time course effects of ovarian steroids on kisspeptin and GnRH/LH systems is not totally clarified. We investigated the temporal relationship among kisspeptin and GnRH mRNA and kisspeptin content in the preoptic area (POA), GnRH content and release in the medial basal hypothalamus (MBH) and plasma LH levels under different steroid treatments. Ovariectomized rats treated with oil (OVOO), oil plus single dose of estradiol (OVOE), oil plus single dose of progesterone (OVOP), estradiol for 3 days plus oil (OVEO) or estradiol for 3 days plus progesterone (OVEP) were hourly decapitated from 10:00 to 17:00 or had the MBH microdialyzed from 09:00 to 19:00. Estradiol and progesterone acutely increased POA kisspeptin content without altering POA kisspeptin mRNA levels. Short-term exposure to both hormones stimulated MBH GnRH content, although no GnRH/LH surges had occurred. Chronic estradiol-treatment increased both kisspeptin mRNA levels and content in the POA, demonstrating that long exposure to estradiol is required to activate the whole kisspeptin synthesis machinery. This was followed by the peak in the GnRH/LH release. In estradiol-primed rats, progesterone further increased POA kisspeptin content, amplified and advanced GnRH/LH surges, with no additional change on POA kisspeptin mRNA. The data show an estradiol-induced temporal association between kisspeptin increase in the POA and GnRH/LH surges. Interestingly, the classic action of progesterone in amplifying and accelerating the GnRH/LH surges seems to occur by a mechanism which involves POA kisspeptin system.

Acute Influences of Bisphenol A Exposure on Hypothalamic Release of Gonadotropin-Releasing Hormone and Kisspeptin in Female Rhesus Monkeys.

Bisphenol A (BPA) is an industrial compound with pervasive distribution in the environments of industrialized countries. The U.S. Centers for Disease Control recently found that greater than 90% of Americans carry detectable levels of BPA, raising concern over the direct influences of this compound on human physiology. Epidemiologic evidence links elevated BPA serum concentrations to human reproductive dysfunction, although controlled studies on the acute effect of BPA exposure on reproductive function are limited, particularly in primates. We evaluated the effect of direct BPA exposure on female primate hypothalamic peptide release. Specifically, using a microdialysis method, we examined the effects of BPA (0.1, 1, and 10nM) directly infused to the stalk-median eminence on the release of GnRH and kisspeptin (KP) in mid to late pubertal ovarian intact female rhesus monkeys. We found that the highest level of BPA exposure (10nM) suppressed both GnRH and KP release, whereas BPA at lower concentrations (0.1 and 1nM) had no apparent effects. In addition, we measured BPA in plasma and hypothalamic dialysates after an iv bolus injection of BPA (100 µg/kg). We found a relatively stable distribution of BPA between the blood and brain (plasma:brain ≅ 5:1) persists across a wide range of blood BPA concentrations (1-620 ng/mL). Findings of this study suggest that persistent, high-level exposures to BPA could impair female reproductive function by directly influencing hypothalamic neuroendocrine function.

The advancement of the onset of vaginal opening in female rats subjected to chronic testosterone treatment occurs independently of hypothalamic Kiss1 and RFRP expression.

OBJECTIVE: The neonatal and/or prepubertal androgen milieu affects sexual maturation. In rodents, neonatal chronic testosterone treatment, which is used as a model of polycystic ovary syndrome (PCOS), results in the onset of vaginal opening occurring earlier in the pubertal period. DESIGN: In the present study, the changes in hypothalamic Kiss1 (a gonadotropin-releasing hormone (GnRH)-stimulating factor) and RF-amide related peptide (RFRP; a GnRH inhibitory factor) mRNA expression induced by testosterone treatment were examined in order to clarify whether these factors are involved in the testosterone-induced acceleration of sexual maturation. RESULTS: The onset of vaginal opening occurred earlier and uterine weight was increased in female rats subjected to chronic (from postnatal day 23 to day 31) testosterone treatment. Contrary to our expectations, the rats' hypothalamic Kiss1 and Kiss1 receptor mRNA levels were not changed, and their serum luteinizing hormone (LH) levels were decreased. Although hypothalamic RFRP mRNA expression was decreased in the testosterone-treated rats, this change was not reflected in their serum LH levels. CONCLUSIONS: These results indicate that the advancement of sexual maturation observed in chronic testosterone-treated rats might be caused by a peripheral, rather than a central, mechanism.

Prenatal androgen excess enhances stimulation of the GNRH pulse in pubertal female rats.

In adolescent girls with polycystic ovary syndrome (PCOS), neuroendocrine derangements manifest after the onset of puberty, characterized by rapid LH pulse frequency. The early mechanism underlying the pubertal regulation of the GNRH/LH pulsatile release in adolescents with PCOS remains uncertain. To determine the effects of prenatal androgen exposure on the activation of GNRH neurons and generation of LH pulse at puberty, we administrated 5α-dihydrotestosterone to pregnant rats and observed serum LH levels and expression of hypothalamic genes in female offspring from postnatal 4 to 8 weeks. The 6-week-old prenatally androgenized (PNA) female rats exhibited an increase in LH pulse frequency. The hypothalamic expression of neurokinin B (Nkb (Tac2)) and Lepr mRNA levels in PNA rats increased remarkably before puberty and remained high during puberty, whereas elevated Kiss1 mRNA levels were detected only after the onset of puberty. Exogenous kisspeptin, NK3R agonist, and leptin triggered tonic stimulation of GNRH neurons and increased LH secretion in 6-week-old PNA rats. Leptin upregulated Kiss1 mRNA levels in the hypothalamus of pubertal PNA rats; however, pretreatment with a kisspeptin antagonist failed to suppress the elevated serum LH stimulated by leptin, indicating that the stimulatory effects of leptin may be conveyed indirectly to GNRH neurons via other neural components within the GNRH neuronal network, rather than through the kisspeptin-GPR54 pathway. These findings validate the hypotheses that NKB and leptin play an essential role in the activation of GNRH neurons and initiation of increased LH pulse frequency in PNA female rats at puberty and that kisspeptin may coordinate their stimulatory effects on LH release.

Probable gamma-aminobutyric acid involvement in bisphenol A effect at the hypothalamic level in adult male rats.

The aim of the present study was to investigate the effects of bisphenol A (BPA) on the neuroendocrine mechanism of control of the reproductive axis in adult male rats exposed to it during pre- and early postnatal periods. Wistar mated rats were treated with either 0.1% ethanol or BPA in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 2.5 mg/kg body weight per day of BPA. After 21 days, the pups were separated from the mother and sacrificed on 70 day of life. Gn-RH and gamma-aminobutyric acid (GABA) release from hypothalamic fragments was measured. LH, FSH, and testosterone concentrations were determined, and histological and morphometrical studies of testis were performed. Gn-RH release decreased significantly, while GABA serum levels were markedly increased by treatment. LH serum levels showed no changes, and FSH and testosterone levels decreased significantly. Histological studies showed abnormalities in the tubular organization of the germinal epithelium. The cytoarchitecture of germinal cells was apparently normal, and a reduction of the nuclear area of Leydig cells but not their number was observed. Taken all together, these results provide evidence of the effect caused by BPA on the adult male reproductive axis when exposed during pre- and postnatal period. Moreover, our findings suggest a probable GABA involvement in its effect at the hypothalamic level.

Organization of two independent kisspeptin systems derived from evolutionary-ancient kiss genes in the brain of zebrafish.

Kisspeptins are new actors in the neuroendocrine regulation of reproduction. In vertebrates, the number of kiss genes varies from none to three. Zebrafish have two kiss genes, kiss1 and kiss2, and two kiss receptors (GPR54), kiss1r and kiss2r. To provide detailed information on the organization of the kiss systems in zebrafish, antibodies were raised against the C terminus of zebrafish preproKiss1 and preproKiss2. Immunohistochemistry fully confirmed in situ hybridization data, showing that kiss1-expressing neurons are only located in the habenular nucleus, while kiss2-expressing neurons are found in the dorsal and ventral hypothalamus. Kiss1-expressing cells project only to the interpeduncular and raphe nuclei and strongly expressed the kiss1r receptor. In contrast, kiss2-expressing cells are mostly present in the dorsal and ventral hypothalamus and project widely into the subpallium, the preoptic area, the thalamus, the ventral and caudal hypothalamus, and the mesencephalon. All these regions strongly expressed the kiss2r messengers. Kiss2 fibers profusely innervate the ventral forebrain and notably made close apposition with GnRH3 neurons. Estrogen treatment of juvenile fish with estradiol causes increase in kiss2 and kiss2r expression. In the pituitary gland, no proKiss2- positive fibers were detected, while positive cells were observed in the pars intermedia. In addition to proposing a successful strategy to develop antibodies to kisspeptins, these data indicate that the kiss2 systems of zebrafish are implicated in reproductive events, while the kiss1 gene would play other functions that remain to be established.

Noradrenergic control of GnRH release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study investigates the influence of alpha(1)-adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates alpha(1)-adrenoreceptor-GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media-alpha (MEM-alpha) at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to specific alpha(1)-adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1-10 mm). The alpha(1)-adrenoreceptor agonist (10 mm) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, alpha(1)-adrenoreceptor agonist (10 mm)-induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus-pituitary sequential double-chamber perifusion. Only after exposure of hypothalamic slices to alpha(1)-adrenoreceptor agonist (10 mm), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 +/- 12.3-36.2 +/- 21.6 ng/ml) confirming that the alpha(1)-adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.

The Effects of GABA on embryonic gonadotropin-releasing hormone neurons in rat hypothalamic primary culture.

Gonadotropin-releasing hormone (GnRH) neurons arise in the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence during embryogenesis. Little information is available concerning the properties of GnRH neurons during the late gestational period when GnRH neurons reach the POA and form neuronal networks, although many studies have examined such properties during earlier developmental stages or the postnatal period. The present study was performed to elucidate the involvement of gamma-aminobutyric acid (GABA), one of the major neurotransmitters modifying GnRH neural activity, in regulation of GnRH gene expression on embryonic day 18.5 (E18.5) using transgenic rats expressing enhanced green fluorescence protein (EGFP) under the control of GnRH promoter. First, using RT-PCR, the mRNA of two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), GAD65 and GAD67 was detected in E18.5 embryonic POA-containing tissues. GAD67-positive cells were also demonstrated in close vicinity to GnRH-positive cells by immunohistochemistry, and immunoreactivity for both the GABA-A and GABA-B receptor subunits was detected in GnRH neurons. Next, primary cultures derived from anterior hypothalamic tissue of E18.5 embryos were prepared, and the effects of GABA and its agonists on GnRH promoter activity were evaluated using EGFP expression as a marker. GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, significantly increased the EGFP-positive/GnRH-positive cell ratio. These results suggest that GABA plays a role in stimulating GnRH gene expression through GABA-A receptors in embryonic GnRH neurons in late gestational stages.

The effect of intracerebroventricular infusions of leptin on the immunoreactivity of neuropeptide Y and gonadotrophin releasing hormone neurons in the hypothalamus of prepubertal sheep in conditions of short fasting.

In the study we evaluated the effects of infusion of exogenous leptin to the third ventricle of the brain on the expression of immunoreactive (ir) neuropeptide Y (NPY) neurons in the hypothalamus and ir gonadotrophin releasing hormone (GnRH) nerve terminals in the median eminence of prepubertal lambs in the conditions of short fasting. Merino female sheep (n=16) were randomly divided into four groups, two fed with standard feeds and two fasted for 72 h. One standard and one fasted groups were infused with Ringer saline (controls), remaining standard and fasted groups with leptin (25 microg/120 microl/h), for 4 h during three consecutive days, and then slaughtered. Ir NPY and ir GnRH were localized by immunohistochemistry using specific polyclonal antibodies. Detection of both hormones was followed by the image analysis and expressed as the percent area stained and integral density of immunostaining. In the hypothalami from all groups the ir NPY perikarya and varicose nerve fibers were localized in three distinct sub-areas, in the arcuate (ARC), paraventricular and periventricular nuclei. In fasted sheep the percent area and integral density for immunoreactivity of NPY increased significantly (P<0.001) in three sub-areas compared to the standard-fed animals. Leptin infusion lowered the both parameters (P<0.001) but solely in the ARC NPY population of fasted sheep. The percent area and integral density of immunostaining for ir GnRH in fasted sheep revealed the augmentation (P<0.001) compared to standard-fed sheep. Leptin infusions diminished (P<0.001) both parameters in fasted, without effects in standard-fed lambs. In conclusion, the enhanced by fasting immunoreactivity of the ARC NPY perikarya and varicose nerve fibers and restrained immunoreaction of GnRH terminals in the median eminence were reversed by exogenous leptin. It is suggested that leptin can affect GnRH release via ARC NPY neurons in conditions of deficit of nutrients in prepubertal, female lambs.

Regulation of gonadotropin-releasing hormone secretion by cannabinoids.

Cannabinoids (CBs) exert untoward effects on reproduction by reducing LH secretion and suppressing gonadal function. Recent evidence suggests these effects are due primarily to hypothalamic dysfunction; however, the mechanism is obscure. Using immortalized hypothalamic GnRH neurons, we find these cells produce and secrete at least two different endocannabinoids. After release, 2-arachidonyl monoacylglycerol and anandamide are rapidly transported into GnRH neurons and are degraded to other lipids by fatty-acid amide hydrolase. The immortalized GnRH neurons also possess CB1 and CB2 receptors that are coupled to Gi/Go proteins whose activation leads to inhibition of GnRH secretion. In perifusion experiments, CBs block pulsatile release of GnRH. When a CB receptor agonist is delivered into the third ventricle of adult female mice, estrous cycles are prolonged by at least 2 d. Although in situ hybridization experiments suggest either that GnRH neurons in vivo do not possess CB1 receptors or that they are very low, transcripts are localized in close proximity to these neurons. Inasmuch as GnRH neurons in vivo possess G protein receptors that are coupled to phospholipase C and increased intracellular Ca2+, these same neurons should also be able to synthesize endocannabinoids. These lipids, in turn, could bind to CB receptors on neighboring cells, and perhaps GnRH neurons, to exert feedback control over GnRH function. This network could serve as a novel mechanism for regulating GnRH secretion where reproductive functions as diverse as the onset of puberty, timing of ovulation, duration of lactational infertility, and initiation/persistence of menopause may be affected.

Expression and differential effects of the activation of glucocorticoid receptors in mouse gonadotropin-releasing hormone neurons.

Prenatal exposure of rodents to glucocorticoids (Gc) affects the sexual development of the offspring, possibly interfering with the differentiation of the hypothalamic-pituitary-gonadal axis. Glucocorticoid receptors (GR) are present on gonadotropin-releasing hormone (GnRH) neurons in the rat hypothalamus, suggesting a direct effect of Gc in the control of the synthesis and/or release of the hormone. In this study, we demonstrate the colocalization of immunoreactive GR with GnRH in a subpopulation of mouse hypothalamic GnRH neurons, confirming the possible involvement of Gc in mouse GnRH neuronal physiology. Receptor-binding assay, RT-PCR, immunocytochemistry, and immunoblotting experiments carried out in GN11 immortalized GnRH neurons show the presence of GR even in the more immature mouse GnRH neurons and confirm the expression of GR in GT1-7 mature GnRH cells. In GN11 cells, the activation of GR with dexamethasone produces nuclear translocation, but does not lead to the inhibition of GnRH gene expression already reported in GT1-7 cells. Long-term exposure of GN11 cells to dexamethasone induces an epithelial-like phenotype with a reorganization of F-actin in stress fibers. Finally, we found that Gc treatment significantly decreases the migratory activity in vitro and the levels of phosphorylated focal adhesion kinase of GN11 immature neurons. In conclusion, these data indicate that GR are expressed in mouse hypothalamic GnRH neurons in vivo as well as in the immature GN11 GnRH neurons in vitro. Moreover, the effects of the GR activation in GN11 and in GT1-7 cells may be related to the neuronal maturational stage of the two cell lines, suggesting a differential role of Gc in neuronal development.

Regulation of cyclic adenosine 3',5'- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons.

Immortalized GnRH neurons (GT1-7) express receptors for estrogen [estrogen receptor-alpha and-13(ERa and ERI3)] and progesterone (progesterone receptor A) and exhibit positive immunostaining for both intracellular and plasma membrane ERs. Exposure of GT1-7 cells to picomolar estradiol concentrations for 5-60 min caused rapid, sustained,and dose-dependent inhibition of cAMP production. In contrast, treatment with nanomolar estradiol concentrations for 60 min increased cAMP production. The inhibitory and stimulatory actions of estradiol on cAMP formation were abolished by the ER antagonist, ICI 182,780. The estradiol-induced inhibition of cAMP production was prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrated an estradiol-regulated stimulatory interaction between ERa and G,3 that was prevented by the ER antagonist, ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physio-logical estradiol levels causes activation of a G, protein and modulates cAMP signaling and neuropeptide secretion.

Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression.

The physiological actions of nitric oxide (NO) as a signaling molecule in endothelial and brain cells and as a toxic molecule used by activated immune cells have been the focus of a wide range of studies. Nevertheless, the downstream effector molecules of this important neuromodulator are not well understood. We have previously demonstrated that expression of the gene for the reproductive neuropeptide, GnRH, is repressed by the glutamate/NO/cyclic GMP (cGMP) signal transduction pathway through cGMP-dependent protein kinase in the hypothalamic GnRH-secreting neuronal cell line GT1-7. This repression localized within a previously characterized 300-bp neuron-specific enhancer. Here, we find that mutation of either of two adjacent elements within the enhancer eliminates repression by this pathway. An AT-rich sequence located at -1695 has homology to the octamer motif known to bind POU-homeodomain proteins, while the adjacent element at -1676 has homology to the C/EBP (CCAAT/enhancer-binding protein) protein family consensus sequence. Antibody supershift assays reveal that one of the proteins bound at the -1695 sequence is Oct-1, and one of the proteins bound to the element at -1676 is C/EBPbeta. These two proteins can bind simultaneously to the adjacent -1695 and -1676 binding sites in vitro. In nuclear extracts of GT1-7 cells treated with an NO donor, the intensity of the Oct-1 complex is increased. However, although Western blot analysis indicates that neither Oct-1 nor C/EBPbeta protein levels are increased, the relative binding affinity of Oct-1 is increased. Dephosphorylation of the nuclear extracts decreases binding of the Oct-1 complex to the -1695 site only in NO donor-treated extracts. Thus, we conclude that Oct-1 and C/EBPbeta are both downstream transcriptional regulators involved in the repression of GnRH gene expression by the glutamate/NO/ cGMP signal transduction pathway.