GnRH Gene Research Articles

In VivoIdentification of a 107-Base Pair Promoter Element Mediating Neuron-Specific Expression of Mouse Gonadotropin-Releasing Hormone

To identify regions of the mouse GnRH (mGnRH) promoter that mediate tissue-specific gene expression, transgenic mice have been generated with fragments of mGnRH promoter fused to the luciferase reporter gene. In this manuscript, we examine transgenic mice, generated with -356/+28 bp and -249/+28 bp of the mGnRH gene. Both fragments of mGnRH promoter target ovarian expression of the luciferase transgene, but neuronal luciferase activity is detected only in the mice bearing the -356-bp fragment, suggesting that the DNA sequences essential for directing neuron-specific expression of the GnRH gene are located between -356 and -249 bp. Two consensus binding sites for Otx2 were identified in this promoter region and were confirmed to be functional. EMSAs demonstrated specific binding of Otx2 to the mGnRH promoter, and overexpression of Otx2 increased transcriptional activity of the mGnRH promoter in transient transfection studies. When both Otx2 binding sites were eliminated, overexpression of Otx2 had no effect. GnRH mRNA expression in immortalized GnRH-secreting cell lines was also found to correlate with Otx2 expression. In addition, transgenic mice, bearing the 356 fragment of the mGnRH gene in which the Otx2 binding sites were eliminated, have significantly lower luciferase activity in the neonatal brain compared with mice generated with intact Otx2 binding sites. Luciferase activity was, however, still present in the ovary. Our findings provide evidence that Otx2 may have a critical role in directing tissue-specific expression of the mGnRH gene to the neuron, but not the ovary.

Two novel GnRHR gene mutations in two siblings with hypogonadotropic hypogonadism

Mutations in the gonadotropin-releasing hormone receptor (GnRHR) gene are the cause of isolated hypogonadotropic hypogonadism (HH). We describe the molecular investigations of the GnRHR gene in two siblings affected by HH and their clinical course. The female was referred at age 14 for pubertal delay with no secondary sexual signs, whereas the male had been followed since prepuberty. Hormonal evaluation showed very low levels of gonadotropins, luteinizing hormone-releasing hormone test (LHRH test) and sexual steroids in both patients, suggesting a possible defect in the mechanism of action of the GnRH gene on its receptor. The GnRHR gene of the two siblings and their parents were analyzed by PCR followed by direct sequencing. Two new single nucleotide substitutions resulting in the T104I and the Y108C substitutions in the first extracellular loop (ECL1) were identified in both siblings. The molecular analysis confirmed the carrier status of the parents. We identified two new missense mutations in the GnRHR gene in two siblings with HH. The nature of the substitutions lying in the ECL1 involved in the ligand-receptor interaction, as well as the high conservation of the two residues in all mammalian GnRHR, are suggestive of some implications in the phenotype observed.

Control of GnRH neuronal activity by metabolic factors: The role of leptin and insulin

Energy balance exerts a critical influence on reproductive function. Leptin and insulin are among the metabolic factors signaling the nutritional status of an individual to the hypothalamus, and their role in the overall modulation of the activity of GnRH neurons is increasingly recognized. The experiments described here were designed to further investigate the central mechanisms of action of these two hormones and the precise hypothalamic pathways implicated in their effects on the reproductive axis. NPY neurons represent a primary target of leptin actions within the hypothalamus We used mice lacking the NPY Y1 receptor (Y1 −/− mice) to investigate the physiological importance of the hypothalamic NPY neuronal system and its downstream pathways involving Y1 in the reproductive effects of leptin. Results point to a crucial role for the NPY Y1 receptor in the control of the onset of puberty and the maintenance of reproductive functions by leptin. A striking finding of these experiments was the observation that juvenile Y1 −/− mice submitted to food restriction can proceed through puberty like normally fed animals, demonstrating that the absence of Y1 impairs the perception of decreasing energy stores by the gonadotrope axis. Next, we used parallel in vivo and in vitro experiments to delineate the role of insulin in the stimulation and maintenance of the activity of the neuroendocrine reproductive axis. First, we observed that the increase in circulating insulin levels achieved during hyperinsulinemic clamp studies in normal male mice was associated with a significant rise in LH secretion. This effect of insulin is likely mediated at the hypothalamic level, as insulin stimulates the secretion and the expression of GnRH by hypothalamic neurons in culture. Using primary neuronal cultures as well as a novel GnRH neuronal cell line obtained by conditional immortalization of adult rat hypothalamic neurons, we have recently demonstrated that this effect of insulin on GnRH gene expression is probably mediated directly at the level of GnRH neurons, and involves the stimulation of the MAP kinase Erk1/2 pathway. Taken together, these results provide new insights into the mechanisms involved in the regulation of GnRH neuronal activity by metabolic factors.

The gonadotropin-releasing hormone (GnRH)-1 gene, the GnRH receptor gene, and their promoters in patients with idiopathic hypogonadotropic hypogonadism with or without resistance to GnRH action

The study included 26 patients with idiopathic hypogonadotropic hypogonadism (24 sporadic and 2 familial). The Pro146Ser mutation was identified in the gonadotropin-releasing hormone receptor (GnRHR) gene in two sisters as well as in their mother, and one polymorphism in the GnRH1 gene (the Trp16Ser) was identified in four patients. No mutations in transcription factor-binding sites of their promoters were identified. Three patients (one male and two sisters) were found with resistance to GnRH action. No mutations were identified in the male, whereas in the females the mutation Pro146Ser in the GnRHR was identified in heterozygosity.

Identification of a novel OCT1 binding site that is necessary for the elaboration of pulses of rat GnRH promoter activity

Recent evidence from our laboratory demonstrated that the OCT1 protein was necessary for GnRH gene promoter pulse activity through its interaction with a specific OCT1 binding site (OCT1BS-a, −1774/−1781). In light of the importance of this POU homeoprotein in pulsatile function, we focused on two other highly conserved OCT1 sites within this region, OCT1BS-b (−1694/−1701, previously AT-b), and OCT1BS-c (−1569/−1562). Mutagenesis of these sites revealed that alteration of OCT1BS-c, but not OCT1BS-b, virtually abolished gene expression pulses in GT1-7 cells. EMSAs confirmed that OCT1 can bind to both sites. Taken together, our findings demonstrate clearly that more than one Oct1 binding site is necessary for GnRH promoter pulses. Moreover, the lack of an influence observed with OCT1BS-b on pulse activity indicates that OCT1 action is not general to all OCT1 sites, but specific to certain octamer sequences in the NSE region of the GnRH promoter.

Feedback Loops Link Odor and Pheromone Signaling with Reproduction

Pheromones can have profound effects on reproductive physiology and behavior in mammals. To investigate the neural circuits underlying these effects, we used a genetic transneuronal tracer to identify neurons that synapse with GnRH (LHRH) neurons, the key regulators of reproduction. We then asked whether the connected neurons are presynaptic or postsynaptic to GnRH neurons and analyzed their responses to chemosensory cues. Surprisingly, these experiments indicate that GnRH neurons receive pheromone signals from both odor and pheromone relays in the brain and may also receive common odor signals. Moreover, feedback loops are evident whereby GnRH neurons could influence both odor and pheromone processing. Remarkably, approximately 800 GnRH neurons communicate with approximately 50,000 neurons in 53 functionally diverse brain areas, with some connections exhibiting sexual dimorphism. These studies reveal a complex interplay between reproduction and other functions in which GnRH neurons appear to integrate information from multiple sources and modulate a variety of brain functions.

Insulin regulation of GnRH gene expression through MAP kinase signaling pathways

In mammals, reproduction is acutely regulated by metabolic status. Insulin is an important nutritional signal from the periphery that may regulate the reproductive axis. To determine whether insulin acts directly on the GnRH neuron, we performed studies in mouse-derived GnRH-expressing cell lines. Both insulin receptor protein and mRNA were detected in these cells. A saturation radioligand binding assay revealed high affinity, low capacity binding sites for insulin in GnRH neurons. Insulin also stimulated GnRH promoter activity in GnRH neurons. This effect was blocked by pretreatment with the MEK inhibitor, PD98059, indicating a role for MAP kinase signaling. In transient transfection studies, insulin treatment stimulated expression of a 1250 bp mouse GnRH gene promoter fragment four-fold when compared to promoter activity in untreated cells. In contrast, insulin did not stimulate activity of a 587 bp fragment of the mGnRH gene promoter, indicating that the promoter elements mediating insulin stimulation of the GnRH promoter are located between −1250 and −587 bp. Our studies suggest that insulin may regulate reproductive function by direct effects on the GnRH neurons and specifically by stimulating GnRH gene expression.

Cloning and functional analysis of promoters of three GnRH genes in a cichlid

Mechanisms regulating gonadotropin-releasing hormone (GnRH) types, a key molecule for reproductive physiology, remain unclear. In the present study, we cloned the promoters of GnRH1, GnRH2, and GnRH3 genes in the tilapia, Oreochromis niloticus; and found putative binding sites for glucocorticoid receptors, Sp1, C/EBP, GATA, and Oct-1, but not for androgen receptors in all three GnRH promoters using computer analysis. The presence of binding sites for progesterone receptors in GnRH1, estrogen receptors in GnRH1 and GnRH2, and thyroid hormone receptors in GnRH1 and GnRH3 suggests direct action of steroid hormones on GnRH types. Our observation of SOX and LINE-like sequences exclusively in GnRH1, COUP in GnRH2, and retinoid X receptors in GnRH3 suggests their role in sexual differentiation, midbrain segmentation, and visual cue integration, respectively. Thus, the characteristic binding sites for nuclear receptors and transcription factors support the notion that each GnRH type is regulated differently and has distinct physiological roles.

Chromosomal Organization, Evolutionary Relationship, and Expression of Zebrafish GnRH Family Members

Multiple forms of gonadotropin-releasing hormone (GnRH) are found in different vertebrates. In this study, we have cloned cDNA encoding the full-length gnrh3 and gnrh2 from zebrafish brain and characterized their structure and expression patterns. We performed phylogenetic analysis and compared conserved syntenies in the region surrounding the GnRH genes from human, chicken, pufferfish, and zebrafish genores. The gnrh3 and gnrh2 genes were mapped to LG17 and LG21, respectively. The zebrafish genome appears to lack an ortholog to human GNRH1, and the human genome appears to lack an ortholog of gnrh3. Expression of gnrh3 began in the olfactory pit at 24-26 h postfertilization and expanded to the olfactory bulb during early larval stage. Expression of gnrh2 is always in the midbrain. In addition, GnRH is also expressed in boundary cells surrounding seminiferous cysts of the testis. Thus, this detailed phylogenetic, chromosomal comparison, and expression study defines the identity and the evolutionary relationship of two zebrafish gnrh genes. We propose a model describing the evolution of gnrh genes involving ancestral duplication of the genes followed by selective loss of one gene in some teleosts.

The Protein Kinase C Pathway Acts through Multiple Transcription Factors to Repress Gonadotropin-Releasing Hormone Gene Expression in Hypothalamic GT1–7 Neuronal Cells

The GnRH gene uses two well-defined regions to target expression to a small population of hypothalamic GnRH neurons: a 173-bp proximal promoter and a 300-bp enhancer localized at approximately -1800 to -1500 bp from the start site. Interaction of multiple factors with the GnRH enhancer and promoter is required to confer neuron-specific expression in vivo and in cells in culture. In addition, the expression of the GnRH gene is regulated by numerous neurotransmitters and hormones. Several of these effectors act through membrane receptors to trigger the protein kinase C pathway, and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a modulator of this pathway, has been shown to suppress GnRH gene expression through the promoter. We find that TPA suppresses expression through the GnRH enhancer as well as the promoter. In the enhancer, an Oct-1 binding site, a Pbx/Prep binding site, Msx/Dlx binding sites, and a previously unidentified protein-binding element at -1793, all contribute to TPA suppression. TPA treatment leads to decreased binding of Oct-1 and Pbx1a/Prep to their sites. However, a complex formed by GT1-7 nuclear extracts on the -1793 site is not affected by TPA treatment. It is known that cooperative interaction among multiple factors is necessary for GnRH gene expression; thus, one mechanism by which TPA suppresses GnRH gene expression is to disengage some of these factors from their cis-regulatory elements.

GnRH is expressed in GnRH-deficient hypogonadal (hpg) mice after GnRH gene transfer with a herpes simplex virus (HSV) amplicon vector

The challenge of gene therapy is to develop non-toxic vectors that can achieve stable transgene expression in target cells. The HSV amplicon is a unique vector that has essentially no toxicity, a large transgene capacity, and the potential to integrate the transgene into a specific site in the genome, thus making it a useful tool for gene therapy. Hpg mice are genetically deficient in GnRH production in the hypothalamus, resulting in many deficits in the reproductive system including infertility. Our goal was to determine neuronal expression of GnRH and the recovery of gonadal function after CNS delivery of an HSV amplicon vector containing the GnRH gene using hpg mice. An HSV amplicon containing the GnRH gene under its cognate promoter was prepared and packaged using a helper virus-free packaging system. This vector was injected into the preoptic area (POA) of the hypothalamus of hpg mice using a stereotaxic apparatus. Following injection, we determined GnRH neuronal expression by immunohistochemistry (IHC) and recovery of gonadal function using vaginal cytology and blood hormonal assays. Eight adult female hpg mice were studied. Five mice were injected with the HSV-GnRH vector in the POA, and 3 mice were injected with vehicle only as control. The HSV-GnRH vector (1× 108 transducing units/ml) was injected into the POA of hpg mice under anesthesia using a stereotaxic apparatus. Mice were sacrificed 10 days (n=4) or 50 days (n=1) after injection. Neuronal expression of GnRH was analyzed by IHC in brain tissue. Plasma FSH and LH levels were measured by radioimmunoassay. The weights of the ovary, uterus and oviduct were compared between the study and control groups. Vaginal cytology was also checked each day after HSV amplicon injection to evaluate estrous cyclicity. GnRH expression was present in neurons near the injection site, as determined by IHC 10 days and 50 days following the vector injections. However, there was no significant difference in plasma FSH (2.0 ± 0.7 ng/ml vs. 4.9 ± 0.9 ng/ml; study vs. control), LH (0.07 ± 0.01 ng/ml vs. 0.06 ± 0.02 ng/ml) levels, or in the weight of reproductive organs (24.2 ± 6.4 mg vs. 17.9 ± 2.7 mg) between the study and control groups. We observed only diestrus specific morphology in the vaginal epithelial cells. Expression of GnRH was observed in the hypothalamic neurons in the POA in GnRH-deficient hpg mice following GnRH gene transfer using an HSV amplicon vector. These data suggest that such HSV vectors may be useful in correcting the reproductive defect in these animals. Further studies are necessary to determine if normal hypothalamic-pituitary-gonadal axis function can be restored in hpg mice by gene therapy using this approach.

The role of Otx2 in the differential expression of neuronal and ovarian GnRH

Our previous data demonstrated mouse GnRH (mGnRH) gene promoter activity in ovarian granulosa cells as well as in hypothalamic neurons. We also identified two binding sites for Otx2, between -356 bp and -249 bp, in the mouse GnRH gene promoter. Our objective was to determine whether binding of Otx2, a homeodomain transcription factor, to these sites in the mouse GnRH gene promoter has a role in the differential expression of neuronal and ovarian GnRH in vivo.

Phylogenetic footprinting reveals evolutionarily conserved regions of the gonadotropin-releasing hormone gene that enhance cell-specific expression.

Reproductive function is controlled by the hypothalamic neuropeptide, GnRH, which serves as the central regulator of the hypothalamic-pituitary-gonadal axis. GnRH expression is limited to a small population of neurons in the hypothalamus. Targeting this minute population of neurons (as few as 800 in the mouse) requires regulatory elements upstream of the GnRH gene that remain to be fully characterized. Previously, we have identified an evolutionarily conserved promoter region (-173 to +1) and an enhancer (-1863 to -1571) in the rat gene that targets a subset of the GnRH neurons in vivo. In the present study, we used phylogenetic sequence comparison between human and rodents and analysis of the transcription factor clusters within conserved regions in an attempt to identify additional upstream regulatory elements. This approach led to the characterization of a new upstream enhancer that regulates expression of GnRH in a cell-specific manner. Within this upstream enhancer are nine binding sites for Octamer-binding transcription factor 1 (OCT1), known to be an important transcriptional regulator of GnRH gene expression. In addition, we have identified nuclear factor I (NF1) binding to multiple elements in the GnRH-regulatory regions, each in close proximity to OCT1. We show that OCT1 and NF1 physically and functionally interact. Moreover, the OCT1 and NF1 binding sites in the regulatory regions appear to be essential for appropriate GnRH gene expression. These findings indicate a role for this upstream enhancer and novel OCT1/NF1 complexes in neuron-restricted expression of the GnRH gene.

Cadmium: An Endocrine Disrupter That Affects Gene Expression in the Liver and Brain of Juvenile Rainbow Trout1

An inhibition of vitellogenesis is observed in fish exposed to cadmium (Cd), either in natural or in experimental conditions. To investigate whether this correlates or not with modifications in the expression of several genes involved in reproduction, we have performed a study on juvenile rainbow trout (Oncorhynchus mykiss) exposed to waterborne Cd in combination with estradiol (E2). A relative reverse transcription-PCR protocol was used to evaluate the effect of Cd exposure on the expression of several genes. We quantified vitellogenin, rainbow trout estradiol receptor alpha (rtERalpha), short and long isoforms (rtERalphaS and rtERalphaL), mRNA levels in liver, and salmon GnRH1, salmon GnRH2, rtERalphaS, and rtERalphaL mRNA levels in the brain. In liver, Cd reduced the E2-stimulated mRNA levels of vitellogenin as well as these of both rtERalpha isoforms in a dose-dependent manner. In brain tissue, our results indicate that rtERalpha mRNA levels are not enhanced by E2. Cd treatments did not modify rtERalphaS isoform expression but reduced rtERalphaL expression in the brain. Focusing on the expression of salmon GnRH (sGnRH) genes, E2 did not affect mRNA levels, but experiments with Cd alone greatly enhanced sGnRH 1 as well as sGnRH 2 gene expression in a dose-dependant manner. This study supports the idea that Cd is an important endocrine disrupter that could act through an inhibition of E2-stimulated genes in the liver and also through a central effect on sGnRH gene expression. Cd may affect a number of E2 signaling pathways but could also affect the reproductive axis by nonestrogenic mechanisms.

Estrogen down-regulates GnRH gene expression in human placental cytotrophoblast cells

This study aims at investigating and characterizing GnRH gene expression and regulation in human placental cytotrophoblasts by estradiol. RT-PCR analysis demonstrates that the cytotrophoblasts expressed the pro-GnRH gene. PCR with two pairs of oligonucleotide primers generated doublets 870 bases apart at the expected molecular weights, indicating that the upstream and downstream transcriptional start sites of the GnRH gene were utilized and that transcripts with and without intron I were both produced. In human placental cytotrophoblasts, the upstream transcription start site of GnRH gene was the major one and gave rise to an mRNA level three times higher than the downstream start site. RT-PCR assay also revealed that estradiol downregulated the expression of GnRH gene in human cytotrophoblasts. Estradiol at the concentration of 10 nM led to a significant (50%) reduction in the mRNA from the upstream transcription start site. Such downregulation was dose-dependent and abolished by the addition of an estrogen antagonist, ICI, indicating that the gene expression was mediated by estrogen receptors. Furthermore, downregulation of GnRH gene expression by estradiol has also been confirmed by luciferase assay.

Sex Differences in Estrogen-Dependent Transcription of Gonadotropin-Releasing Hormone (GnRH) Gene Revealed in GnRH Transgenic Mice

The mechanisms through which gonadal steroids exert feedback actions on the activity of the GnRH neurons are not understood. Using a series of GnRH-LacZ transgenic mice we have examined the manner in which gonadal steroids suppress GnRH mRNA expression in male and female mice. The long-term gonadectomy of 5.5-GNZ-3.5 transgenic mice resulted in significant increases in cellular GnRH mRNA expression (P < 0.05) and plasma LH concentrations (P < 0.01) in both sexes. However, cellular levels of LacZ mRNA and beta-galactosidase, which provide an index of GnRH gene transcription, were only elevated in males after gonadectomy. This sexually differentiated response was also observed in mice gonadectomized for 2 wk. Estrogen replacement in gonadectomized males returned transgene expression to intact levels. Experiments in transgenic mice with 3' and 5' deleted GnRH-LacZ constructs revealed that the suppressive influence of estrogen on LacZ transcription in the male required a critical element located between -5.2 and -1.7 kb of the GnRH promoter. These studies show that the suppression of GnRH mRNA expression by estrogen in the male involves a decrease in GnRH gene transcription that is dependent on a distal GnRH promoter element. The same mechanism does not exist in females, indicating that gonadal steroids suppress GnRH mRNA levels in a sexually dimorphic manner.

Direct and indirect regulation of gonadotropin-releasing hormone neurons by estradiol.

Estrogen signaling to GnRH neurons is critical for coordinating the preovulatory surge release of LH with follicular maturation. Until recently it was thought that estrogen signaled GnRH neurons only indirectly through numerous afferent systems. This minireview presents new evidence indicating that GnRH neurons are directly regulated by estradiol (E2), primarily through estrogen receptor (ER)-beta, and indirectly through E2-sensitive neurons in the anteroventral periventricular (AVPV) region. The data described suggest that E2 generally represses GnRH gene expression but that this repression is transiently overcome by indirect E2-dependent signals relayed by AVPV neurons. We also present evidence that the AVPV neurons responsible for relaying E2 signals to GnRH neurons are multifunctional gamma aminobutyric acid-ergic/glutamatergic/neuropeptidergic neurons.

Autosomal recessive idiopathic hypogonadotropic hypogonadism: genetic analysis excludes mutations in the gonadotropin-releasing hormone (GnRH) and GnRH receptor genes.

Failure of the normal pattern of episodic secretion of GnRH from the hypothalamus results in the clinical syndrome of idiopathic hypogonadotropic hypogonadism (IHH), with failure of pubertal development and infertility. The only gene that has been implicated in normosmic IHH is the GnRH receptor gene (GNRHR), which accounts for 10% of cases. This report presents four families with autosomal recessive IHH, including a consanguineous pedigree from the Middle East. Defects within the genomic coding sequence of the GNRHR, and the GnRH gene itself, GNRH1, were excluded by temperature gradient gel electrophoresis, direct sequencing, and haplotypes created from simple sequence polymorphisms flanking the GNRH1 and GNRHR loci. We concluded that: 1) genetic analysis has excluded sequence variations in GNRH1 and GNRHR in four families with recessive IHH, suggesting the existence of a novel, as-yet-undiscovered gene for this condition, and 2) because mutation analysis of genomic coding sequence will fail to detect mutations deep within introns or regulatory regions, haplotype analysis is the preferred genetic methodology to eliminate the role of specific candidate genes.

Overexpression of glutamic acid decarboxylase-67 (GAD-67) in gonadotropin-releasing hormone neurons disrupts migratory fate and female reproductive function in mice.

gamma-Aminobutyric acid (GABA) inhibits the embryonic migration of GnRH neurons and regulates hypothalamic GnRH release. A subset of GnRH neurons expresses GABA along their migratory route in the nasal compartment before entering the brain, suggesting that GABA produced by GnRH neurons may help regulate the migratory process. To examine this hypothesis and the possibility that persistence of GABA production by GnRH neurons may affect subsequent reproductive function, we generated transgenic mice in which the expression of glutamic acid decarboxylase-67 (GAD-67), a key enzyme in GABA synthesis, is targeted to GnRH neurons under the control of the GnRH gene promoter. On embryonic d 15, when GnRH neurons are still migrating, the transgenic animals had more GnRH neurons in aberrant locations in the cerebral cortex and fewer neurons reaching the hypothalamic-preoptic region, whereas migration into the brain was not affected. Hypothalamic GnRH content in mutant mice was low during the first week of postnatal life, increasing to normal values during infantile development (second week after birth) in the presence of increased pulsatile GnRH release. Consistent with these changes, serum LH and FSH levels were also elevated. Gonadotropin release returned to normal values by the time steroid negative feedback became established (fourth week of life). Ovariectomy at this time demonstrated an enhanced gonadotropin response in transgenic animals. Although the onset of puberty, as assessed by the age at vaginal opening and first ovulation, was not affected in the mutant mice, estrous cyclicity and adult reproductive capacity were disrupted. Mutant mice had reduced litter sizes, increased time intervals between deliveries of litters, and a shorter reproductive life span. Thus, GABA produced within GnRH neurons does not delay GnRH neuronal migration, but instead serves as a developmental cue that increases the positional diversity of these neurons within the basal forebrain. In addition, the results suggest that the timely termination of GABA production within the GnRH neuronal network is a prerequisite for normal reproductive function. The possibility arises that similar abnormalities in GABA homeostasis may contribute to syndromes of hypothalamic amenorrhea/oligomenorrhea in humans.

Developmental changes in hypothalamic insulin-like growth factor-1: relationship to gonadotropin-releasing hormone neurons.

Reproductive development in vertebrates is controlled by changes in hypothalamic GnRH neurons and their inputs from other neurons and glia. One factor involved in the regulation of the GnRH system is the neurotrophic factor, IGF-1. To better understand the regulation of GnRH neurons by hypothalamic IGF-1, we quantified levels of IGF-1 mRNA in hypothalamic and preoptic regions containing GnRH cells, studied the effects of IGF-1 on GnRH gene expression, and examined the neuroanatomical relationship between GnRH neurons and hypothalamic IGF-1 in neonatal, peripubertal, and reproductively mature mice. Our results indicated that IGF-1 mRNA levels in the preoptic area and anterior hypothalamus peaked at postnatal day (P) 5, decreased through P20, and then increased through peripubertal and adult development. Second, IGF-1 had stimulatory effects on GnRH gene expression in explanted preoptic area-anterior hypothalamuses of P5 and peripubertal mice, with results varying by sex and duration of treatment. In contrast, IGF-1 had no effect or even inhibited GnRH gene expression in adult P60 mice. Third, GnRH perikarya coexpressed IGF-1, and this increased throughout sexual maturation. Taken together, the results suggest that IGF-1 can modulate GnRH neurons, that the sensitivity of GnRH neurons to IGF-1 changes developmentally, and that GnRH cells coexpress IGF-1.