Gonadotropin-releasing Hormone Biosynthesis Research Articles

Effect of short-term and prolonged stress on the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamus and GnRHR in the pituitary of ewes during various physiological states

Using an ELISA assay, the levels of GnRH and GnRHR were analysed in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk/median eminence (SME); and GnRHR in the anterior pituitary gland (AP) of non-breeding and breeding sheep subjected to short-term or prolonged stress. The ELISA study was supplemented with an analysis of plasma LH concentration. Short-term footshock stimulation significantly increased GnRH levels in hypothalamus in both seasons. Prolonged stress elevated or decreased GnRH concentrations in the POA and the VM, respectively during anoestrus, and lowered GnRH amount in the POA-hypothalamus of follicular-phase sheep. An up-regulation of GnRHR levels was noted in both, anoestrous and follicular-phase animals. In the non-breeding period, a prolonged stress procedure increased GnRHR biosynthesis in the VM and decreased it in the SME and AP, while in the breeding time the quantities of GnRHR were significantly lower in the whole hypothalamus. In follicular-phase ewes the fluctuations of GnRH and GnRHR levels under short-term and prolonged stress were reflected in the changes of LH secretion, suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release in this period.The study showed that stress was capable of modulating the biosynthesis of GnRH and GnRHR; the pattern of changes was dependent upon the animal’s physiological state and on the time course of stressor application. The obtained results indicate that the disturbances of gonadotropin secretion under stress conditions in sheep may be due to a dysfunction of GnRH and GnRHR biosynthetic pathways.

Central Injection of Exogenous IL‐1β in the Control Activities of Hypothalamic–Pituitary–Gonadal Axis in Anestrous Ewes

This study was performed to determine the effect of intracerebroventricular (icv) injection of interleukin (IL)-1β on the gene expression, translation and release of gonadotropin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) gene expression in the hypothalamus of anestrous ewes. In the anterior pituitary gland (AP), the expression of genes encoding: GnRHR, β subunits of luteinizing hormone (LH) and folliculotropic hormone (FSH) was determined as well as the effect of IL-1β on pituitary gonadotropins release. The relative mRNA level was determined by real-time PCR, GnRH concentration in the cerebrospinal fluid (CSF) was assayed by ELISA and the plasma concentration of LH and FSH were determined by radioimmunoassay. Our results showed that icv injection of IL-1β (10 or 50 μg/animal) decreased the GnRH mRNA level in the pre-optic area (POA) (35% and 40% respectively; p ≤ 0.01) and median eminence (ME) (75% and 70% respectively; p ≤ 0.01) and GnRHR gene expression in ME (55% and 50% respectively; p ≤ 0.01). A significant decrease in GnRHR mRNA level in the AP in the group treated with the 50 μg (60%; p ≤ 0.01) but not with the 10 μg dose was observed. The centrally administrated IL-1β lowered also GnRH concentration in the CSF (60%; p ≤ 0.01) and reduced the intensity of GnRH translation in the POA (p ≤ 0.01). It was not found any effect of icv IL-1β injection upon the release of LH and FSH. However, the central injection of IL-1β strongly decreased the LHβ mRNA level (41% and 50%; p ≤ 0.01; respectively) and FSHβ mRNA in the case of the 50 μg dose (49%; p ≤ 0.01) in the pituitary of anestrous ewes. These results demonstrate that the central IL-1β is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Neuroendocrine control of reproductive aging: roles of GnRH neurons

The process of reproductive senescence in many female mammals, including humans, is characterized by a gradual transition from regular reproductive cycles to irregular cycles to eventual acyclicity, and ultimately a loss of fertility. In the present review, the role of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons is considered in this context. GnRH neurons provide the primary driving force upon the other levels of the reproductive axis. With respect to aging, GnRH cells undergo changes in biosynthesis, processing and release of the GnRH decapeptide. GnRH neurons also exhibit morphologic and ultrastructural alterations that appear to underlie these biosynthetic properties. Thus, functional and morphologic changes in the GnRH neurosecretory system may play causal roles in the transition to acyclicity. In addition, GnRH neurons are regulated by numerous inputs from neurotransmitters, neuromodulators and glia. The relationship among GnRH cells and their inputs at the cell body (thereby affecting GnRH biosynthesis) and the neuroterminal (thereby affecting GnRH neurosecretion) is crucial to the function of the GnRH system, with age-related changes in these relationships contributing to the reproductive senescent process. Therefore, the aging hypothalamus is characterized by changes intrinsic to the GnRH cell, as well as its regulatory inputs, which summate to contribute to a loss of reproductive competence in aging females.

Excision of the First Intron from the Gonadotropin-releasing Hormone (GnRH) Transcript Serves as a Key Regulatory Step for GnRH Biosynthesis

The mammalian gonadotropin-releasing hormone (GnRH) gene consists of four short exons (denoted as 1, 2, 3, and 4) and three intervening introns (A, B, and C). Recently, we demonstrated that excision of the first intron (intron A) from the GnRH transcript is regulated in a tissue- and developmental stage-specific fashion and is severely attenuated in hypogonadal (hpg) mouse because of its lack of exonic splicing enhancers (ESE) 3 and 4. In the present study, we examined the influence of intron A on translational efficiency, thereby establishing a post-transcriptional control over GnRH biosynthesis. First, we verified that an intron A-retained GnRH transcript is a splicing variant but not a splicing intermediate. Intron A-retained transcripts can be transported to the cytoplasm in contrast to intron B-containing transcripts, which are restricted to the nucleus. This result implicates the intron A-retained GnRH transcript as a splicing variant; it has a long 5'-untranslated region, as the GnRH prohormone open reading frame (ORF) begins on exon 2. We investigated whether an intron A-retained GnRH transcript can properly initiate translation at the appropriate start codon and found that intron A completely blocks the translation initiation of its downstream reporter ORF both in vivo and in vitro. The inhibition of translation initiation appears to be due to the presence of a tandem repeat of ATG sequences within intron A. Constructs bearing mutations of ATGs to AAGs restored translation initiation at the downstream start codon; the extent of this restoration correlated with the number of mutated ATGs. Besides the failure in the translation initiation of GnRH-coding region in the intron A-containing variant, the present study also suggests that the interference between mature GnRH mRNA and intron A-retained splicing variant could occur to lower the efficiency of GnRH biosynthesis in the GT1-1-immortalized GnRH-producing cell line. Therefore, our results indicate that the precise and efficient excision of intron A and the joining of adjacent exons may be a critical regulatory step for the post-transcriptional regulation of GnRH biosynthesis.

A novel mechanism for endocrine-disrupting effects of polychlorinated biphenyls: direct effects on gonadotropin-releasing hormone neurones.

Polychlorinated biphenyls (PCBs) cause abnormal development and physiology of the reproductive system. We hypothesized that these effects may be mediated, at least in part, by neuroendocrine cells in the hypothalamus that integrate inputs to and outputs from the central nervous system and reproductive systems. The effects of two PCB mixtures, Aroclor 1221 and Aroclor 1254, were tested on the hypothalamic GT1-7 cells, which synthesize and secrete the key hypothalamic hormone, gonadotropin-releasing hormone (GnRH). GT1-7 cells were treated for 24 h in dose-response experiments and GnRH gene expression and release were quantified. Aroclor 1221 was stimulatory to GnRH gene expression, particularly at post-transcriptional levels (GnRH cytoplasmic mRNA), and increased GnRH peptide levels, suggesting a post-translational regulation of GnRH biosynthesis. It also caused a qualitative increase in GT1-7 neurite outgrowth and cell confluency. Aroclor 1254 had very different effects from Aroclor 1221. It inhibited GnRH nuclear mRNA levels at high dosages, and stimulated GnRH mRNA at low doses, suggesting a post-transcriptional mechanism of regulation. Aroclor 1254 did not alter GnRH peptide levels. Qualitatively, Aroclor 1254 caused a retraction of GT1-7 cell processes and neurotoxicity at high dosages. In order to gauge the involvement of the oestrogen receptor in these responses, the oestrogen receptor antagonist, ICI 182,780 (ICI) was coadministered in other studies with the PCBs. While effects of Aroclor 1221 on GnRH gene expression were not blocked by ICI, its effects on GnRH peptide levels were blocked by ICI, indicating that some but not all of the effects of Aroclor 1221 are mediated by the classical oestrogen receptor alpha and/or beta. The inhibitory effects of Aroclor 1254 on GnRH gene expression were not prevented by ICI, although ICI itself had stimulatory effects on GnRH gene expression that were blocked by cotreatment with Aroclor 1254. These results demonstrate a novel mechanism for effects of the two PCBs directly on GnRH gene expression, and indicate a hypothalamic level for endocrine disruption by these environmental toxicants.

Organochlorine pesticides directly regulate gonadotropin-releasing hormone gene expression and biosynthesis in the GT1-7 hypothalamic cell line

Environmental toxicants profoundly affect growth and developmental processes. In the present study, we hypothesized that hypothalamic gonadotropin-releasing hormone (GnRH) neurons, which regulate the reproductive axis, are targets of environmental endocrine disrupting chemicals. Two organochlorine pesticides (methoxychlor and chlorpyrifos) were tested for their effects on GnRH gene expression and biosynthesis in the immortalized hypothalamic GT1-7 cells, which synthesize and secrete GnRH. GT1-7 cells were treated with methoxychlor or chlorpyrifos for 24 h in dose-response experiments, and GnRH gene expression and peptide levels were quantified. In order to examine whether these pesticides affect GnRH biosynthesis through the estrogen receptor (ER), in other experiments their effects were compared to those of estrogen, or they were co-administered with the ER antagonist, ICI 182,780 (ICI). Both methoxychlor and chlorpyrifos had significant effects on GnRH gene transcription and GnRH mRNA levels. These effects were not consistently blocked by ICI, nor did the effects of these pesticides consistently mimic those of estrogen, suggesting a mechanism independent of the ER. Chlorpyrifos and methoxychlor slightly stimulated peptide levels, and this effect was blocked by ICI, suggesting that the ER may mediate effects of pesticides on GnRH release. These results indicate that chlorpyrifos and methoxychlor alter GnRH biosynthesis in this hypothalamic cell line in vitro, suggesting that they may have endocrine disrupting effects on GnRH neurons in vivo.

Reduction of luteinzing hormone secretion induced by long-term feed restriction in male rats is associated with increased expression of GABA-synthesizing enzymes without alterations of GnRH gene expression.

In rats, fasting or restriction of feed intake impairs the activity of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator which results in reduced luteinizing hormone (LH) secretion. It is still unknown which neurotransmitters are involved in this phenomenon. However, it is known that increased GABA concentrations in the hypothalamus reduce GnRH biosynthesis and release. Therefore, we examined whether 17 days of feed restriction in male rats affected the hypothalamic gene expression of GnRH and the GABA-synthesizing enzymes glutaminase (GLS) and glutamic acid decarboxylase-which exists in two forms, GAD67 and GAD65-in the mammalian brain. Furthermore, the expression of the GnRH receptor (GnRH-R) and the GABA transporter 1 (GAT-1) were investigated. Feed restriction resulted in a 75% reduction in body weight (b.w.) compared to rats fed ad libitum. Serum concentrations of LH and testosterone in the feed restricted group were significantly reduced to approximately 15% of that of rats fed ad libitum, while the FSH concentration remained unchanged. In the mediobasal hypothalamus (MBH) where GnRH is released into the portal vessels, mRNA levels of GAD67 and GLS were increased twofold compared to rats fed ad libitum while no changes were observed in the preoptic area of the hypothalamus (POA) where GnRH is biosynthesised. Neither the expression of preoptic GnRH mRNA nor the expression of GAD65 and of GnRH-R mRNA in both hypothalamic structures was affected by feed restriction. In the anterior pituitary, a significant reduction of the expression of GnRH-R, LH-beta and the alpha subunit was observed in the feed restricted rats, whereas FSH-beta mRNA levels remained constant. Thus, feed restriction selectively increased the expression of GABA-synthesizing enzymes in the MBH but did not modify GnRH expression in the POA. However, the reduced expression of the LH-beta- and alpha-subunit and of the GnRH-R in the anterior pituitary indicates that pulsatile GnRH release may have been attenuated or even abolished. We suggest, that enhanced expression of GABA-synthesizing enzymes reflects increased GABAergic neurotransmission and thereby reducing GnRH release from the MBH.

Regulation of Gonadotropin-Releasing Hormone Gene Expressionin Vivoandin Vitro

The pulsatile release of gonadotropin-releasing hormone (GnRH) into the portal vasculature is responsible for the maintenance of reproductive function. Levels of GnRH decapeptide available for this process can be regulated at transcriptional, posttranscriptional, and posttranslational levels. In the immortalized neuronal GT1 cell lines which synthesize and secrete GnRH, regulation of GnRH biosynthesis has been studied using activators of the protein kinase A (PKA), protein kinase C (PKC), and calcium second messenger systems. These substances, while stimulating GnRH release, cause a universal inhibition of all biosynthetic indices measured to date, including decreases in transcription of the proGnRH gene, GnRH mRNA levels, mRNA stability, and translational efficiency. In contrast, in the animal, the mechanism for the regulation of GnRH gene expression appears to be primarily posttranscriptional, since changes in GnRH mRNA levels often occur in the absence of changes in GnRH primary transcript levels, an index of GnRH gene transcription. For example, GnRH mRNA levels increase in response to stimulation with glutamate analogs, while GnRH primary transcript levels are unchanged. However, parallel changes in GnRH mRNA and primary transcript have been observed on proestrus prior to the LH/GnRH surge, suggesting that the regulation of GnRH mRNA levelsin vivoinvolves a complex interplay of transcriptional and posttranscriptional processes.

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells

1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence. 2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis. 3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression. 4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.

Activation of central GABAA-but not of GABAB-receptors rapidly reduces pituitary LH release and GnRH gene expression in the preoptic/anterior hypothalamic area of ovariectomized rats.

gamma-Aminobutyric acid (GABA) exerts an inhibitory action on gonadotropin-releasing hormone (GnRH) release from the hypothalamus. In vivo, this inhibitory action appears to be mediated via the GABAA receptor since in ovariectomized (ovx) rats and sheep direct application of muscimol (MUS), a GABAA agonist, into the preoptic area (POA), the site were the GnRH cell bodies are located, caused an immediate reduction of LH release. This effect may be the result of an inhibition of GnRH release but also GnRH biosynthesis may be affected. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) we now addressed the question, whether an acute inhibition of the GnRH pulse generator in ovx rats by GABA involves reduction of GnRH biosynthesis as determined by GnRH mRNA levels in micropunches of the POA. To activate either the GABAA or GABAB receptor, we injected intraventricularly (icv) MUS or baclofen (BAC). Intracerebroventricular injection of 10 nmol MUS caused a rapid and lasting inhibition of LH release from about 7.5 ng/ml (pretreatment value) to approximately 1.5 ng/ml. Neither application of BAC or saline (control injections) affected LH secretion. Two hours after icv injections, rats were decapitated and GnRH mRNA levels were determined. MUS induced a pronounced decrease of GnRH levels in the POA (control rats: 2.26 pg GnRH mRNA; MUS-treated rats: 0.85 pg, n = 10/group). BAC was without any effect on GnRH mRNA levels. Thus, we confirm the inhibitory action of GABA on LH release in vivo which is exerted via the A-subtype of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of 5β-pregnan-3α-ol-20-one on gonadotropin-releasing hormone gene expression in the male rat

Neuroanatomical data have documented the existence of synaptic contacts between γ-aminobutyric acid (GABA) terminals and preoptic gonadotropin-releasing hormone (GnRH) neurons in the rat anterior hypothalamus. In addition, pharmacological studies have suggested that the GABAergic system may be involved in the control of gonadotropin release. Moreover, it has been shown that some progesterone metabolites such as 5β-pregnan-3α-ol-20-one (5β3αP) are able to interact with the GABA A receptor complex. In the present study, we have investigated the effects of chronic (5 days) treatment with the GABA A-positive ligand 5β3αP (20 mg/kg body weight i.p., twice a day) or with the GABA A agonist muscimol (1 mg/kg body weight i.p., twice a day) alone or in combination on GnRH mRNA levels in the preoptic area of the male anterior hypothalamus as measured by in situ hybridization. Treatment with 5β3αP produced a 30% decrease in the number of grains overlying labelled cells, while muscimol treatment decreased the hybridization signal by 36%. The concomitant administration of 5β3αP and muscimol resulted in a 46% decrease in the GnRH mRNA levels. This inhibitory effect was completely antagonized by the concomitant administration of picrotoxin (4 mg/kg body weight i.p., twice a day). These data suggest that the GABA A receptor complex and steroids that interact positively with this GABA A receptor complex may play an important role in the regulation of GnRH biosynthesis by hypothalamic neurons.

Production and characterization of antibodies to gonadotropin-releasing hormone receptor.

Antibodies to the gonadotropin-releasing hormone (GnRH) receptor of bovine pituitary membranes have been raised in rabbits by immunization with affinity-purified receptor preparations. These antibodies did not compete with 125I-labeled GnRH analog (Buserelin) for binding to the receptors but did precipitate rat and bovine solubilized receptors labeled with 125I-Buserelin. Binding of the antibodies to the receptors was also demonstrated by immunoprecipitation of 125I-labeled purified receptors and photoaffinity-labeled receptors. The antibodies did not have a GnRH-like activity but rather inhibited, in a dose-dependent manner, GnRH-stimulated luteinizing hormone release from cultured rat pituitary cells. In addition, the antibodies did not inhibit luteinizing hormone release stimulated by high K+ concentration. This suggests that the antibodies recognize domains of the receptor other than the binding site of the hormone and thereby inhibit the biological response. These GnRH receptor antibodies provide a useful tool for studying GnRH receptor structure, function, localization, and biosynthesis.