Gonadotropin-releasing Hormone mRNA Levels Research Articles

Role of nitric oxide in the regulation of gonadotropin-releasing hormone and tyrosine hydroxylase gene expression in the male rat brain

It has been recently demonstrated that nitric oxide (NO), a free radical gas which may act as neurotransmitter in the brain, can stimulate the in vivo release of luteinizing hormone as well as the in vitro hypothalamic release of gonadotropin-releasing hormone (GnRH). In order to study the influence of NO on GnRH mRNA expression, two inhibitors of NO synthase (NOS) N G-monomethyl- l-arginine (NMMA) and HP-228, were microinjected into the left lateral ventricle of sham-operated and castrated male rats 4 h before sacrifice. Since the dopaminergic system can positively influence GnRH gene expression, we have also measured in the same animals tyrosine hydroxylase (TH) mRNA in tuberoinfundibular dopamine (TIDA) neurons. GnRH and TH mRNA levels were measured at the cellular level by quantitative in situ hybridization. The injection of HP-228 or NMMA induced a similar decrease (−19.5%) in GnRH mRNA. In castrated animals, the hybridization signal was 88% higher than that observed in sham-operated animals. Both HP-228 and NMMA produced in castrated animals a 39% decrease in GnRH mRNA. In contrast the injection of NOS inhibitors resulted in an increase in the amount of TH mRNA in TIDA neurons. The stimulating effect was more striking in HP-228-treated (+60%) than in NMMA-treated (+32%) animals. Castration did not induce any changes in the number of silver grains overlying TIDA neurons, while the administration of either HP-228 or NMMA induced a 43% increase in castrated animals. These results together with previous ones on GnRH release in vitro suggest that NO exerts a positive influence not only on the secretion but also on the biosynthesis of GnRH. Since NO appears to play a role in the negative regulation of dopamine, it is likely that the increase in GnRH mRNA expression is not mediated by the TIDA system.

Retinoic acid regulates gonadotropin-releasing hormone (GnRH) release and gene expression in the rat hypothalamic fragments and GT1-1 neuronal cells in vitro

The present study attempts to examine the possible involvement of retinoic acid (RA) in the regulation of gonadotropin-releasing hormone (GnRH) release and gene expression in the rat hypothalamic fragments and GT1-1 neuronal cells in vitro. During a short-term period (2 h), RA (0.01–1 μM) increased GnRH release in a dose-related manner. Time-course experiments showed that RA rapidly increased GnRH release by 30 min in both cells. RA-induced GnRH release was slowly attenuated in the next incubation period in hypothalamic fragments, but rapidly returned to control levels in GT1-1 cells. In hypothalamic fragments, GnRH mRNA levels decreased, but in GT1-1 cells, no change in GnRH mRNA levels was observed. We then extended the incubation time to see any changes in GnRH mRNA levels by RA in GT1-1 cells. In a long term (up to 48 h), RA increased GnRH mRNA levels in a dose- and time-related manner. Significant increase in GnRH mRNA levels by RA (at higher than 10 nM) was observed within 12 h. Transient transfection experiments with a luciferase reporter vector containing more than 3 kb of the rat GnRH 5′-flanking region (−3002 to +88) revealed that RA also increased the rat GnRH promoter activity in a similar dose- and time-dependent manner, suggesting that increases in GnRH mRNA levels are attributable, at least in part, to the enhanced gene transcription. The promoter analysis with the 5′-deletional constructs demonstrated that cis-elements responsible for the RA action may reside within −1640/−1438 of the rat GnRH promoter, where multiple direct or palindromic arrangements of the AGGTCA-related sequences exist. We also showed that GT1-1 cells as well as the hypothalamic tissues express mRNA for multiple subtypes of retinoid receptors, and that reporter plasmids with three copies of the strong retinoic acid response element (RARE) were activated by 80 folds upon treatment with RA in GT1-1 cells, suggesting that retinoid receptors in GT1-1 cells are functional. Taken together, the present study strongly suggests that RA is an important regulator of the GnRH neurons.

Temporal Patterns of Gonadotropin-Releasing Hormone (GnRH), c-fos, and Galanin Gene Expression in GnRH Neurons Relative to the Luteinizing Hormone Surge in the Rat

Gonadotropin-releasing hormone (GnRH) neurons increase their expression of Fos and galanin coincident with the luteinizing hormone (LH) surge in the female rat. To define the temporal relationships between the expression of these genes and the GnRH gene itself and to gain insight about the possible functional interactions of these processes, we compared levels of c-fos, galanin, and GnRH mRNA in GnRH neurons and plasma levels of LH in the rat, beginning 6 hr before and continuing for 24 hr after a sex steroid-induced LH surge. LH levels were increased significantly by 1600 hr. They increased twofold further by 1800 hr and then returned to baseline by 2400 hr. Using in situ hybridization, we determined that levels of c-fos mRNA in GnRH neurons were elevated significantly at 1600 hr only, whereas levels of galanin mRNA in GnRH neurons first increased twofold by 1800 hr, increased an additional twofold by 2400 hr, and remained elevated at all time points sampled thereafter. There were no significant changes in cellular levels of GnRH mRNA over the time points sampled. These results are consistent with the hypothesis that the induction of c-fos gene expression in GnRH neurons leads to an increase in galanin gene expression, and that the sustained increase in galanin mRNA levels in GnRH neurons reflects either the need to replenish galanin stores that are depleted at the time of the LH surge or the involvement of galanin with physiological events that occur on the day of estrus.

Differential regulation of gonadotropin-releasing hormone (GnRH) receptor expression in the posterior mediobasal hypothalamus by steroid hormones: implication of GnRH neuronal activity

The present study is designed to evaluate the relationship between gonadotropin-releasing hormone (GnRH) and GnRH receptor ( GnRHR) gene expression during the steroid-induced LH surge. One week after ovariectomy (OVX), a capsule containing 17β-estradiol (E) or vehicle (V) was implanted into OVX rats, and 2 days later a single injection of progesterone (P) or V was administered s.c. at 10:00 h. Poly(A)-rich RNA samples were isolated from the micropunches of the preoptic area (POA) and the posterior mediobasal hypothalamus (pMBH) from both sides of individual brain slices. Using competitive reverse transcription–polymerase chain reaction (RT-PCR) procedures, three parameters (POA GnRH, pMBH GnRHR and pituitary GnRHR mRNA levels) were simultaneously determined in each individual animal. POA GnRH mRNA and pituitary GnRHR mRNA levels were decreased by treatment with E, but increased by a combination of E and P. In contrast, pMBH GnRHR mRNA levels were clearly augmented by treatment with E, and decreased by the combination of E and P. Temporal changes in such parameters were determined in OVX+E+V- and OVX+E+P-treated rats. P augmented POA GnRH mRNA levels at the time of the LH surge (17:00 h) and the increased GnRH mRNA levels were remained until 22:00 h, while E alone failed to alter POA GnRH mRNA levels. In the pMBH micropunch samples, P substantially decreased E-induced increase in GnRHR mRNA levels at 17:00 h and further lowered those until 22:00 h. Antisense oligonucleotides of GnRHR mRNA administered into the lateral ventricle of OVX+E-treated rats blocked the E-induced increase in pMBH GnRHR mRNA levels. The antisense oligonucleotides also prevented the LH surge as well as the increase in pituitary GnRHR mRNA levels in the OVX+E+P-treated group. However, administration of this antisense oligonucleotides failed to alter POA GnRH mRNA levels. In conclusion, the present study demonstrated that there is an inverse relationship between POA GnRH mRNA levels and pMBH GnRHR mRNA levels in response to E and/or P, and that the blockade of the E-induced increase in pMBH GnRHR mRNA levels effectively nullified the P-induced LH surge. These results indicate that pMBH GnRHR gene expression is involved in synchronizing the GnRH neuronal activity, which is crucial for the generation of the LH surge.

Autocrine regulation of gonadotropin‐releasing hormone (GnRH) operates at multiple control levels of GnRH gene expression in GT1–1 neuronal cells

We previously found that a potent gonadotropin‐releasing hormone (GnRH) agonist, buserelin, decreases GnRH promoter activity together with GnRH mRNA level, providing evidence for an autoregulatory mechanism operating at the level of GnRH gene transcription in immortalized GT1–1 neuronal cells. To examine whether agonist‐induced decrease in GnRH mRNA level requires the continuous presence of buserelin, we performed a pulse‐chase experiment of buserelin treatment. Short‐term exposure (15min) of GT1–1 neuronal cells to buserelin (10 μM) was able to decrease GnRH mRNA levels when determined 24 h later. When GT1–1 cells were treated with buserelin (10 uM) for 30min and then incubated for 1, 3, 6, 12, 24, and 48 h after buserelin removal, a significant decrease in GnRH mRNA levels was observed after the 12 h incubation period. These data indicate that inhibitory signaling upon buserelin treatment may occur rapidly, but requires a long time (at least 12 h) to significantly decrease the GnRH mRNA level. To examin...

Suppression of GnRH gene expression in GT1-1 hypothalamic neuronal cells: action of protein kinase C.

We attempted to elucidate molecular mechanisms of gonadotropin-releasing hormone (GnRH) gene regulation by the protein kinase C (PKC) pathway in GT1-1 cells. Activation of PKC with 12-tetra-decanoylphorbol-13-acetate (TPA) or inhibition with staurosporine or calphostin C down-regulated GnRH mRNA levels. A serial deletion mutant analysis revealed that this suppression was mediated by the proximal region (-187/-69) of the mouse GnRH promoter. TPA transiently induced c-fos mRNA, whereas staurosporine or calphostin C failed to do so. However, PKC inhibitors blocked the TPA-evoked c-fos induction. Over-expression of PKC alpha down-regulated GnRH promoter activity, indicating that PKC activation was sufficient to inhibit GnRH gene expression. These results suggest that both activation and inhibition of PKC decrease the GnRH gene expression in the GT1-1 cells probably through different signal cascade mechanisms.

Evidence for autocrine inhibition of gonadotropin-releasing hormone (GnRH) gene transcription by GnRH in hypothalamic GT1-1 neuronal cells

To examine whether an ultrashort feedback mechanism of gonadotropin-releasing hormone (GnRH) operates at the level of gene transcription, we studied the effects of GnRH analogs on GnRH promoter activity and GnRH mRNA level in hypothalamic GT1–1 neuronal cells. Treatment of GT1–1 cells with buserelin, a GnRH agonist, or native GnRH for 24 h significantly decreased GnRH promoter activity and its mRNA level, whereas that with GnRH antagonists, antide or [ D-Phe 2, D-Ala 6]-GnRH, showed no effect. The inhibitory effects of buserelin on GnRH gene transcription and GnRH mRNA level were dose-related, and a significant inhibition was observed in cells treated with buserelin at concentrations higher than 0.1 μM. Time-course experiments showed that significant decreases in GnRH promoter-driven luciferase activity and GnRH mRNA level were observed within 12 h and sustained up to 48 h. Moreover, treatment with GnRH agonist for 12 h significantly decreased the transcription rate of the mouse GnRH gene, as revealed by nuclear run-on transcription assay. The promoter analysis with the 5′-deletional constructs demonstrated that cis-acting elements important for GnRH autoregulation by GnRH agonist reside within −854 bp upstream from the transcription start site. These data clearly demonstrate that GnRH can exert autocrine regulation at the level of GnRH gene transcription.

Role of adrenal and gonadal steroids in the response of GnRH gene expression to the endogenous benzodiazepine receptor ligand octadecaneuropeptide in the male rat brain

We have recently demonstrated that the inhibitory influence of the endogenous benzodiazepine receptor ligand octadecaneuropeptide (ODN) on gonadotropin-releasing hormone (GnRH) gene expression could be prevented by specific inhibitors of 3β-hydroxysteroid dehydrogenase and 5α-reductase in adrenalectomized and castrated male rats, then suggesting an involvement of neurosteroids in the action of ODN on GnRH neurons. In order to study in detail the role of circulating steroids in the effect of ODN, we have evaluated the influence of adrenalectomy, castration and the combination of adrenalectomy and castration as well as the effect of dexamethasone administration in the response of GnRH gene expression to ODN in the male rat. The intracerebroventricular injection of ODN (4 h before sacrifice) produced a 36% decrease in the hybridization signal. Adrenalectomy induced a 21% decrease in GnRH mRNA levels. In the adrenalectomized rats, the injection of ODN increased by 11% the amounts of mRNA. As previously reported by our group, castration was found to enhance GnRH mRNA (15% over control values). In castrated animals, ODN produced an inhibitory effect in the hybridization signal which was of the same amplitude as that observed in sham-operated animals. Finally, the combination of adrenalectomy and castration resulted in a small but significant decrease in the hybridization signal, the values being intermediary between those observed after adrenalectomy and those obtained after castration. In these animals, ODN induced a 38% decrease in the amounts of GnRH mRNA. In animals that had been castrated and adrenalectomized, dexamethasone treatment during 4 days produced a 19% increase in hybridization signal. In these dexamethasone-treated animals, ODN produced the usual decrease (33%) in GnRH mRNA. These results demonstrate that gonadal hormones do not play a major role in the activation of the GABA A receptor complex by ODN. On the other hand, it clearly appears that glucocorticoids exert a tonic stimulatory influence on GnRH neuronal activity and are involved in the inhibitory effect of ODN. The mechanism of action of glucocorticoids, which seems complex since the influence of adrenalectomy on the ODN action can be prevented by orchidectomy, remains to be fully elucidated.

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.

Phorbol ester regulation of the gonadotropin-releasing hormone (GnRH) gene in GnRH-secreting cell lines: a molecular basis for species differences.

Phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) regulation of the GnRH gene was studied in two mouse GnRH neuronal cell lines, Gn11 and NLT. TPA treatment of NLT cells grown in low serum conditions did not alter endogenous mouse GnRH mRNA levels, indicating that the endogenous mouse gene is not regulated by phorbol esters under these conditions. This result is confirmed in transfection studies in which TPA treatment did not change expression of a mouse GnRH-luciferase reporter gene construct. In contrast, TPA treatment stimulated expression of a human GnRH-luciferase reporter construct, correlating with the expression of the protoon-cogenes c-fos and c-jun. TPA stimulation of the human GnRH gene is mediated by a consensus AP-1 site located at -402 to -396 bp, TGACTCA, which specifically binds c-fos and c-jun in Gn11 and NLT cells and recombinant c-jun in gel mobility shift studies. In contrast, the rodent GnRH genes, when aligned for maximum homology, contain a DNA sequence with a 1-bp difference, TGTCTCA from the human gene. In gel mobility shift studies, this DNA sequence does not form a complex with Gn11 or NLT nuclear extract or with recombinant c-jun. This is the first demonstration of species-specific differences in phorbol ester regulation of GnRH gene transcription and could, in part, explain differences in reproductive function among mammals.

Gonadotropin-Releasing Hormone and NMDA Receptor Gene Expression and Colocalization Change during Puberty in Female Rats

During development, an increase in gonadotropin-releasing hormone (GnRH) release occurs that is critical for the initiation of puberty. This increase is attributable, at least in part, to activation of the GnRH neurosecretory system by inputs from neurotransmitters, such as glutamate, acting via NMDA receptors. We examined changes in GnRH and NMDA-R1 gene expression by RNase protection assay of preoptic area-anterior hypothalamic (POA-AH) dissections of female rats undergoing normal puberty or in which precocious puberty was induced by treatment with the glutamate agonist NMA. GnRH mRNA levels increased significantly throughout normal development; this was accelerated by treatment with NMA. NMDA-R1 mRNA levels increased only between P10 and P20. The acceleration of the elevation in GnRH mRNA levels by NMDA suggests that a stimulation of GnRH gene expression may be a rate-limiting factor for the onset of puberty. This is attributable to a post-transcriptional mechanism because GnRH primary transcript levels, an index of proGnRH gene transcription, were not observed to change during puberty. Alterations in the colocalization of GnRH neurons with the NMDA-R1 subunit during puberty also were assessed immunocytochemically. The percentage of GnRH neurons that double-labeled with NMDA-R1 was 2% in prepubertal rats and 3% in pubertal rats; this increased to 19% in postpubertal rats. Taken together, these studies suggest that an increase in glutamatergic input to GnRH neurons plays a role in the increase in GnRH release and gene expression that occurs at the initiation of puberty.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on gonadotropin-releasing hormone and somatostatin gene expression in the rat brain

Pituitary adenylate cyclase-activating peptide (PACAP) is a 38-amino acid polypeptide, first isolated from ovine hypothalamus, which directly stimulates the release of several pituitary hormones, including GH, ACTH, and LH. The presence of PACAP receptors in several brain areas, including the hypothalamus, suggests that this peptide might play a role as neurotransmitter/neuromodulator. We have thus investigated the effects of intracerebroventricular (i.c.v.) and intravenous (i.v.) injections of PACAP and the potent PACAP antagonist PACAP(6–38) on gonadotropin-releasing hormone (GnRH) and somatostatin ( SS) gene expression in the male rat hypothalamus. The levels of mRNA were measured at the cellular level by quantitative in-situ hybridization. The i.c.v. injection of PACAP produced a 12.5% increase in the GnRH mRNA levels, an effect which was completely prevented by the concomitant administration of the PACAP antagonist. The administration of the PACAP antagonist induced by itself a 12.9% decrease in the hybridization signal. The i.v. administration of the same peptides induced modifications in GnRH gene expression which were completely opposite to those produced by i.c.v. administration. In somatostatinergic neurons located in the periventricular nucleus, the i.c.v. injection of the peptides induced modification in SS gene expression which were very similar to those observed for GnRH gene expression, although the changes were less striking. The i.v. administration of PACAP or its antagonist did not induce any change in the levels of SS mRNA. These results then strongly suggest that PACAP might be involved in a positive regulation of two neuropeptides involved in the control of anterior pituitary secretion via central specific receptors. The inverse influence of PACAP on GnRH gene expression after the i.v. injection might be explained by the short feedback effect induced by the direct stimulation of gonadotropin hormone release following the systemic injection of the peptide.

Estrogen regulation of gonadotropin-releasing hormone receptor messenger RNA in female rat pituitary tissue.

Gonadotropin releasing hormone (GnRH) is crucial in regulating the reproductive system of female vertebrates. In the present study we have analyzed the estrogen regulation of the GnRH receptor mRNA at the cellular level in Sprague-Dawley female rats. Northern blot analysis detected 3 species (5.0, 4.5 and 1.4 kb) of GnRH receptor mRNA in pituitary tissues. The GnRH receptor mRNA levels of these 3 species were increased by estrogen. By in situ hybridization we observed a 3.5-fold increase in GnRH receptor mRNA levels after 48 h of estrogen treatment when compared to ovariectomized (OVX) rats, 12 h of estrogen treatment did not change the GnRH mRNA levels. Similar increases in GnRH receptor mRNA levels by estrogen were also found in Wistar-Imamichi female rat pituitary tissue. In situ hybridization analysis identified clusters of anterior pituitary cells that expressed the GnRH receptor mRNA. The estradiol effect depends on increased mRNA levels in these clusters. Moreover, a significant increase in the number of pituitary cells that expressed GnRH receptor was observed after 48 h of estrogen treatment. These findings suggest that the mechanisms for estrogen regulation of GnRH receptor include changing levels of GnRH receptor mRNA in the rat pituitary.

5. Locally advanced non-small cell lung cancer: The “local” issue

There exists maternal immunological modification in maternal immune organs during early pregnancy in mammals. Gonadotropin releasing hormone (GnRH) is widely distributed in vertebrate tissues, including immune organs. However, it is unclear that early pregnancy induces expression of GnRH and GnRH receptor (GnRHR) in ovine immune organs. The objective of this study was to explore the expression of GnRH and GnRHR in main immune organs (thymus, lymph node, spleen, and liver) during early pregnancy in sheep. Ovine thymus, lymph node, spleen and liver were sampled at day 16 of estrous cycle, and days 13, 16, and 25 of pregnancy. The expression of GnRH and GnRHR was detected through real-time quantitative PCR, Western blot and immunohistochemistry analysis. The results indicated that early pregnancy induced upregulation of mRNA and protein levels of GnRH and GnRHR in the maternal lymph node, spleen and liver, and mRNA and protein of GnRH in the maternal thymus, but mRNA and protein of GnRHR decreased in the maternal thymus during early pregnancy. In summary, the mRNA and protein levels of GnRH and GnRHR were changed in maternal thymus, lymph node, spleen and liver in a tissue specific manner during early pregnancy in sheep.

Further studies on the mechanism of action of the endogenous benzodiazepine receptor ligand octadecaneuropeptide on gonadotropin-releasing hormone gene expression in the rat brain.

We have recently reported that the intracerebroventricular injection of the endogenous benzodiazepine (BZD) receptor ligand octadecaneuropeptide (ODN) (30 micrograms/kg b.w.) could induce a decrease in gonadotropin-releasing hormone (GnRH) mRNA levels in the male rat brain. This inhibitory effect could be completely reversed by the concomitant administration of the GABAA antagonist picrotoxin. In order to further investigate the mechanism of action of ODN, we investigated the effects of intracerebroventricular injection of a smaller dose of ODN (3 micrograms/kg b.w.) and the influence of the GABAA receptor agonist muscimol and the antagonist to BZD receptors flumazenil on GnRH gene expression. Treatment with ODN induced a 40% decrease in mRNA levels, an effect which was potentiated by the concomitant administration of muscimol. The administration of flumazenil produced a small increase in GnRH mRNA while the inhibitory effect of ODN on GnRH mRNA levels was completely prevented by the administration of this BZD antagonist. Moreover, the intravenous administration of increasing doses of ODN also induced a marked decrease of 33, 32 and 38% for 75, 150 and 300 micrograms/kg b.w., an effect which was completely abolished by picrotoxin. These data clearly indicate that an endogenous neuropeptide can activate the GABAA BZD receptor complex to negatively modulate the activity of GnRH neurons. They also suggest that this peptide or other endogenous activators of the GABAA BZD receptors might exert a tonic inhibitory influence on GnRH gene expression.

Involvement of serotonin in the regulation of GnRh gene expression in the male rat brain

The role of serotonin (5-HT) in the regulation of the hypothalamo-pituitary-gonadal axis is still controversial. In order to evaluate the influence of 5-HT on gonadotropin-releasing hormone (GnRH) neurons, we have investigated the effects of repeated administration (during 2 days) of 5-HT, the 5-HT1+2 receptor antagonist methysergide, the 5-HT2 receptor antagonist ketanserin, and the 5-HT3 receptor antagonist ondansetron on GnRH mRNA levels in the male rat medial preoptic area (MPOA), as measured by quantitative in situ hybridization. The treatment with 5-HT decreased by 32% the number of silver grains overlying labelled neurons. The administration of methysergide and ketanserin increased the hybridization signal by 32% and 29%, respectively. On the other hand, the 5-HT3 receptor antagonist did not modify GnRH mRNA levels. The present results clearly indicate that the serotoninergic system exerts a negative tonic influence on the biosynthesis of GnRH as evaluated by mRNA level measurements. They also strongly suggest that the influence of 5-HT in the regulation of GnRH neuronal activity is mediated via activation of 5-HT2 receptor, although an involvement of 5-HT1 receptors cannot be totally excluded.

Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons

We recently demonstrated that immortalized GT1-7 neurons co-express luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor and gonadotropin releasing hormone (GnRH) genes. Treatment of GT1-7 neurons with LH/hCG resulted in a transcriptional inhibition of GnRH gene. In the present study, we investigated the signaling and transacting factors involved in the action of hCG. Eight-bromo-cyclic AMP can mimic the down-regulating action of hCG on GnRH mRNA levels. H-89, a protein kinase (PK) A inhibitor, but not bisindolylmaleimide, a PKC inhibitor, blocked the down-regulating actions of hCG as well as of 8-bromo-cyclic AMP Treatment with the PKA inhibitor alone modestly decreased GnRH mRNA levels suggesting that PKA signaling also controls the basal expression of the GnRH gene. The direct measurement of PK activities revealed that hCG treatment of GT1-7 neurons increased the PKA but not the PKC activity. New protein synthesis is required for the down-regulating action of hCG on GnRH mRNA levels. Since some of the new proteins could be nuclear transcription or transacting factors, we investigated the effects of hCG on cyclic AMP response element binding protein (CREB), c-Fos and c-Jun protein levels, Treatment of GT1-7 neurons with hCG resulted in an increase of 43 kDa phosphorylated CREB, 50 kDa c-Fos and 40 kDa c-Jun proteins compared to the corresponding controls. The kinetics of increases were different and in all cases the increases of the proteins preceded the decrease of GnRH mRNA levels. In summary, PKA signaling and transacting factors such as CREB, Fos and Jun are probably involved in transcriptional inhibition of GnRH gene by hCG in GT1-7 neurons.

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)

Hypothalamic gonadotropin-releasing hormone gene expression during rat estrous cycle.

Expression of gonadotropin-releasing hormone (GnRH) gene in the hypothalamus of female rats was studied by the quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. During the estrous cycle, the GnRH mRNA level did not change from diestrus I to II, and then increased with a peak at 1100 h on proestrus. In the afternoon of proestrus, GnRH mRNA decreased rapidly with a nadir at 1600 h, and thereafter increased again and reached a peak at 1100 h on estrus. Since hypothalamic GnRH mRNA was found to be increased by subcutaneous implantation of estradiol-containing silastic tubing in ovariectomized rats, the peak of GnRH gene expression in the proestrous morning might be due to an increase in circulating estrogen in this phase of the estrous cycle. The surge of luteinizing hormone in the proestrous afternoon and the subsequent increase in GnRH mRNA were completely blocked by the injection of MK801, an antagonist for NMDA receptors, suggesting that the excitation of GnRH neurons leads to an increase in GnRH gene expression. This was further supported by the in vitro observation that high K(+)-induced membrane depolarization markedly increased GnRH mRNA in hypothalamic slices. This increase in GnRH mRNA due to neuronal excitation does not seem to require newly synthesized proteins because anisomycin, a protein synthesis inhibitor at the level of translation, did not affect GnRH gene expression. These results suggest that the hypothalamic GnRH mRNA level reached two peaks during the rat estrous cycle, i.e., in proestrous morning and estrous morning, and that estrogen and GnRH neuronal excitation play pivotal roles in regulating GnRH gene expression.

Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells

The present study investigated the expression and regulation of GnRH and GnRH receptor (GnRHR) messenger RNAs (mRNAs) in human granulosa-luteal cells using reverse transcription-polymerase chain reaction (RT-PCR). Granulosa-luteal cells were aspirated from preovulatory follicles obtained from women undergoing in vitro fertilization. Two sets of primers derived from human hypothalamic GnRHR complementary DNA (cDNA) were used to amplify cDNAs from granulosa-luteal cells. PCR products corresponding to the expected sizes of GnRH were obtained from granulosa-luteal cells as well as the brain, but not from skeletal muscle cDNA. The authenticity of the PCR products was confirmed by Southern blot hybridization with internal oligonucleotide probes and by subsequent cloning and sequencing. Similarly, using four sets of primers specific for the human pituitary GnRHR cDNA, PCR products with the expected sizes were detected from both brain and granulosa-luteal cells, but not from skeletal muscle. PCR products were subsequently confirmed by Southern blot hybridization using an internal oligonucleotide probe or a cDNA probe which was obtained from screening a human pituitary cDNA library. Cloning and sequencing of the PCR product in the 3'-untranslated region revealed identical sequence with the reported human pituitary GnRHR cDNA sequence. RNA samples obtained from cells immediately after dissociation or after 2, 5, and 8 days of culture were analyzed by RT-PCR, and in all cases, both GnRH and GnRHR mRNA were detected. To investigate how gene expression of GnRH and GnRHR is regulated, we examined the effect of GnRH and hCG on GnRH and GnRHR mRNA levels in cultured human granulosa-luteal cells. Treatment with different concentrations of GnRH induced biphasic responses. Both GnRH and GnRHR mRNA were significantly increased by 1 nM, but slightly decreased by 1 microM GnRH; 1 nM GnRH also significantly inhibited progesterone production, whereas higher doses had no effect. Treatment with hCG (1 IU/ml) decreased GnRHR mRNA levels without altering the expression of the GnRH gene. These results demonstrate for the first time that 1) both GnRH and GnRHR mRNAs are expressed in human granulosa-luteal cells; 2) GnRH mRNA levels are autoregulated by GnRH; and 3) GnRHR gene expression is up-regulated by GnRH, but down-regulated by hCG. These findings provide strong evidence that GnRH is an autocrine regulator in the human ovary.