Gonadotropin-releasing Hormone Gene Transcription Research Articles

Reproductive axis gene regulation during photostimulation and photorefractoriness in Yangzhou goose ganders

BackgroundThe Yangzhou goose is a long-day breeding bird that has been increasingly produced in China. Artificial lighting programs are used for controlling its reproductive activities. This study investigated the regulations of photostimulation and photorefractoriness that govern the onset and cessation of the breeding period.ResultsIncreasing the daily photoperiod from 8 to 12 h rapidly stimulated testis development and increased plasma testosterone concentrations, with peak levels being reached 2 months after the photoperiod increase. Subsequently, testicular activities, testicular weight, spermatogenesis, and plasma testosterone concentrations declined steadily and reached to the nadir at 5 months after the 12-hour photoperiod. Throughout the experiment, plasma concentrations of triiodothyronine and thyroxine changed in reciprocal fashions to that of testosterone. The stimulation of reproductive activities caused by the increasing photoperiod was associated with increases in gonadotropin-releasing hormone (GnRH), but decreases in gonadotropin-inhibitory hormone (GnIH) and vasoactive intestinal peptide (VIP) gene messenger RNA (mRNA) levels in the hypothalamus. In the pituitary gland, the levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mRNA abruptly increased during the longer 12-hour photoperiod. The occurrence of photorefractoriness was associated with increased GnIH gene transcription by over 250-fold, together with increased VIP mRNA levels in the hypothalamus, and then prolactin and thyroid-stimulating hormone in the pituitary gland. FSH receptor, LH receptor, and StAR mRNA levels in the testis changed in ways paralleling those of testicular weight and testosterone concentrations.ConclusionsThe seasonal reproductive activities in Yangzhou geese were directly stimulated by a long photoperiod via upregulation of GnRH gene transcription, downregulation of GnIH, VIP gene transcription, and stimulation of gonadotrophin. Development of photorefractoriness was characterized by hyper-regulation of GnIH gene transcription in the hypothalamus, in addition of upregulation of VIP and TRH gene transcription, and that of their receptors, in the pituitary gland.

Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation

Pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH) is essential for pituitary gonadotrope function. Although the importance of pulsatile GnRH secretion has been recognized for several decades, the mechanisms underlying GnRH pulse generation in hypothalamic neural networks remain elusive. Here, we demonstrate the ultradian rhythm of GnRH gene transcription in single GnRH neurons using cultured hypothalamic slices prepared from transgenic mice expressing a GnRH promoter-driven destabilized luciferase reporter. Although GnRH promoter activity in each GnRH neuron exhibited an ultradian pattern of oscillations with a period of ∼10 h, GnRH neuronal cultures exhibited partially synchronized bursts of GnRH transcriptional activity at ∼2-h intervals. Surprisingly, pulsatile administration of kisspeptin, a potent GnRH secretagogue, evoked dramatic synchronous activation of GnRH gene transcription with robust stimulation of pulsatile GnRH secretion. We also addressed the issue of hierarchical interaction between the circadian and ultradian rhythms by using Bmal1-deficient mice with defective circadian clocks. The circadian molecular oscillator barely affected basal ultradian oscillation of GnRH transcription but was heavily involved in kisspeptin-evoked responses of GnRH neurons. In conclusion, we have clearly shown synchronous bursts of GnRH gene transcription in the hypothalamic GnRH neuronal population in association with episodic neurohormone secretion, thereby providing insight into GnRH pulse generation.

Androgen receptor repression of gonadotropin-releasing hormone gene transcription via enhancer 1

Gonadotropin-releasing hormone (GnRH) plays a major role in the hypothalamic-pituitary–gonadal (HPG) axis, and synthesis and secretion of GnRH are regulated by gonadal steroid hormones. Disruptions in androgen levels are involved in a number of reproductive defects, including hypogonadotropic hypogonadism and polycystic ovarian syndrome. Androgens down-regulate GnRH mRNA synthesis in vivo and in vitro via an androgen receptor (AR)-dependent mechanism. Methyltrienolone (R1881), a synthetic AR agonist, represses GnRH expression through multiple sites in the proximal promoter. In this study, we show AR also represses GnRH transcription via the major enhancer (GnRH-E1). A multimer of the −1800/−1766 region was repressed by R1881 treatment. Mutation of two bases, −1792 and −1791, resulted in decreased basal activity and a loss of AR-mediated repression. AR bound to the −1796/−1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region. We conclude that AR repression of GnRH-E1 acts via multiple AR-responsive regions, including the site at −1792/−1791.

Otx2 Induction of the Gonadotropin-releasing Hormone Promoter Is Modulated by Direct Interactions with Grg Co-repressors

Hormonal communication between the hypothalamus, pituitary, and gonads orchestrates the development and regulation of mammalian reproductive function. In mice, gonadotropin-releasing hormone (GnRH) expression is limited to approximately 1000 neurons that originate in the olfactory placode then migrate to specific positions scattered throughout the hypothalamus. Coordination of the hypothalamic-pituitary-gonadal axis is dependent upon correct migration of GnRH neurons into the hypothalamus followed by the appropriate synthesis and pulsatile secretion of GnRH. Defects in any one of these processes can cause infertility. Recently, substantial progress has been made in identifying transcription factors, and their cofactors, that regulate not only adult expression of GnRH, but also the maturation of GnRH neurons. Here, we show that expression of Otx2, a homeodomain protein required for the formation of the forebrain, is dramatically up-regulated during GnRH neuronal maturation and that overexpression of Otx2 increases GnRH promoter activity in GnRH neuronal cell lines. Furthermore, Otx2 transcriptional activity is modulated by Grg4, a member of the Groucho-related-gene (Grg) family. Using mutational analysis, we show that a WRPW peptide motif within the Otx2 protein is required for physical interaction between Otx2 and Grg4. Without this physical interaction, Grg4 cannot repress Otx2-dependent activation of GnRH gene transcription. Taken together, these data show that Otx2 is important for GnRH expression and that direct interaction between Otx2 and Grg co-repressors regulates GnRH gene expression in hypothalamic neurons.

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

Insulin increases gonadotropin-releasing hormone (GnRH) gene expression in in vitro models of GnRH neurons. Early growth response-1 (Egr-1) is a transcription factor that mediates the effect of insulin on target genes. In the GN11 cell line – an immortalized GnRH-secreting neuronal cell line – insulin maximally increases Egr-1 mRNA after 30 min of treatment and Egr-1 protein and GnRH mRNA after 60 min of treatment. Egr-1 small interfering RNA blocks the insulin-induced increase in GnRH promoter activity, measured as luciferase expression. Chromatin immunoprecipitation using Egr-1 antibody precipitates DNA in a proximal region of the GnRH promoter but not DNA in a distal region. Mutagenesis of a putative Egr-1 binding site within the proximal region blocks the insulin-induced increase in GnRH promoter activity. Thus, Egr-1 binds the GnRH promoter at a site between −67 and −76 bp from the transcriptional start site to mediate the insulin-induced increase in GnRH gene transcription.

The Groucho-related Gene Family Regulates the Gonadotropin-releasing Hormone Gene through Interaction with the Homeodomain Proteins MSX1 and OCT1

Gonadotropin-releasing hormone (GnRH) is exclusively expressed in a unique population of hypothalamic neurons that controls reproductive function. GnRH gene expression is highly dynamic. Its transcriptional activity is regulated in a complex spatiotemporal manner during embryonic development and postnatal life. Although a variety of transcription factors have been identified as regulators of GnRH transcription, most are promiscuous in their DNA-binding requirements, and none are solely expressed in GnRH neurons. Their specific activity is probably determined by interactions with distinct cofactors. Here we find that the Groucho-related gene (GRG) family of co-repressors is expressed in a model cell line for the GnRH neuron and co-expresses with GnRH during prenatal development. GRG proteins associate in vivo with the GnRH promoter. Furthermore, GRG proteins interact with two regulators of GnRH transcription, the homeodomain proteins MSX1 and OCT1. Co-transfection experiments indicate that GRG proteins regulate GnRH promoter activity. The long GRG forms enhance MSX1 repression and counteract OCT1 activation of the GnRH gene. In contrast, the short form, GRG5, has a dominant-negative effect on MSX1-dependent repression. Taken together, these data suggest that the dynamic switch between activation and repression of GnRH transcription is mediated by recruitment of the GRG co-regulators.

Effects of Ovariectomy on the Neuroendocrine Axes Regulating Reproduction and Energy Balance in Young Cynomolgus Macaques

Degeneration of the ovary in middle-aged women results in castrate levels of ovarian steroids and increased gonadotropin secretion from the anterior pituitary gland. Ageing in women is also accompanied by significant changes in energy homeostasis. We have observed alterations in hypothalamic morphology and gene expression in older women, including hypertrophy and increased gene expression of neurokinin B (NKB) neurones, elevated levels of gonadotropin releasing-hormone (GnRH) mRNA and decreased numbers of neurones expressing pro-opiomelanocortin (POMC) mRNA. To determine if loss of ovarian steroids could produce comparable changes in gene expression in young primates, we measured the effects of ovariectomy on NKB, GnRH and POMC gene expression in young cynomolgus monkeys. We also measured serum leptin and body weight to examine the consequences of ovariectomy on energy balance. NKB neurones in the infundibular nucleus of ovariectomized monkeys were larger, more numerous and displayed increased levels of NKB mRNA compared to those of intact controls. Moreover, ovariectomy increased the number of neurones expressing GnRH gene transcripts and elevated serum luteinizing hormone. By contrast, several parameters related to energy balance, including POMC gene expression, serum leptin and body weights, were unchanged by ovariectomy. Thus, the rise in NKB and GnRH gene expression in older women was simulated by ovariectomy in monkeys, but the changes in POMC gene expression and energy balance were not. This study provides strong support for the hypothesis that ovarian failure contributes to the increased NKB and GnRH gene expression observed in postmenopausal women.

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.

9- cis-Retinoic acid represses transcription of the gonadotropin-releasing hormone (GnRH) gene via proximal promoter region that is distinct from all- trans-retinoic acid response element

We previously reported an enhancing effect of all- trans-retinoic acid (all- trans-RA) on gonadotropin-releasing hormone (GnRH) gene transcription via distal promoter elements of the rat GnRH gene. The present study examined the effects of another biologically active retinoid, 9- cis-retinoic acid (9- cis-RA), on GnRH transcription in GT1-1 cells. Similar to the action of all- trans-RA, 9- cis-RA significantly induced the luciferase activity of the strong retinoic acid response element (RARE) reporter construct, 3XβRARE-Luc, by about 60-fold, indicating that GT1-1 cells are also responsive to 9- cis-RA. In contrast to the stimulatory effect of all- trans-RA on GnRH transcription, 9- cis-RA inhibited the GnRH promoter activity in a dose- and time-dependent manner. Significant inhibition by 9- cis-RA required at least an 18 h treatment and a further decrease of GnRH promoter-driven luciferase activity was observed up to 48 h of incubation. Accordingly, GnRH mRNA levels were decreased by 9- cis-RA treatment in a similar dose- and time-related manner, indicating that mouse GnRH expression is also negatively regulated by 9- cis-RA. Transient transfections of serial deletion constructs of the rat GnRH promoter revealed that the −230/−110 sequence of the rat GnRH promoter is responsible for 9- cis-RA-induced inhibition of GnRH transcription. Within this region, however, no consensus retinoid X receptor response element was found. To gain insights into the role of retinoid X receptors (RXRs) in GnRH expression, we examined the effects of RXR overexpression on GnRH transcriptional activity. Interestingly, co-transfection of RXR overexpression vectors significantly increased the GnRH promoter-driven luciferase activity, while treatment with 9- cis-RA not only nullified the enhancing effect of RXR overexpression but also decreased the basal GnRH promoter-driven luciferase activity by 50% compared to vehicle-treated controls. This implies that RXRs in the absence of its cognate ligand 9- cis-RA contribute to the maintenance of basal GnRH gene transcription. Northern blot analysis revealed that 9- cis-RA, but not all- trans-RA, down-regulated RXRβ expression in GT1-1 cells, suggesting that one possible mechanism of 9- cis-RA-induced repression involves down-regulation of RXR expression. In conclusion, the present study clearly demonstrates that 9- cis-RA is a negative regulator of GnRH gene expression in immortalized GnRH neurons.

Regulation of Gonadotropin-Releasing Hormone (GnRH) Gene Expression during GnRH Neuron Migration in the Mouse

The mechanisms underlying the migration of the gonadotropin-releasing hormone (GnRH) neurons from the nose into the forebrain are not resolved. In an attempt to characterize further the migrating GnRH neurons, we have employed in situ hybridization techniques and transgenic mouse models to examine levels of GnRH mRNA and GnRH gene transcription in GnRH neurons during migration in the mouse. In the first experiment, cellular levels of GnRH mRNA in neurons located throughout the nose and forebrain were examined in embryonic day (E) 12.5, 14.5, 16.5 and 19.5 mice using in situ hybridization. The GnRH mRNA content of cells located in both the nose (p < 0.01) and forebrain (p < 0.05) was found to increase significantly from E12.5 to E19.5 and from E14.5 to E19.5, respectively. However, cellular levels of GnRH mRNA were not significantly different in neurons located in the nose compared with the brain at each developmental age. In the second experiment, levels of GnRH gene transcription were investigated at E14.5 using two different GNLZ transgenic mouse lines in which 13.5 kb of GnRH gene sequences direct the expression of the LacZ reporter to the nucleus of GnRH neurons. Migrating GnRH neurons displayed up to a 3-fold increase (p < 0.01) in transgene expression, an index of GnRH transcription, precisely as they approached and entered the forebrain. These results indicate that GnRH gene expression in migrating GnRH neurons is likely regulated by temporal as well as spatial factors and that, as found postnatally, this may involve both transcriptional and post-transcriptional regulatory mechanisms.

Neuroendocrine mechanisms for reproductive senescence in the female rat: gonadotropin-releasing hormone neurons.

Reproductive aging in female rats is characterized by profound alterations in the neuroendocrine axis. The preovulatory luteinizing hormone (LH) surge is attenuated, and preovulatory expression of the immediate early gene fos in gonadotropin-releasing hormone (GnRH) neurons is substantially reduced in middle-aged compared with young rats. We tested the hypothesis that alterations in GnRH gene expression may be correlated with the attenuation of the LH surge and may be a possible mechanism involved in neuroendocrine senescent changes. Sprague-Dawley rats ages 4 to 5 mo (young), 12-14 mo (middle-aged), or 25 to 26 mo (old) were killed at 10:00 AM or 3:00 PM on proestrus, the day of the LH surge, or diestrus I in cycling rats, and on persistent estrus or persistent diestrus in acyclic rats. RNase protection assays of GnRH mRNA and GnRH primary transcript were performed. GnRH mRNA levels increased significantly with age, whereas GnRH primary transcript levels, an index of GnRH gene transcription, decreased in old compared to young and middle-aged rats. This latter result suggests that an age-related change in GnRH mRNA levels occurs independently of a change in gene transcription, indicating a potential posttranscriptional mechanism. On proestrus, GnRH mRNA levels increased significantly from 10:00 AM to 3:00 PM in young rats. This was in contrast to proestrous middle-aged rats, in which this afternoon increase in GnRH mRNA levels was not observed. Thus, the normal afternoon increase in GnRH mRNA levels on proestrus is disrupted by middle age and may represent a substrate for the attenuation of the preovulatory GnRH/LH surge that occurs in rats of this age, prior to reproductive failure.

Regulation of gonadotropin-releasing hormone gene transcription

The GT1-7 cell line, derived from gonadotropin-releasing hormone (GnRH) neurons of the mouse hypothalamus, has provided a useful system for the analysis of GnRH gene regulation. We have used these cells to examine the mechanism of glucocorticoid repression of GnRH gene transcription. One GnRH negative glucocorticoid response element (nGRE) that contributes to glucocorticoid repression is not bound directly by the glucocorticoid receptor (GR). Rather, GR is tethered to this nGRE by virtue of its interaction with a DNA-bound POU domain transcription factor (i.e. Oct-1). DNA-dependent conformational changes in Oct-1 play a major role in recruiting GR to the distal nGRE and impacts transcriptional repression brought about by either glucocorticoids or tumor-promoting phorbol esters. GT1-7 cell-specific transcription of the mouse GnRH gene is controlled by an enhancer element that shares a high degree of sequence homology with the rat GnRH gene enhancer. As in the rat gene, Oct-1 is important for mGnRH enhancer activity. Furthermore, enhancer activity appears to be influenced by the DNA-dependent conformation adopted by bound Oct-1. Thus, the precise sequence recognized by Oct-1 appears to play a important role in both cell-specific and hormonal regulation of GnRH gene transcription.

The Glucocorticoid Receptor Is Tethered to DNA-bound Oct-1 at the Mouse Gonadotropin-releasing Hormone Distal Negative Glucocorticoid Response Element

An element required for glucocorticoid repression of mouse gonadotropin-releasing hormone (GnRH) gene transcription, the distal negative glucocorticoid response element (nGRE), is not bound directly by glucocorticoid receptors (GRs) but is recognized by Oct-1 present in GT1-7 cell nuclear extracts or by Oct-1 purified from HeLa cells. Furthermore, purified full-length GRs interact directly with purified Oct-1 bound to the distal nGRE. Increasing the extent of distal nGRE match to an Oct-1 consensus site not only increases the affinity of Oct-1 binding, but also alters the conformation of DNA-bound Oct-1 and the pattern of protein DNA complexes formed in vitro with GT1-7 cell nuclear extracts. In addition, the interaction of purified GR with DNA-bound Oct-1 is altered when Oct-1 is bound to the consensus Oct-1 site. Mutation of the distal nGRE to a consensus Oct-1 site is also associated with reduced glucocorticoid repression in transfected GT1-7 cells. Furthermore, repression of GnRH gene transcription by 12-O-tetradecanoylphorbol-13-acetate, which utilizes sequences that overlap with the nGRE, is reversed by this distal nGRE mutation leading to activation of GnRH gene transcription. Thus, changes in the assembly of multi-protein complexes at the distal nGRE can influence the regulation of GnRH gene transcription.

Diurnal Rhythmicity of Gonadotropin-Releasing Hormone Gene Expression in the Rat

The release of the decapeptide gonadotropin-releasing hormone (GnRH) from neuroterminals in the median eminence varies depending upon the stage of development, time of day, reproductive season, and day of the ovarian cycle. With respect to the biosynthetic events responsible for such alterations in GnRH release, we have reported that increases in GnRH mRNA occur concomitantly with or precede the proestrous GnRH/LH surge in female rats, and the pubertal increase in GnRH. Because a hallmark of puberty is the development of nocturnal increases in gonadotropin/GnRH release, in the present study, we examined whether a rhythm in GnRH gene expression occurs that may be responsible for this rhythm of neurosecretion. Additionally, we extended the previous finding of diurnal rhythms of GnRH gene expression in cycling females to adult male rats. The mechanism for these changes, i.e. GnRH gene transcription, RNA processing or a posttranscriptional mechanisms was also elucidated. In experiment I, GnRH gene expression was measured in female rats at different stages of puberty [P25 (juvenile); ∼P37 (day of vaginal opening); and P45 (postpubertal)] at 10.00, 14.00, 18.00, 22.00, 2.00 and 6.00 h. GnRH mRNA levels increased significantly during pubertal development, independent of any changes in GnRH gene transcription or RNA processing, indicating a posttranscriptional mechanism. GnRH mRNA levels did not vary with time of day in these pubertal rats, indicating that the diurnal rhythm of GnRH release that develops during puberty is probably a posttranslational event. In experiment II, GnRH gene expression was measured in adult male rats at the same 4-hour intervals. GnRH mRNA levels varied significantly with time of day: they increased from 10.00 to 14.00 h, remained elevated through 22.00 h, then decreased significantly to the nadir at 10.00 h. The mechanism for these changes appears to involve processing or stability of the GnRH mRNA in the nucleus. These results indicate that GnRH mRNA levels are regulated differentially with time of day depending upon age and gender.

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...

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.

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.

Characterization of gonadotropin-releasing hormone gene transcripts in a mouse hypothalamic neuronal GT1 cell line

We have characterized the nuclear and cytoplasmic RNA transcripts derived from the gonadotropin releasing hormone (GnRH) gene in a mouse hypothalamic neuronal GT1 cell line. Analyses of nuclear GnRH RNA precursors present in the GT1 cells by RNase protection assay show that there is no particular order of intron excision, suggesting the existence of multiple processing pathways. A similar pattern is observed in mouse preoptic area–anterior hypothalamus (POA-AH). In GT1 cells, approximately 5% of the total GnRH RNA transcripts are found in the nucleus. In contrast, in the POA-AH of mice, nuclear transcripts comprise 40% of the total GnRH transcripts. Thus the GT1 cells, while similar in overall GnRH RNA processing to mouse hypothalamic GnRH neurons, do not exhibit the high abundance of nuclear GnRH RNA transcripts seen in the rodent GnRH neuron in vivo. Quantitative analysis of the nuclear RNA species shows that the GnRH primary transcript comprises more than 90% of the total nuclear GnRH mRNA precursors in both GT1 cells and mouse POA-AH and thus GnRH processing intermediates account for fewer than 10% of these precursors. Using these probes, we have examined changes in GnRH primary transcript expression in GT1-7 cells. In the presence of RNA synthesis inhibitors, the half-life of the GnRH primary transcript was found to be quite short, ∼ 18 min, suggesting that the level of primary transcript would reflect levels of GnRH gene transcription. When GT1-7 cells are treated with the phorbol ester PMA (phorbol, 12-myristate, 13-acetate) for 1 h, GnRH primary transcript levels decrease by ∼ 70%. Supporting the hypothesis that GnRH primary transcript is a good indicator of GnRH gene transcription is the finding that 1 h of PMA treatment results in a similar (∼ 50%) decrease in GnRH gene transcription, as assayed by nuclear run-on assay. Our observation that GT1 cells resemble mouse hypothalamic GnRH neurons in their pattern of intron excision and in the ratio of primary transcript to other nuclear transcripts emphasizes the utility of these cells for studying the regulation of GnRH gene expression in this immortalized hypothalamic cell line.

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.

Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor.

We have identified two regions of the mouse gonadotropin-releasing hormone (GnRH) promoter, one between -237 and -201 (distal element) and the other between -184 and -150 (proximal element), which are required for glucocorticoid repression in transiently transfected GT1-7 cells. These sequences show no similarity to known positive or negative glucocorticoid response elements (nGREs) and do not function when placed upstream of heterologous viral promoters. The glucocorticoid receptor (GR) does not bind directly to the distal or proximal promoter elements but may participate in glucocorticoid repression of GnRH gene transcription by virtue of its association within multiprotein complexes at these nGREs. Electrophoretic mobility shift assays with GT1-7 nuclear extract demonstrate the presence of GR-containing protein complexes on GnRH nGREs. One protein that co-occupies the distal nGRE in vitro along with GR is the POU domain transcription factor Oct-1. Thus, the tethering of GR to the GnRH distal nGRE, by virtue of a direct or indirect association with DNA-bound Oct-1, could play a role in hormone-dependent transcriptional repression of the GnRH gene. In contrast, Oct-1 does not appear to be a component of the GR-containing protein complex that is bound to the proximal nGRE.