ABSTRACT
Results from our previous studies have demonstrated regulatory effects of estradiol on human gonadotropin-releasing hormone (GnRH) gene expression in human placental cells. The present study was designed to determine the molecular mechanisms whereby estrogens regulate the human GnRH gene expression in the placenta. The effects of estradiol on human GnRH upstream promoter activity in JEG-3 cells depends on the amounts of estrogen receptor (ER) alpha expression vector co-transfected, with the maximal effect obtained at the amount of 1.0 microg of ER expression vector cotransfected. Estriol, an isoform of estradiol, also possesses a regulatory effect on the upstream promoter activity, while estrone, another isoform, does not. Serial deletion studies revealed two estrogen responsive elements in the GnRH upstream promoter region. One element (-987 to -968 bp, E4 element) confers a negative estradiol response, while another one (-827 to -730 bp) is responsible for a positive estradiol effect. Replacement of these two elements with unrelated DNA sequences could abolish the responsiveness to estradiol treatment. Furthermore, footprinting and gel shift assays demonstrated that nuclear protein from estradiol-treated JEG-3 cells, but not from control cells, could bind to a 41 bp DNA fragment (-824 to -784 bp) within the estrogen positive responsive element. Results of gel-shift assay demonstrated that other protein(s) might also be involved in interacting the E4 element to mediate the negative effect of estradiol on the hGnRH upstream promoter activity in JEG-3 cells.
Subject(s)
Estrogens/pharmacology , Gonadotropin-Releasing Hormone/genetics , Placenta/cytology , Promoter Regions, Genetic/genetics , Response Elements/physiology , Dose-Response Relationship, Drug , Estradiol/pharmacology , Estrogens/physiology , Gene Expression Regulation/drug effects , Humans , Mutagenesis, Site-Directed , Placenta/metabolism , Promoter Regions, Genetic/drug effects , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Response Elements/drug effects , Transfection , Tumor Cells, CulturedABSTRACT
In previous studies, we have localized four specific nuclear protein-binding elements in the human GnRH upstream promoter. To test whether these four elements are reproductive tissue specific, we placed the four elements upstream to a thymidine kinase (TK) promoter/luciferase reporter gene, and transfected the constructs into human placental choriocarcinoma (JEG-3) cells. The 272-bp fragment (-994 to -723) containing the four elements can drive heterologous TK promoter expression in JEG-3 cells about 15 times more than that of basal TK promoter activity. Deletion of element 4 (E4, -987/-968) significantly decreased (4-fold) the luciferase activity. Further deletion of the other elements (E3 individual, -960/-940 or E3 and E2 in combination, -919/-896) only slightly decreased the luciferase activity. In contrast, deletion of element 1 (E1, -876/-851) caused a 2-fold loss of luciferase activity and elimination of E2 and E3 only lost less than 2-fold of the luciferase activity. Study performed with 5' end deletion of this region confirmed these observations. Furthermore, E4 DNA-protein complex can be supershifted by Oct-1 antibody, indicating that Oct-1 binds to E4. These results clearly demonstrated that all four elements are required to confer tissue-specific expression of the hGnRH gene in JEG-3 cells. However, the E4 is the most important for the tissue-specific expression of the hGnRH gene in JEG-3 cells. Oct-1 factor binds with E4 element and may be involved in the mediation of the human GnRH upstream promoter activity.
Subject(s)
DNA-Binding Proteins/metabolism , Gonadotropin-Releasing Hormone/genetics , Homeodomain Proteins/metabolism , Promoter Regions, Genetic/physiology , Transcription Factors/metabolism , Gene Deletion , Gene Expression/physiology , Genes, Regulator/physiology , Host Cell Factor C1 , Humans , Octamer Transcription Factor-1 , Stereoisomerism , Thymidine Kinase/genetics , Tumor Cells, CulturedABSTRACT
OBJECTIVE: This study was undertaken to clone and express a recombinant human zona pellucida protein 3 and to characterize its biologic activities as a sperm ligand and an inducer of the acrosome reaction. STUDY DESIGN: Human ovarian teratocarcinoma (PA-1) cells were transfected with an expression vector containing human zona pellucida protein 3 complementary deoxyribonucleic acid with a sequence coding for a 6-histidine tail introduced into its 3' end. Purification of the secreted glycoprotein was performed by sequential affinity (lectin and nickel--nitrilotriacetic acid) and ion-exchange chromatography. RESULTS: Western blot analysis confirmed a molecular weight of approximately 65 kd for the purified product. A cell-free translation system revealed a correctly sized protein backbone of 47 kd. The recombinant human zona pellucida protein 3 demonstrated specific, potent, and dose-dependent competitive inhibition of sperm-zona pellucida binding in vitro under hemizona assay conditions. Recombinant human zona pellucida protein 3 also stimulated the acrosome reaction of live sperm. This effect was fast, dose dependent, and capacitation time dependent. Furthermore, advance incubation with pertussis toxin, an inactivator of heterotrimeric G proteins, blocked recombinant human zona pellucida protein 3--induced acrosomal exocytosis. CONCLUSION: The recombinant human zona pellucida protein 3 expressed in PA-1 cells manifested the full spectrum of expected biologic activities. It therefore represents a valuable tool for examination of human fertilization and the design of new strategies in diagnosis of male factor infertility and in contraception.
Subject(s)
Egg Proteins/pharmacology , Membrane Glycoproteins/pharmacology , Ovary/physiology , Receptors, Cell Surface , Sperm-Ovum Interactions/drug effects , Acrosome Reaction/drug effects , Acrosome Reaction/physiology , Blotting, Western , Chromatography, Affinity , Chromatography, Ion Exchange , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Egg Proteins/biosynthesis , Egg Proteins/genetics , Female , Humans , Male , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Ovarian Neoplasms , Ovary/metabolism , Pertussis Toxin , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sperm Capacitation/drug effects , Sperm Capacitation/physiology , Sperm Motility/drug effects , Sperm-Ovum Interactions/physiology , Spermatozoa/physiology , Transfection , Tumor Cells, Cultured , Virulence Factors, Bordetella/pharmacology , Zona Pellucida GlycoproteinsABSTRACT
The postnatal pattern of renal endothelial nitric oxide synthase (eNOS) is unknown. The purpose of this study was to characterize eNOS expression during maturation and compare this to neuronal NOS (nNOS). The experiments measured whole kidney eNOS mRNA expression by RT-PCR and protein content by Western blot, as well as cortical and medullary protein content in piglets at selected postnatal ages and in adult pigs. Whole kidney eNOS mRNA was compared with nNOS. Whole kidney eNOS expression decreased from the newborn to its lowest at 7 days, returning by 14 days to adult levels. This eNOS mRNA pattern contrasted with nNOS, which was highest at birth, and progressively decreased to its lowest level in the adult. At birth, cortical eNOS protein was greater than medullary, contrasting with the adult pattern of equivalent levels. In conclusion eNOS is developmentally regulated during early renal maturation and may critically participate in renal function during this period. The eNOS developmental pattern differs from nNOS, suggesting that these isoforms may have different regulatory factors and functional contributions in the postnatal kidney.
Subject(s)
Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Kidney/growth & development , Nitric Oxide Synthase/genetics , Transcription, Genetic , Aging , Animals , Animals, Newborn , Kidney/enzymology , Kidney Cortex/enzymology , Kidney Cortex/growth & development , Kidney Medulla/enzymology , Kidney Medulla/growth & development , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type III , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , SwineABSTRACT
To determine if the developing kidney differs from the adult in the expression of the neuronal nitric oxide synthase, NOS I, these experiments measured mRNA gene expression by RNase protection assay and protein content by Western blot of NOS I in piglets at ages newborn and 3, 7, 10, 14, and 21 days and adult pigs. Whole kidney NOS I mRNA was greatest at birth and decreased progressively during renal maturation to adult levels. NOS I protein content paralleled this developmental pattern. Cortical NOS I protein was equivalent in newborn and 14-day-old piglets and was greater at both ages than the adult. Medullary NOS I protein was relatively greater than cortical in both immature ages and decreased from a peak at birth to adult levels. We conclude the following. 1) During postnatal maturation, renal NOS I mRNA and protein content show a pattern that is developmentally regulated. 2) This developmental pattern of NOS I after birth may, in part, contribute to the enhanced functional role of NO during renal maturation.
Subject(s)
Kidney Cortex/enzymology , Kidney Medulla/enzymology , Nitric Oxide Synthase/genetics , Age Factors , Animals , Animals, Suckling , Base Sequence , Blotting, Western , DNA, Complementary , Gene Expression Regulation, Developmental/physiology , Gene Expression Regulation, Enzymologic/physiology , Kidney Cortex/growth & development , Kidney Medulla/growth & development , Molecular Sequence Data , Nitric Oxide/metabolism , Nitric Oxide Synthase/analysis , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type I , RNA, Messenger/analysis , SwineABSTRACT
We have isolated a second goldfish estrogen receptor (ER) beta-subtype (gfER-beta2) cDNA which is distinct from the liver-derived ER-beta (gfER-beta1) cDNA reported previously. The 2650-bp cDNA, isolated from a goldfish pituitary and brain cDNA library, encodes a 610 amino acid (aa) protein which shows only a 53% aa sequence identity with gfER-beta1 in overall structure. RT-PCR analysis showed that mRNA of gfER-beta2, in contrast to that of gfER-beta1, was predominantly expressed in pituitary, telencephalon and hypothalamus as well as in liver of female goldfish. The existence of a second distinct ER-beta subtype opens new dimensions for studying tissue-specific regulation of gene expression by estrogen in the tetraploid goldfish.
Subject(s)
Goldfish/genetics , Receptors, Estrogen/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Brain/metabolism , Cloning, Molecular , DNA, Complementary/isolation & purification , Estrogen Receptor beta , Female , Gene Library , Liver/metabolism , Molecular Sequence Data , Phylogeny , Pituitary Gland/metabolism , RNA/isolation & purification , Receptors, Estrogen/chemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In the baboon, estrogen regulated 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzed metabolism of cortisol and cortisone by the placenta is an important component in the sequence of events regulating the fetal pituitary-adrenocortical axis. The present study was designed to isolate and sequence the promoter region of the baboon 11beta-HSD-2 gene and to produce constructs of this gene and the 1.7 kb fragment of 5'-flanking region of baboon 11beta-HSD-1 isolated previously in order to determine whether the promoters of these two genes were activated in human placental JEG-3 cells and whether expression could be modulated by estradiol. The 11beta-HSD-2 genomic DNA was isolated from a baboon kidney genomic library using a human 11beta-HSD-2 cDNA as a probe. The sequence of a 1.2 kb fragment of the 5'-flanking region showed extensive homology with that published by others for human 11beta-HSD-2, particularly in exon 1 (>95%) and in the proximal promoter (>90%). Primer extension confirmed that the baboon 11beta-HSD-2 gene has multiple transcriptional start sites which are preceded by a GC box. To determine promoter activity of 11beta-HSD-2 and -1, the 5'-flanking regions of these genes were subcloned into luciferase reporter pGL3 vectors, transiently transfected into human placental JEG-3 cells, and then incubated for 16-18 h in the presence or absence of 10-8 M 17beta-estradiol or 17alpha-estradiol. To augment the low level of estrogen receptor (ER) in JEG cells, promoter activity studies were also performed in JEG cells co-transfected with an expression vector containing the human ER cDNA. The promoters of both 11beta-HSD-1 and -2 were activated following transient transfection into JEG-3 cells although basal activity of 11beta-HSD-2 (87+/-21 RLU/microg protein) always exceeded (P<0.05) that of 11beta-HSD-1 (37+/-7). In the absence of co-transfected ER, basal promoter activities of both 11beta-HSD genes were not altered by 17beta-estradiol. In contrast, in cells co-transfected with ER, 17beta-estradiol but not 17alpha-estradiol increased (P<0.05) basal promoter activities of 11beta-HSD-1 and -2 by 8.1+/-1.5 and 8.3+/-2. 0 fold, respectively. Collectively, these findings indicate that the promoter region of the baboon 11beta-HSD-2 gene is comparable to that in the human and that the 5'-flanking region of both the baboon 11beta-HSD-1 and -2 genes were active when transiently transfected into JEG-3 cells and that activation could be enhanced by estradiol in the presence of an estrogen receptor.
Subject(s)
Estradiol/pharmacology , Hydroxysteroid Dehydrogenases/genetics , Promoter Regions, Genetic/drug effects , 11-beta-Hydroxysteroid Dehydrogenases , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , DNA/chemistry , Gene Expression Regulation/drug effects , Humans , Hydroxysteroid Dehydrogenases/biosynthesis , Kidney/metabolism , Molecular Sequence Data , Papio , Placenta/metabolism , RNA/isolation & purification , Receptors, Estrogen/biosynthesis , Receptors, Estrogen/genetics , Sequence Homology, Nucleic Acid , TransfectionABSTRACT
To examine the transcriptional regulation of gonadotropin-releasing hormone (GnRH) gene in reproductive tissues, the expression of human GnRH gene promoter in cultured human granulosa cells and a Chinese hamster ovary-derived CHO-K1 tumor cells was studied. Transfection of luciferase reporter gene construct containing either upstream (hU) or downstream (hD) human GnRH gene promoter into both human granulosa and CHO-K1 cells showed that the upstream promoter, hU, was more active than hD in directing luciferase expression in these ovarian tissues. CHO-K1 cells transfected with either hU or hD construct showed insignificant changes in luciferase activity in response to 17beta-estradiol and GnRH. However, cotransfection of hU construct with a vector expressing a human estrogen receptor-alpha (ER-alpha) cDNA results in dose-dependent decreases in luciferase activity in response to both 17beta-estradiol and a GnRH agonist. By functional analysis of a series of deletion constructs, the ER-mediated suppression of GnRH promoter activity by 17beta-estradiol was localized to a region between -169 and -548 bp 5' of the upstream transcription start site of the human GnRH gene. Results of this study demonstrated that estrogen receptor can mediate the negative feedback regulation of human GnRH upstream promoter activity by both estrogen and GnRH in the ovary.
Subject(s)
Gonadotropin-Releasing Hormone/metabolism , Promoter Regions, Genetic , Receptors, Estrogen/metabolism , Animals , Base Sequence , CHO Cells , Cells, Cultured , Cricetinae , Estradiol/pharmacology , Female , Genes, Reporter/genetics , Granulosa Cells/metabolism , Humans , Luciferases/genetics , Mice , Molecular Sequence Data , TransfectionABSTRACT
Many peripheral reproductive tissues have been found to contain gonadotrophin-releasing hormone (GnRH) and express the GnRH gene at low levels, presumably because the hormone functions in a paracrine/autocrine fashion. This study was designed to investigate and characterize GnRH gene expression in human endometrial tissue at different stages of the endometrial cycle. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis together with Southern blot assay demonstrated that human endometrial tissue expresses the proGnRH gene. RNA samples from endometrial tissue were analysed with two pairs of oligonucleotide primers. Both gave a doublet 870 bases apart at the expected sizes, indicating that both the upstream and downstream transcriptional start sites of the GnRH gene are used in endometrial tissue and that transcripts with and without intron 1 were produced. Our data also demonstrated that utilization of the two promoters varies with the stage of the endometrial cycle. The largest difference came from the mRNA transcribed from the downstream promoter and without intron 1. This mRNA was expressed at a very low level during the proliferative phase and dramatically increased almost 10-fold (P < 0.01) during the early secretory phase, and subsequently decreased 5-fold during the late secretory stage. The presence of GnRH mRNA in the endometrium, as well as the differential expression of the GnRH gene during the early secretory phase provides physiological evidence that human GnRH may play a paracrine/autocrine function in the human uterus.
Subject(s)
Endometrium/metabolism , Gonadotropin-Releasing Hormone/genetics , Adult , Base Sequence , DNA Primers/genetics , Female , Gene Expression , Humans , Menstrual Cycle/genetics , Protein Precursors/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
PURPOSE: Our purpose was to examine implantation of singleton pregnancies achieved following various assisted reproductive technologies (ARTs) through the appearance and rising titers of serum human chorionic gonadotropin (hCG) levels. METHODS: A total of 114 singleton pregnancies resulting from in vitro fertilization and intrauterine insemination was analyzed. Patients were divided into five groups according to the type of ovarian stimulation protocol [gonadotropin stimulation with/without the use of gonadotropin-releasing hormone agonist (GnRHa), long protocol, or flare-up technique] and to the day of embryo transfer (day 2 or day 3 after oocyte retrieval). Serial serum hCG levels were measured between 10 and 25 days after fertilization and log-transformed. Linear regression analyses were performed and extrapolated to hCG = 10 mIU/ml (hCG10), which was used as an estimate of detectable implantation. The slopes of the regression lines were used to estimate the rising speed of hCG. RESULTS: There were no significant differences in the days of hCG in maternal serum to reach 10 mIU/ml (implantation) or in the slopes of the regression lines for all five studied groups. CONCLUSIONS: The appearance of hCG in maternal serum was used to assess the time of clinically detectable implantation. Furthermore, because hCG production is a marker of trophoblastic activity, its serum doubling time was used as an indicator of embryo quality. Results showed that in various ART protocols with and without GnRHa, there were no significant differences in implantation time or embryo quality. Embryo development in early pregnancy follows a preprogrammed-timing schedule and depends mainly on the embryonic age of the healthy, successfully implanted conceptus.
Subject(s)
Chorionic Gonadotropin/blood , Embryo Implantation/physiology , Fertilization in Vitro , Insemination, Artificial , Adult , Female , Fertility Agents, Female/therapeutic use , Follicle Stimulating Hormone/therapeutic use , Humans , Immunoenzyme Techniques , Leuprolide/therapeutic use , Linear Models , Male , Menotropins/therapeutic use , Microspheres , Pregnancy , Pregnancy Outcome , Radioimmunoassay , Regression Analysis , Retrospective StudiesABSTRACT
A human gonadotrophin releasing hormone (GnRH) upstream promoter/luciferase reporter gene construct (H2 construct) was generated by inserting a 1.7 kb XbaI/AflII fragment containing the human GnRH upstream promoter region only into a promoter-less luciferase reporter vector. When JEG-3 cells were transiently transfected with this construct and treated with cortisol or its synthetic analogue dexamethasone, a stimulatory effect on the upstream promoter activity was observed. This stimulation was dependent on the cotransfection of a glucocorticoid receptor (GR) cDNA expression vector due to the low level of GR in JEG-3 cells and could be completely abolished by RU486, a glucocorticoid antagonist. Moreover, the cortisol actions could be modulated to a different extent by oestradiol. Thus, since the human placenta contains GRs and the increase in cortisol metabolism near term is regulated by oestrogen, the current findings suggest that cortisol may be physiologically involved in the regulation of GnRH gene expression in the human placenta.
Subject(s)
Gene Expression Regulation/physiology , Glucocorticoids/pharmacology , Gonadotropin-Releasing Hormone/genetics , Placenta/metabolism , Promoter Regions, Genetic/genetics , Cell Line , Dexamethasone/pharmacology , Estradiol/pharmacology , Gene Expression Regulation/drug effects , Genes, Reporter/genetics , Glucocorticoids/antagonists & inhibitors , Hormone Antagonists/pharmacology , Humans , Hydrocortisone/pharmacology , Luciferases/genetics , Mifepristone/pharmacology , Placenta/cytology , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/physiology , Recombinant Fusion Proteins , TransfectionABSTRACT
Two promoters directing tissue-specific expression of GnRH gene in neuronal and reproductive tissues were characterized by functional analyses of GnRH promoter-luciferase (LUC) constructs in transfected placental cells (JEG) and hypothalamic neuronal cells (GT1-7). Results indicate that the downstream promoter directs the expression in a neuronal cell-specific manner, whereas the upstream promoter is fully active in the nonneural placental cell line. Transfection studies carried out in several tumor cell lines derived from human reproductive tissues verified that the upstream GnRH promoter construct was much more active in directing luciferase expression in reproductive tissue. The use of both upstream and downstream promoters in various human tumor cell lines derived from reproductive tissues were demonstrated by RT-PCR. Our studies also demonstrate that the reproductive tissue-specific messenger RNA transcribed from upstream promoter is capable of directing synthesis of preproGnRH protein. Serial deletion studies localized a cell-specific upstream promoter region that directs reproductive tissue expression. DNase I footprint analysis using nuclear extract obtained from the JEG cells indicated DNA/protein interactions in four specific sequence elements of the upstream promoter region. The interaction between nuclear binding proteins present in the JEG cells (but not the GT1-7 cells) and the four specific sequences in the upstream promoter region was confirmed by gel mobility shift analysis.
Subject(s)
Gene Expression Regulation , Gonadotropin-Releasing Hormone/genetics , Promoter Regions, Genetic , Animals , Base Sequence , Cell-Free System , Cells, Cultured , Gonadotropin-Releasing Hormone/biosynthesis , Humans , Mice , Molecular Sequence Data , Protein Biosynthesis , Protein Precursors/biosynthesis , Protein Precursors/genetics , RNA, Messenger/metabolism , Restriction Mapping , TransfectionABSTRACT
In the baboon, the 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-catalyzed metabolism of maternal cortisol and cortisone by the placenta is an important component in the sequence of events regulating the function of the fetal pituitary-adrenocortical axis. The present study was designed to isolate and sequence the promoter region of the baboon 11beta-HSD-1 gene. The 11beta-HSD-1 genomic DNA was isolated from a baboon kidney genomic library using a human 11beta-HSD-1 cDNA as a probe. The sequence of a 1.7 kb fragment of the 5'-flanking region showed extensive homology (> 95) to that published by others for human 11beta-HSD-1 particularly in exons I and II (> 95%) and in the proximal promoter ( > 98%). Using total RNA from adult baboon liver annealed with a 22 bp antisense primer located at the 3' end of Exon I, parallel genomic sequencing reactions with the same primer confirmed that the baboon 11beta-HSD-1 gene has two transcriptional start sites 93 nucleotides apart and that both start sites are preceded by a CAAT box but not a TATA box. RNase protection assays confirmed that both transcription start sites are utilized in liver and near term baboon placenta and that transcripts emanating from the downstream start site dominate in the placenta in contrast to the preferential utilization of the upstream start site in adult liver.
Subject(s)
Gene Expression Regulation, Enzymologic/genetics , Hydroxysteroid Dehydrogenases/genetics , Papio/genetics , Promoter Regions, Genetic/genetics , 11-beta-Hydroxysteroid Dehydrogenases , Animals , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Developmental , Genes/genetics , Humans , Kidney , Liver/chemistry , Liver/embryology , Molecular Sequence Data , Organ Specificity , RNA, Messenger/analysis , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
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, approximately 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 approximately 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 (approximately 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.
Subject(s)
Gene Expression/genetics , Gonadotropin-Releasing Hormone/genetics , Hypothalamus/metabolism , Transcription, Genetic/genetics , Animals , Cell Line , Mice , Mice, Inbred Strains , RNA/metabolismABSTRACT
In humans, transcription of the gonadotropin-releasing hormone (GnRH) gene can be initiated at two transcription start sites to produce different GnRH mRNAs. The upstream transcription start site is used only in reproductive tissues and tumors. To determine if a similar pattern of GnRH gene expression exists in non-human primates, we cloned GnRH cDNA from rhesus monkey hypothalamic RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) and the 5' flanking region of the monkey GnRH gene by PCR. A 96% similarity between monkey and human GnRH cDNA was found with 94% similarity in the upstream promoter region. An upstream transcriptional start site, was identified in cynomolgus monkey testicular mRNA, 504 base pairs upstream from the hypothalamic site, which was different from that identified in the human GnRH gene. Various cynomolgus monkey reproductive tissues were found to utilize this upstream transcriptional start site. In contrast, no evidence was found for the use of upstream transcriptional start sites in rat testis or placenta, suggesting that the reproductive tissue specificity of the upstream transcription start site may be a primate specific feature.
Subject(s)
Gene Expression Regulation , Gonadotropin-Releasing Hormone/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Humans , Hypothalamus/metabolism , Macaca fascicularis , Macaca mulatta , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger , Rats , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
Two fragments of the human gonadotropin-releasing hormone (GnRH) promoter, one containing 0.6 kb of the downstream promoter sequence (H-1) and another 1.8 kb fragment (H-2) containing the upstream promoter region with a deletion of the downstream promoter sequence, were fused to a promoterless luciferase (Luc) reporter construct and transfected into the human placental (JEG) cells. JEG cells transfected with both constructs showed insignificant changes in luciferase activity in response to estradiol. However, cotransfection of H-2-Luc construct with a vector expressing a human estrogen receptor (ER) cDNA results in dose-dependent decreases in luciferase activity in response to estradiol. This ER mediated down-regulation of promoter activity was retained in constructs with the GnRH promoter deleted to position 548 bp 5' to the upstream transcription start site. Further deletion of upstream promoter sequence to 169 bp reversed the estrogen responsiveness from inhibitory to stimulatory. Thus, this study demonstrated that the upstream GnRH promoter region can be regulated by estrogen in transfected JEG cells.
Subject(s)
Estrogens/pharmacology , Gene Expression Regulation , Gonadotropin-Releasing Hormone/genetics , Placenta/metabolism , Promoter Regions, Genetic , Receptors, Estrogen/metabolism , Dose-Response Relationship, Drug , Estradiol/pharmacology , Humans , Placenta/cytology , Placenta/drug effects , Transfection , Tumor Cells, CulturedABSTRACT
In mice, expression of the transcription factor Oct-3 and the proto-oncogene c-mos is limited to germ cells, suggesting a specific role for these factors in gamete physiology and early embryonic development. We have studied the expression pattern of Oct-3 and c-mos in various reproductive as well as control tissues in the cynomolgus monkey, using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern analysis. Analogously with the data from the mouse model, strong expression of Oct-3 and c-mos could be detected in monkey ovary and oocytes. Unexpectedly, strong expression of c-mos was demonstrable in the pituitary gland and the amount of mRNA expression in the pituitary was roughly equal to that found in the ovary. Of the tissues examined, the testicular expression of c-mos was the most intense. Weak signal for c-mos mRNA was also seen in hypothalamus and brain; however, all other tissue types examined were negative for c-mos expression. In addition to the oocytes, expression of Oct-3 mRNA was detected in the ovarian granulosa cells, fallopian tube, myometrium, cervix, breast, liver, adrenal gland, pituitary, hypothalamus, brain cortex, prostate, and in testis. Thus, in the cynomolgus monkey, Oct-3 is predominantly, but not specifically, expressed in reproductive tissues. In the female monkey reproductive organs, the expression of c-mos seems to be germ cell specific. Therefore, further characterization of c-mos and Oct-3 functions in primate reproductive physiology, especially in gametogenesis and early embryonic development, is highly warranted.
Subject(s)
DNA-Binding Proteins/analysis , Germ Cells/metabolism , Proto-Oncogene Proteins c-mos/analysis , RNA, Messenger/analysis , Transcription Factors/analysis , Animals , Base Sequence , DNA-Binding Proteins/biosynthesis , Female , Gene Expression Regulation , Macaca fascicularis , Male , Molecular Sequence Data , Octamer Transcription Factor-3 , Organ Specificity , Proto-Oncogene Proteins c-mos/biosynthesis , Transcription Factors/biosynthesisABSTRACT
Using a transgenic mouse derived GnRH expressing neuronal cell line, GT1-3, we studied the effects of activation of cAMP, Ca2+ and protein kinase C pathways by forskolin, ionomycin and the phorbol ester phorbol 12-myristate 13-acetate (PMA), respectively, upon gonadotropin-releasing hormone (GnRH) secretion, cellular peptide content, mRNA and RNA primary transcript levels. Forskolin, ionomycin and phorbol ester all caused an increase in GnRH secretion in GT1-3 cells in a time and dose-dependent manner during a short-term (1 h) static incubation. Prolonged treatment with forskolin (10 microM), ionomycin (1 microM) and PMA (10 nM) for 12 or 24 h resulted in significant decreases in GnRH mRNA levels. Time-course studies showed that the increases in GnRH secretion stimulated by forskolin, ionomycin and PMA were gradually attenuated over time in parallel with the decreases in mRNA expression. In contrast, there were only small and variable changes in the GnRH cellular content. Studies using a GnRH antagonist (100 microM) suggested that the released GnRH has a negative feedback effect on its own secretion. However, co-incubation with the GnRH antagonist did not alter the inhibitory effects on GnRH mRNA levels by the secretagogues. Further studies on the transcriptional effects of forskolin, ionomycin and PMA on GnRH gene expression in GT1-3 cells revealed that all three secretagogues suppressed GnRH RNA primary transcript levels, with forskolin having a slower time course of action. Thus, the inhibition of cytoplasmic GnRH mRNA, and presumably its synthesis, after 12-24 h of secretagogue treatment may be due at least in part to a suppression of GnRH gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Gonadotropin-Releasing Hormone/genetics , Hypothalamus/cytology , RNA, Messenger/biosynthesis , RNA/biosynthesis , Second Messenger Systems , Animals , Cell Line, Transformed , Colforsin/pharmacology , Cyclic AMP/physiology , Gene Expression Regulation , Gonadotropin-Releasing Hormone/biosynthesis , Gonadotropin-Releasing Hormone/drug effects , Gonadotropin-Releasing Hormone/metabolism , Hypothalamus/physiology , Ionomycin/pharmacology , Mice , Mice, Transgenic , Protein Kinase C/physiology , RNA/drug effects , RNA, Messenger/drug effects , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The synthesis and release of GnRH within a specific subset of neurons in the hypothalamus, which serves as the primary drive to the hypothalamic-pituitary-gonadal (HPG) axis, is subject to various levels of control. Although a number of direct synaptic connections to GnRH-containing neurons have been identified, which presumably provide some regulatory inputs, the mechanisms responsible for hormonal regulation of GnRH synthesis and release mediated by either cell surface or intracellular receptors remain controversial. The recent demonstration that a subset of GnRH-containing neurons in the rat hypothalamus possesses immunoreactive glucocorticoid receptors (GR) implies that this class of steroid hormones could exert a direct effect to regulate the functioning of these neurons and perhaps the HPG axis. We used the GT1-3 and GT1-7 cell lines of immortalized GnRH-secreting hypothalamic neurons as a model to study the direct effects of glucocorticoids on GnRH gene expression. We demonstrated that these cell lines possess GR that bind the synthetic glucocorticoid, dexamethasone, in vitro with high affinity (Kd = 2-3 nM). These receptors are functional, as indicated by their ability to activate transcription from exogenously introduced heterologous glucocorticoid-responsive promoters. Furthermore, dexamethasone represses both the endogenous mouse GnRH gene, decreasing steady state levels of GnRH mRNA, and the transcriptional activity of transfected rat GnRH promoter-reporter gene vectors. Glucocorticoid repression of rat GnRH promoter activity appears to be mediated by sequences contained within the promoter proximal 459 basepairs and not be influenced by the relative basal activity of the GnRH promoter. Thus, our results provide the first direct demonstration of glucocorticoid repression of transcription in a hypothalamic cell line and suggest that GR acting directly within GnRH neurons could be at least partly responsible for negative regulation of the HPG axis by glucocorticoids.