Chang et al. (1989) isolated a member of the steroid receptor superfamily, which they called TR3, from a human
prostate cDNA library by use of an oligonucleotide probe to the DNA-binding domain common to members of the
steroid receptor superfamily. Sequence analysis of the TR3 cDNA revealed that it encodes a 598-amino acid protein
with domains homologous to the DNA-binding and hormone-binding domains of other members of the steroid receptor
superfamily. Chang et al. (1989) found that the TR3 receptor shares about 20% amino acid homology with the estrogen
receptor and less than 15% homology with other known receptors. The authors noted that the TR3 gene may be the
human homolog of the mouse nur77 gene, with which it shares 91% amino acid identity.
This gene was found to be regulated by gonadotropins in DNA arrays.
NCBI Summary:
This gene encodes a member of the steroid-thyroid hormone-retinoid receptor superfamily. Expression is induced by phytohemagglutinin in human lymphocytes and by serum stimulation of arrested fibroblasts. The encoded protein acts as a nuclear transcription factor. Translocation of the protein from the nucleus to mitochondria induces apoptosis. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2011]
General function
Nucleic acid binding, DNA binding, Transcription factor
The involvement of NR4A1 and NR4A2 in the regulation of the luteal function in rats. Qi L et al. (2018) The nuclear receptor 4A (NR4A) members play important roles in cellular proliferation, differentiation and apoptosis. The current study first evaluate the expression of ovarian NR4A1 during different luteal stages in rats. Immature rats aged 28 days were treated with sequential Pregnant mare serum gonadotropin (PMSG) (D -2) / human chorionic gonadotropin (hCG) (D 0) to induce pseudopregnancy. Serum progesterone (P4) and ovarian expression of NR4A1 were detected by RIA and WB, respectively, at follicle stage (D 0), early (D 2), middle (D 7) and late (D 14 and D 20) luteal stages. To confirm the role of NR4A1 during the luteal regression, rats were treated with prostaglandin F2α analog (PGF) for 0-8 h on D 7 to detect the expressions of NR4A1 and NR4A2. RIA result showed that serum P4 reached highest level on D 7 and then declined. WB results showed that there were two types of NR4A1 (NR4A1-L and NR4A1-S) expressed in the ovary. The ovarian NR4A1-L decreased at the late luteal stage (D 20). However, the NR4A1-S increased at the late luteal stage (D 14). After PGF treatment on D 7, the expression of NR4A1-S increased which peaked at 0.5-1 h and then declined; while NR4A1-L expression did not change within 8 h. Real-time PCR results showed that the ovarian NR4A1 mRNA increased within 0.5 h, maintained high at 1 h and then declined. The NR4A2 mRNA expression exhibited a similar pattern to that of NR4A1 mRNA, though its abundance was not as high as NR4A1. IHC results revealed that NR4A1-L was expressed mainly in the cytoplasm of luteal steroidogenic cells, faintly expressed in the follicle theca cells, oocytes and the pericytes; while NR4A2 was primarily localized in the cytoplasm of luteal steroidogenic cells. In conclusion, all these results demonstrate that NR4A2 as well as NR4A1 might be involved in the luteal development and luteolysis in rats.//////////////////
Stocco CO, et al 2000 reported that Prostaglandin F-2 alpha-induced expression of 20
alpha-hydroxysteroid dehydrogenase involves the transcription
factor NUR77.
Prostaglandin F(2)alpha (PGF(2)alpha) binding to its receptor on the rat
corpus luteum triggers various signal transduction pathways that lead to the
activation of a steroidogenic enzyme, 20 alpha -hydroxysteroid dehydrogenase
(20 alpha -HSD), which in turn catabolizes progesterone. Using mice lacking PGF(2)alpha
receptor and pregnant rats, the authors showed that PGF(2)alpha is responsible for the rapid and
massive expression of the 20 alpha -HSD gene at the end of pregnancy leading
to a decrease in progesterone secretion. They also present evidence that
PGF(2)alpha enhances 20 alpha -HSD promoter activity and have determined a
region upstream of the -1590 position in the 20 alpha -HSD promoter that
confers regulation by PGF(2)alpha in ovarian primary cells. This region
encompasses a unique transcription factor-binding site with a sequence of a
NUR77 response element. Deletion of this motif or overexpression of a NUR77
dominant negative protein caused a complete loss of 20 alpha -HSD promoter
activation by PGF(2)alpha. NUR77 also transactivated the 20a-HSD promoter in
transient transfection experiments in corpus luteum-derived cells (GG-CL),
This induction required the NUR77-transactivating domain. the authors show that
PGF(2)alpha induces a very rapid expression of NUR77 that binds to a distal
response element located at -1599/ -1606 but does not interact with another
proximal putative NUR77 response element located downstream in the promoter. A
rapid increase in NUR77 mRNA was observed in mice corpora lutea just before
parturition at a time when 20 alpha -HSD becomes expressed. This increase in
the expression of both genes was not seen in PGF(2)alpha receptor knockout
mice.
247 CANDIDATE mRNA REGULATING MEIOTIC RESUMPTION IN BOVINE CUMULUS-OOCYTE COMPLEXES. Hicks JE et al. When cumulus-oocyte complexes (COC) are cultured with follicle-stimulating hormone (FSH), germinal vesicle breakdown (GVBD) occurs and is transcriptionally dependent. In mice, expression of mRNA for nuclear receptor subfamily 4, member 1 (Nr4A1), and early growth response 1 (Egr1) is reduced when GVBD is blocked by the transcriptional inhibitor DRB. The objectives of this study were to define the period required for transcription initiation in bovine COC, determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COC, and reduce Nr4A1 mRNA expression using small interfering RNA (siRNA) to determine the effect on GVBD. In Experiment 1, pools of COC were randomly assigned to mature in TCM-199 supplemented with 10% estrous cow serum, 1 mug mL(-1) estradiol, 200 nm pyruvate, 5 mug mL(-1) FSH, and 50 mug mL(-1) gentamicin. The DRB (120 mum) was added at 0, 30, 60, 90, 120, 150, or 180 min of culture (n = 27 +/- 2 COC/treatment per replication; n = 5 replications). The COC were harvested at 20 h and assessed for meiotic stage. In Experiment 2, COC were randomly assigned to maturation medium with FSH and were harvested at 0, 30, 60, 90, and 180 min of culture. Semiquantitative RT-PCR was used to assess Nr4A1, Egr1, and GAPDH (housekeeper) mRNA levels (n = 39 +/- 2 COC/treatment per replication; n = 10 replications). In Experiment 3a, COC were matured for 30 min with either FSH, FSH + DRB, or FSH + 25 nm, 50 nm, or 100 nm siRNA for bovine Nr4A1 (siNr4A1). Relative levels of Nr4A1 mRNA were assessed (58 +/- 4 COC/treatment per replication; n = 4 replications). In Experiment 3b, COC were matured 9 h in medium containing either FSH, FSH + DRB, FSH + 50 nm siNr4A1, or FSH + 50 nm nonspecific siRNA (siNS). Oocytes were assessed for meiotic stage (n = 13 +/- 1 COC/treatment per replication; n = 4 replications). Data were analyzed by one-way ANOVA and Duncan's test. For Experiment 1, when DRB was added after the start of culture, progressively more oocytes underwent GVBD (least squares mean +/- SEM; 99 +/- 7%(A), 6 +/- 7%(E), 33 +/- 7%(D), 64 +/- 7%(C), 77 +/- 7%(B,C), 91 +/- 7%(A,B), 87 +/- 7%(A,B), 90 +/- 7%(A,B) for control, 0 + DRB, 30 + DRB, 60 + DRB, 90 + DRB, 120 + DRB, 150 + DRB, 180 + DRB; (A,B,C,D,E)P < 0.05). Thus, the gene transcription required for GVBD occurred between 0 and 60 min of culture. For Experiment 2, relative levels of Nr4A1 mRNA increased (P < 0.05) at 30 min of culture (100 +/- 18% v. 192 +/- 18%, for 0 v. 30 min). Relative levels of Egr1 mRNA did not change during culture. For Experiment 3a, the relative expression of Nr4A1 mRNA was decreased in cultures containing siNr4A1 (100 +/- 6%(A), 32 +/- 6%(D), 62 +/- 6%(C), 59 +/- 6%(C,D), 86 +/- 6%(B) for FSH, FSH + DRB, FSH + 25 nm siNr4A1, FSH + 50 nm siNr4A1, FSH + 100 nm siNr4A1; (A,B,C,D)P < 0.05). For Experiment 3b, there were no differences in the proportions of COC undergoing GVBD at 9 h of culture in the FSH and FSH + siNS treatments. There was a decreased percentage of GVBD oocytes at 9 h of culture with either DRB or 50 nm siNr4A1 (95 +/- 4%(A), 4 +/- 4%(C), 65 +/- 4%(B), 97 +/- 4%(A) for FSH, FSH + DRB, FSH + 50 nm siNr4A1, FSH + 50 nm siNS; (A,B,C)P < 0.05). These data are consistent with the hypothesis that Nr4A1 may play a role in regulating GVBD in bovine COC.
FSH modulates the expression of inhibin-alpha and the secretion of inhibins via orphan nuclear receptor NUR77 in ovarian granulosa cells. He QY 2013 et al.
It has been previously reported that follicle-stimulating hormone (FSH) regulates the expression of inhibin-alpha in human granulosa cells, but the precise molecular pathway remains unknown. In the present study, we investigated the role of the orphan nuclear receptor, NUR77, in both the transcriptional regulation of the inhibin a-subunit gene and the secretion of inhibins. Our results showed that in a human granulosa cell tumor-derived cell line (KGN) and in human granulosa-lutein cells (hGL), FSH induced the expression of NUR77 and inhibin-alpha, although inhibin-alpha expression did not increased following FSH treatment if NUR77 was knocked down. Furthermore, simply overexpressing or reducing NUR77 levels affected inhibin-a expression, while NUR77 overexpression improved the secretion of inhibin A and B from human granulosa cells. In addition, the chromatin immunoprecipitation-PCR, avidin-biotin-conjugated DNA precipitation, and luciferase reporter assays confirmed that NUR77 directly regulated the transcription of the inhibin-alpha gene through the specific NGFI-B response element located within its promoter. In the ovarian granulosa cells of the Nur77 knockout mice, the mRNA levels of inhibin-alpha were decreased relative to wild-type mice. These data indicate a role of NUR77 in the regulation of inhibin-alpha in ovarian granulosa cells. Mol. Reprod. Dev. 2013 Wiley Periodicals, Inc.
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Divergence of intracellular signaling pathways and early response genes of two closely related fibroblast growth factors, FGF8 and FGF18, in bovine ovarian granulosa cells. Jiang Z et al. Fibroblast growth factors (FGFs) modulate ovarian function, including FGF8 and FGF18. These FGFs activate the same receptors, although FGF18 is unusual in that it increases apoptosis in ovarian granulosa cells whereas the 'typical' response to FGF is increased proliferation. The objective of the present study was to determine which early response genes and pathways are activated by FGF8 and FGF18 in bovine granulosa cells. FGF8 increased abundance of mRNA encoding the FGF-responsive genes SPRY1, SPRY2, SPRY4, NR4A1 and NR4A3 whereas FGF18 did not. FGF8 increased but FGF18 decreased levels of mRNA encoding the growth arrest associated protein, GADD45B. FGF8 increased ERK1/2 phosphorylation but FGF18 did not. Microarray analysis identified EGR1, FOS, FOSL1, BAMBI, XIRP1 and PLK2 as other FGF8 immediate-early response genes, and FGF18 stimulated EGR1, FOSL1, BAMBI and PLK2, but not FOS or XIRP1. This study demonstrates that FGF8 and FGF18 signal through divergent pathways in ovarian granulosa cells, despite reportedly similar receptor activation patterns.
Differential actions of fibroblast growth factors on intracellular pathways and target gene expression in bovine ovarian granulosa cells. Jiang Z et al. Several fibroblast growth factors (FGFs) alter ovarian granulosa cell function, including FGF1, FGF4 and FGF10. These ligands exhibit different patterns of receptor activation, and their mechanisms of action on granulosa cells remain unknown. The objective of the present study was to identify the major pathways and target genes activated by FGF1, FGF4 and FGF10 in primary oestrogenic granulosa cells cultured under serum-free conditions. FGF1 and FGF4 increased levels of mRNA encoding Sprouty family members, SPRY2 and SPRY4, and the orphan nuclear receptors NR4A1 and NR4A3. Both FGF1 and FGF4 decreased levels of mRNA encoding SPRY3 and the pro-apoptotic factor BAX. FGF1 but not FGF4 stimulated expression of the cell cycle regulator, GADD45B. In contrast, FGF10 altered the expression of none of these genes. Western blot demonstrated that FGF4 activated ERK1/2 and Akt signalling rapidly and transiently, whereas FGF10 elicited a modest and delayed activation of ERK1/2. These data show that FGF1 and FGF4 activate typical FGF signalling pathways in granulosa cells, whereas FGF10 activates atypical pathways.
Orphan nuclear receptor nur77 regulates androgen receptor gene expression in mouse ovary. Dai A et al. The androgen receptor (AR) is a nuclear receptor that is expressed in growing follicles and involved in folliculogenesis and follicle growth. The orphan nuclear receptor, Nur77, also has an important role in steroid signaling and follicle maturation. We hypothesized that AR levels and androgen signaling through AR are regulated by Nur77 in the ovary. In the ovaries of Nur77 knockout mice (n?=?5), real-time PCR results showed that the mRNA levels of AR and an androgen signaling target gene, Kitl, were decreased by 35% and 24%, respectively, relative to wild-type mice (n?=?5), which suggested transcriptional regulation of AR by Nur77 in vivo. In cultured mouse granulosa cells and a steroidogenic human ovarian granulosa-like tumor cell line, KGN, mRNA and protein expression levels of AR were increased by overexpressing Nur77 but decreased by knocking down endogenous Nur77. Consistent with increased AR expression, chromatin immunoprecipitation showed that Nur77 bound to the NGFI-B response element (NBRE) in the AR promoter sequence. AR promoter activity was stimulated by Nur77 in HEK293T cells and attenuated in Nur77 knockout mouse granulosa cells (luciferase assay). Overexpression of Nur77 enhanced the androgenic induction of Kitl (200 nM; 48h), while knockout of Nur77 attenuated this induction. These results demonstrate that AR is regulated by Nur77 in the ovaries, and they suggest that the participation of Nur77 in androgen signaling may be essential for normal follicular development.
NGFI-B, ALSO CALLED nur77, is one of the immediate-early genes originally identified by virtue
of its rapid activation by nerve growth factor in PC12 pheochromocytoma cells and by serum in
fibroblasts . As with many other immediate-early genes that encode transcription factors, the
NGFI-B protein presumably regulates the expression of other genes, ultimately culminating in
phenotypic changes. The NGFI-B gene encodes a member of the steroid-thyroid hormone
superfamily, a class of ligand-dependent transcriptional modulator proteins.
Jae-Il Park, et al 2001 reported that Northern blot analysis of ovaries obtained from
prepubertal rats revealed the increased expression of NGFI-B during prepubertal development. Treatment of immature rats
with PMSG, however, decreased ovarian NGFI-B expression. The major cell types expressing NGFI-B messenger RNA
were thecal cells of follicles in different sizes. In contrast, treatment of PMSG-primed rats with human (h) CG resulted in the
rapid and transient stimulation of ovarian NGFI-B messenger RNA, reaching a peak within 1 h. In situ hybridization analysis
revealed that hCG treatment induced the expression of NGFI-B in granulosa cells of preovulatory follicles. Treatment of
cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of NGFI-B
messenger RNA and protein. Furthermore, treatment of adult cycling rats with pentobarbital abolished NGFI-B expression
on proestrus, and exogenous administration of hCG restored it, indicating the role of the preovulatory surge of LH in the
stimulation of NGFI-B expression. These results demonstrate the cell type-specific expression and gonadotropin induction of
NGFI-B in granulosa cells of preovulatory follicles and suggest a role for NGFI-B in the ovulatory process.
Park JI, et al 2001 reported the gonadotropin regulation of NGFI-B messenger ribonucleic acid expression during ovarian follicle development in the rat.
NGFI-B is an immediate-early gene that encodes an orphan nuclear receptor. Northern blot analysis of
ovaries obtained from prepubertal rats revealed the increased expression of
NGFI-B during prepubertal development. Treatment of immature rats with PMSG,
however, decreased ovarian NGFI-B expression. The major cell types expressing
NGFI-B messenger RNA were thecal cells of follicles in different sizes. In contrast, treatment of PMSG-primed rats with human (h) CG resulted in the rapid and transient stimulation of ovarian NGFI-B messenger RNA, reaching a peak
within 1 h. In situ hybridization analysis revealed that hCG treatment induced the expression of NGFI-B in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and
dose-dependent stimulation of NGFI-B messenger RNA and protein.
Furthermore, treatment
of adult cycling rats with pentobarbital abolished NGFI-B expression on
proestrus, and exogenous administration of hCG restored it, indicating the role of
the preovulatory surge of LH in the stimulation of NGFI-B expression. These
results demonstrate the cell type-specific expression and gonadotropin induction
of NGFI-B in granulosa cells of preovulatory follicles and suggest a role for
NGFI-B in the ovulatory process.
Regulation of protein tyrosine phosphatase 4a1, B-cell translocation gene 2, nuclear receptor subfamily 4a1 and diacylglycerol O-acyltransferase 1 by follicle stimulating hormone in the rat ovary. Schmidt J et al. Ovarian response to follicle stimulating hormone (FSH) and luteinising hormone (LH) leads to the formation of a mature follicle that is eventually ovulated. FSH and LH are essential for this process because they direct changes in somatic cells associated with folliculogenesis by regulating the expression of multiple genes. We hypothesised that genes induced by FSH in rat Sertoli cells would also show hormonal regulation during rat folliculogenesis. The objective of this study was to determine the expression patterns of diacylglycerol O-acyltransferase 1 (Dgat1), nuclear receptor subfamily 4a1 (Nr4a1), an anti-proliferative gene (Btg2) and a protein tyrosine phosphatase (Ptp4a1) in the ovaries of pregnant mare serum gonadotrophin (PMSG)-treated and human chorionic gonadotrophin (hCG)-treated rats. Expression of Dgat1, Nr4a1 and Ptp4a1 was induced in ovaries 4 h post PMSG treatment. When rats were treated with hCG, Dgat1, Nr4a1 and Ptp4a1 expression was induced by 12 h. Expression of Nr4a1 protein increases 12-24 h after induction of gene expression. Nr4a1 protein was observed in the granulosa, theca and luteal cells post PMSG and hCG treatment. These findings should increase our knowledge of mechanisms regulating folliculogenesis and luteinisation and demonstrate the diverse proteins that are important in ovarian functionFSH modulates the expression of inhibin-alpha and the secretion of inhibins via orphan nuclear receptor NUR77 in ovarian granulosa cells. He QY 2013 et al.
It has been previously reported that follicle-stimulating hormone (FSH) regulates the expression of inhibin-alpha in human granulosa cells, but the precise molecular pathway remains unknown. In the present study, we investigated the role of the orphan nuclear receptor, NUR77, in both the transcriptional regulation of the inhibin a-subunit gene and the secretion of inhibins. Our results showed that in a human granulosa cell tumor-derived cell line (KGN) and in human granulosa-lutein cells (hGL), FSH induced the expression of NUR77 and inhibin-alpha, although inhibin-alpha expression did not increased following FSH treatment if NUR77 was knocked down. Furthermore, simply overexpressing or reducing NUR77 levels affected inhibin-a expression, while NUR77 overexpression improved the secretion of inhibin A and B from human granulosa cells. In addition, the chromatin immunoprecipitation-PCR, avidin-biotin-conjugated DNA precipitation, and luciferase reporter assays confirmed that NUR77 directly regulated the transcription of the inhibin-alpha gene through the specific NGFI-B response element located within its promoter. In the ovarian granulosa cells of the Nur77 knockout mice, the mRNA levels of inhibin-alpha were decreased relative to wild-type mice. These data indicate a role of NUR77 in the regulation of inhibin-alpha in ovarian granulosa cells. Mol. Reprod. Dev. 2013 Wiley Periodicals, Inc.
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Ovarian localization
Granulosa, Theca, Luteal cells
Comment
Orphan Nuclear Receptor NR4A1 Is a Negative Regulator of DHT-Induced Rat Preantral Follicular Growth. Xue K et al. Nuclear receptor subfamily 4 group A member1 (NR4A1), an orphan nuclear receptor, is involved in the transcriptional regulation of thecal cell androgen biosynthesis and paracrine factor insulin-like 3 (INSL3) expression. Androgens are known to play an important regulatory role in ovarian follicle growth. Using a chronically androgenized rat model, a preantral follicle culture model and virus-mediated gene delivery, we examined the role and regulation of NR4A1 in the androgenic control of preantral follicular growth. In the present study, Ki67 staining was increased in preantral follicles on ovarian sections from 5a-dihydrotestosterone (DHT)-treated rats. Preantral follicles from DHT-treated rats cultured for 4 d exhibited increased growth and up-regulation of mRNA abundance of G(1)/S-specific cyclin-D2 (Ccnd2) and FSH receptor (Fshr). Similarly, DHT (1 ?m) increased preantral follicular growth and Ccnd2 and Fshr mRNA abundance in vitro. The NR4A1 expression was high in theca cells and was down-regulated by DHT in vivo and in vitro. Forced expression of NR4A1 augmented preantral follicular growth, androstenedione production, and Insl3 expression in vitro. Inhibiting the action of androgen (with androgen receptor antagonist flutamide) or INSL3 (with INSL3 receptor antagonist INSL3 B-chain) reduced NR4A1-induced preantral follicular growth. Furthermore, NR4A1 overexpression enhanced DHT-induced preantral follicular growth, a response attenuated by inhibiting INSL3. In conclusion, DHT promotes preantral follicular growth and attenuates thecal NR4A1 expression in vivo and in vitro. Our findings are consistent with the notion that NR4A1 serves as an important point of negative feedback to minimize the excessive preantral follicle growth in hyperandrogenism.
The Orphan Nuclear Receptor NR4A1 Regulates Transcription of Key Steroidogenic Enzymes in Ovarian Theca Cells. Li M et al. Orphan nuclear receptor NR4A1, a member of the nuclear receptor superfamily, is widely expressed in different cell types and mediates diverse biological processes. Recent emerging evidence suggests that NR4A1 is involved in the transcriptional regulation of several steroidogenic enzyme genes in gonads and adrenals. However, its function in ovarian theca cells remains to be defined. In the present study, immunohistochemical staining of NR4A1 in healthy human ovaries indicate that it is expressed in theca cells and granulosa cells. In an effort to explore the function of NR4A1 in the transcript regulation of steroidogenic enzyme genes responsible for ovarian theca cell steroidogenesis, we constructed recombinant adenovirus AdCMV-NR4A1 and AdH1-NR4A1 to enhance or knockdown the expression of NR4A1 in theca cells, respectively. The expression patterns of StAR, CYP11A1, CYP17 and HSD3B2 were subsequently analyzed by real-time RT-PCR. Moreover, concentrations of testosterone in the spent medium were measured by radioimmunoassay. Our results show that overexpression of NR4A1 in theca cells stimulates the expression of StAR, CYP11A1, CYP17 and HSD3B2, leading to increased testosterone production. Conversely, knockdown of the endogenous NR4A1 exhibits a significant decrease in StAR, CYP11A1, CYP17 and HSD3B2 expression and testosterone production. Since expression of NR4A1 in the endocrine organs is known to be regulated by both cAMP/PKA mediated hormones, ACTH and LH, forskolin (FSK), an activator of cAMP/PKA pathway, was applied to the cultured follicles. FSK rapidly increases the NR4A1 mRNA levels followed by an increase in StAR, CYP11A1, CYP17 and HSD3B2. Collectively, our results outline a previously unrecognized role for NR4A1 in the transcriptional regulation of StAR, CYP11A1, CYP17 and HSD3B2 in ovarian theca cells. Modulation of these steroidogenic enzymes by NR4A1 could influence the capacity of the ovarian theca cells to produce androgen.
The Orphan Nuclear Receptors NURR1 and NGFI-B Modulate Aromatase Gene Expression in Ovarian Granulosa Cells: a Possible Mechanism for Repression of Aromatase Expression upon Luteinizing Hormone Surge Wu Y, et al .
Ovarian granulosa cells play pivotal roles in many aspects of ovary functions including folliculogenesis and steroidogenesis. In response to follicle stimulating hormone (FSH) and LH (LH), the elevation of intracellular cyclic AMP (cAMP) level in granulosa cells leads to activation of multiple ovarian genes. Here we report findings from a genome-wide study of the cAMP-responsive gene expression profiles in a human granulosa-like tumor cell line, KGN. The study identified 140 genes that are either activated or repressed by 2-fold or greater following stimulation by the adenylyl cyclase activator forskolin. The induction patterns of some cAMP-responsive genes were further analyzed by quantitative real time PCR. Consistent with previous observations, the LH-responsive genes such as the nuclear receptor 4A subfamily (NURR1, NGFI-B, and NOR-1) were rapidly but transiently induced, whereas the FSH-responsive gene CYP19 encoding aromatase was induced in a delayed fashion. Interestingly, ectopic expression of NURR1 or NGFI-B severely attenuated the cAMP-responsive activation of the ovary-specific aromatase promoter. Reduction of the endogenous NURR1 or NGFI-B by small interfering RNA (siRNA) significantly elevated aromatase gene expression. The cis-elements responsible for NURR1/NGFI-B-mediated repression were mapped to the minimal aromatase promoter sequence that confers cAMP-responsiveness. Furthermore, the DNA-binding domain of NURR1 was required for the repression. Taken together, these results strongly suggest a causal relationship between the rapid decline of aromatase mRNA and induction of NR4A expression that concomitantly occur upon LH surge at the later stages of ovarian follicular development.
Regulation of NGFI-B/Nur77 gene expression in the rat ovary and in leydig tumor cells MA-10. Inaoka Y et al. NR4A1, also called NGFI-B in the rat, Nur77 in the mouse and TR3 in humans, belongs to the orphan nuclear steroid hormone receptor superfamily and is one of the immediate-early genes. In the endocrine organs, including the gonads, NGFI-B/Nur77 gene expression is rapidly induced by pituitary hormones. NGFI-B/Nur77 expression was found to be rapidly reduced by an estrogenic endocrine disrupter, diethylstilbestrol (DES) in theca interna cells of immature rat ovaries. DES treatment also triggered a rapid decrease of serum luteinizing hormone (LH) levels, suggesting that DES acts on the hypothalamo-pituitary axis to suppress LH secretion from the pituitary. The transcriptional regulation of NGFI-B/Nur77 by LH/human chorionic gonadotropin (hCG) or 8-bromoadenosine 3'-5'-cyclic monophosphate (8 Br-cAMP) was examined in mouse Leydig tumor cells MA-10. Luciferase assays using NGFI-B/Nur77 promoter constructs and electric mobility shift assays (EMSA) showed that NGFI-B/Nur77 gene expression was mediated through three of the four activator protein-1 (AP-1)-like sites, namely the -233 AP-1, -213 AP-1 and -69 AP-1 sites adjacent to the transcription start site of the NGFI-B/Nur77 promoter. We also demonstrated here that both the Jun family and cAMP-responsive element binding (CREB) proteins bind to the -233 AP-1 site, whereas the main binding protein to the -213 AP-1 site was CREB, and Jun family protein to the -69 AP-1 site, respectively. The rapid induction of NGFI-B/Nur77 gene expression by LH/hCG in MA-10 cells appears to be mediated by both CREB and Jun family proteins through the cAMP-protein kinase A (PKA) pathway. Mol. Reprod. Dev. (c) 2007 Wiley-Liss, Inc.
Follicle stages
Antral, Preovulatory, Corpus luteum
Comment
xyz
Phenotypes
Mutations
1 mutations
Species: None
Mutation name: None
type: naturally occurring fertility: fertile Comment: Molecular cloning, tissue expression and association of porcine NR4A1 gene with reproductive traits. Liu LQ et al. Nuclear receptor subfamily 4, group A, member 1 (NR4A1), other aliase NGFI-B, is an immediate-early gene that encodes an orphan nuclear receptor, which play a potential role in the ovulatory process. In this study, a 4,870 bp fragment covered the complete coding region (CDS) and its unique intron sequences of porcine NR4A1 gene was obtained. The reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that NR4A1 was highly expressed in ovary, uterus, kidney, heart but at very low level in oviduct and not expressed in other tissues. Compared the sequence of CDS and its unique intron of Large White and Meishan pigs, a A/G mutation in intron 5 was found and a PCR-Dde1-RFLP genotyping assay was developed. Association of the SNP and litter size was assessed in two populations [purebred Large White and an experimental synthetic Line (DIV) sows]. Statistical analysis demonstrated that, in the first parity, AG animals in experimental synthetic Line (DIV) sows had 1.805 more piglets born compared to the GG animals (P < 0.05). For all parities, in the purebred Large White pigs, those with the GG genotype had an additional 0.877 piglets born and 0.780 piglets born alive compared to the AA animals (P < 0.05), those with the AG genotype had additional 0.780 piglets born compared to the AA animals (P < 0.05). In addition, significant additive effect of 0.438 +/- 0.182 piglets/litter and 0.368 +/- 0.165 piglets/litter on piglets born and piglets born alive were detected in the purebred Large White lines (P < 0.05), respectively. Therefore, NR4A1 gene was significantly associated with litter size in two populations and could be a useful molecular marker in selection for increasing litter size in pigs.