| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Biochemistry, Fukui Medical University, Shimoaizuki, Matsuoka, Fukui 910-1193and
b CREST, JST (Japan Science and Technology), Japan
c Department of Obstetrics and Gynecology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
follicular development, gene regulation, granulosa cells, luteinizing hormone, ovary
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The LH receptor gene expression is primarily regulated by specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. The promoter of the rat LH receptor gene lacks TATA and CAAT motifs, is GC rich, and initiates transcription at multiple sites [19–21]. Several studies have shown several trans-acting factor-binding sequences that have a consensus to these, including steroidogenic factor-1 (SF-1), Sp1, and AP-2, and that the proximal 155 base pairs (bp; relative to the translation initiation codon) of the 5'-flanking region represents a minimal promoter that accounts for basal expression of this receptor in rat granulosa cells [22]. In the present study, we also demonstrated, by means of luciferase assays, that the region between -171 and -137 bp is essential for basal expression of the LH receptor gene. To identify factors that interact with the region between -171 and -137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated as a result of the screening, was found to encode a transcription factor early growth response gene-1 (Egr-1).
The Egr-1 [23], also known as NGFI-A [24], Krox-24 [25], and Zif268 [26], is a (Cys)2-(His)2-type, zinc-finger phosphoprotein that was originally identified as an immediate-early serum response or nerve growth factor response gene product. Egr-1 recognizes the sequence GCG(T/G)(G/A)GG(C/A/T)G(G/T) [27] and is rapidly induced in many cell types during growth, differentiation, and apoptotic stimuli. An analysis of Egr-1 knockout mice demonstrated that these mice were deficient in LH-ß in the pituitary as well as in the LH receptors in the ovary, which, in turn, caused female infertility [28, 29]. With respect to the LH-ß promoter, a canonical Egr-1 site in the promoter exists, which has been shown to be essential for activation of the gene via the synergistic action of Egr-1 and SF-1 [30]. However, to our knowledge, studies concerning the relationship between Egr-1 and LH receptor gene expression have not yet been reported. The ovarian expression of the Egr-1 gene was also demonstrated during the early stage of the ovulatory process in gonadotropin-primed, immature rats [31].
In the present study, we report an analysis concerning the regulation of LH receptor gene expression with respect to Egr-1 using an LH receptor promoter assay and an electrophoretic mobility shift assay (EMSA). The analysis reveals, to our knowledge for the first time, that Egr-1 binds to the region between -171 and -137 bp of the LH receptor promoter region and increases transcription of the LH receptor gene. We also demonstrate that Egr-1 mRNA, as well as its protein, is expressed and induced in differentiated granulosa cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The yeast one-hybrid system and the pACT2 vector were purchased from Clontech (Palo Alto, CA). The QuikChange Site-Directed Mutagenesis Kit was purchased from Stratagene (La Jolla, CA). FuGENE 6 Transfection Reagent was obtained from Roche Molecular Biochemicals Mannheim (Indianapolis, IN). The dual luciferase reporter assay system, the pGEM-T Easy vector, and the pGL3-Basic and pRL-SV40 vectors were purchased from Promega (Madison, WI). The cytomegalovirus promoter/enhancer-directed expression vector pcDNA3 was purchased from Invitrogen (Carlsbad, CA). The Trizol regent was purchased from Gibco BRL Life Technologies (Grand Island, NY). The TaKaRa Bca BEST Labeling Kit was purchased from TaKaRa Shuzo (Kyoto, Japan). The [
-32P]deoxycytidine triphosphate (111 TBq/mmol) was obtained from NEN Life Science Products (Wilmington, DE). The anti-Sp1 (sc-59-G) and anti-Egr-1 (sc-110) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The protein assay reagent was purchased from Bio-Rad (Hercules, CA). The enhanced chemiluminescence kit was purchased from Amersham Pharmacia Biotech (ECL-Plus; Arlington Heights, IL). Ovine FSH (oFSH-20, 4453 IU/mg) and hCG were obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). The DES and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) were purchased from Sigma Chemical Co (St. Louis, MO).
Animals and Rat Granulosa Cell Culture
Immature Wistar female rats (age, 21 days) were used. The rats were treated with 2 mg of DES in 0.1 ml of sesame oil once daily for 4 days to stimulate the proliferation of ovarian granulosa cells. At all times, the animals were treated according to National Institutes of Health guidelines. The ovaries were then excised, and the granulosa cells were isolated by puncturing the follicles with a 26-gauge needle. The cells were washed and collected by brief centrifugation at 500 x g for 5 min at room temperature, and cell viability was determined by trypan blue staining. Cell viability was more than 90%. The granulosa cells were then cultured in Ham F-12:Dulbecco modified Eagle medium (1:1, v:v) supplemented with antibiotics and 0.1% BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2 and 95% air at 37°C [32].
Yeast One-Hybrid Screening
The pRW95-1 vector was a gift from Dr. M. Schweizer [33]. The pLacZiB1 vector has been previously described [34]. Oligonucleotides with two CGGGGGTGGGGG boxes, a longLH sense strand (5'- CTAGCGGGGGTGGGGGGCCGGGGGTGGGGGGC-3') and a longLH antisense strand (5'-CTAGGCCCCCCACCCCCGGCCCCCCACCCCCG-3'), were synthesized to construct the reporter plasmids. These oligonucleotides were annealed, phosphorylated, and ligated into the SpeI site of the pRW95-1 or the XbaI site of the pLacZiB1 to give the HIS3-based or LacZ-based reporter plasmids, respectively. As a result, 2 copies of the double-stranded oligonucleotides were tandemly incorporated into the vectors. This constitutes 4 tandem repeats of CGGGGGTGGGGG boxes in the reporter constructs. A rat granulosa cell cDNA library in the pACT2 vector was constructed as previously described [34]. The YM4271 yeast cells were sequentially transformed with both of the reporter plasmids, namely pRW95-1-LHR and pLacZiB1-LHR, to give a reporter yeast strain. The reporter yeast strain was then transformed with the rat granulosa cell cDNA library using a high-efficiency transformation method [35]. When 7.2 x 106 clones were screened, one positive clone was obtained. A plasmid, pACT2-Egr-1 that contains an insert of 3.2 kilobases in length was isolated from the yeast, and its nucleotide sequence was determined. To verify the interactions, the transformants were streaked on a leucine-, tryptophan-, and uracil-depleted synthetic dextrose plate and a leucine-, tryptophan-, uracil-, and histidine-depleted synthetic dextrose plate.
Plasmids
Reporter plasmids that contained different lengths of the LH receptor 5' flanking region (-478/+1, -281/+1, -171/+1, and -137/+1) were constructed using the pGL3-Basic luciferase vector, which lacks both elements of the eukaryotic promoter and enhancer sequences. Promoter DNA fragments containing substituted nucleotide sequences, namely mut-a, mut-b, and mut-ab, were generated by polymerase chain reaction (PCR) using primers with the following nucleotide substitutions: mut-a-S, 
The resulting mutant promoters contained the same LH receptor upstream region (-281/+1) with different point mutations (mut-a, mut-b, or mut-ab). These mutated DNA fragments were ligated into a pGL3-Basic luciferase vector. All the reporter plasmids were authenticated by DNA sequencing. A rat Egr-1 cDNA containing the entire coding region was generated by reverse transcription-PCR and subcloned into the expression vector, pcDNA3.
In Vitro Translation and EMSA
One microgram of the Egr-1/pcDNA3 plasmid was incubated at 30°C for 60 min with the T7 TNT quick-coupled transcription/translation system (Promega) in the presence of methionine. The in vitro-synthesized product was then subjected to EMSA, which was performed as described elsewhere [36] with minor modifications. Briefly, 1 µl of one third-diluted, in vitro-translated product was added to the binding mixture with an -32P-labeled probe. For a competition analysis, a 50- or 200-fold molar excess of competitor DNAs was added to the binding mixture. After completion of the binding, the mixture was subjected to electrophoresis on a 6% polyacrylamide gel (19:1 [w/w] = acrylamide:bis-acrylamide) in 44.5 mM Tris-HCl (pH 8.0), 44.5 mM boric acid, and 1 mM EDTA at 200 V for 1 h, after which the gels were dried and exposed at -80°C for 16 h to Kodak X-AR films (Eastman Kodak, Rochester, NY).
Transient Transfection and Luciferase Assay
Granulosa cells were dispensed into 48-well plates and cultured for 24 h in hormone-free conditions before transfection. DNA samples that contained each reporter plasmid and pRL Renilla luciferase control vector (for normalization) were mixed with 0.375 µl of FuGENE 6, and the resulting mixture was added to the cells. Forty-four hours later, the cells were treated with FSH (30 ng/ml) for 4 h. Cells were harvested 48 h after transfection. MA-10 cells, originated from a mouse Leydig cell tumor, were maintained in Waymouth medium supplemented with 15% horse serum and antibiotics. Cells were dispensed into 24-well plates and cultured until 70% confluence was achieved. DNA samples that contained each reporter plasmid and the pRL Renilla luciferase control vector (for normalization) with or without expression plasmid (Egr-1/pcDNA3) were mixed with 1.5 µl of FuGENE 6, and the resulting mixture was added to the cells. The total amount of DNA (µg) was adjusted, if required, by adding pcDNA3 plasmid. Twenty-four hours later, the cells were treated with 8-Br-cAMP (1 mM) for 12 h. Cells were harvested 36 h after transfection. Measurements were made using a Lumat LB9501 luminometer (Berthold, Wildbad, Germany) in a single tube, with the firefly luciferase assay first and the Renilla luciferase assay second. Firefly luciferase activities (relative light units) were normalized to Renilla luciferase activities. Data are presented as the ratio of firefly luciferase to Renilla luciferase and represent the mean ± SEM of 4 independent experiments.
Preparation and Analysis of RNA
Granulosa cells were cultured for 24 h under hormone-free conditions in 35-mm dishes containing 2.5 x 106 viable cells in 2.5 ml of medium, and cells were then treated with ovine FSH (30 ng/ml) for 48 h, followed by further treatment with hCG (30 ng/ml) or 8-Br-cAMP (2 mM) for periods of 0, 1, 2, 4, 8, and 24 h. Cells were then harvested, and total RNA was extracted with the Trizol reagent. Northern blot analysis was performed as reported previously [34]. Ten micrograms of total RNA were used.
Western Blot Analysis of Egr-1
Granulosa cells were cultured in 60-mm dishes containing 5 x 106 viable cells in 5 ml of medium, and reagents were added to the medium 24 h after plating. At the end of the selected culture time, the cells were collected by a scraper and washed with 10 ml of Tris-buffered saline (TBS). The resulting cells were suspended in 1 ml of TBS, transferred to an Eppendorf tube, and pelleted by centrifugation at 1500 x g for 10 min at 4°C. Nuclei from granulosa cells were prepared by the method of Schreiber et al. [37] with minor modifications. The cell pellet was resuspended in 600 µl of cold buffer A (10 mM Hepes, pH 7.9; 10 mM KCl; 1 mM EDTA; 0.5 mM EGTA; 1 mM DTT [dithiothreitol]; and 0.5 mM PMSF) by gentle pipetting. The cells were allowed to swell on ice for 15 min, after which 37.5 µl of a 10% solution of Nonidet P-40 (Fluka, St. Louis, MO) were added and the tube vigorously vortexed for 10 sec. The homogenate was centrifuged at 17 000 x g for 5 min at 4°C. The supernatant, which contained cytoplasm and RNA, was removed, and the nuclear pellet was resuspended in 40 µl of ice-cold buffer C (20 mM Hepes, pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; and 1 mM PMSF) and the tube vigorously shaken at 4°C for 15 min on a shaking-platform. The mixture was then centrifuged for 15 min at 17 000 x g at 4°C, and the supernatant, which contained the nuclear extract, was used for protein analysis. The nuclear extracts (10 µg) were electrophoresed by SDS-PAGE on a 10% acrylamide gel under reducing conditions. Western blot analysis was performed, and immunoreactive Egr-1 protein was then detected using the ECL-Plus kit according to the manufacturer's instructions.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To determine the 5'-flanking region that is essential for the regulation of LH receptor gene expression, various deletion mutants were constructed upstream of the rat LH receptor gene and linked to a luciferase vector (LHR-luc). Granulosa cells were transfected with LHR-luc reporter plasmids, and luciferase activities were then measured. The effects of FSH on luciferase activity were also examined. As shown in Figure 1, the luciferase activity of the shortest construct, containing 137 bp upstream of the translational start site, was markedly reduced from that of the construct containing 171 bp. This indicates that one or more elements between -171 and -137 bp must exert positive effects on LH receptor gene expression. Figure 1 also shows the presence of one or more negative elements between -478 and -171 bp. The ratios of FSH-induced to noninduced luciferase activities were nearly constant, regardless of the length of the 5'-upstream of LH receptor gene in the reporter constructs.
|
Identification of DNA-Binding Protein by Yeast One Hybrid System
Because the region between -171 and -137 bp was shown to be essential for the regulation of LH receptor gene expression, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system to identify proteins that bind to this region. A rat granulosa cell cDNA library containing 7.2 x 106 independent clones was screened. One clone, which was double-positive to the HIS3- and LacZ-based assays, was obtained. A nucleotide sequence analysis revealed that the clone encodes for rat Egr-1. Although the region used in this study also includes consensus sequences for several transcription factors, such as Sp1 or WT1, clones encoding such transcription factors were not obtained in the present experiments.
Analysis of Egr-1 Consensus Element in the 5'-Upstream of LH Receptor Gene
To determine the sequence to which Egr-1 protein binds, we employed EMSA using the 5'-upstream region of the LH receptor gene between -157 and -136 bp as a probe. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. When the probe was incubated with in vitro-synthesized Egr-1, a DNA-protein complex was observed, as shown in Figure 2. In the competition experiments, the formation of the complex was blocked by addition of a 50- or 200-fold molar excess of the unlabeled, wild-type oligonucleotide. Complex formation was also blocked by addition of oligonucleotides with mutations at -152/-151 (mut-b) or at -142/-141 (mut-a). On the other hand, complex formation was not blocked by oligonucleotide with mutations at both sites (mut-ab), suggesting that the two possible sites shown in Figure 2 are able to independently recognize Egr-1. Accordingly, the in vitro-translated Egr-1 specifically binds to the 5'-flanking region of LH receptor gene at 2 independent sites between -157 and -136 bp.
|
Effects of Egr-1 on Regulation of LH Receptor Gene Expression
MA-10 is a tumor cell line derived from mouse Leydig cells that intrinsically expresses LH receptors. The effects of Egr-1 on the regulation of LH receptor gene expression were examined by transiently transfecting and overexpressing Egr-1 in MA-10 cells. As shown in Figure 3, the overexpression of Egr-1 led to an increase in luciferase activities in all constructs except the shortest one (LHR137-Luc). Probably because of the absence of the Egr-1 binding site, the basal luciferase activity was reduced, and no response to Egr-1 was observed in the case of the shortest construct (LHR137-Luc). However, the ratios of cAMP-induced to noninduced activities were essentially unchanged even in the shortest construct, suggesting that Egr-1 is not directly involved in the cAMP responsiveness of the LH receptor gene.
|
To define the effects of Egr-1 precisely, plasmids that contained mutations in either or both of the 2 Egr-1-binding sites were prepared. As shown in Figure 4, luciferase activities were markedly reduced in all constructs that contained mutations within the Egr-1-binding sites, even when the Egr-1 protein was overexpressed. These data clearly demonstrate that mutations in either of the Egr-1-binding sites attenuate LH receptor transcription. Accordingly Egr-1 protein binds to the 5'-upstream region of the LH receptor gene in a sequence-dependent manner, thus positively regulating LH receptor gene expression.
|
Analysis of Egr-1 mRNA and Egr-1 Protein in Granulosa Cells
Finally, to examine the expression of Egr-1 with reference to mRNA and protein levels, Northern and Western blot analyses were performed using granulosa cell extracts. Granulosa cells were prepared from DES-treated immature rats and, after an overnight culture, were then treated with FSH. After incubation with FSH for 48 h, the cells were further treated with hCG. As shown in Figure 5A, Northern blot analysis revealed that the onset of Egr-1 mRNA expression began at some point between 0 and 4 h after hCG stimulus. Subsequently, at 8 h after hCG treatment, Egr-1 gene expression declined to a level that was not significantly different from the 0-h control value. Essentially the same pattern was obtained when cells were treated with 8-Br-cAMP after pretreatment with FSH for 48 h. Treatment with FSH for 48 h was a prerequisite for the hCG- or 8-Br-cAMP-induced expression of the Egr-1 gene. Cholera toxin also had similar effects on Egr-1 gene expression (data not shown). However, treatment with FSH for 48 h alone did not result in the induction of Egr-1 gene expression in these cells.
|
As shown in Figure 5B, Western blot analysis revealed that translation of the Egr-1 gene product coincided with the temporal pattern of Egr-1 mRNA expression as evidenced by Northern blot analysis. Negligible translation at 0 h after hCG treatment was observed, and the strongest signal was at 2 h. The size of the translated protein was 84 kDa.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The upstream region between -171 and -137 bp contains two overlapping Egr-1 consensus sequences (Fig. 2). We next examined the issue of whether Egr-1 binds to either or both of the 2 consensus sites. As shown in Figure 2, competition analysis revealed that Egr-1 was able to bind to both sites in an independent fashion. In addition, Egr-1 actually enhanced LH receptor gene expression (Fig. 3). Mutation analysis of the 2 Egr-1 sites clearly showed that both sites are important for the expression of LH receptor gene (Fig. 4). However, we also showed that increases in luciferase activity by cAMP are not associated with Egr-1 (Figs. 3 and 4). This is not inconsistent with the observations reported by Chen et al. [22], who concluded that Sp1 sites in the promoter region were important for cAMP-induced LH receptor transcription.
It has been reported that Sp1 binds to the upstream region between -143 and -138 bp and positively regulates expression of the gene [22, 41]. In the present study, however, our findings show that Egr-1 also binds to the region, which overlaps with the Sp1 site, and positively regulates LH receptor gene expression. Several investigators have reported that Egr-1 and Sp1 bind to the same or an overlapping region. Biesiada et al. [42] reported that Egr-1 and Sp1 bind to the same site in the upstream region of the basic fibroblast growth factor (bFGF) gene, that Sp1 is required for basal expression, and that both Sp1 and Egr-1 mediate the inducible expression of the bFGF gene by phorbol esters or by serum treatment. In this case, however, cAMP is an inducer of LH receptor gene transcription. As shown in Figures 3 and 4, the observed increase in luciferase activities by cAMP was not dependent on the region between -171 and -137 bp in terms of induction ratio (i.e., cAMP-unstimulated to cAMP-stimulated), suggesting that, in this case, both Sp1 and Egr-1 may not be directly involved in the induced expression of the LH receptor gene.
The Egr-1 itself is induced by diverse exogenous stimuli very early in the apoptotic process [24, 28]. In differentiated granulosa cells that were pretreated with FSH for 48 h, both mRNA and protein levels of Egr-1 were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and returning to basal levels 8 h thereafter. Practically no Egr-1 mRNA or protein was detected on the undifferentiated granulosa cells, even after stimulation with cAMP (data not shown). Similar results were obtained when cholera toxin was used to induce Egr-1 mRNA expression (data not shown), suggesting that, in this case, stimulatory subunit of G protein may be involved in the Egr-1 gene expression. These results suggest that Egr-1 functions only in the luteinized granulosa cells after a hormonal stimulus, such as by hCG or cAMP. This is consistent with in vivo observations by Lawrence et al. [31], who reported that Egr-1 was induced in ovaries by hCG only after they had been differentiated by the eCG pretreatment.
The Egr-1 knockout mice show a deficiency of LH-ß in pituitary glands and reduced LH receptor expression on the surface of granulosa cells; moreover, both the male and female mice are infertile, which is thought to be secondary to LH-ß or the LH receptor [28, 29]. The importance of Egr-1 in expression of the LH receptor has been demonstrated using knockout female mice, the injection of which with hCG did not cause ovulation and subsequent luteinization in the ovary. This suggests that expression of the LH receptor is limited by Egr-1. The present study strengthens the observations mentioned above, and it clearly shows that Egr-1 plays direct and necessary roles in expression of the LH receptor.
In conclusion, the present findings show that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. They also show that Egr-1 expression is observed only in the luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further evidence in support of the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by grants from the Smoking Research Foundation, Kanzawa Medical Research Foundation, and CREST, JST (Japan Science and Technology), Japan. 
2 Correspondence: Kaoru Miyamoto, Department of Biochemistry, Fukui Medical University, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. FAX: 81 776 61 8102; kmiyamot{at}fmsrsa.fukui-med.ac.jp
Accepted: January 9, 2002.
Received: October 1, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y. Kobayashi, M. Nakamura, T. Sunobe, T. Usami, T. Kobayashi, H. Manabe, B. Paul-Prasanth, N. Suzuki, and Y. Nagahama Sex Change in the Gobiid Fish Is Mediated through Rapid Switching of Gonadotropin Receptors from Ovarian to Testicular Portion or Vice Versa Endocrinology, March 1, 2009; 150(3): 1503 - 1511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hamel, I. Dufort, C. Robert, C. Gravel, M.-C. Leveille, A. Leader, and M.-A. Sirard Identification of differentially expressed markers in human follicular cells associated with competent oocytes Hum. Reprod., May 1, 2008; 23(5): 1118 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sayasith, K. A Brown, J. G Lussier, M. Dore, and J. Sirois Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation. J. Mol. Endocrinol., October 1, 2006; 37(2): 239 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. X. Donadeu and M. Ascoli The Differential Effects of the Gonadotropin Receptors on Aromatase Expression in Primary Cultures of Immature Rat Granulosa Cells Are Highly Dependent on the Density of Receptors Expressed and the Activation of the Inositol Phosphate Cascade Endocrinology, September 1, 2005; 146(9): 3907 - 3916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-S. Ge, Q. Dong, C. M. Sottas, H. Chen, B. R. Zirkin, and M. P. Hardy Gene Expression in Rat Leydig Cells During Development from the Progenitor to Adult Stage: A Cluster Analysis Biol Reprod, June 1, 2005; 72(6): 1405 - 1415. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Youn, Y. Koo, I. Ji, and T. H. Ji An Upstream Initiator-Like Element Suppresses Transcription of the Rat Luteinizing Hormone Receptor Gene Mol. Endocrinol., May 1, 2005; 19(5): 1318 - 1328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kajitani, T. Mizutani, K. Yamada, T. Yazawa, T. Sekiguchi, M. Yoshino, H. Kawata, and K. Miyamoto Cloning and Characterization of Granulosa Cell High-Mobility Group (HMG)-Box Protein-1, a Novel HMG-Box Transcriptional Regulator Strongly Expressed in Rat Ovarian Granulosa Cells Endocrinology, May 1, 2004; 145(5): 2307 - 2318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yamada, H. Kawata, T. Mizutani, T. Arima, T. Yazawa, K. Matsuura, Z. Shou, T. Sekiguchi, M. Yoshino, T. Kajitani, et al. Gene Expression of Basic Helix-Loop-Helix Transcription Factor, SHARP-2, Is Regulated by Gonadotropins in the Rat Ovary and MA-10 Cells Biol Reprod, January 1, 2004; 70(1): 76 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yamada, H. Kawata, Z. Shou, T. Mizutani, T. Noguchi, and K. Miyamoto Insulin Induces the Expression of the SHARP-2/Stra13/DEC1 Gene via a Phosphoinositide 3-Kinase Pathway J. Biol. Chem., August 15, 2003; 278(33): 30719 - 30724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yazawa, T. Mizutani, K. Yamada, H. Kawata, T. Sekiguchi, M. Yoshino, T. Kajitani, Z. Shou, and K. Miyamoto Involvement of Cyclic Adenosine 5'-Monophosphate Response Element-Binding Protein, Steroidogenic Factor 1, and Dax-1 in the Regulation of Gonadotropin-Inducible Ovarian Transcription Factor 1 Gene Expression by Follicle-Stimulating Hormone in Ovarian Granulosa Cells Endocrinology, May 1, 2003; 144(5): 1920 - 1930. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Russell, K. M. H. Doyle, I. Gonzales-Robayna, C. Pipaon, and J. S. Richards Egr-1 Induction in Rat Granulosa Cells by Follicle-Stimulating Hormone and Luteinizing Hormone: Combinatorial Regulation By Transcription Factors Cyclic Adenosine 3',5'-Monophosphate Regulatory Element Binding Protein, Serum Response Factor, Sp1, and Early Growth Response Factor-1 Mol. Endocrinol., April 1, 2003; 17(4): 520 - 533. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
