help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chiang, C.-H.
Right arrow Articles by Leung, P. C. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiang, C.-H.
Right arrow Articles by Leung, P. C. K.
Right arrowPubmed/NCBI databases
*OMIM
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CHORIONIC GONADOTROPIN
*PROGESTERONE
Medline Plus Health Information
*Hormones
The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 10 3828-3839
Copyright © 2000 by The Endocrine Society

Original Studies

Hormonal Regulation of Estrogen Receptor {alpha} and ß Gene Expression in Human Granulosa-Luteal Cells in Vitro1

Chi-Hsin Chiang2, Kwai Wa Cheng2, Shigeo Igarashi, Parimal S. Nathwani and Peter C. K. Leung3

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen is one of the major sex steroid hormones that is produced from the human ovary, and its actions are established to be a receptor-mediated process. Despite the demonstration of estrogen receptor (ER) expression, little is known regarding the regulation of ER in the human ovary. In the present study we investigated the expression and hormonal regulation of ER{alpha} and ERß in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ER{alpha} and ERß messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ER{alpha} is expressed at a relatively lower level than ERß. Basal expression studies indicated that ER{alpha} mRNA levels remain unchanged, whereas ERß mRNA levels increased with time in culture in vitro, suggesting that ERß is likely to play a dynamic role in mediating estrogen action in hGLCs.

The regulation of ER{alpha} and ERß expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ER{alpha} (45%; P < 0.01) and ERß (40%; P < 0.01) mRNA levels. The hCG-induced decrease in ER{alpha} and ERß expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10 µmol/L) treatment. Additional studies using a specific protein kinase A (PKA) inhibitor (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt) and an adenylate cyclase inhibitor (SQ 22536) further implicated the involvement of the cAMP/PKA signaling pathway in hCG action in these cells. The hCG-induced decrease in ER{alpha} and ERß mRNA levels was prevented in the presence of these inhibitors. Next, the effect of GnRH on ER expression was studied. Sixty-eight percent (P < 0.001) and 60% (P < 0.001) decreases in ER{alpha} and ERß mRNA levels, respectively, were observed after treatment with 0.1 µmol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHainduced down-regulation of ER{alpha} and ERß expression, suggesting the involvement of PKC in GnRH signal transduction in hGLCs. In agreement with the semiquantitative RT-PCR results, Western blot analysis detected a decrease in ER{alpha} and ERß proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 µmol/L), 8-bromo-cAMP (1 mmol/L), forskolin (10 µmol/L), or phorbol 12-myristate 13 acetate (10 µmol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17ß-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 µmol/L GnRHa for 24 h before 17ß-estradiol administration.

In summary, we observed a differential expression of ER{alpha} and ERß mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ER{alpha} and ERß gene expression suggests that hCG and GnRH may contribute to the control of granulosa-luteal cell function. Furthermore, our data suggest that the effects of hCG and GnRH on ER{alpha} and ERß expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL documented that estrogen plays an important role in regulating reproductive function. Until recently, the classical estrogen receptor (now known as ER{alpha}) was thought to be the only form of nuclear receptor that binds estradiol and mediates its hormonal effects. However, the recent cloning of second form of estrogen receptor (ERß) in rat (1), mouse (2), and human (3) has opened a new era in the study of estrogen signaling. Studies using in situ hybridization and immunohistochemistry have revealed that ER{alpha} is localized primarily in the ovarian stromal cells and thecal cells of the rat (4), whereas ERß is concentrated predominately in the granulosa cells of small, developing, and preovulatory follicles in rat and bovine ovary (4, 5, 6). The development of ER{alpha} knockout ({alpha}ERKO) mice (7) and ERß knockout (ßERKO) mice (8) provides an excellent model to study ER{alpha}- and ERß-mediated events. Female {alpha}ERKO mice are anovulatory and infertile even in the presence of significant levels of ERß expression (7, 9), whereas ovarian follicular development and ovulation are partially compromised in ßERKO mice (8). The observation that the {alpha}ERKO ovary contains primary and secondary follicles implicates a role for ERß in early follicular development. On the other hand, final follicle maturation is hampered in the {alpha}ERKO, suggesting an interaction between ER{alpha} and ERß in controlling the later phases of folliculogenesis. Although both ER{alpha} and ERß have been detected in the rat corpus luteum (CL) (5, 10, 11), the precise role of estrogen and its receptor in the CL remains unclear.

Several studies have suggested that estrogen may regulate ovarian function in humans, including modulation of steroidogenesis from human granulosa-luteal cells (hGLCs) (12), inhibition of 3ß-hydroxysteroid dehydrogenase in lu-teal cells (13), and inhibition of luteal progesterone production (13, 14). These observations suggest the presence of functionally active ERs within the human ovary. The recent demonstration of ER{alpha} and ERß messenger ribonucleic acid (mRNA) in the human CL and hGLCs further corroborate this idea (15, 16). Despite the expression of ER{alpha} and ERß in the human ovary, little is known about the hormonal regulation of ER{alpha} and ERß in the human ovary. The present study was designed to investigate the regulation of ER{alpha} and ERß, at the mRNA and protein levels, by hCG and GnRH in hGLCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
hGLC culture

The use of hGLCs was approved by the clinical screening committee for research and other studies involving human subjects of the University of British Columbia. GLCs were harvested in conjunction with oocyte collection in the University of British Columbia’s in vitro fertilization program. Throughout the precollection period, follicular development was monitored using estradiol assays and ultrasonography. After pituitary down-regulation with GnRH analog (Synarel, Syntex, Montreal, Canada), follicular development was stimulated with hMG (75 IU FSH and 75 IU LH, Humegon ,Organon, Scarborough, Canada; or 75 IU FSH, Fertinorm Serono, Oakville, Canada). Final maturation was induced with hCG (10,000 IU; Serono) when two or more follicles had reached diameters of 16–18 mm. Follicles were harvested 36 h later using a transvaginal approach. Human GLCs from the follicular fluid were prepared and cultured as described previously (17). The isolated granulosa cells were cultured at a density of 2 x 105 cells/mL in medium 199 (Life Technologies, Inc., Burlington, Canada) supplemented with 10% FBS, 10 U/mL penicillin, and 10 µg/mL streptomycin (Life Technologies, Inc.) in a 37 C humidified environment with 5% CO2.

RNA isolation and RT-PCR amplification

hGLC total RNA was prepared as previously described (18). The RNA concentration was determined based on the absorbance at 260 nm, and its integrity was confirmed by agarose-formaldehyde gel electrophoresis. One microgram of total RNA isolated from hGLCs was reverse transcribed into first strand complementary DNA (cDNA) in a total volume of 15 µL using First Strand cDNA Synthesis Kit (Pharmacia Biotech, Morgan, Canada), following the manufacturer’s suggested procedure. PCR primers specific for ER{alpha} (forward, 5'-ATGACCATGACCCTCAACACCAA-3'; reverse, 5'-CTTGGCAGATTCCATAGCCATAC-3') and ERß (forward, 5'-TACAGCATTCCCAGCAATGTCAC-3'; reverse, 5'-GAAGTGAGCATCCCTCTTTGAAC-3') were designed based on the published sequence of human ER{alpha} and ERß cDNAs, respectively (3, 19). The cDNA was amplified in a 50 µL PCR reaction containing 2.5 U Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mmol/L MgCl2, 10 mmol/L of each deoxy-NTP, and 25 pmol of the respective specific primers. Thirty-five cycles of amplification were carried out by denaturing at 94 C for 1 min, annealing for 1 min at 55 C, extension for 90 s at 72 C, and a final extension for 15 min at 72 C. Several controls were performed to determine the accuracy of the PCR. First, PCR was performed in the absence of cDNA to examine the cross-contamination of sample. Second, PCR for ß-actin (sense, 5'-GGACCTGACTGACTACCTAATGAA-3'; antisense, 5'-TGATCCACATCTGCTGGAAGGTGG-3') was run in parallel to rule out the possibility of RNA degradation and to normalize the levels of ER{alpha} and ERß PCR products. Finally, as all primer pairs used span at least one intron, the sizes of the predicted PCR products ruled out the presence of contaminating genomic DNA in the RNA sample.

The amplified ER{alpha} and ERß PCR products were cloned into PCR II vector using the TA Cloning Kit (Invitrogen, San Diego, CA), and the putative ER{alpha} and ERß cDNAs were sequenced by the dideoxy nucleotide chain termination method using the T7 DNA Polymerase Sequencing Kit (Amersham Pharmacia Biotech, Morgan, Canada). Semiquantitative PCR for ER{alpha} and ERß mRNA levels were performed with 30 and 27 cycles of amplifications, respectively. The expression levels PCR for ER{alpha} and ERß in hGLCs were normalized against ß-actin levels.

Northern and Southern blot analyses

Five to 40 µg total RNA isolated from hGLCs were resolved by formaldehyde denaturing agarose gel electrophoresis and prepared for Northern blot analysis of ER{alpha} and ERß mRNA. Radiolabeled ER{alpha} and ERß probes were prepared using the Random Labeling Kit (Life Technologies, Inc.). The membrane was prehybridized and hybridized in standard hybridization solution [50% formamide, 5 x SSPE (0.09 M NaCl, 5 mM NaH2PO4, 5 mM EDTA [pH 7.4]), 5 x Denhardt’s solution, 0.5% SDS, and 100 µg/mL denatured herring sperm DNA] at 42 C. The membrane was washed at high stringency conditions (0.1 x SSPE and 0.1% SDS at 65 C for 10 min) and exposed to Kodak Omat x-ray film (Eastman Kodak Co., Rochester, NY).

For quantitation of human ER{alpha} and ERß mRNA levels, PCR products were separated by agarose gel electrophoresis and transferred to nylon membranes, which were hybridized with digoxigenin-labeled ER{alpha} and ERß cDNA probes (Roche Molecular Biochemicals, Laval, Canada). After washing at high stringency conditions, the membranes were exposed to Kodak Omat x-ray film. The radioautograms were then scanned and quantified with Scion Image-Released Beta 3b (Scion Corp., Bethesda, MD).

Pharmacological treatments

Pharmacological reagents, including D-Ala6-GnRH, which is a GnRH agonist (GnRHa), hCG, forskolin, phorbol 12-myristate 13 acetate (TPA), and 17ß-estradiol (E2) were purchased from Sigma-Aldrich Corp. (Oakville, Ontario, Canada). The protein kinase C (PKC) inhibitor (PKCI), bisindolymaleimide I (GF109203X); the protein kinase A (PKA) inhibitor (PKAI), adenosine 3',5'-cyclic monophosphorothioate Rp-isomer triethylammonium salt; and the adenylate cyclase inhibitor (ACI), SQ 22536 were obtained from Calbiochem (La Jolla, CA). In experiments in which the effects of GnRHa, hCG, forskolin, and TPA on ER{alpha} and ERß expression were studied, the cells were treated for 24 h before total RNA or total cellular protein isolation. To study the PKA and PKC signaling pathway, hGLCs were pretreated with 20 µmol/L PKAI, 0.5 mmol/L ACI, or 10 µmol/L PKCI 30 min before the addition of hCG or GnRHa.

Western blot analysis

For Western blot analysis, hGLCs were incubated in 75 µL cell lysis buffer RIPA [containing 1 x PBS (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/mL phenylmethylsulfonylfluoride, 30 µg/mL aprotinin, and 10 µg/mL leupeptin] for 15 min on ice. The cell lysates were centrifuged to remove cellular debris. The supernatant was used in Western blot analysis. The protein concentration in the cell lysates was determined using a modified Bradford assay (Bio-Rad Laboratories, Inc., Richmond, CA). An aliquot (35 µg) was taken from the total cell lysates and subjected to SDS-PAGE under reducing conditions. The stacking gels contained 5% (wt/vol) acrylamide, and the separating gels were composed of 8% (wt/vol) acrylamide. The proteins were electrophoretically transferred from the gels onto nitrocellulose paper (Hybond-C, Amersham Pharmacia Biotech). The nitrocellulose blots were blocked with 5% (wt/vol) nonfat milk in Tris buffer saline, containing 20 mmol/L Tris-Cl (pH 8.0), 140 mmol/L NaCl, and 0.05% (wt/vol) Tween-20. The membranes were then probed with a mouse monoclonal antibody (1:2000) directed against human ER{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; catalogue no. sc-8002) or goat polyclonal antibody (1:1000) against human ERß (Santa Cruz Biotechnology, Inc., catalogue no. sc-6822). All antibody incubations and washes were performed in Tris-buffered saline with 0.05% Tween-20. The Amersham Pharmacia Biotech enhanced chemiluminescence system (ECL) was used for detection. Membranes were visualized by exposure to Kodak X-Omat film. The radioautograms were then scanned and quantified with Scion Image-Released Beta 3b (Scion Corp.).

Progesterone RIAs

Human GCs were seeded at a density of 5 x 105 cells in 35-mm dishes. On days 3 and 6 in culture, hGLCs were washed twice with medium and treated with 1 nmol/L, 0.1 µM 17ß-estradiol, or vehicle in triplicate for 24 h before collection of sample medium. To study the effect of 17ß-estradiol on progesterone secretion after down-regulation of ERs by hCG and GnRHa, hGLCs on day 5 were pretreated with 10 IU/mL hCG, 0.1 µmol/L GnRHa, or vehicle for 24 h. The cells were then washed twice with medium and further incubated in the presence of 0.1 µmol/L 17ß-estradiol for an additional 24 h. The medium was removed and stored at -20 C before being assayed for progesterone content. The cells were lysed with 100 µL RIPA, and the total protein concentration was used to standardize the progesterone secretion. The RIA for progesterone was preformed as previously described (20). The standard curve and samples were assayed in triplicate. Inter- and intraassay coefficients of variation were less then 10%.

Data analysis

Data are shown as the mean ± SE from four independent experiments from four patients. The data were analyzed by one-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison test using SPSS computer software (version 9.0, SPSS, Inc., Chicago, IL). Data were considered statistically significant different from controls when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ER{alpha} and ERß mRNA in cultured hGLCs

The expression of ER{alpha} and ERß mRNA in cultured human granulosa-luteal cells was examined by RT-PCR. Using PCR primers designed from the published ER{alpha} and ERß cDNA sequences, the expected 540-bp product for ER{alpha} and the 279-bp product for ERß were observed by agarose gel electrophoresis and ethidium bromide staining (Fig. 1AGo). Sequence analysis of the PCR products revealed that they were identical to the published human ER{alpha} and ERß sequences, respectively (3, 19). In addition, Northern blot analysis revealed a 6.5-kb ER{alpha} transcript, from 40 µg total RNA isolated from hGLCs, only after a long exposure period (10 days). The same blot was stripped and rehybridized with ERß probe. Multiple transcripts for ERß ranging from 1.35–9.5 kb were observed after 2 days of exposure (Fig. 1BGo), and these signals could be detected from as low as 15 µg total RNA (data not shown). These results confirm the expression of ER{alpha} and ERß in hGLCs and suggest that a higher level of ERß mRNA is expressed in these cells.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 1. Expression of ER{alpha} and ERß mRNA in hGLCs. A, One microgram of hGLCs total RNA was reverse transcribed and amplified for 35 cycles of PCR. The PCR products were fractionated by 1% agarose gel electrophoresis. The expected sizes of the amplified cDNA fragment for ER{alpha} (left panel) and ERß (right panel) were 540 and 279 bp, respectively. B, Northern blot analysis of ER{alpha} and ERß mRNA in hGLCs. Forty micrograms of total RNA were extracted from hGLCs and hybridized with 32P-labeled ER{alpha} and ERß cDNA probes, respectively. A 6.5-kb ER{alpha} transcript (left panel) was detected. ERß shows multiple transcripts from 1.3–10 kb (right panel).

 
Changes in ER{alpha} and ERß mRNA levels in hGLCs in vitro

Due to the low levels of endogenous ER{alpha} mRNA in hGLCs and the limited availability of cells for Northern blot analysis, RT-PCR amplification was employed to quantify the relative ER{alpha} and ERß mRNA levels in subsequent experiments. PCR optimization showed a linear amplification of ER{alpha} or ERß cDNA up to 35 and 30 cycles of amplification, respectively (Fig. 2AGo). Similarly, a linear amplification for ß-actin was obtained from 15–25 cycles (data not shown). As a result, 30 and 27 cycles of amplification for ER{alpha} and ERß cDNA, respectively, were performed. In addition, 18 cycles for ß-actin were performed to standardize the ER expression.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. A, Validation of semiquantitative RT-PCR for ER{alpha} and ERß. To determine the linear phase of PCR amplification, ER{alpha} and ERß were amplified from hGLCs cDNA with increasing number of cycles. A linear relationship between cycle number and optical density was observed between 25–35 cycles for ER{alpha} (left panel) and 20–30 cycles for ERß (right panel). B, Changes in ER{alpha} and ERß expression with spontaneous lutenization in vitro. Total RNA was extracted from hGLCs, isolated from four patients, on days 1, 4, 7, and 10 of culture. One microgram of total RNA was reverse transcribed and amplified by PCR. The amounts of ER{alpha} and ERß transcripts were quantified by RT-PCR and normalized against ß-actin. Data were expressed as a percentage of the control value (day 1) and as the mean ± SE (n = 4). a, P < 0.05 vs. day 1 culture. b, P < 0.01 vs. day 4 culture.

 
To determine the changes in ER{alpha} and ERß mRNA levels in vitro, the isolated hGLCs were cultured for 1, 4, 7, and 10 days before RNA extraction and RT-PCR analysis. The relative mRNA levels of ER{alpha} and ERß were expressed as the percent change with respect to the day 1 mRNA levels. As shown in Fig. 2BGo, no statistically significant change in ER{alpha} mRNA levels was observed between days of culture. In contrast, ERß mRNA levels gradually increased in vitro. Thirty percent (P < 0.05), 60% (P < 0.01), and 58% (P < 0.01) increases in ERß mRNA levels were observed on days 4, 7, and 10 of culture, respectively.

Regulation of ER{alpha} and ERß mRNA levels by hCG and PKA pathway in hGLCs

As hCG is a major physiological regulator of ovarian function (21), we examined the effect of hCG in modulating ER{alpha} and ERß expression in hGLCs. As shown in Fig. 3Go, no significant change in ER{alpha} and ERß mRNA levels was observed in day 1 cultured hGLCs after hCG treatment. The levels of ER{alpha} and ERß mRNA were decreased to 55% (P < 0.01 vs. control) and 60% (P < 0.01 vs. control), respectively, in response to a 24-h treatment with 10 IU/mL hCG on day 7 of culture. These inhibitory effects were also observed in day 10 cultures. Further studies using hGLCs on day 7 in culture revealed a dose- and time-dependent decrease in ER{alpha} and ERß mRNA levels after hCG treatment (Fig. 4Go). A significant decrease in ER mRNA levels was observed when cells were treated with 1 and 10 IU/mL hCG for 24 h (Fig. 4AGo). Prolonged treatment with 10 IU/mL hCG (48 h) did not further decrease the mRNA levels of ERs compared to the effect of 24-h treatment (Fig. 4BGo). To investigate the potential role of the hCG-stimulated PKA signaling pathway in regulating ER{alpha} and ERß gene expression, hGLCs on day 7 in culture were treated with 1 mmol/L 8-bromo-cAMP and 10 µmol/L forskolin for 24 h. As shown in Fig. 5Go, a significant decrease (P < 0.01) in both ER{alpha} and ERß mRNA was observed after hCG (10 IU/mL), 8-bromo-cAMP (1 mmol/L), or forskolin (10 µmol/L) treatment. The role of the PKA signaling pathway in regulating ERs gene expression was further examined pharmacologically by the use of a specific PKA or adenylate cyclase inhibitor. Day 7 hGLC culture was treated with vehicle, 10 IU/mL hCG, 20 µmol/L PKAI, or 0.5 mmol/L ACI alone or with 10 IU/mL hCG in the presence of 20 µmol/L PKAI or 0.5 mmol/L ACI. The hCG-induced decrease in ER{alpha} and ERß was abolished in the presence of PKAI or ACI, whereas PKCI or ACI alone did not significantly affect the expression of ERs (Fig. 6Go). These data suggest that activation of the PKA pathway by hCG results in a down-regulation of ER gene expression in hGLCs.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. Effects of hCG on ER{alpha} (A) and ERß (B) mRNA expression in cultured hGLC. Cells were cultured for 1, 7, and 10 days and treated with vehicle or 10 IU/mL hCG for 24 h before total RNA isolation. The amounts of ER{alpha} and ERß transcripts were quantified by RT-PCR and normalized against ß-actin. Data were presented as a percentage of the control and as the mean ± SE (n = 4). *, P < 0.01 vs. control.

 


View larger version (36K):
[in this window]
[in a new window]
  		Figure 4. Dose- and time-dependent effects of hCG on ER{alpha} and ERß expression in hGLC. ER{alpha} and ERß mRNA levels were estimated by semiquantitative RT-PCR and normalized against ß-actin mRNA. A, hGLC were cultured for 7 days before treatment with vehicle and various doses of hCG (0.1–10 IU/mL) for 24 h. B, Day 7 cultured hGLCs were treated with vehicle or 10 IU/mL hCG for 0, 12, 24, and 48 h. Data were presented as a percentage of the control value and as the mean ± SE (n = 4). a, P < 0.01 vs. control; b, P < 0.05 vs. the immediately adjacent group.

 


View larger version (32K):
[in this window]
[in a new window]
  		Figure 5. Effects of hCG, 8-bromo-cAMP, and forskolin on ER{alpha} and ERß expression. hGLC were cultured for 7 days and treated with vehicle, 10 IU/mL hCG, 1 mmol/L 8-bromo-cAMP, or 10 µmol/L forskolin for 24 h. The amounts of ER{alpha} and ERß transcripts were quantified by RT-PCR and normalized against ß-actin. Data were presented as a percentage of the control and as the mean ± SE (n = 4). *, P < 0.01 vs. control.

 


View larger version (56K):
[in this window]
[in a new window]
  		Figure 6. Effects of hCG, PKAI, adenylate cyclase inhibitor (ACI), and hCG plus PKAI or ACI on ER{alpha} (A) and ERß (B) expression in hGLC. Day 7 cultured hGLCs were treated with vehicle, 10 IU/mL hCG, 20 µmol/L PKAI, 0.5 mmol/L ACI, or 10 IU/mL hCG in the presence of 20 µmol/L PKAI or 0.5 mmol/L ACI for 24 h. The amounts of ER{alpha} and ERß transcript were quantified by RT-PCR and normalized against ß-actin. The radioautograms of ER{alpha}, ERß, and ß-actin are shown above the graph. Data were presented as a percentage of the control and as the mean ± SE (n = 4). *, P < 0.01 vs. control.

 
Regulation of ER{alpha} and ERß mRNA levels by GnRH and PKC pathway in hGLCs

The expression of GnRH and its receptor and the demonstration of direct effects of GnRH on progesterone production in hGLCs have suggested an autocrine/paracrine role for GnRH in the ovary (17, 22). To investigate the ability of GnRH to alter ER{alpha} and ERß gene expression, hGLCs on day 1, 7, or 10 in culture were treated with 0.1 µmol/L GnRHa for 24 h. ER{alpha} and ERß mRNA levels were examined by semiquantitative RT-PCR. As shown in Fig. 7Go, GnRHa failed to affect ER{alpha} and ERß mRNA levels in day 1 and 10 cultured hGLCs, but caused 68% (P < 0.001 vs. control) and 60% (P < 0.001 vs. control) decreases in ER{alpha} and ERß mRNA, respectively, in day 7 cultured hGLCs. These results suggest a temporal regulation of ER{alpha} and ERß mRNA by GnRHa in hGLCs. Further studies using day 7 cultured hGLCs revealed a dose- and time-dependent regulation of ER{alpha} and ERß mRNA levels (Fig. 8Go). A significant decrease (P < 0.01) in ER{alpha} and ERß mRNA levels was observed when cells were treated with 0.1 µmol/L GnRHa for 24 h (Fig. 8AGo). No further decrease in ER mRNA levels was observed when the GnRHa concentration was increased to 1 µmol/L. The decreases in ER{alpha} and ERß mRNA levels were observed after 12- and 24-h treatment with 0.1 µmol/L GnRHa (Fig. 8BGo). It has been well documented that binding of GnRH receptor to its ligands activates the PKC pathway. In the human ovary, however, the second messenger pathway involved in GnRH action remains unclear. To determine the potential role of the PKC signaling pathway on GnRH action in regulating ER{alpha} and ERß mRNA in hGLCs, a specific PKC inhibitor, GF109203X, was used. Day 7 cultures of hGLCs were treated with vehicle, 0.1 µmol/L GnRHa, 1 µmol/L PKC inhibitor, and PKC inhibitor plus GnRHa for 24 h (Fig. 9Go). PKC inhibitor alone had no effect on ER{alpha} and ERß gene expression. However, GnRHa-mediated down-regulation of ER gene expression was inhibited in the presence of PKCI (Fig. 9Go). The effect of GnRHa-mediated down-regulation of ER{alpha} and ERß mRNA was mimicked by TPA treatment (data not shown), further implicating a role of the PKC pathway in regulating ER gene expression.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. Effects of D-Ala6-GnRH (GnRHa) on ER{alpha} (A) and ERß (B) mRNA expression in human granulosa-luteal cells. Cells were cultured for 1, 7, and 10 days and were treated with vehicle or 0.1 µmol/L GnRHa for 24 h before total RNA isolation. The amounts of ER{alpha} and ERß transcript were quantified by RT-PCR and normalized against ß-actin. Data were presented as the percent change from the control value and as the mean ± SE (n = 4). *, P < 0.001 vs. control.

 


View larger version (38K):
[in this window]
[in a new window]
  		Figure 8. Dose- and time-dependent effect of D-Ala6-GnRH (GnRHa) on ER{alpha} and ERß mRNA expression. ER{alpha} and ERß mRNA levels were estimated by semiquantitative RT-PCR and normalized against ßactin mRNA. A, hGLC were cultured for 7 days before being treated with vehicle and various doses of GnRHa (0–1 µmol/L) for 24 h. B, Day 7 cultured hGLCs were treated with vehicle or 0.1 µmol/L GnRHa for 0, 6, 12, and 24 h. Data were presented as a percentage of the control value and as the mean ± SE (n = 4). *, P < 0.01 vs. control.

 


View larger version (51K):
[in this window]
[in a new window]
  		Figure 9. Effects of D-Ala6-GnRH (GnRHa), PKCI, and PKCI plus GnRHa on ER{alpha} (A) and ERß (B) expression in hGLC. Day 7 cultured hGLCs were treated with vehicle, 0.1 µmol/L GnRHa, 10 µmol/L PKCI, or 10 µmol/L PKCI plus 0.1 µmol/L GnRHa for 24 h. The radioautograms of ER{alpha}, ERß, and ß-actin are shown above the graph. Data were presented as a percentage of the control and as the mean ± SE (n = 4). *, P < 0.01 vs. control.

 
Effects of GnRH, TPA, hCG, 8-bromo-cAMP, and forskolin on ER{alpha} and ERß protein levels

Western blot analysis of hGLCs using antibodies directed against ER{alpha} and ERß, respectively, revealed a 68-kDa ER{alpha} and a 55-kDa ERß protein species in all of the cellular extracts (Fig. 10Go). In agreement with the results from RT-PCR analysis, 45% (P < 0.01) and 40% (P < 0.01) decreases in ER{alpha} and ERß protein levels, respectively, were observed after 0.1 µmol/L GnRHa treatment. Similarly, hCG treatment (10 IU/mL) resulted in a significant decrease in ER{alpha} and ERß protein levels to 60% (P < 0.01) and 57% (P < 0.01) of control levels. These reductions in ER{alpha} and ERß protein levels were also observed after TPA, 8-bromo-cAMP, and forskolin treatments.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 10. Western blot analysis of ER{alpha} (A) and ERß (B) in hGLC. Protein (35 µg) extracted from day 7 cultured hGLCs after 24-h treatment with vehicle, 0.1 µmol/L GnRHa, 10 IU/mL hCG, 1 mmol/L 8-bromo cAMP, 10 µmol/L TPA, or 10 µmol/L forskolin was separated on a SDS-polyacrylamide gel. The radioautogram of ER{alpha} and ERß were scanned and quantified. The results were presented as a percentage of the control and as the mean ± SE (n = 4). *, P < 0.01 vs. control.

 
Effects of hCG and GnRHa pretreatment on 17ß-estradiol-mediated progesterone production

By the use of day 4 and day 7 hGLCs in culture, the effect of 17ß-estradiol on progesterone production was studied. Thirty percent (P < 0.01) and 34% (P < 0.01) decreases in progesterone secretion from day 4 hGLCs were observed after 1 nmol/L and 0.1 µmol/L 17ß-estradiol treatments, respectively (Fig. 11AGo). Similarly, 40% (P < 0.01) and 56% (P < 0.01) decreases in progesterone secretion were observed in day 7 hGLC in culture after 1 nmol/L and 0.1 µmol/L 17ß-estradiol treatments, respectively (Fig. 11AGo). This inhibitory effect could be blocked by the cotreatment of tamoxifen (data not shown), suggesting that it was an ER-mediated process. The effect of down-regulating ER expression on the E2-mediated decrease in progesterone secretion was examined in hGLCs on day 7 of culture. Pretreatment of hGLCs with 10 IU/mL hCG or 0.1 µmol/L GnRHa for 24 h before the addition of 0.1 µmol/L 17ß-estradiol eliminated the E2-induced decrease in progesterone secretion (Fig. 11BGo). This inhibition was shown to be specific, as hCG- and GnRHa-pretreated hGLCs retained the ability to produce progesterone when stimulated by 1 µmol/L forskolin (Fig. 11BGo).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 11. Effects of E2 in progesterone secretion in hGLCs in vitro. A, Progesterone secretion (over 24 h) in response to vehicle (control), 1 nmol/L or 0.1 µmol/L E2 in day 4 and day 7 cultured hGLCs. The results are presented as a percentage of the control and as the mean ± SE (n = 4). a, P < 0.01 vs. control; b, P < 0.05 vs. 1 nmol/L E2. B, Progesterone secretion (over 24 h) in response to vehicle (control), 1 µmol/L forskolin, or 0.1 µmol/L E2 in day 7 cultured hGLCs. hGLCs were pretreated with 10 IU/mL hCG or 0.1 µmol/L GnRHa for 24 h to down-regulate ER expression before the addition of forskolin or E2. The progesterone level was normalized by the protein concentration and is presented as the mean ± SE (n = 4). a, P < 0.001 vs. control; b, P < 0.01 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of ER in the ovary, especially in the human, is controversial. Several reports have suggested that ER was not expressed in granulosa cells and CL. For example, an immunohistochemical study showed no specific staining for ER in the human CL (23). ER mRNA was not detected by RT-PCR in hGLCs (24) or granulosa cells or CL of the baboon (25). However, others have reported the identification of ER in hGLCs cells and human CL immunologically (26, 27). Recent studies have demonstrated the expression of ER{alpha} and ERß mRNA in the human ovary (15, 16, 28, 29). The present study, using RT-PCR and Northern blot analysis, further supported the idea that ER{alpha} and ERß are expressed in hGLCs. However, there appears to be a quantitative difference in the expression of ER{alpha} vs. ERß in the human ovary. By the use of Northern blotting analysis, the ER{alpha} transcript was only detected with 40 µg total RNA isolated from hGLCs, whereas ERß can be detected with as little as 15 µg total RNA. These results suggest that the expression of ER{alpha} is at a relatively lower level compared with that of ERß in hGLCs. Similar results observed in rat ovarian RNA by Northern blot analysis further support the differential expression of ERs in the ovary (30). Immunohistochemical studies have shown that ERß is the primary ER subtype expressed in rat and bovine ovaries, particularly in granulosa cells, whereas ER{alpha} was mainly in the germinal epithelium, interstitial cells, and thecal cells of rat ovary (4). These findings may explain the failure of several studies (23, 24, 25) to detect ER mRNA and protein in granulosa cells by PCR and antibody directed against the ER{alpha} subclass. Interestingly, others have reported comparable expression of human ER{alpha} and ERß mRNA levels by Northern blot analysis and ribonuclease protection assay in normal human ovaries (15, 31). Competitive RT-PCR from human CL revealed similar levels of ER{alpha} and ERß mRNA expression during the luteal phase (16). One possible explanation is that in these studies using human ovarian tissues, the results could be confounded by other cell types, such as thecal cells, which may express higher levels of ER. This possibility is supported by the differential spatial expression of ER observed in the rat ovary (4). In the present study we also found that ER{alpha} and ERß mRNA displayed different temporal patterns of expression depending on the day of culture. The expression of ERß mRNA levels appears to increase significantly with spontaneous luteinization in vitro. In contrast, ER{alpha} expression did not change substantially throughout in vitro culture. These data suggest that ERß, more so than ER{alpha}, plays a dynamic role in regulating CL function in the human.

Gonadotropins are the major regulators of steroidogenesis in the human ovary (32, 33). As a first attempt to investigate the hormonal regulation of ER expression, we examined the effect of hCG on ER{alpha} and ERß mRNA levels in hGLCs. No significant change in either ER{alpha} or ERß mRNA levels was observed on day 1 of culture. As the granulosa-luteal cells were retrieved from in vitro fertilization patients after exposure to high doses of GnRHa and hCG in vivo, it is possible that the hCG/LH receptor was already down-regulated, which may explain the lack of responsiveness on day 1 in culture. A similar lag period has been observed for the hCG effect on the expression of several other genes in primary cultures of hGLCs (34, 35, 36). In day 7 cultured hGLCs, hCG produced a time- and concentration-dependent downregulation of both ER{alpha} and ERß mRNA levels. This down-regulation was mimicked by 8-bromo-cAMP and forskolin treatments, suggesting that the effect of hCG is mediated via the activation of the PKA pathway. Similar results have been reported in the rat, where hCG treatment of granulosa cells induced a down-regulation of ERß mRNA, and this effect was mimicked by 8-bromo-cAMP and TPA treatment (30). The participation of PKA signaling pathway in regulating human ER gene expression in hGLCs was further examined pharmacologically using a specific PKAI and ACI. The hCG-induced down-regulation of ER{alpha} and ERß expression was abolished in the presence of PKAI or ACI. These results strongly support the idea that down-regulation of ER{alpha} and ERß gene expression in the ovary by hCG was mediated through the PKA signaling pathway.

GnRH is primarily recognized for its role in the regulation of LH and FSH release from the pituitary gland. However, GnRH is also thought to be a potential paracrine/autocrine regulator in the gonads (17, 22, 37). GnRH and its agonists have been shown to exert various effects on ovarian function (17, 22, 37, 38, 39), and the expression of both GnRH and its receptor in cultured hGLCs (17) further corroborates a putative role for GnRH as an intraovarian hormone. The present study demonstrated that GnRH was able to down-regulate both ER{alpha} and ERß mRNA in hGLCs. In the rat ovary it is well established that the signal transduction pathway for GnRHR is primarily through activation of phospholipase C (40). It has been shown that TPA significantly down-regulates the expression of ERß in rat granulosa cells (30) and ER{alpha} in a human mammary adenocarcinoma MCF-7 cell (41). In the human ovary, the second messenger pathway(s) involved in GnRH action remains unclear. We have shown that GnRH-mediated down-regulation of ER{alpha} and ERß mRNA levels was inhibited in the presence of a PKC inhibitor and was mimicked by TPA administration. These results strongly implicate the activation of the PKC pathway by GnRH in the human ovary and the involvement of the PKC pathway in regulating the expression of ERs in hGLCs.

Although the role of estrogen in human ovarian function in the luteal phase remains unclear, several studies have indicated that estrogen could be an intraovarian regulator of luteolysis (42). This is supported by the observation that high concentrations of estrogen significantly inhibited luteal steroidogenesis (12). Administration of tamoxifen from day 18 of the menstrual cycle to the onset of menstruation resulted in a significant prolongation of the luteal phase and an elevation of progesterone levels in human (43). Similar results were observed in the present study; E2 treatment resulted in a significant decrease in progesterone secretion from the hGLCs in vitro, which could be block by the coadministration of tamoxifen. Furthermore, hCG and GnRHa pretreatment abolished the E2-induced decrease in progesterone secretion, suggesting that the regulation of ER expression by hCG and GnRHa was physiologically important.

Estrogen produced by the primate CL may cause luteolysis (44) by increasing PGF2{alpha} levels in the ovary (45). In the rat and guinea pig, estradiol was found to stimulate the synthesis of PGF2{alpha} (46, 47). Estradiol has been shown to increase the activity of phospholipase A2 (48) and increase the expression (49) of PG synthase, which control the production of PGF2{alpha}. A possible paracrine interaction of estradiol, oxytocin, and PGF2{alpha} within the primate ovary may promote luteolysis (50). It is possible that a similar mechanism exists in the human ovary. It has been demonstrated that estrogen induces PGF2{alpha} and oxytocin production and oxytocin receptor expression in various human tissues (51, 52, 53). Increasing ER{alpha} and ERß levels in the human CL may lead to increased susceptibility to the luteolytic effect of estrogen. On the other hand, down-regulation of ER subtypes in the mid- or late luteal phase may prevent the regression of hGLCs or the CL. Thus, regulation of ER{alpha} and ERß by LH/hCG and GnRH may be important to the function of hGLCs and/or CL.

In summary, we observed a differential temporal expression of ER{alpha} and ERß mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ER{alpha} and ERß gene expression suggests a role for these hormones, which may contribute to the control of hGLC and/or CL function. Furthermore, our results indicate that the down-regulation of ER{alpha} and ERß by hCG and GnRH in hGLCs is mediated by activation of the PKA and PKC signaling pathways, respectively.


   Footnotes
 
Address all correspondences and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Room 2H30-4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5.

1 This work was supported by the Medical Research Council of Canada, a postdoctoral fellowship from the Chang Gung Memorial Hospital (Taipei, Taiwan; to C.-H.C.), a postdoctoral fellowship from Gunma University (Gunma, Japan; to S.I.), and a studentship award from British Columbia Research Institute of Children’s and Women’s Health (to P.S.N.). Back

2 C.-H.C. and K.W.C. contributed equally and should be considered as first authors. Back

3 Career investigator with the British Columbia Research Institute for Children’s and Women’s Health. Back

Received December 30, 1999.

Revised June 28, 2000.

Accepted July 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA. 93:5925–5930.[Abstract/Free Full Text]
  2. Tremblay GB, Tremblay A, Copeland NG, et al. 1997 Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol Endocrinol. 11:353–365.
  3. Mosselman S, Polman J, Dijkema R. 1996 ER ß: identification and characterization of a novel human estrogen receptor. FEBS Lett. 392:49–53.[CrossRef][Medline]
  4. Sar M, Welsch F. 1999 Differential expression of estrogen receptor-ß and estrogen receptor-{alpha} in the rat ovary. Endocrinology. 140:963–971.[Abstract/Free Full Text]
  5. Fitzpatrick SL, Funkhouser JM, Sindoni DM, et al. 1999 Expression of estrogen receptor-ß protein in rodent ovary. Endocrinology. 140:2581–2591.[Abstract/Free Full Text]
  6. Rosenfeld CS, Yuan X, Manikkam M, Calder MD, Garverick HA, Lubahn DB. 1999 Cloning, sequencing, and localization of bovine estrogen receptor-ß within the ovarian follicle. Biol Reprod. 60:691–697.[Abstract/Free Full Text]
  7. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA. 90:11162–11166.[Abstract/Free Full Text]
  8. Krege JH, Hodgin JB, Couse JF, et al. 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA. 95:15677–15682.[Abstract/Free Full Text]
  9. Korach KS. 1994 Insights from the study of animals lacking functional estrogen receptor. Science. 266:1524–1527.[Abstract/Free Full Text]
  10. Tetsuka M, Milne M, Hillier SG. 1998 Expression of oestrogen receptor isoforms in relation to enzymes of oestrogen synthesis in rat ovary. Mol Cell Endocrinol. 141:29–35.[CrossRef][Medline]
  11. Telleria CM, Zhong L, Deb S, et al. 1998 Differential expression of the estrogen receptors {alpha} and ß in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology. 139:2432–2442.[Abstract/Free Full Text]
  12. Endo T, Henmi H, Goto T, et al. 1998 Effects of estradiol and an aromatase inhibitor on progesterone production in human cultured luteal cells. Gynecol Endocrinol. 12:29–34.[Medline]
  13. Vega M, Devoto L, Castro O, Kohen P. 1994 Progesterone synthesis by human luteal cells: modulation by estradiol. J Clin Endocrinol Metab. 79:466–469.[Abstract]
  14. Fisch B, Rose MP, Elder MG, Winston RM, Margara RA, Hillier SG. 1994 Effects of oestrogen on progesterone synthesis and arachidonic acid metabolism in human luteal cells. Clin Endocrinol (Oxf). 40:21–32.[Medline]
  15. Brandenberger AW, Tee MK, Jaffe RB. 1998 Estrogen receptor {alpha} (ER-[alpha) and ß (ER-ß) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ER-ß in neoplastic tissues. J Clin Endocrinol Metab. 83:1025–1028.[Abstract/Free Full Text]
  16. Misao R, Nakanishi Y, Sun WS, et al. 1999 Expression of oestrogen receptor {alpha} and ß mRNA in corpus luteum of human subjects. Mol Hum Reprod. 5:17–21.[Abstract/Free Full Text]
  17. Peng C, Fan NC, Ligier M, Vaananen J, Leung PC. 1994 Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology. 135:1740–1746.[Abstract]
  18. Yin H, Cheng KW, Hwa HL, Peng C, Auersperg N, Leung PCK. 1998 Expression of the messenger RNA for gonadotropin-releasing hormone and its receptor in human cancer cell lines. Life Sci. 62:2015–2023.[CrossRef][Medline]
  19. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P. 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 320:134–139.[CrossRef][Medline]
  20. Li W, Khorashen S, Ho Yeun B, Ling N, Leung PCK. 1993 Stimulation of progesterone secretion by recombinant follistatin-288 in human granulosa cells. Endocrinology. 132:1750–1756.[Abstract]
  21. Hunter MG, Baker TG. 1981 Effect of hCG, cAMP and FSH on steroidogenesis by human corpora lutea in vitro. J Reprod Fertil. 63:285–288.[Abstract]
  22. Gaetje R. 1994 Influence of gonadotropin releasing hormone (GnRH) and a GnRH-agonist on granulosa cell sterodiogenesis Clin Exp Obstet Gynecol. 21:164–169.
  23. Iwai T, Nanbu Y, Iwai M, Taii S, Fujii S, Mori T. 1990 Immunohistochemical localization of oestrogen receptors and progesterone receptors in the human ovary throughout the menstrual cycle. Virchows Arch A Pathol Anat Histopathol. 417:369–375.[CrossRef][Medline]
  24. Wu TCJ, Wang L, Wan YJW. 1993 Detection of estrogen receptor messenger ribonucleic acid in human oocytes and cumulus-oocyte complexes using reverse transcriptase-polymerase chain reaction. Fertil Steril. 59:54–59.[Medline]
  25. Hild-Petito S, Fazleabas AT. 1997 Expression of steroid receptors and steroidogenic enzymes in the baboon (Papio anubis) corpus luteum during the menstrual cycle and early pregnancy. J Clin Endocrinol Metab. 82:955–962.[Abstract/Free Full Text]
  26. Al-Timimi A, Buckley CH, Fox H. 1985 An immunohistochemical study of the incidence and significance of sex steroid hormone binding sites in normal and neoplastic human ovarian tissue. Int J Gynecol Pathol. 4:24–41.[Medline]
  27. Anderson RE, Holt JA. 1989 Binding of radiolabeled estrogens by human cells in vitro: implications to the development of a new diagnostic and therapeutic modality in the treatment of malignancies with estrogen receptors. Gynecol Oncol. 34:80–83.[CrossRef][Medline]
  28. Hurst BS, Zilberstein M, Chou JY, Litman B, Stephens J, Leslie KK. 1995 Estrogen receptors are present in human granulosa cells. J Clin Endocrinol Metab. 80:229–232.[Abstract]
  29. Moore JT, McKee DD, Slentz-Kesler K, et al. 1998 Cloning and characterization of human estrogen receptor ß isoforms. Biochem Biophys Res Commun. 247:75–78.[CrossRef][Medline]
  30. Byers M, Kuiper GG, Gustafsson JA, Park-Sarge OK. 1997 Estrogen receptor-ß mRNA expression in rat ovary: down-regulation by gonadotropins. Mol Endocrinol. 11:172–182.[Abstract/Free Full Text]
  31. Enmark E, Pelto-Huikko M, Grandien K, et al. 1997 Human estrogen receptor ß-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab. 82:4258–4265.[Abstract/Free Full Text]
  32. Hanson FW, Powell JE, Stevens VC. 1971 Effects of hCG and human pituitary LH on steroid secretion and functional life of the human corpus luteum. J Clin Endocrinol Metab. 32:211–215.[Medline]
  33. Nishimori K, Dunkel L, Hsueh AJW, Yamoto M, Nakano R. 1995 Expression of luteinizing hormone and chorionic gonadotropin receptor messenger ribonucleic acid in human corpora lutea during menstrual cycle and pregnancy. J Clin Endocrinol Metab. 80:1444–1448.[Abstract]
  34. Eramaa M, Tuuri T, Hilden K, Ritvos O. 1994 Regulation of inhibin {alpha}- and ß A-subunit messenger ribonucleic acid levels by chorionic gonadotropin and recombinant follicle-stimulating hormone in cultured human granulosa-luteal cells. J Clin Endocrinol Metab. 79:1670–1677.[Abstract]
  35. Laitinen M, Rutanen EM, Ritvos O. 1995 Expression of c-kit ligand messenger ribonucleic acids in human ovaries and regulation of their steady state levels by gonadotropins in cultured granulosa-luteal cells. Endocrinology. 136:4407–4414.[Abstract]
  36. Ristimaki A, Jaatinen R, Ritvos O. 1997 Regulation of prostaglandin F2{alpha} receptor expression in cultured human granulosa-luteal cells. Endocrinology. 138:191–195.[Abstract/Free Full Text]
  37. Chamson-Reig A, Pignataro OP, Libertun C, Lux-Lantos VA. 1999 Alterations in intracellular messengers mobilized by gonadotropin-releasing hormone in an experimental ovarian tumor. Endocrinology. 140:3573–3580.[Abstract/Free Full Text]
  38. Casper RF, Erickson GF, Yen SS. 1984 Studies on the effect of gonadotropin-releasing hormone and its agonist on human luteal steroidogenesis in vitro. Fertil Steril. 42:39–43.[Medline]
  39. Bussenot I, Azoulay-Barjonet C, Parinaud J. 1993 Modulation of the steroidogenesis of cultured human granulosa-lutein cells by gonadotropin-releasing hormone analogs. J Clin Endocrinol Metab. 76:1376–1379.[Abstract]
  40. Wang J, Leung PCK. 1989 Arachidonic acid as a stimulatory mediator of luteinizing hormone-releasing hormone action in the rat ovary. Endocrinology. 124:1973–1979.[Abstract]
  41. Ree AH, Knutsen HK, Landmark BF, Eskild W, Hansson V. 1992 Down-regulation of messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) by phorbol ester requires ongoing RNA synthesis but not protein synthesis. Is hormonal control of ER mRNA degradation mediated by an RNA molecule? Endocrinology. 131:1810–1814.[Abstract]
  42. Auletta FJ, Flint AP. 1988 Mechanisms controlling corpus luteum function in sheep, cows, nonhuman primates, and women especially in relation to the time of luteolysis. Endocr Rev. 9:88–105.[Medline]
  43. Swahn ML, Bygdeman M, Matlin SA, Wu ZY. 1989 The effect of tamoxifen on the function and lifespan of the corpus luteum and on subsequent ovarian function. Acta Endocrinol (Copenh). 121:417–425.[Medline]
  44. Knobil E. 1973 On the regulation of the primate corpus luteum. Biol Reprod. 8:246–258.
  45. Auletta FJ, Caldwell BV, Speroff L. 1976 Estrogen-induced luteolysis in rhesus monkey: reversal with indomethacin. Prostaglandins. 11:745–752.[CrossRef][Medline]
  46. Blatchley FR, Donovan BT, Poyser NL, Horton EW, Thompson CJ, Los M. 1971 Identification of prostaglandin F2{alpha} in the utero-ovarian blood of guinea-pig after treatment with oestrogen. Nature. 230:243–244.[CrossRef][Medline]
  47. Castracane VD, Jordan VC. 1975 The effect of estrogen and progestrone on uterine prostaglandin biosynthesis in the ovariectomized rat. Biol Reprod. 13:587–596.[Abstract]
  48. Bonney RC, Qizilbash ST, Frank S. 1987 Endometrial phospholipase A2 enzyme and their regualtion by steroid hormones. J Steroid Biochem. 27:1057–1064.[CrossRef][Medline]
  49. Charpigny G, Reinaud P, Tamby JP, et al. 1997 Expression of cyclo-oxygenase-1 and -2 in ovine endometrium during the estrous cuycle and early pregnancy. Endocrinology. 138:2163–2171.
  50. Mass S, Jarry H, Teichmann A, Rath W, Kuhn W, Wuttke W. 1992 Paracrine actions of oxytocin, prostaglandin F2{alpha} and estradiol within the human corpus luteum. J Clin Endocrinol Metab. 74:306–310.[Abstract]
  51. Schatz F, Markiewicz L, Barg P, Gurpide E. 1985 In vitro effects of ovarian steroids on prostaglandin F2{alpha} output by human endometrium and endometrial epithelial cells. J Clin Endocrinol Metab. 61:361–367.[Abstract]
  52. Chibbar R, Wong S, Miller FD, Mitchell BF. 1995 Estrogen stimulates oxytocin gene expression in human chorio-decidua. J Clin Endocrinol Metab. 80:567–572.[Abstract]
  53. Bale TL, Dorsa DM. 1995 Sex differences in and effects of estrogen on oxytocin receptor messenger ribonucleic acid expression in the ventromedial hypothalamus. Endocrinology. 136:27–32.[Abstract]



This article has been cited by other articles:


Home page
 ReproductionHome page
S. van den Driesche, V. M Smith, M. Myers, and W C. Duncan
Expression and regulation of oestrogen receptors in the human corpus luteum
Reproduction, April 1, 2008; 135(4): 509 - 517.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
P. Galluzzo, F. Caiazza, S. Moreno, and M. Marino
Role of ER{beta} palmitoylation in the inhibition of human colon cancer cell proliferation
Endocr. Relat. Cancer, March 1, 2007; 14(1): 153 - 167.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
S. A. Milne, T. A. Henderson, R. W. Kelly, P. T. Saunders, D. T. Baird, and H. O. D. Critchley
Leukocyte Populations and Steroid Receptor Expression in Human First-Trimester Decidua; Regulation by Antiprogestin and Prostaglandin E Analog
J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4315 - 4321.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. Yang, J. Wang, Y. Shen, and S. K. Roy
Developmental Expression of Estrogen Receptor (ER) {alpha} and ER{beta} in the Hamster Ovary: Regulation by Follicle-Stimulating Hormone
Endocrinology, December 1, 2004; 145(12): 5757 - 5766.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. J. Pinzone, H. Stevenson, J. S. Strobl, and P. E. Berg
Molecular and Cellular Determinants of Estrogen Receptor {alpha} Expression
Mol. Cell. Biol., June 1, 2004; 24(11): 4605 - 4612.
[Full Text] [PDF]