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Endocrinology Vol. 141, No. 5 1754-1763
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation of Gonadotropin-Releasing Hormone and Its Receptor Gene Expression by 17ß-Estradiol in Cultured Human Granulosa-Luteal Cells1

Parimal S. Nathwani, Sung Keun Kang, Kwai Wa Cheng, Kyung-Chul Choi and Peter C. K. Leung2

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

Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynaecology, University of British Columbia, Room 2H30–4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is evidence that GnRH and its binding sites are expressed in numerous extrapituitary tissues, including the primate ovary. However, the factors that regulate ovarian GnRH and its receptor (GnRH-R) remain poorly characterized. Since gonadal steroids are key regulators of ovarian functions, the present study investigated the role of 17ß-estradiol (E2) in regulating GnRH and GnRH-R messenger RNA (mRNA) from human granulosa-luteal cells (hGLCs). RT-PCR was used to isolate the ovarian GnRH-R transcript equivalent to the full-length coding region in the pituitary from hGLCs. Sequence analysis revealed that the ovarian GnRH-R mRNA is identical to its pituitary counterpart. Basal expression studies indicated that GnRH and GnRH-R mRNA levels significantly increased with time in vitro, reaching levels of 160% and 170% on day 8 and 10 of culture, respectively (P < 0.05). Treatment with various concentrations of estradiol (1–100 nM) for 24 h resulted in a dose-dependent decrease (P < 0.05) in GnRH and GnRH-R mRNA levels. Time course studies indicated that short-term treatment (6 h) with E2 (1 nM) had no significant effect on GnRH mRNA levels, while long-term treatment (48 h) with E2 resulted in a 40% decrease (P < 0.001) in GnRH mRNA levels. In contrast, GnRH-R mRNA levels exhibited a biphasic pattern, such that a short-term treatment (6 h) with E2 increased GnRH-R mRNA levels by 20% (P < 0.05), whereas long-term treatment (48 h) resulted in a 60% decrease (P < 0.001) in GnRH-R expression in hGLCs. Cotreatment of estradiol and tamoxifen blocked the E2 induced-regulation of GnRH and its receptor mRNAs, indicating that the E2 effect was mediated through its receptor. In summary, our studies demonstrate that the ovary possesses an intrinsic GnRH axis that is regulated during luteinization in vitro, and that E2 is capable of regulating GnRH and its receptor in the human ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADOTROPIN-RELEASING HORMONE (GnRH) is a key regulator of reproductive functions in mammals. Synthesized in hypothalamic neurons, GnRH is secreted into the hypothalamo-hypophyseal portal system and travels to the anterior pituitary gland, where it binds to a G protein-coupled receptor and stimulates the biosynthesis and secretion of gonadotropins (1, 2, 3). In addition to its role at the level of the pituitary, GnRH has been implicated as an autocrine/paracrine regulator in several extrapituitary tissues, including the gonads (4, 5, 6, 7, 8). During the earlier stages of follicular maturation, GnRH exerts antigonadotrophic effects by inhibiting granulosa cell differentiation and directly induces apoptotic cell death of granulosa cells, reminiscent of follicular atresia (9). During the periovulatory period, GnRH seems to play an important role in follicular rupture and ovulation by inducing transcription of tissue plasminogen activator (10), PG endoperoxidase synthase type 2 (11), and progesterone receptor (12). In contrast, during the luteal phase, the functional role of GnRH is poorly understood. Recent evidence indicates that rat ovarian GnRH messenger RNA (mRNA) levels and serum progesterone levels are inversely related during pregnancy and parturition, thereby providing evidence that GnRH may regulate ovarian steroidogenesis (13). Previous reports have demonstrated that GnRH modulates gonadotropin-stimulated steroidogenesis in the ovary (1, 6, 15), while others have shown that GnRH directly inhibits progesterone production in human granulosa-luteal cells (hGLCs) (5, 14). In the primate ovary, induced luteolysis resulted in an impairment of the steroidogenic pathway (16). Hence, locally produced GnRH may play a role in corpus luteum regression.

It is well documented that the pituitary GnRH receptor (GnRH-R) is regulated by GnRH and gonadal steroids. In general, homologous regulation of the GnRH-R by its ligand exhibits a biphasic pattern of expression in the pituitary gland, such that chronic administration of high doses leads to receptor down-regulation, whereas pulsatile treatments of GnRH lead to receptor up-regulation (17, 18, 19, 20, 21, 22). Gonadal steroids also play an important role in regulating GnRH and GnRH-R levels (23, 24, 25, 26, 27, 28, 29, 30, 31, 32). GnRH levels are inversely related to estrogen profiles during the estrous cycle (33). Also, GnRH-R mRNA levels are highly regulated during the estrous cycle in the rat (34), thereby providing strong evidence that gonadal steroids are key regulators of GnRH and GnRH-R expression at the hypothalamic and pituitary levels. However, regulation of ovarian GnRH and its receptor is poorly characterized. Like the pituitary GnRH-R, the ovarian GnRH-R appears to be highly regulated during the rat estrous cycle, peaking in proestrus and estrus (7). Gonadotropins also seem to play a role in regulating the intrinsic ovarian GnRH axis. In both the rat (4) and human ovary (5), LH/human (h)CG down-regulated GnRH-R mRNA levels. However, the effect of gonadal steroids on GnRH and GnRH-R expression in the human ovary remains to be elucidated. The present study was designed to investigate the role of E2 in regulating GnRH and GnRH-R expression in human granulosa-luteal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Granulosa-luteal cell culture and pharmacological treatments
The use of human granulosa-luteal cells in vitro was approved by the Clinical Screening Committee for Research and Other Studies involving Human Subjects of the University of British Columbia. Follicular aspirates were collected during oocyte retrieval from women undergoing in vitro fertilization at the University of British Columbia and granulosa-luteal cells were prepared as previously described (5), with some modifications. Briefly, the follicular contents were centrifuged at 1,000 x g and the supernatant was removed. The cells were resuspended in 6 ml of medium 199 (M199) (Life Technologies, Inc., Burlington, Ontario, Canada) and layered onto Ficoll Paque (Amersham Pharmacia Biotech, Bale D’Urfé, Québec, Canada) and centrifuged to remove red blood cells. Cells in the interface were collected and rinsed twice with M199 supplemented with 100 U/ml penicillin G and 100 µg/ml streptomycin. The cell pellet was resuspended in M199 supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin at a density of 1 x 105 cells/ml. The cells were seeded at a density of 2 x 105 cells in 35-mm culture dishes and were allowed to adhere for 48 h at 37 C in a humidified atmosphere of 5% CO2-95% air. Subsequently, the cells were rinsed with HBSS, and on day 4 in culture the cells were cultured in phenol red-free M199 supplemented with 2% charcoal-stripped FBS. Cells were treated with estradiol in a time course and dose-dependent manner in phenol red-free M199 on day 5 in culture. Control cultures were treated with vehicle (0.01% wt/vol of ethanol). In addition, tamoxifen treatment alone was also performed in the appropriate studies.

Total RNA extraction and first strand cDNA synthesis
Total RNA was extracted from cultured hGLCs using the RNaid Kit (Bio/Can Scientific, Mississauga, Ontario, Canada) as per the manufacturer’s protocol. Briefly, cells were disrupted in lysis buffer [4 M guanidine thiocynate, 25 mM sodium citrate (pH 7.0), 0.5% N-lauroyl sarcosine, and 0.1 M ß-mercaptoethanol] and subsequently acid phenol extracted. RNA was purified from the aqueous phase on an RNA matrix and eluted into ribonuclease-free water. The concentration of RNA was determined by the absorbance at 260 nm, and the integrity was confirmed by agarose-formaldehyde gel electrophoresis.

Cloning and sequencing of the full-length GnRH-R cDNA
One microgram of total RNA was reverse transcribed into first-strand cDNA in a total volume of 15 µl using the first-strand cDNA synthesis kit (Pharmacia Biotech). Based on the published pituitary sequence for the human GnRH-R, two primers were designed to amplify the full-length GnRH-R from hGLCs. The sense and antisense primers were 5'-AATATGGCAAACAGTGCCTCTC-3' (P48–2) and 5'-CAATCACA-GAGAAAAATATCCA-3' (P48R), respectively. The cDNA was amplified in a 50 µl PCR reaction containing 2.5 U of Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mM MgCl2, 10 mM each deoxynucleoside triphosphate (dNTP), and 50 pmol of each primer. The PCR product was separated by agarose gel electrophoresis and visualized with ethidium bromide staining and UV light. The PCR product was transferred to a nylon membrane and probed with a digoxigenin-labeled GnRH-R cDNA probe (Roche Molecular Biochemicals, Laval, Québec, Canada). All probes used for Southern blot analysis were internal to the oligonucleotide primers used for PCR amplification to avoid nonspecific binding. After high-stringency washes, the membrane was exposed to Omat x-ray film (Eastman Kodak, Rochester, NY). The putative full-length GnRH-R cDNA was cloned into PCRII Vector (Invitrogen, San Diego, CA ) and sequenced by the dideoxy nucleotide chain termination method using the T7 DNA Polymerase Sequencing Kit (Pharmacia Biotech).

RT-PCR and quantification of GnRH mRNA from hGLCs
Using a semiquantitative PCR system, GnRH mRNA levels were quantitated. PCR amplification was carried out in 50 µl reactions containing 2.5 U of Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mM MgCl2, 2 mM each dNTP, and 50 pmol of sense and antisense primer. The primers for GnRH were designed based on the published sequence for human hypothalamic GnRH. The forward and reverse primers were 5'-ATTCTACTGACTTGGTGCGTG-3' (F1) and 5'-GGAATATGTGCAACTTGGTGT-3' (R1), respectively. PCR amplification was carried out for 26 cycles with denaturing at 94 C for 60 sec, annealing at 53 C for 35 sec, and extension at 72 C for 90 sec, followed by a final extension at 72 C for 15 min. To standardize for first-strand cDNA synthesis efficiency, PCR for ß-actin was performed. Primers for ß-actin were designed from the human published sequence (35). Amplified PCR products were separated by agarose gel electrophoresis and subjected to Southern blot analysis. Quantitation was performed using a visual light densitometer (model 620, Bio-Rad Laboratories, Inc., Richmond, CA).

Construction of the native (target) and mutant (competitive) cDNA for GnRH-R
Using an internal primer pair (P44F and P45R) based on the human pituitary GnRH-R cDNA, a 347-bp fragment of native GnRH-R was obtained by PCR amplification from human pituitary cDNA. The primers were sense 5'-GTATGCTGGAGAGTTACTCTGCA-3' (P44F) and antisense 5'-GGATGATGAAGAGGCAGCTGAAG-3' (P45R). The expected 347-bp PCR product was separated by agarose gel electrophoresis and confirmed to be hGnRH-R by Southern blot analysis. The PCR product was cloned into PCRII cloning vector (Invitrogen). Sequence analysis was performed to confirm the identity of the cloned fragment. Subsequently, the fragment was subcloned into pBSKII (Stratagene, La Jolla, CA) at SacI and XhoI sites. The mutant competitor cDNA was generated by digesting the subcloned fragment with HindIII and StyI and self-ligating the resulting clone. The 227-bp hGnRH-R cDNA fragment contained a 120-bp deletion from the native clone. However, the competitor retained the identical primer binding sites as the native GnRH-R cDNA.

RT-PCR and quantification of GnRH-R mRNA from hGLCs
For competitive PCR, 4 µl of first-strand cDNA from 1 µg of total RNA were coamplified with 0.08 pg of mutant GnRH-R cDNA. PCR amplification was carried out in 50 µl reactions containing 2.5 U of Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mM MgCl2, 2 mM each dNTP, and 50 pmol of sense (P44F) and antisense primer (P45R). Amplified PCR products were separated by agarose gel electrophoresis and subjected to Southern blot analysis. The PCR products were hybridized with a digoxigenin-labeled GnRH-R cDNA probe (Roche Molecular Biochemicals, Laval, Quebec, Canada). After stringency washes, the membranes were exposed to Omat x-ray film (Eastman Kodak). Quantitation was performed using a visual light densitometer (model 620, Bio-Rad Laboratories, Inc.).

Data analysis
Relative GnRH mRNA levels were represented as the ratio of GnRH to ß-actin. The amount of GnRH-R transcript was calculated based on the ratio of the target to competitive cDNA. The data are represented as the percent change relative to control. Data are depicted as the mean ± SE. Each experiment was conducted with four different patient samples. The data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (PRISM GraphPad version 2, GraphPad Software, Inc., San Diego, CA). A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and Cloning of the ovarian GnRH-R cDNA from hGLCs
RT-PCR amplification was used to investigate the expression of the ovarian GnRH-R transcript equivalent to the pituitary form from hGLCs. A pair of primers encompassing the start and stop codons was designed based on the human pituitary sequence of GnRH-R cDNA (Fig. 1AGo). As shown in Fig. 1BGo, PCR amplification yielded a 1003-bp fragment. Upon transfer of the PCR product to a nylon membrane and hybridization with a specific cDNA probe, a signal was detected, thereby confirming the identity of the PCR product (Fig. 1BGo, lower panel). The possibility of cross-contamination was ruled out, as no PCR product was detected in the negative control (Fig. 1BGo, lane 1). Since the primers were located in different exons, the amplified product was not due to genomic DNA contamination, but rather specific amplification of mRNA. Sequencing analysis revealed that the GnRH-R from hGLCs was identical to the published human pituitary cDNA sequence (data not shown).



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  		Figure 1. Molecular characterization of the full-length GnRH-R cDNA from hGLCs. A, Schematic representation of the GnRH-R gene and cDNA. The positions and sequences of the primers used for RT-PCR amplification of the coding region of GnRH-R are depicted. TM, Transmembrane domain. B, RT-PCR amplification and Southern blot analysis of the GnRH-R from hGLCs. One microgram of total RNA was reverse transcribed and amplified for 35 cycles. A negative control without cDNA template was used to control for PCR contamination (lane 1). The PCR product was resolved on a 1% agarose gel stained with ethidium bromide and a 1003-bp fragment was visualized (lane 3) (upper panel). MW, Molecular weight marker (lane 2). The 1-kb fragment was confirmed to be GnRH-R by Southern blot analysis using a digoxigenin-labeled cDNA probe (lower panel).

 
Validation of semiquantitative and competitive RT-PCR for GnRH and GnRH-R
Using semiquantitative RT-PCR with primers derived from the human hypothalamic GnRH cDNA (Fig. 2AGo), the levels of GnRH mRNA were examined in hGLCs. To determine where PCR amplification for GnRH and ß-actin mRNA were in the logarithmic phase, 1 µg of total RNA was reverse transcribed and amplified under different cycle numbers (Fig. 2Go). Southern blot analysis revealed a 380-bp PCR product corresponding to GnRH (Fig. 2BGo), and a 525-bp PCR product for ß-actin (Fig. 2CGo). A linear relationship was observed for GnRH (Fig. 2BGo, lower panel) and ß-actin (Fig. 2CGo, lower panel) mRNA amplification with varying cycle numbers. Consequently, 26 cycles for GnRH and 18 cycles for ß-actin were employed for quantification purposes. GnRH-R mRNA levels were analyzed using competitive RT-PCR. Coamplification of the native and mutant GnRH-R cDNA yielded two PCR products at 347 bp and 227 bp, respectively (Fig. 3BGo). A standard curve for GnRH-R was constructed by coamplification of a fixed amount of mutant GnRH-R cDNA with serial dilutions of the native GnRH-R construct. Increasing the amount of native construct resulted in a decreased amplification of the mutant internal standard (Fig. 3CGo). When the mutant GnRH-R cDNA was plotted as a function of the ratio of the mutant and native GnRH-R construct, a linear relationship was observed (Fig. 3DGo). To determine the optimal concentration of the competitor for quantification purposes, a fixed amount of first-strand cDNA (4 µl synthesized from 1 µg of total RNA) was coamplified with serial dilutions of the competitor (data not shown). Increasing the amount of mutant construct resulted in a decline of native GnRH-R amplification from the cDNA sample. For quantitation purposes, 0.08 pg of mutant construct was employed for competitive RT-PCR for GnRH-R.



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  		Figure 2. Validation of semiquantitative RT-PCR for GnRH and ß-actin from hGLCs. A, Schematic representation of the GnRH cDNA, including primer positions and sequences. SP, Signal peptide; GAP, GnRH-associated peptide. B, Total RNA was extracted from hGLCs and 1 µg was reverse transcribed. Subsequent to PCR amplification with primers F1/R1, a 380-bp fragment was detected by agarose gel electrophoresis. To determine the linear phase of PCR amplification, GnRH was amplified from hGLCs cDNA under increasing cycle numbers. A linear relationship between the cycle number and optical density was observed between 25–30 cycles. Hence, 26 cycles were used for quantitation of GnRH mRNA from hGLCs. C, PCR amplification for ß-actin was performed with increasing cycle numbers. A linear relationship between the cycle number and optical density was observed between 15–20 cycles. Thus, 18 cycles were used for quantitation purposes.

 


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  		Figure 3. Construction of the native and mutant GnRH-R cDNA and validation of GnRH-R competitive RT-PCR. A, Internal primers (P44F and P45R) were designed based on the published human GnRH-R cDNA sequence from the pituitary gland. RT-PCR amplification from a 760-bp human pituitary cDNA template resulted in a 347-bp fragment corresponding to the native GnRH-R cDNA construct. The fragment was subcloned into pBSK II (Stratagene) at SacI and XhoI restriction enzyme sites. The mutant was generated by digesting the native GnRH-R cDNA with HindIII and StyI, followed by self-ligation. B, PCR amplification of the native and mutant GnRH-R cDNA with P44 and P45 resulted in the expected 347-bp and 227-bp fragments, respectively (lanes 1 and 2). Also note that both the native and mutant GnRH-R were coamplified in the same PCR reaction (lane 3). C, The standard curve for GnRH-R competitive PCR was constructed by coamplification of a fixed amount mutant GnRH-R cDNA and varying concentrations of the native GnRH-R cDNA. PCR amplification was carried out for 33 cycles, and the products were analyzed by Southern blot analysis. D, A linear relationship was observed when the ratio of the native and mutant GnRH-R was plotted against the concentration of native cDNA.

 
Expression of GnRH and GnRH-R during spontaneous luteinization in vitro
To examine the changes in GnRH and GnRH-R mRNA levels with time in culture, hGLCs were cultured for 1, 4, 8, and 10 days followed by RNA extraction and RT-PCR quantitative analysis. As seen in Fig. 4Go, GnRH and its receptor mRNA levels significantly (P < 0.05) increased with time in culture. For quantitative purposes, all time points were standardized to day 1 mRNA levels. Hence, the day 1 cultures acted as controls. For GnRH mRNA levels (Fig. 4AGo), a 65% increase (P < 0.05) was observed on day 10 compared with day 1 cultures. When day 4 cultures were compared with those on days 8 and 10, there was a significant increase in GnRH mRNA levels with increasing time in culture. However, there was no significant difference between day 8 and day 10 cultures for GnRH mRNA levels. A similar trend was observed for GnRH-R mRNA levels (Fig. 4BGo). There was a 55% and 63% (P < 0.05) increase in GnRH-R mRNA levels on day 8 and 10 cultures compared with day 1 cultures, respectively.



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  		Figure 4. Changes in GnRH (A) and GnRH-R (B) expression with spontaneous luteinization in culture. Total RNA was extracted from hGLCs (n = 4) on day 1, 4, 8, and 10 in culture. One microgram of total RNA was reverse transcribed. GnRH and GnRH-R mRNA levels were estimated by semiquantitative and competitive RT-PCR, respectively, as described in Materials and Methods. GnRH mRNA levels were normalized against ß-actin (18 cycles) mRNA. The amount of GnRH-R transcript was calculated from the ratio of the native and mutant GnRH-R cDNA. Data were expressed as percent change relative to control and represent the mean ± SE of four different experiments from four different patients. a, P < 0.05, significantly different from the control day 1 cultures.

 
Effects of E2 on GnRH and GnRH-R expression in hGLCs
Human granulosa-luteal cells were treated in a dose- and time-dependent fashion to examine the effects of estradiol on GnRH and GnRH-R expression (Figs. 5Go and 6Go). As seen in Fig. 5AGo, doses as low as 1 nM decreased GnRH mRNA levels by 45% (P < 0.05) relative to control values. A 70% decrease (P < 0.001) in GnRH mRNA levels was observed with a dose of 100 nM E2. Similarly, as seen in Fig. 5BGo, estradiol had an inhibitory effect on GnRH-R mRNA levels. A significant decrease in GnRH-R mRNA levels (40%; P < 0.05) was observed with a 1 nM E2 treatment for 24 h. A 65% decrease (P < 0.01) in GnRH-R mRNA levels was observed at a 100 nM E2 treatment. For the time course studies, hGLCs were treated with 1 nM E2 for 0, 6, 12, 24, and 48 h, followed by RNA extraction and RT-PCR quantitation. For each time point a control was included to take into account the changes during time in culture (data not shown). Relative changes of GnRH and GnRH-R mRNA levels were plotted as seen in Fig. 6Go. Short-term treatment (6 h) with E2 had no significant effect on GnRH mRNA levels. However, 24 h treatment significantly decreased GnRH mRNA levels (P < 0.001) compared with control. Long-term culture (48 h) had no further effect on GnRH mRNA levels compared with 24 h (P > 0.05). Hence, maximal inhibition was observed after 24 h treatment with a 1 nM dose of E2 (Fig. 6AGo). In contrast, a short-term treatment (6 h) with E2 significantly increased GnRH-R mRNA levels in hGLCs (P < 0.05), followed by a significant decrease (45%; P < 0.05) after a 24-h treatment. Long-term treatment (48 h) further decreased GnRH-R mRNA levels by 25% (P < 0.05) when compared with the 24-h treatment (Fig. 6BGo).



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  		Figure 5. The effects of different concentrations of E2 on GnRH (A) and GnRH-R (B) mRNA in cultured hGLCs. Cells were precultured for 4 days and on day 5 were treated with various doses (0–100 nM) of E2 for 24 h. GnRH and GnRH-R mRNA levels were estimated by semiquantitative and competitive RT-PCR, respectively, as described in Materials and Methods. GnRH mRNA levels were normalized against ß-actin (18 cycles) mRNA. The amount of GnRH-R transcript was calculated from the ratio of the native and mutant GnRH-R cDNA. Data were expressed as percent change relative to control and represent the mean ± SE of four different experiments from four different patients. a, P < 0.05, significantly different from the control.

 


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  		Figure 6. Time-dependent effects of E2 on GnRH (A) and GnRH-R mRNA (B) levels in cultured hGLCs. Cells were precultured for 4 days and on day 5 were treated with a 1 nM dose of E2 for 0, 6, 12, 24, and 48 h. GnRH and GnRH-R mRNA levels were estimated by semiquantitative and competitive RT-PCR, respectively, as described in Materials and Methods. GnRH mRNA levels were normalized against ß-actin (18 cycles) mRNA. The amount of GnRH-R transcript was calculated from the ratio of the native and mutant GnRH-R cDNA. Data were expressed as percent change relative to control and represent the mean ± SE from four different experiments from four different patients. a, P < 0.05, significantly different from the control; b, P < 0.05, significantly different from the 6-h treatment group; c, P < 0.05, significantly different from the 24-h treatment group.

 
Effects of cotreatment with E2 and tamoxifen
To determine whether the estradiol-induced regulation of GnRH and GnRH-R was a receptor-mediated event, hGLCs were treated with E2 (100 nM), alone or in combination with tamoxifen (10–100 nM), for 24 h. As seen in Fig. 7Go, E2 significantly decreased GnRH and GnRH-R mRNA levels (P < 0.001). Tamoxifen alone had no significant effect on GnRH and GnRH-R mRNA levels (data not shown). Cotreatment with E2 and tamoxifen reversed the response in a dose-dependent fashion. When hGLCs were treated with E2 and 10 nM tamoxifen, the mean GnRH and GnRH-R mRNA levels increased but were not statistically significant compared with E2 treatment alone (P > 0.05).



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  		Figure 7. The effect of E2 and tamoxifen cotreatment on GnRH (A) and GnRH-R mRNA in cultured hGLCs. Cells were precultured for 4 days and on day 5 were treated with a 100 nM dose of E2 in combination with varying doses of tamoxifen (0–100 nM) for 24 h. GnRH and GnRH-R mRNA levels were estimated by semiquantitative and competitive RT-PCR, respectively, as described in Materials and Methods. GnRH mRNA levels were normalized against ß-actin (18 cycles) mRNA. The amount of GnRH-R transcript was calculated from the ratio of the native and mutant GnRH-R cDNA. Data were expressed as percent change relative to control and represent the mean ± SE of four different experiments from four different patients. a, P < 0.001, significantly different from the control. b, P < 0.01, significantly different from the E2 treatment.

 
However, when the cells were treated with equimolar doses of E2 and tamoxifen (100 nM), the E2-induced down-regulation of GnRH and GnRH-R was reversed. As depicted in Fig. 7Go, there was no statistical difference between the equimolar treatment group and the control group. However, there was a significant difference between the E2 treatment alone and equimolar treatment of E2 and tamoxifen (P < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Luteinization is a complex differentiation process that is under the influence of many gonadal factors such as steroids and growth factors. However, the exact regulatory mechanisms underlying luteinization and regression remain unknown. GnRH has been implicated as an autocrine/paracrine regulator of luteal function and luteolysis (36). Since GnRH actions are mediated through a G protein-coupled receptor, the expression of the GnRH-R is crucial for GnRH actions. Since cultured hGLCs express the components of the GnRH system (5, 14), they provide an excellent model to study the autocrine/paracrine function and regulation of GnRH in the ovary. The hGLC culture system employed in this study has many advantages. First, since it is an in vitro system, the confounding factors from the hypothalamo-pituitary axis do not influence our results. Furthermore, since the hGLCs are cultured in the absence of an androgen substrate, the exogenous effects of E2 are not influenced by the endogenous production. This idea is further corroborated in this study as tamoxifen treatment alone had no significant effect on GnRH and GnRH-R gene expression (data not shown). Finally, this culture system is commonly used to study ovarian endocrinology in the human (5, 14, 15).

While expression of the GnRH-R from the human ovary has been previously reported (5), molecular characterization of the ovarian transcript equivalent to the full-length GnRH-R in the pituitary has not been documented. The present study isolated the entire coding region of the GnRH-R from hGLCs by RT-PCR amplification. Sequence analysis of the 1-kb fragment revealed that the ovarian GnRH-R is identical to the pituitary cDNA sequence (37). However, the binding affinity for the ovarian GnRH-R is lower than that of the pituitary (38, 39). This may be explained by differential posttranslational modifications as opposed to distinct molecular structures.

Results from the present study indicate that GnRH and its receptor mRNA levels significantly increase with spontaneous luteinization in vitro. Changes in GnRH mRNA levels are also seen during postnatal development and through puberty. Increases in GnRH mRNA levels during development are hypothesized to play a crucial role in regulating the onset of puberty (40). Although the mechanism of increased GnRH and GnRH-R mRNA in the ovary during luteinization remains unknown, in the mouse changes in GnRH mRNA levels during development are attributed to gene transcription and altered mRNA stability (40). The increase in GnRH and its receptor mRNA levels in our culture system may be due to the endogenous production of ovarian factors.

As gonadal steroids are key regulators of reproduction, the effects of E2 and progesterone on GnRH and its receptor gene expression at the hypothalamo-pituitary level are well documented (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). For example, during the rat estrous cycle, GnRH mRNA levels in the anterior hypothalamus were inversely correlated to plasma estrogen levels, providing evidence that E2 may directly or indirectly inhibit hypothalamic GnRH mRNA levels (33). However, other groups have documented that E2 increases GnRH gene expression in the rat (23), which may contribute to the gonadotropin surge before ovulation. The discrepancies observed between different groups may be due to the anatomical regions analyzed, time points during the estrous cycle and/or differences in sensitivities of techniques. No information is available on the role of steroids in regulating GnRH and its receptor in the ovary, especially in the human. In this study we demonstrate for the first time that estradiol negatively regulates GnRH mRNA in cultured hGLCs, and significant effects were seen at physiological doses of 1 nM after 24 h treatment.

Like GnRH, regulation of GnRH-R by E2 in the pituitary is well documented. In the sheep, mouse, and rat, estradiol increased GnRH-R mRNA levels (25, 27, 28, 29, 30, 31, 32). However, other groups have shown that E2 negatively regulates GnRH-R mRNA levels in the rat pituitary gland (20). Differences in steroid-induced regulation of GnRH-R also display tissue specificity. For example, in the rat hippocampus, E2 significantly decreased GnRH-R levels (41, 42). Taken together, these data suggest that the GnRH-R is differentially regulated depending on the tissue and species. Our study demonstrates that GnRH-R mRNA displays a biphasic regulatory pattern. Short-term treatment with E2 (6 h) resulted in an increase in GnRH-R mRNA levels, whereas a long-term treatment resulted in a significant decrease in GnRH-R mRNA levels.

The exact mechanism by which E2 regulates GnRH and its receptor mRNAs in the ovary remains uncertain. Others have documented that the ovary expresses estrogen receptor {alpha} and ß (43), and our studies confirm that the E2-mediated regulation of GnRH and GnRH-R is a receptor-mediated event as cotreatment with tamoxifen blocked the E2-induced decrease in GnRH and GnRH-R mRNAs. Similar results were also obtained in the pituitary (25). However, after binding to its receptor, E2 may modulate GnRH and GnRH-R directly or indirectly. Analysis of the human GnRH and GnRH-R promoter region reveals no consensus sequence for an estrogen response element, ERE (44). However, E2 can directly modulate transcription of the GnRH gene (44, 45), and preliminary evidence from our laboratory suggests that E2 may modulate GnRH-R promoter activity. Interestingly, previous studies have shown that E2 decreased GnRH promoter activity in placental cells (45), providing further support for the E2-induced down-regulation of GnRH mRNA in the extrapituitary hGLCs. Alternatively, E2 may act through indirect pathways to regulate GnRH and its receptor in hGLCs. It has been demonstrated that E2 can mobilize intracellular Ca2+ in chicken granulosa cells (46), which may lead to activation of the protein kinase C pathway. There is evidence that activation of the protein kinase C pathway can modulate GnRH-R gene expression (47). The present study indicates that the E2-induced down-regulation of GnRH and its receptor is a receptor-mediated event.

Activation of the GnRH-R in extrapituitary tissues, in contrast to pituitary cells, may activate the programmed cell death pathways by interacting with the Fas/Fas ligand pathway (48). It has been demonstrated that the FasL is capable of inducing apoptotic cell death in rat luteal cells and hGLCs (49, 50). In rat granulosa cells, GnRH directly induced apoptosis and may be an important factor in regulating follicular atresia (9). Sridaran et al. (51) have reported that GnRH decreased serum progesterone levels and induced apoptotic cell death in the corpus luteum of rats during early pregnancy. However, since this was an in vivo study, the direct effects of GnRH on the ovary cannot be dissected from the influence of the hypothalamo-pituitary axis. Thus, the role of GnRH during the luteal phase remains poorly characterized. Nevertheless, previous reports have implicated GnRH as a luteolytic factor. For example, LH/hCG decreased GnRH-R expression in the rat and human ovary (4, 5). Human CG is an important rescue factor for the maintenance of the corpus luteum during the early stages of pregnancy. Hence, the down-regulation of GnRH-R by LH/hCG may be involved in the maintenance of the corpus luteum during pregnancy. Our study demonstrates an increase in GnRH and its receptor mRNAs during spontaneous luteinization in vitro, which suggests that GnRH, in combination with other factors, may play an important role in corpus luteum regression. Indeed, it has been suggested that GnRH potentiates the antigonadotropic effects of PGF2{alpha} in hGLCs (15). GnRH has been shown to inhibit the gonadotropin-induced response in hGLCs, which may be clinically linked to the low follicular development rates seen in patients treated with GnRH agonists for ovulation induction (52). This idea is further corroborated by the decreased steroidogenic potential, a biochemical marker of functional luteolysis (16), with GnRH treatment in hGLCs (5, 14). In contrast, in rabbits and humans, E2 increased progesterone production during the early luteal phase (53, 54, 55). Furthermore, deprivation of E2 in the rabbit corpus luteum resulted in a time-dependent induction of apoptosis (56). Therefore, the E2-induced down regulation of GnRH and its receptor in hGLCs may play an important role in the maintenance of the corpus luteum.

Clinically, since a GnRH regimen is a common practice in assisted reproductive technologies, the direct ovarian effects of GnRH agonists on follicle development and quality have been raised (57, 58). Thus, understanding the dynamics of the intrinsic ovarian GnRH axis may play an important role in the discernment of assisted reproductive technologies outcomes.

In summary, we have isolated the ovarian GnRH-R transcript that is equivalent to the full-length GnRH-R cDNA from the pituitary in cultured hGLCs and determined that the cDNA sequence is identical to its counterpart in the pituitary gland. In addition, we observed an increase in GnRH and GnRH-R expression with spontaneous luteinization in culture, suggesting that GnRH may play a role in controlling corpus luteum function. Furthermore, E2 induced a down regulation of GnRH and its receptor mRNA in hGLCs. Our findings demonstrate, for the first time, that a gonadal steroid can regulate the expression of GnRH and GnRH-R in the human ovary. Taken together, these data strongly suggest that GnRH plays an autocrine role in the hGLCs, and that the E2-induced down-regulation GnRH and its receptor may contribute to the appropriate development of the corpus luteum.


   Acknowledgments
 
We thank the University of British Columbia’s in vitro fertilization program for collecting the follicular aspirate for hGLCs isolation.


   Footnotes
 
1 Presented in part at the 32nd Annual Meeting of the Society for the Study of Reproduction, Pullman, Washington, 1999. This work was supported by the Medical Research Council of Canada. Back

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

Received December 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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