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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 663-672
Copyright © 2003 by The Endocrine Society

Differential Regulation of Gonadotropin-Releasing Hormone (GnRH)I and GnRHII Messenger Ribonucleic Acid by Gonadal Steroids in Human Granulosa Luteal Cells

Shahram Khosravi and Peter C. K. Leung

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

Address all correspondence 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. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In humans, reproduction was generally believed to be controlled by only one form of GnRH (called mammalian GnRH or GnRHI). However, recently, a second form of GnRH, analogous to chicken GnRHII, was discovered in several tissues, including the human ovary. The regulation and function of GnRHI in the hypothalamus has been well studied. However, the function and regulation of GnRHI, and particularly GnRHII in the ovary, is less well understood. Because gonadal sex steroids are one of the main regulators of reproduction, we investigated, in the present study, the regulation of GnRHI and GnRHII mRNA expression by 17ß-estradiol (E2) and RU486 (a progesterone antagonist) in human granulosa luteal cells (hGLCs).

The levels of the mRNA transcripts encoding the two GnRH forms were examined using semiquantitative RT-PCR followed by Southern blot analysis. With time in culture, GnRHI and GnRHII mRNA levels significantly increased, by 120% and 210%, at d 8 and d 1, respectively. The levels remained elevated until the termination of these experiments at d 10. A 24-h treatment of hGLCs with E2 (10-9 to 10-7 M) resulted in a dose-dependent decrease and increase in mRNA expression of GnRHI and GnRHII, respectively. E2 (10-9 M) significantly decreased GnRHI mRNA levels (by 55%) and increased GnRHII mRNA levels (by 294%). Time-course studies demonstrated that E2 (10-9 M) significantly decreased GnRHI mRNA levels in a time-dependent manner, with maximal inhibition of 77% at 48 h. In contrast, GnRHII mRNA levels significantly increased in a time-dependent fashion, reaching a maximum level of 280% at 24 h. Cotreatment of hGLCs with E2 and tamoxifen (an E2 antagonist) reversed the inhibitory and stimulatory effects of E2 on the mRNA expression of GnRHI and GnRHII, respectively. Time- and dose-dependent treatment with RU486 did not affect GnRHI mRNA levels in hGLCs. In contrast, RU486 treatment significantly increased GnRHII mRNA levels in hGLCs in a time- and dose-dependent fashion, with a maximum increase being observed at 24 h (with 10-5M RU486). In summary, the present study demonstrated that the expression of GnRHI and GnRHII at the transcriptional level is differently regulated by E2 and P4 in hGLCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A KEY REGULATOR of reproduction in mammals, GnRH was originally isolated from the mammalian hypothalamus (1). In the brain, this decapeptide is secreted from the hypothalamus in a pulsatile manner and stimulates the synthesis and release of gonadotropins, FSH and LH. Gonadotropins, in turn, promote steroidogenesis and gametogenesis (2, 3). In more than 80 vertebrate species, there are at least 2 GnRH forms present (4). Besides the classical form (GnRHI or GnRH), humans recently were also known to have a second form of GnRH called chicken GnRH (cGnRHII or GnRHII) (5). GnRHII is the most ubiquitous variant of GnRH (5).

In addition to the hypothalamus, the presence of GnRHI and GnRHII and their receptor has also been detected in extrapituitary tissues, including the ovary (6, 7). Depending on the stage of the ovarian cycle, GnRH seems to have different biological functions. During the follicular phase, the highest expression of GnRH receptor was demonstrated in atretic rat follicles, and no GnRH receptor was detected in primordial follicles or oocytes (8). Because GnRH has been shown to induce apoptosis in cultured rat granulosa cells (GCs), GnRH might be involved in the process of follicular atresia through the induction of apoptosis (9). During the periovulatory period in the rat, GnRH may play a role in follicular rupture and oocyte maturation. This is probably done by the GnRH-induced increase in the transcription of certain genes, such as prostaglandin endoperoxidase synthase II (10), plasminogen activator (11), and progesterone (P4) receptor (PR) (12). GnRH is thought to be involved in luteolysis during the luteal phase. GnRH was demonstrated to stimulate the expression of matrix metalloproteinase 1 and 2 (MMP-1 and MMP-2) in the corpus luteum of rats. These enzymes are involved in degrading collagens and remodeling extracellular matrix, leading to luteolysis (13). In cultured human granulosa luteal cells (hGLCs), both GnRHI and GnRHII have been shown to decrease P4 secretion (7, 14).

Contradictory results have been reported regarding the regulatory effects of estrogen [17ß-estradiol (E2)] on GnRH secretion. During the rat estrous cycle, the levels of GnRH mRNA in the hypothalamus and E2 in the plasma were shown to be inversely related (15). Administration of E2, for 7 d, to ovariectomized rats increased the GnRH mRNA expression, but E2 administration for 2 d decreased GnRH mRNA levels (15). Park et al. (16) demonstrated that, in the rat, E2 increased GnRH gene expression and this increase resulted in gonadotropin surge before ovulation. The differences in GnRH regulation by E2 may be owing to methods used to measure GnRH secretion, dose, and duration of treatments.

The regulation of GnRHII expression by gonadal steroids is even less clear. Experiments done on juvenile and adult castrated tilapia indicated no effect of E2 and P4 on hypothalamic GnRHII mRNA expression (17, 18, 19, 20). Similarly, steroids in chicken had no significant effect on the expression of GnRHII (21). Taken together, these observations suggest that GnRHII hypothalamic neurons may not be targets for the feedback effects of steroid hormones. In contrast, in the European eel, androgens in combination with E2 could decrease the expression of GnRHII (22). Furthermore, studies in the musk shrew showed that GnRHII was modified by ovulation, suggesting that ovarian steroids might modulate the release of GnRHII (23). The controversy concerning GnRHII regulation by steroids may result from species difference or different experimental conditions. We have previously shown that E2 inhibited GnRHI mRNA level in hGLCs (24). However, the effect of gonadal steroids on GnRHII expression in the human ovary remains to be elucidated. The present study was designed to compare the regulation of GnRHI and GnRHII mRNA expression by E2 and P4 in hGLCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Granulosa luteal cell culture and treatments with E2, tamoxifen, and RU486 (a P4 antagonist)

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. Follicular aspirates were collected during oocyte retrieval from women undergoing in vitro fertilization at the University of British Columbia. The hGLCs were prepared according to methods described by Peng et al. (14), with some modifications. Briefly, the follicular aspirates were centrifuged at 1000 x g for 10 min, and the supernatant was removed. The cells were resuspended in 6 ml culture medium (M199; Life Technologies, Inc., Burlington, Ontario, Canada). Granulosa cells were then separated from red blood cells by centrifugation through an equal volume of Ficoll Paque (Pharmacia Biotech, Morgan, Canada) at 1000 x g for 20 min. Cells at the interface were collected and washed once with M199. After brief centrifugation, the cell pellet was resuspended in M199 supplemented with10% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin at a density of 1 x 105 cells/ml. The cells were plated at a density of approximately 2 x 105 cells per dish, in 35-mm culture dishes. The dishes were incubated at 37 C, under a water-saturated atmosphere of 5% CO2 in air.

After 4 d of culture, the cells were transferred into phenol-red-free M199 supplemented with 2% charcoal stripped fetal bovine serum for 24 h. The cells were then serum starved for 4 h and treated with the appropriate hormones (E2, tamoxifen, RU486) in a dose- and time-dependent fashion. To examine the effects of E2 or RU486, in a dose-dependent manner, on the expression of GnRHI and GnRHII mRNA in cultured hGLCs, 5-d-old cultured cells obtained from each patient were treated with different concentrations of E2 (0, 10-9, 10-8, and 10-7 M) or RU486 (0, 10-11, 10-9, 10-7, and 10-5 M) for 24 h. To examine the effects of E2 or RU486, in a time-dependent manner, on the expression of GnRHI and GnRHII mRNA in cultured hGLCs, 5-d-old cultured cells obtained from each patient were treated with 10-9 M E2 or 10-5 M RU486 for 0, 6, 12, 24, and 48 h. For each time point, a control was included to take into account the changes during time in culture. To determine whether the regulation of GnRHI and GnRHII by E2 is a receptor-mediated process, 5-d-old cultured hGLCs obtained from each patient were treated with 10-7 M E2 in combination with varying doses of tamoxifen (0, 10-8, and 10-7 M) for 24 h. To examine the regulation of GnRHI and GnRHII mRNA with time in culture, hGLCs were cultured for 1, 4, 8, and 10 d with no treatment. For all the experiments, 0.01% ethanol was used as the vehicle.

Total RNA extraction and first-strand cDNA synthesis

All the cells were subjected to RNA extraction. An Rnaid kit (Bio/Can Scientific, Mississauga, Canada) was used to extract total RNA from cultured hGLCs, according to the manufacturer’s protocol. Briefly, the cells were lysed and subjected to acid phenol extraction. RNA was purified from the aqueous phase, on an RNA matrix, and eluted into ribonuclease-free water. One microgram of total RNA was reverse transcribed into first-strand cDNA in a total vol of 15 µl, using the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Oakville, Ontario, Canada). RNA concentration was determined at 260 nm, using a spectrophotometer.

Semiquantitative PCR and Southern blot analysis

After completing total RNA extraction and first-0strand cDNA synthesis, the resultant products were subjected to PCR and Southern blot analysis. In each trial, some of the first-strand cDNA resulting from the cells obtained from each patient were subjected to PCR for GnRHI, and some for GnRHII. PCR amplifications were carried out in 50-µl reactions containing 2 µl cDNA, 2.5 U Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mM MgCl2, 2 mM deoxynucleotide triphosphate, and 50 pmol forward and reverse primers. Primers for GnRHI were designed based on the published sequence for human hypothalamic GnRH. The forward and reverse primers were 5'-ATTCTACTGACTTGGTGCGTG-3' and 5'-GGAATATGTGCAACTTGGTGT-3', respectively. Forward and reverse primers for GnRHII were 5'-GCCCACCTTGGACCCTCAGAG-3' and 5'-CCAATAAAGTGTGAGGTTCTCCG-3', respectively (24). PCR amplification for GnRHI 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. PCR for GnRHII was carried out with denaturing for 1 min at 94 C, annealing for 65 sec at 62 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C for 26 cycles (24). The PCR products were sequenced to assure authenticity. All probes used for Southern blot analysis were internal to the oligonucleotide primers used for PCR amplification, to avoid nonspecific binding. The sizes for GnRHII and GnRHI cDNAs were 327 and 380 bp, respectively. After electrophoresis, PCR products were transferred to a nylon membrane and fixed using UV irradiation. The blotted membranes were prehybridized for 3 h at 42 C in prehybridization solution containing 50% formamide, 5x SSC, 0.1% N-lauroyl sarcosine, 0.02% sodium dodecyl sulfate (SDS), and 2% blocking solution. The prehybridized membranes were hybridized overnight at 42 C with digoxigenin-labeled GnRHII or GnRHI cDNA probes. Membranes were then washed 2 times (10 min each time) in ample 2x SSC and 0.1% SDS at room temperature and 2 times (15 min each time) in 0.1x SSC and 0.1% SDS at 68 C, respectively. The hybridized membranes were detected with a luminescence method (Roche Molecular Biochemicals, Laval, Quebec, Canada) and exposed to Omat x-ray film (Eastman Kodak Co., Rochester, NY). The specific bands were scanned and quantified using a computerized visual light densitometer (model 620; Bio-Rad Laboratories, Inc., Richmond, CA). To standardize for the first-strand cDNA synthesis efficiency, PCR for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed for 18 cycles. Primers for GAPDH were designed according to the published sequence (25).

Statistical analysis

Each experiment was done with three different patient samples. The data were shown as mean ± SD and were represented as the percentage change, relative to the control. The raw data were analyzed by one-way ANOVA, followed by the Tukey’s multiple-comparison test, before transformation to percentage (PRISM Graphed version 2; Graphed Software, Inc., San Diego, CA). P value no greater than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validation of semiquantitative RT-PCR for GAPDH, GnRHI, and GnRHII

Semiquantitative RT-PCR was used to examine the mRNA levels of GnRHI and GnRHII in hGLCs. Southern blot analysis revealed a 372-bp product corresponding to GAPDH (Fig. 1AGo), a 380-bp product corresponding to GnRHI (Fig. 1BGo), and a 327-bp product (Fig. 1CGo) corresponding to GnRHII. A linear relationship was found between the cycle numbers, 12 to 21, 23 to 32, 23 to 35, and optical density for GAPDH (Fig. 1AGo), GnRHI (Fig. 1BGo), and GnRHII (Fig. 1CGo), respectively. As a result, 26 cycles for GnRHI and GnRHII and 18 cycles for GAPDH were chosen for quantification.



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  		Figure 1. Validation of semiquantitative RT-PCR for GAPDH, GnRHI, and GnRHII from hGLCs. Total RNA was extracted from the cells, and 1 µg of the RNA was subjected to RT. Subsequent to PCR amplification, 372-, 380-, and 327-bp fragments were obtained, by agarose gel electrophoresis, for GAPDH (A), GnRHI (B), and GnRHII (C), respectively. To determine the linear phase of PCR amplification, GAPDH, GnRHI, and GnRHII were amplified from hGLC cDNA under increasing cycle numbers. A linear relationship between the cycle number and optical density was observed between 12–21, 23–32, and 23–35 cycles for GAPDH, GnRHI, and GnRHII, respectively. Hence, 18 cycles for GAPDH and 26 cycles for GnRHI and GnRHII were chosen for quantitation purposes.

 
Changes in GnRHI and GnRHII mRNA expression with time in culture

For quantitative purposes, all time points were standardized to d 1 mRNA levels. There were 120% (P < 0.05) and 195% (P < 0.05) increases in GnRHI mRNA levels on d 8 and 10, compared with d 1 cultures, respectively (Fig. 2AGo). A similar trend was observed for GnRHII mRNA levels. There were 210% (P < 0.05) and 220% (P < 0.05) increases in GnRHII mRNA levels on d 8 and 10, compared with d 1 cultures, respectively (Fig. 2BGo). When d 4 cultures were compared with those of d 8 and 10, there were significant increases in GnRHI and GnRHII mRNA levels with increasing time in culture. However, there was no significant difference between either d 8 and d 10 cultures or d 4 and d 1 cultures for GnRHI and GnRHII mRNA levels.



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  		Figure 2. Changes in GnRHI (A) and GnRHII (B) mRNA expression with time in culture. Total RNA was extracted from hGLCs (n = 3) on d 1, 4, 8, and 10 in culture. One microgram of total RNA was reverse transcribed. GnRHI and GnRHII mRNA levels were estimated by semiquantitative RT-PCR and were normalized against the GAPDH mRNA. Data were expressed as percentage change, relative to control, and represent the mean ± SD of three different patients. a, P < 0.05, significantly different from the control d 1 cultures; b, P < 0.05, significantly different from the d 4 cultures.

 
Dose- and time-dependent effects of E2 on GnRHI and GnRHII mRNA levels in cultured hGLCs

In the dose-dependent studies, after 24 h, GnRHI mRNA levels from all treatment groups decreased significantly, compared with the control. However, there was no significant difference among different treatment groups (Fig. 3AGo). E2 (10-9 M) decreased GnRHI mRNA levels by 55% (P < 0.05), relative to the control. E2 (10-8 M and 10-7 M doses) resulted in decreases of 62% (P < 0.05) and 71% (P < 0.05) in GnRHI mRNA levels, compared with the control (Fig. 3AGo). In contrast, as a result of a 24-h dose-dependent treatment with E2, GnRHII mRNA levels of different treatment groups increased significantly, compared with the control (Fig. 3BGo). Increases of 294% (P < 0.05), 382% (P < 0.05), and 156% (P < 0.05) in GnRHII mRNA level were observed with 10-9 M, 10-8 M, and 10-7 M E2, respectively. The mRNA level of the 10-7 M treatment group was significantly lower than those of the other two treatment groups (Fig. 3BGo).



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  		Figure 3. The effects of varying concentrations of E2 on GnRHI (A) and GnRHII (B) mRNA levels in cultured hGLCs. On d 5 of culture, cells were treated, for 24 h, with different doses of E2 (0–10-7 M). GnRHI and GnRHII mRNA levels were estimated by semiquantitative RT-PCR and were normalized against GAPDH mRNA. Data were expressed as percentage change, relative to control, and represent the mean ± SD of three different experiments from three different patients. a, P < 0.05, significantly different from control; b, P < 0.05, significantly different from 10-9 M treatment group; c, P < 0.05, significantly different from 10-8 M treatment group.

 
In the time-dependent studies, 6-h and 12-h treatments with 10-9 M E2 had no significant effect on GnRHI mRNA levels. GnRHI mRNA levels decreased significantly, compared with the 0-h treatment group, only 24 and 48 h after treatment (Fig. 4AGo). Twenty-four-hour and 48-h treatments with 10-9 M E2 resulted in 48% (P < 0.05) and 77% (P < 0.05) decreases in GnRHI mRNA level, compared with the 0-h treatment group (Fig. 4AGo). The GnRHI mRNA level of the 48-h treatment group was not significantly different, compared with the 24-h treatment group (Fig. 4AGo). In contrast, the GnRHII mRNA level increased significantly for every treatment group, compared with the 0-h treatment group (Fig. 4BGo). Treatment with 10-9 M of E2 for 6 h, 12 h, 24 h, and 48 h resulted in 105% (P < 0.05), 204% (P < 0.05), 280% (P < 0.05), and 150% (P < 0.05) increases, respectively, in GnRHII mRNA level, compared with the 0-h treatment group (Fig. 4BGo). In addition, the GnRHII mRNA levels of the 12-h and 24-h treatment groups were significantly higher, compared with the 6-h treatment group. The GnRHII mRNA level of the 48-h treatment group was significantly lower, compared with the 24-h treatment group, but still significantly higher than the 0-h treatment group (Fig. 4BGo).



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  		Figure 4. Time-dependent effects of E2 on GnRHI (A) and GnRHII (B) mRNA levels in cultured hGLCs. On d 5 of culture, cells were treated with 10-9 M E2 for 0, 6, 12, 24, and 48 h. GnRHI and GnRHII mRNA levels were estimated by semiquantitative RT-PCR and were normalized against GAPDH mRNA. Data were expressed as percentage change, relative to control, and represent the mean ± SD of three different experiments from three different patients. a, P < 0.05, significantly different from 0 h treatment group; b, P < 0.05, significantly different from 6 h treatment group; c, P < 0.05, significantly different from 12-h treatment group.

 
Dose- and time-dependent effects of RU486 on GnRHI and GnRHII mRNA levels in cultured hGLCs

In the dose-dependent studies, after 24 h, there was no significant difference between the vehicle-treated and RU486-treated groups, in terms of GnRHI mRNA levels (Fig. 5AGo). In contrast, GnRHII mRNA level showed a dose-dependent increase with an increasing concentration of RU486 (Fig. 5BGo). Increases of 75% (P < 0.05), 220% (P < 0.05), 189% (P < 0.05), and 330% (P < 0.05) in GnRHII mRNA level were observed at doses of 10-11 M, 10-9 M, 10-7 M, and 10-5 M of RU486, respectively (Fig. 5BGo). The 10-9 M and 10-7 M treatment groups had a significantly higher GnRHII mRNA level, compared with the 10-11 M treatment group as well, compared with the control group. The level of GnRHII mRNA in the 10-5 M treatment group was also significantly higher than in all other treatment groups (Fig. 5BGo).



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  		Figure 5. The effects of varying concentrations of RU486 on GnRHI (A) and GnRHII (B) mRNA levels in cultured hGLCs. On d 5 of culture, cells were treated for 24 h with different doses of RU486 (0–10-5 M). GnRHI and GnRHII mRNA levels were estimated by semiquantitative RT-PCR and were normalized against GAPDH mRNA. Data were expressed as percentage change, relative to control, and represent the mean ± SD of three different experiments from three different patients.

 
In the time course studies, there was no significant difference between different RU486-treated groups and the 0-h treatment group in terms of GnRHI mRNA level (Fig. 6AGo). However, all RU486-treated groups had a significantly higher level of GnRHII mRNA, compared with the 0-h treatment group (Fig. 6BGo). Increase of 243% (P < 0.05), 191% (P < 0.05), 362% (P < 0.05), and 414% (P < 0.05), respectively, were observed in GnRHII mRNA level after 6, 12, 24, and 48 h of treatment with 10-5 M RU486 (Fig. 6BGo). The 24- and 48-h treatment groups had a significantly higher level of GnRHII mRNA, compared with the 6- and 12-h treatment groups as well, compared with the 0-h treatment group (Fig. 6BGo).



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  		Figure 6. Time-dependent effects of RU486 on GnRHI (A) and GnRHII (B) mRNA levels in cultured hGLCs. On d 5 of culture, cells were treated with 10-5 M RU486 for 0, 6, 12, 24, and 48 h. GnRHI and GnRHII mRNA levels were estimated by semiquantitative RT-PCR and were normalized against GAPDH mRNA. Data were expressed as percentage change, relative to control, and represent the mean ± SD of three different experiments from three different patients.

 
Effects of cotreatment with E2 and tamoxifen on GnRHI and GnRHII mRNA levels in cultured hGLCs

Treatment with 10-7 M E2 significantly decreased GnRHI mRNA level, compared with the no-treatment group (Fig. 7AGo). When the cells were cotreated with 10-8 M tamoxifen and 10-7 M E2, the mean GnRHI mRNA level increased, but no significant change was observed when compared with the E2-only treatment group. However, equimolar treatment with E2 and tamoxifen reversed the down-regulating effect of E2 (Fig. 7AGo). Therefore, no significant difference was observed between the equimolar treatment group and the no-treatment group, but there was a significant difference between the E2-only treatment group and the equimolar treatment group (Fig. 7AGo). Treatment with 10-7 M E2 significantly increased GnRHII mRNA level, compared with the no-treatment group (Fig. 7BGo). When the cells were cotreated with 10-8 M tamoxifen and 10-7 M E2, there was no significant difference between this treatment group and the E2-only treatment group; however, the mean mRNA level of GnRHII was lower, compared with the E2-only treatment group. The GnRHII mRNA level of this cotreatment group was significantly higher, compared with the no-treatment group (Fig. 7BGo). The GnRHII mRNA level of the equimolar cotreatment group (10-7 M tamoxifen plus 10-7 M E2) was not significantly different from the no-treatment group. However, the mRNA level of the equimolar cotreatment group was significantly lower than those of the other cotreatment group and the E2-only treatment group (Fig. 7BGo). Tamoxifen alone had no significant effect on GnRHI and GnRHII mRNA levels (data not shown).



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  		Figure 7. The effect of E2 and tamoxifen (Txf) cotreatment on GnRHI (A) and GnRHII (B) mRNA levels in cultured hGLCs. On d 5, cells were treated with 10-7 M E2, in combination with varying doses of Txf (0–10-7 M), for 24 h. GnRHI and GnRHII mRNA levels were estimated by semiquantitative RT-PCR and were normalized against GAPDH mRNA. Data were expressed as percentage change, relative to control, and represent the mean ± SD of three different experiments from three different patients. a, P < 0.05, significantly different from the control group; b, P < 0.05, significantly different from the E2 treatment group; c, P < 0.05, significantly different from the 10-7/10-8 M treatment group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that GnRHI and GnRHII were differentially regulated by E2 and P4 in the human ovary. Differential regulation of two forms of GnRH has been observed before. In hGLCs, FSH and LH were shown to down-regulate GnRHI mRNA and up-regulate GnRHII mRNA (7). In the brain of female silver eel, E2 up-regulated GnRHI, and testosterone down-regulated GnRHII (22). In the goldfish, the ratio between salmon GnRH and GnRHII changed with sexual maturation. A smaller increase in the level of GnRHII than in salmon GnRH was observed in the pituitary (26). In the chicken, castration led to a change in the level of only cGnRHI but not GnRHII (27). Taken together, the differential regulation of two forms of GnRH, by E2 and P4, in the present study, suggests that GnRHI and GnRHII may be temporally regulated by E2 and P4 during different phases of the menstrual cycle.

Hypothalamic GnRH mRNA levels have been demonstrated to increase steadily during postnatal development and puberty. This increase in GnRH mRNA levels is thought to be important for regulating the onset of puberty, given that premature administration of GnRH causes precocious puberty in immature animals (28). In the present study, the increase in the mRNA levels of the two GnRH forms in hGLCs with time in culture suggests that these hormones may have an autocrine/paracrine role, such as regulating luteolysis during the luteal phase of the menstrual cycle. Although the mechanism of this increase in hGLCs is not known, differential mechanisms in the mouse, such as gene transcription and mRNA stability, are thought to play a role in determining GnRH mRNA levels during different stages of development (29).

There are a number of studies available regarding the regulation of GnRH gene expression by E2. It was shown that, in the GT1–7 GnRH neurons, 1 nM E2 down-regulated GnRH mRNA levels to approximately 55% of basal level, over a 48-h time course. This effect seemed to happen via an estrogen receptor (ER)-mediated mechanism, because ICI 182,780, a complete ER antagonist, blocked the repression of GnRH mRNA levels by E2. The same study indicated that GT1–7 cells expressed both ER{alpha} and ERß (30). According to Kang et al. (31), treatment with E2 induced a significant down-regulation of GnRH mRNA in the ovarian cancer cell lines but not in normal human ovarian surface epithelial cells. The same study showed that tamoxifen (an E2 antagonist) reversed the effect of E2, suggesting that E2 action was mediated via ERs. Wierman et al. (32) showed that E2 could negatively regulate the rat GnRH promoter activity in placental tumor cells. Similarly, Dong et al. (33) demonstrated that E2 negatively regulated the GnRH promoter activity in a dose-dependent manner in human placental cells. These results, particularly the ones from experiments on extrapituitary tissues, support our findings demonstrating the down-regulation of GnRHI mRNA by E2 in hGLCs. There is only one study available regarding the regulatory effects of E2 on GnRH in hGLCs in vitro. In that study, Nathawani et al. (24) demonstrated that E2 decreased GnRHI mRNA levels in a time- and dose-dependent manner, supporting the results obtained in our experiments. Quite recently, differential regulation of GnRHI and GnRHII by E2 was demonstrated in human neuronal cell lines (34). That study demonstrated that E2 increased endogenous GnRHII mRNA levels and decreased endogenous GnRHI mRNA levels. These findings agree with the results obtained in the present study, suggesting that regulation of GnRHI and GnRHII by E2 in humans may be similar in the brain and in the ovary.

In the present study, to investigate the function and regulation of GnRHI and GnRHII in the human ovary, primary cultures of hGLCs were used as the model system. The method used to culture the granulosa cells has been highly optimized to separate granulosa cells from blood cells and theca cells. Previous studies have shown that hGLCs in culture express the components of the GnRH system and are a good model for studying the regulation and the autocrine/paracrine function of GnRH in the ovary (14, 35). Because antibodies against GnRHI and GnRHII are not readily available, all the measurements in the present study were done only at the mRNA level.

The biological effects of E2 are mediated through several different pathways (36). Through the classical mechanism of E2 action, E2 diffuses through the plasma membrane and forms complexes with specific cytosolic or nuclear receptors. The E2-ER complexes then bind to estrogen response elements (EREs) in target promoters, leading to an up- or down-regulation of gene transcription and subsequent tissue responses (37). The effects of E2 can also be mediated through ERE-independent genomic actions of ER. Through this mechanism, agonist-bound ER can lead to gene regulation in the absence of direct DNA binding. E2-ER complexes alter transcription of genes containing alternative response elements (such as AP-1) through association with other DNA-bound transcription factors (such as Fos and Jun), which tether the activated ER to DNA, resulting in an up-regulation of gene expression (38, 39, 40). Another potentially important pathway of E2 action is constituted by the very rapid, so-called, nongenomic effects of E2. E2 activates a putative membrane-associated binding site, linked to intracellular signal transduction pathways that generate rapid tissue responses (41, 42, 43). In the present study, tamoxifen inhibited the effects of E2 on the regulation of GnRHI and GnRHII in hGLCs. This suggests that the E2-induced regulation of GnRHI and GnRHII in hGLCs is a receptor-mediated event, indicating genomic effects of E2. This idea is further supported by the presence of both types of nuclear ERs in hGLCs (44, 45). Similarly, studies on mice and rats indicate that also the hypothalamic GnRH-producing neurons possess nuclear ERs (46, 47, 48). Furthermore, the discovery of one ERE on the human GnRHI gene in the hypothalamus (49) suggests that, as in the ovary, this gene in the brain may also be regulated via genomic action of E2.

The reason why E2 differentially regulates the genes for GnRHI and GnRHII is not clear yet. It is possible that one type of ER is primarily responsible for the gene regulation of GnRHI; and another type of ER, for GnRHII; and vice versa. Further investigation in this regard is necessary to differentiate between the two ERs, using selective agonist/antagonists available. In human neuronal cell lines, the human GnRH (hGnRH)II promoter has been activated by E2, but the activity of hGnRHI promoter has been inhibited by E2 (34). In these cells, analysis of hGnRHII promoter has revealed only a partial putative ERE. However, hGnRHII promoter has been shown to possess an exact SP1 site and a cAMP response element site with one mismatch (34). These elements have been known to be able to interact with ER (50, 51). Consequently, E2 may mediate part of its stimulatory effect on this promoter via one or both of these sites. However, to our knowledge, hGnRHI promoter has not been indicated to possess these sites, which might explain the differential regulation of GnRHI and GnRHII by E2. In the human neuronal cells, an area with high homology to the known EREs was found in the 5' flanking GnRHI DNA (49). In this area, the DNA sequence between positions -441 and -428 is similar to EREs of the rat LHß (52) and rat prolactin (53). In human GCs, the ER-mediated suppression of GnRHI promoter activity by E2 has been localized to a region between -169 and -548 bp 5' of the upstream transcription start site of the human GnRH gene (54). To our knowledge, there is yet no information available regarding the promoter region of GnRHII in the ovary.

GnRH regulation by P4 has also been studied at the hypothalamic-pituitary level. Studies done on the rat showed that P4 decreased the expression of hypothalamic GnRH mRNA levels (55). In the ewe, at the hypothalamic-pituitary level, P4 removal accelerated the GnRH pulse frequency, and P4 administration slowed down the GnRH pulse frequency. These effects were shown to be mediated by P4 itself and not by its hydroxylated metabolites. The same experiments showed that P4 exerted its effects by interacting with PRs (56). The present study examined the role of P4 in regulating ovarian GnRHI and GnRHII. The expression of PR by hGLCs has already been shown by immunohistochemical localization (57). Preliminary experiments from our laboratory, measuring changes in GnRH mRNA levels as a result of treatment with P4, produced inconsistent results (data not shown). Because hGLCs have all the enzymes for de novo synthesis of P4, the variable results may have been a consequence of variations in endogenous production of P4 from different patients. Consequently, we used RU486 to indirectly examine the effects of P4 on the expression of GnRHI and GnRHII mRNA in hGLCs. Our findings indicated that RU486 had no significant effects on the mRNA expression of GnRHI. In contrast, there was an increase in GnRHII mRNA levels in response to RU486, suggesting that endogenous P4 decreased GnRHII mRNA in cultured hGLCs. The exact regulatory mechanism of the P4-induced regulation of GnRHII in hGLCs is not known. However, in the GT1–7 neuronal cells transfected with PR, P4 was shown to inhibit GnRHI transcription via direct PR binding on nonconsensus DNA sequences in the proximal GnRH promoter (58). The differences between the P4-induced regulation of GnRHI in hGLCs and in rat GT1–7 cells may be attributed to tissue-specific regulatory mechanisms or species differences. It is important to note that RU486 is not only a P4 antagonist but a glucocorticoid antagonist as well (59). There are different reports regarding the effect of glucocorticoids on the expression of GnRHI in the animals’ brains. In the ovine, no colocalization between GnRHI and glucocorticoid receptors was observed at any level of the brain (60). In the rat, glucocorticoids inhibit GnRHI by acting directly at the hypothalamic level (61). In dairy cows, RU486 is unable to reverse stress-induced delays in the LH surge (59). However, so far, there are no published data regarding the effect of glucocorticoids on GnRHI and GnRHII in hGLCs.

GnRHII is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions (62, 63), such as regulating K+ channels, in bullfrog sympathetic ganglion (64). GnRHI has been shown to have direct effects on reproductive behavior and sexual arousal in rodents, independent of its stimulation of sex hormone production (63, 65). Quick changes in GnRHI content of brain areas and cell size and number, in response to different stimulants of sexual behavior (such as olfactory and visual stimulants) have been observed in reptiles, fish, amphibians, and mammals (62, 63, 65). Moreover, GnRHII is much more effective than mammalian GnRHI in stimulating sexual behavior in ring doves (62) and sparrows (66). There is great concurrence of the GnRHII receptor in the amygdala, medial preoptic area, putamen, temporal lobe, dorsomedial nucleus, ventromedial nucleus, periventricular nucleus of the monkey, and human brain, with effects of stimulation of these areas on reproductive behaviors, such as sexual interest, erection, intromission, thrusting, and ejaculation in rats, dogs, cats, monkeys, and humans (65, 67). Another possible function of GnRHII is specific stimulation of FSH release. GnRHI and GnRHII, in general, stimulate the release of both FSH and LH (68, 69). However, recently, some specific FSH-releasing factors, such as cGnRHII and lamprey GnRHIII, have been reported (70, 71). In placenta, GnRH stimulates the synthesis and secretion of human chorionic gonadotropin (hCG) (72, 73, 74). However, the physiological role of GnRHI and GnRHII in hGLCs remains to be elucidated.

There are a number of reports that demonstrate the ability of GnRHI and GnRHII to induce apoptosis in various reproductive tissues. For example, in goldfish, GnRHII (10-7 M) induced apoptosis in follicle-enclosed oocytes (75). GnRH has also been indicated to induce apoptosis in eutopic endometrial cells from patients with endometriosis (76). In hypophysectomized E2-treated rats, treatment with a GnRH agonist induced ovarian apoptotic DNA fragmentation with or without cotreatment with FSH. In situ hybridization indicated that apoptotic cell death was confined to the GCs (9). The same group indicated that GnRH agonist treatment increased DNA fragmentation in isolated GCs as well (9). Induction of apoptosis in rat and porcine cultured GCs, as a result of GnRH treatment, was similarly shown by Yano et al. (77) and Zhao et al. (78). GnRH was also shown to induce apoptosis in hGLCs in vitro (78). It is known that, in a number of cell types (including hGLCs), Fas (a cell surface receptor protein) triggers apoptosis when cross-linked with its ligand (called Fas ligand) (79, 80). Additionally, GnRH was shown to induce apoptosis in GnRH receptor-bearing tumors, including ovarian carcinoma cells, by increasing Fas ligand expression (81, 82). Thus, it can be envisaged that GnRH may promote apoptosis in hGLCs, which, in turn, contributes to luteolysis. GnRH receptor has been demonstrated to be expressed in hGLCs (14). The present study showed an increase in GnRHI and GnRHII mRNA levels with time in cultured hGLCs. Additionally, GnRHI and GnRHII have been demonstrated to be capable of inducing apoptosis in the ovary (78, 75). Previous reports have implicated GnRH as a luteolytic factor. For example, hCG decreased the expression of GnRH receptor mRNA in the human and rat ovary (14, 83). During the early stages of pregnancy, hCG is an important rescue factor for the maintenance of the corpus luteum. Thus, the down-regulation of GnRH receptor by hCG might be involved in the maintenance of the corpus luteum during pregnancy. Furthermore, GnRHI and PGF2{alpha} have been suggested to have a very similar or complementary role in the ovary. Indeed, GnRHI was shown to enhance the luteolytic effects of PGF2{alpha} in hGLCs (84). Taken together, these observations suggest that the increase of GnRHI and GnRHII during the luteal phase of menstrual cycle and, subsequently, the induction of apoptosis in hGLCs may be one of the contributing factors to the degradation of the corpus luteum during the process of luteolysis.

In summary, we demonstrated an increase in GnRHI and GnRHII mRNA levels with time in cultured hGLCs, suggesting that the two GnRH forms may play a role in controlling granulosa luteal function. We also demonstrated, for the first time, that gonadal sex steroids differentially regulate GnRHI and GnRHII in the human ovary. Because GnRHI and GnRHII have been shown to be capable of inducing apoptosis in the ovary and these GnRH forms are dynamically regulated by E2 and P4, the balance between E2 and P4, and consequently the up- and down-regulation of GnRHI and GnRHII, may play a role in regulating the fate of the corpus luteum. Further study is necessary to elucidate the mechanism of the differential regulation of GnRHI and GnRHII by sex steroids.


   Acknowledgments
 
We thank Dr. Margo Fluker and the Genesis Fertility Center (Vancouver, Canada) for providing human granulosa luteal cells. In addition, we are grateful to Dr. Kyung-Chul Choi for insightful comments and discussion for the studies.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research. P.C.K.L. is a distinguished scholar of the Michael Smith Foundation of Health Research.

Abbreviations: cGnRH, Chicken GnRH; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GC, granulosa cell; hCG, human chorionic gonadotropin; hGLC, human granulosa luteal cell; hGnRH, human GnRH; MMP, matrix metalloproteinase; P4, progesterone; PR, progesterone receptor; SDS, sodium dodecyl sulfate.

Received June 4, 2002.

Accepted October 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Matsuo H, Baba Y, Nair RMG, Arimura A, Schally AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334–1339[CrossRef][Medline]
  2. Conn PM 1994 The molecular mechanism of gonadotropin-releasing hormone action in the pituitary. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press Ltd.; 1815–1826
  3. Noar Z, Shacham S, Harris D, Seger R, Reiss N 1995 Signal transduction of the gonadotropin releasing hormone receptor: Cross-talk of calcium, protein kinase C and arachidonic acid. Cell Mol Neurobiol 15:527–544[CrossRef][Medline]
  4. Von Schalburg KR, Warby CM, Sherwood NM 1999 Evidence for GnRH peptides in the ovary and testis of rainbow trout. Biol Reprod 60:1338–1344[Abstract/Free Full Text]
  5. White RB, Eisen JA, Kasten TL, Fernald RD 1998 Second gene for GnRH in humans. Proc Natl Acad Sci USA 95:305–309[Abstract/Free Full Text]
  6. Dong KW, Yu KL, Roberts JL 1993 Identification of a major up-stream transcription start site for the human progonadotropin-releasing hormone gene used in reproductive tissues and cell lines. Mol Endocrinol 7:1654–1666[Abstract/Free Full Text]
  7. Kang SK, Tai CJ, Nathwani PS, Leung PCK 2001 Differential hormonal regulation of two forms of GnRH mRNA in cultured human granulosa luteal cells. Endocrinology 142:182–192[Abstract/Free Full Text]
  8. Whitelaw PF, Edine KA, Seller R, Smyth CD, Hillier SG 1995 GnRH receptor mRNA expression in rat ovary. Endocrinology 136:172–179[Abstract]
  9. Billig H, Furuta I, Hsueh AJW 1994 Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology 134:245–252[Abstract/Free Full Text]
  10. Wong WY, Richards JS 1992 Induction of prostaglandin H synthase in rat preovulatory follicles by GnRH. Endocrinology 130:3512–3521[Abstract/Free Full Text]
  11. Ny T, Liu YX, Ohlsson M, Jones PB, Hsueh AJW 1987 Regulation of tissue-type plasminogen activator activity and mRNA levels by GnRH gene in cultured rat granulosa cells and cumulus-oocyte complexes. J Biol Chem 262:11790–11793[Abstract/Free Full Text]
  12. Natraj U, Richards JS 1993 Hormonal regulation, localization and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology 133:761–769[Abstract/Free Full Text]
  13. Goto T, Endo T, Henmi H, Kitajima Y, Nishikawa A, Manase K, Sato H, Kudo R 1999 Gonadotropin-releasing hormone agonist has the ability to induce increased matrix metalloproteinase (MMP)-2 and membrane type 1-MMP expression in corpora lutea, and structural luteolysis in rats. J Endocrinol 161:393–402[Abstract]
  14. Peng C, Fan NC, Ligier M, Vaananen J, Leung PCK 1994 Expression and regulation of GnRH and GnRH receptor mRNA in human granulosa luteal cells. Endocrinology 135:1740–1746[Abstract]
  15. Zoeller RT, Young WS 1988 Changes in cellular levels of messenger ribonucleic acid encoding gonadotropin-releasing hormone in the anterior hypothalamus of female rats during the estrus cycle. Endocrinology 123:1688–1689[Abstract/Free Full Text]
  16. Park Ok, Gugneja S, Mayo KE 1990 GnRH gene expression during the rat estrus cycle: effect of pentobarbital and ovarian steroids. Endocrinology 127:365–372[Abstract/Free Full Text]
  17. Parhar IS, Soga T, Sakuma Y 2000 Thyroid hormone and estrogen regulate brain region specific mRNA encoding three GnRH genes in sexually immature male fish, oreochromis niloticus. Endocrinology 141:1618–1626[Abstract/Free Full Text]
  18. Soga T, Sakuma Y, Parhar IS 1998 Testosterone differentially regulates expression of GnRH mRNAs in the terminal nerves, preoptic and midbrain of male tilapia. Brain Res Mol Brain Res 60:13–20[Medline]
  19. Parhar IS, Soga T, Sakuma Y 1998 Quantitative in situ hybridization of three GnRH hormone-encoding mRNAs in castrated and progesterone-treated tilapia. Gen Comp Endocrinol 112:406–414[CrossRef][Medline]
  20. Parhar IS, Soga T, Sakuma Y 1996 In situ hybridization for two differentially expressed GnRH genes following estrogen and triiodothyronine treatment in the brains of juvenile tilapia. Neurosci Lett 218:135–138[CrossRef][Medline]
  21. Wilson SC, Gladwell RT, Cunningham FJ 1990 Differential responses of hypothalamic LHRH-I and LHRH-II to castration and gonadal steroid or tamoxifen treatment in cockerels. J Endocrinol 125:139–146[Abstract/Free Full Text]
  22. Montero M, Le Belle N, King JA, Millar RP, Dufour S 1995 Differential regulation of the two forms of gonadotropin-releasing hormone (mGnRHI and cGnRHII) by sex steroids in the European female silver eel. Neuroendocrinology 61:525–535[Medline]
  23. Rissman EF, Li X 1998 Sex differences in mammalian and chicken-II GnRH immunoreactivity in musk shrew brain. Gen Comp Endocrinol 112:346–355[CrossRef][Medline]
  24. Nathwani PS, Kang SK, Cheng KW, Choi KC, Leung PCK 2000 Regulation of gonadotropin releasing hormone (GnRH) and its receptor gene expression by 17ß-estradiol in cultured hGLCs. Endocrinology 141:1754–1763[Abstract/Free Full Text]
  25. Tokunaga K, Nakamura Y, Sakata K, Fujimori K, Ohkubo M, Sawada K, Skiyama S 1987 Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers. Cancer Res 47:5616–5619[Abstract/Free Full Text]
  26. Rosenblum PM, Goos HJT, Peter RE 1994 Regional distribution and in vitro secretion of salmon and chicken II gonadotropin-releasing hormones from the brain and pituitary of juvenile and adult goldfish. Gen Comp Endocrinol 93:369–379[CrossRef][Medline]
  27. Sharp PJ, Talbot RT, Main GM, Dunn IC, Fraser HM, Huskisson NS 1990 Physiological roles of chicken LHRHI and LHRHII in the control of gonadotropin release in the domestic chicken. J Endocrinol 125:291–299
  28. Wildt L, Marshall G, Knobil E 1980 Experimental induction of puberty in the infantile rhesus monkey. Science 207:1373–1375
  29. Gore AC, Roberts JL, Gibson MJ 1999 Mechanisms for the regulation of GnRH gene expression in the developing mouse. Endocrinology 140:2280–2287[Abstract/Free Full Text]
  30. Roy D, Angelini NL, Belsham DD 1999 Estrogen directly represses GnRH gene expression in estrogen receptor-{alpha} (ER{alpha})-and ERß-expressing GT1–7 GnRH neurons. Endocrinology 140:5045–5053[Abstract/Free Full Text]
  31. Kang SK, Choi KC, Tai CJ, Auersperg N, Leung PCK 2001 Estradiol regulates gonadotropin-releasing hormone (GnRH) and its receptor gene expression and antagonizes the growth inhibitory effects of GnRH in human ovarian surface epithelial and ovarian cancer cells. Endocrinology 142:580–588[Abstract/Free Full Text]
  32. Wierman ME, Kepa JK, Sun W, Gordon DF, Wood WM 1992 Estrogen negatively regulates rat gonadotropin releasing hormone (rGnRH) promoter activity in transfected placental cells. Mol Cell Endocrinol 86:1–10[CrossRef][Medline]
  33. Dong KW, Chen ZG, Cheng KW, Yu KL 1996 Evidence for estrogen receptor-mediated regulation of human GnRH promoter in human placental cells. Mol Cell Endocrinol 117:241–246[CrossRef][Medline]
  34. Chen A, Zi K, Laskar-Levy O, Koch Y 2002 The transcription of the hGnRHI and hGnRHII genes in human neuronal cells is differentially regulated by estrogen. J Mol Neurosci 18:67–76[Medline]
  35. Tureck RW, Mastroianni LJ, Blasco L, Strauss JF 1982 Inhibition of human granulosa cell progesterone secretion by GnRH agonist. J Clin Endocrinol Metab 54:1078–1080[Abstract/Free Full Text]
  36. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA 2001 Mechanisms of estrogen action. Physiol Rev 81:1535–1565[Abstract/Free Full Text]
  37. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Steroid receptor family: structure and functions. Endocr Rev 11:201–220[Abstract/Free Full Text]
  38. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 277:1508–1510[Abstract/Free Full Text]
  39. Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/AP1 pathway: potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9:443–456[Abstract/Free Full Text]
  40. Webb P, Nguyen P, Valentine C, Kushner PJ 1999 The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. Mol Endocrinol 13:1672–1685[Abstract/Free Full Text]
  41. Monje P, Boland R 1997 Characterization of membrane estrogen binding proteins from rabbit uterus. Mol Cell Endocrinol 147:75–84
  42. Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen receptors identified by multiple antibody labeling and impede-ligand binding. FASEB J 9:404–410[Abstract/Free Full Text]
  43. Watson CS, Campbell C, Gametchu B 1999 Membrane oestrogen receptors on rat pituitary tumor cells: immuno-identification and response to oestradiol and xenoestrogens. Exp Physiol 84:1013–1022[Abstract]
  44. Misao R, Nakanishi Y, Sun WS, Fujimoto J, Iwagaki S, Hirose R, Tamaya T 1999 Expression of estrogen receptors mRNA in corpus luteum of human subjects. Mol Hum Reprod 5:17–21[Abstract/Free Full Text]
  45. Chiang CH, Cheng KW, Igarashi S, Nathwani PS, Leung PCK 2000 Hormonal regulation of estrogen receptor alpha and beta gene expression in human granulosa-luteal cells in vitro. J Clin Endocrinol Metab 85:3828–3839[Abstract/Free Full Text]
  46. Skynner MJ, Sim JA, Herbison AE 1999 Detection of estrogen receptor alpha and beta messenger ribonucleic acids in adult gonadotropin-releasing hormone neurons. Endocrinology 140:5195–5201[Abstract/Free Full Text]
  47. Butler JA, Sjoberg M, Coen CW 1999 Evidence for oestrogen receptor alpha-immunoreactivity in gonadotrophin-releasing hormone-expressing neurones. J Neuroendocrinol 11:331–335[CrossRef][Medline]
  48. Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszan T, Carpenter CD, Liposits Z, Petersen SL 2000 Detection of estrogen receptor-beta messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology 141:3506–3509[Abstract/Free Full Text]
  49. Radovic S, Ticknor CM, Nakayama Y, Notides AC, Rahman A, Weintraub BD, Cutler Jr GB, Wondisford FE 1991 Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene. J Clin Invest 88:1649–1655
  50. Sabbah M, Courilleau D, Mester J, Redeuilh G 1999 Estrogen induction of the cyclin D1 promoter: involvement of a cAMP response-like element. Proc Natl Acad Sci USA 96:11217–11222[Abstract/Free Full Text]
  51. Xie W, Duan R, Safe S 1999 Estrogen induces adenosine deaminase gene expression in MCF-7 human breast cancer cells: role of estrogen receptor-Sp1 interactions. Endocrinology 140:219–227[Abstract/Free Full Text]
  52. Shupnik MA, Weinmann CM, Notides AC, Chin WW 1989 An upstream region of the rat luteinizing hormone beta gene binds estrogen receptor and confers estrogen responsiveness. J Biol Chem 264:80–86[Abstract/Free Full Text]
  53. Maurer RA, Notides AC 1987 Identification of an estrogen-responsive element from the 5'-flanking region of the rat prolactin gene. Mol Cell Biol 7:4247–4254[Abstract/Free Full Text]
  54. Chen ZG, Yu KL, Zheng HM, Dong KW 1999 Estrogen receptor-mediated repression of gonadotropin-releasing hormone (gnRH) promoter activity in transfected CHO-K1 cells. Mol Cell Endocrinol 158:131–142[CrossRef][Medline]
  55. Toranzo D, Dupont E, Simard J, Labrie C, Couet J, Labrie F, Pelletier G 1989 Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats. Mol Endocrinol 3:1748–1756[Abstract/Free Full Text]
  56. Chabbert-Buffet N, Skinner DC, Caraty A, Bouchard P 2000 Neuroendocrine effects of progesterone. Steroids 65:613–620[CrossRef][Medline]
  57. 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]
  58. Kepa JK, Jacobsen BM, Boen EA, Prendergast P, Edwards DP, Takimoto G, Wierman ME 1996 Direct binding of progesterone receptor to nonconsensus DNA sequences represses rat GnRH. Mol Cell Endocrinol 117:27–39[CrossRef][Medline]
  59. Dobson H, Smith RF 2000 What is stress, and how does it affect reproduction? Anim Reprod Sci 60–61:743–752
  60. Dufourny L, Skinner DC 2002 Type II glucocorticoid receptors in the ovine hypothalamus: distribution, influence of estrogen and absence of co-localization with GnRH. Brain Res 946:79–86[CrossRef][Medline]
  61. Calogero AE, Burrello N, Bosboom AM, Garofalo MR, Weber RF, D’Agata R 1999 Glucocorticoids inhibit gonadotropin-releasing hormone by acting directly at the hypothalamic level. J Endocrinol Invest 22:666–670[Medline]
  62. King JA, Millar RP 1997 GnRH neurones. In: Parhar I, Sakuma Y, eds. Gene to behaviour. Tokyo: Brain Shuppan; 51–77
  63. Sherwood NM, Lovejoy DA, Coe IR 1993 Origin of mammalian gonadotropin-releasing hormone. Endocr Rev 14:241–254[Abstract/Free Full Text]
  64. Bosma MM, Bernheim L, Leibowitz MD, Pfaffinger PJ, Hille B 1990 Modulation of M current in frog sympathetic ganglion cells. Soc Gen Physiol Ser 45:43–59[Medline]
  65. Slimp JC, Hart BL, Goy RW 1978 Heterosexual, autosexual and social behavior of adult male rhesus monkeys with medial preoptic-anterior hypothalamic lesions. Brain Res 142:105–122[CrossRef][Medline]
  66. Maney DL, Richardson RD, Wingfield JC 1997 Central administration of chicken gonadotropin-releasing hormone-II enhances courtship behavior in a female sparrow. Horm Behav 32:11–18[CrossRef][Medline]
  67. Smalley K, Eisen T 2000 The involvement of p38 mitogen-activated protein kinase in the alpha-melanocyte. Stimulating hormone (alpha-MSH)-induced melanogenic and anti-proliferative effects in B16 murine melanoma cells. FEBS Lett 476:198–202[CrossRef][Medline]
  68. Lumpkin MD, Moltz JH, Yu WH, Samson WK, McCann SM 1987 Purification of FSH-releasing factor: its dissimilarity from LHRH of mammalian, avian, and piscian origin. Brain Res Bull 18:175–178[CrossRef][Medline]
  69. Padmanabhan V, McNeilly AS 2001 Is there an FSH-releasing factor? Reproduction 121:21–30[Abstract]
  70. Yu WH, Karanth S, Walczewska A, Sower SA, McCann SM 1997 A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat. Proc Natl Acad Sci USA 94:9499–9503[Abstract/Free Full Text]
  71. Yu WH, Karanth S, Sower SA, Parlow AF, McCann SM 2000 The similarity of FSH-releasing factor to lamprey gonadotropin-releasing hormone III (l-GnRH-III). Proc Soc Exp Biol Med 224:87–92[Abstract/Free Full Text]
  72. Siler-Khodr TM, Khodr GS, Valenzuela G, Rhode J 1986 Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin. Biol Reprod 34:245–254[Abstract]
  73. Wide M, Persson H, Lundkvist O, Wide L 1988 Localization of mRNA for the beta-subunit of placental hCG by in situ hybridization. Acta Endocrinol (Copenh) 119:69–74
  74. Currie WD, Steele GL, Yuen BH, Kordon C, Gautron JP, Leung PC 1993 LHRH- and (hydroxyproline9) LHRH-stimulated hCG secretion from perifused first-trimester placental cells. Recent Prog Horm Res 48:505–509
  75. Andreu-Vieyra CV, Habibi HR 2000 Factors controlling ovarian apoptosis. Can J Physiol Pharmacol 78:1003–1012[CrossRef][Medline]
  76. Imai A, Takagi A, Tamaya T 2000 GnRH analog repairs reduced endometrial cell apoptosis in endometriosis in vitro. Am J Obstet Gynecol 182:1142–1146[CrossRef][Medline]
  77. Yano T, Yano N, Matsumi H, Morita Y, Tsutsumi O, Schally AV, Taketani Y 1997 Effect of luteinizing hormone releasing hormone analogues on the rat ovarian follicle development. Horm Res 48:35–41
  78. Zhao S, Saito H, Wang X, Saito T, Kaneko T, Hiroi MT 2000 Effects of gonadotropin-releasing hormone agonist on the incidence of apoptosis in porcine and human granulosa cells. Gynecol Obstet Invest 49:52–56[CrossRef][Medline]
  79. Quirk SM, Cowan RG, Joshi SG, Henrikson KP 1995 Fas antigen-mediated apoptosis in human granulosa luteal cells. Biol Reprod 52:279–287[Abstract]
  80. Roughton SA, Lareu RR, Bittles AH, Dharmarajan AM 1999 Fas and Fas ligand mRNA and protein expression in the rat corpus luteum during apoptosis-mediated luteolysis. Biol Reprod 60:797–804[Abstract/Free Full Text]
  81. Imai A, Takagi A, Horibe S, Takagi H, Tamaya T 1998 Evidence for tight coupling of GnRH receptor to stimulated Fas ligand expression in reproductive tract tumors: possible mechanism for hormonal control of apoptotic cell death. J Clin Endocrinol Metab 83:427–431[Abstract/Free Full Text]
  82. Imai A, Takagi A, Horibe S, Takagi H, Tamaya T 1998 Fas and Fas ligand system may mediate antiproliferative activity of GnRH receptor in endometrial cancer cells. Int J Oncol 13:97–100[Medline]
  83. Olofsson JI, Norjavaara E, Selstam G 1992 Synthesis of prostaglandin F2{alpha}, E2 and prostacyclin in isolated corpora lutea of adult pseudopregnant rats throughout the luteal life span. Prostaglandins Leukot Essent Fatty Acids 46:151–161[CrossRef][Medline]
  84. Vaananen JE, Tong BLI, Vaananen CCM, Chan IHY, Yuen BH, Leung PCK 1997 Interaction of prostaglandin F2{forall} and gonadotropin-releasing hormone on progesterone and estradiol production in human granulosa-luteal cells. Biol Reprod 57:1346–1353[Abstract]



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C. Metallinou, B. Asimakopoulos, A. Schroer, and N. Nikolettos
Gonadotropin-Releasing Hormone in the Ovary
Reproductive Sciences, December 1, 2007; 14(8): 737 - 749.
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 Endocr Relat CancerHome page
S Darby, J Stockley, M M Khan, C N Robson, H Y Leung, and V J Gnanapragasam
Expression of GnRH type II is regulated by the androgen receptor in prostate cancer
Endocr. Relat. Cancer, September 1, 2007; 14(3): 613 - 624.
[Abstract] [Full Text] [PDF]

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 Hum ReprodHome page
R. L. Gustofson, J. H. Segars, and F. W. Larsen
Ganirelix acetate causes a rapid reduction in estradiol levels without adversely affecting oocyte maturation in women pretreated with leuprolide acetate who are at risk of ovarian hyperstimulation syndrome
Hum. Reprod., November 1, 2006; 21(11): 2830 - 2837.
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 Endocr Relat CancerHome page
J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C K Leung
Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells.
Endocr. Relat. Cancer, June 1, 2006; 13(2): 641 - 651.
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 Hum ReprodHome page
C. Morimoto, Y. Osuga, T. Yano, Y. Takemura, M. Harada, T. Hirata, Y. Hirota, O. Yoshino, K. Koga, K. Kugu, et al.
GnRH II as a possible cytostatic regulator in the development of endometriosis
Hum. Reprod., November 1, 2005; 20(11): 3212 - 3218.
[Abstract] [Full Text] [PDF]

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 Endocr. Rev.Home page
C. K. Cheng and P. C. K. Leung
Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans
Endocr. Rev., April 1, 2005; 26(2): 283 - 306.
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 Biol. Reprod.Home page
F. Parborell, G. Irusta, A. Vitale, O. Gonzalez, A. Pecci, and M. Tesone
Gonadotropin-Releasing Hormone Antagonist Antide Inhibits Apoptosis of Preovulatory Follicle Cells in Rat Ovary
Biol Reprod, March 1, 2005; 72(3): 659 - 666.
[Abstract] [Full Text] [PDF]

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 J. Clin. Endocrinol. Metab.Home page
B.-S. An, J.-H. Choi, K.-C. Choi, and P. C. K. Leung
Differential Role of Progesterone Receptor Isoforms in the Transcriptional Regulation of Human Gonadotropin-Releasing Hormone I (GnRH I) Receptor, GnRH I, and GnRH II
J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1106 - 1113.
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 J. Clin. Endocrinol. Metab.Home page
T. M. Siler-Khodr, F.-Q. Yu, P. Wei, S.-X. Tao, and Y.-X. Liu
Contraceptive Action of a Gonadotropin-Releasing Hormone II Analog in the Rhesus Monkey
J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4513 - 4520.
[Abstract] [Full Text] [PDF]

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