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Endocrinology Vol. 141, No. 1 72-80
Copyright © 2000 by The Endocrine Society

ARTICLES

Role of Gonadotropin-Releasing Hormone as an Autocrine Growth Factor in Human Ovarian Surface Epithelium1

Sung Keun Kang, Kyung-Chul Choi, Kwai Wa Cheng, Parimal S. Nathwani, Nelly Auersperg 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: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 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
 
Epithelial ovarian cancer, which accounts for 80–90% of all ovarian cancers, is the most common cause of death from gynecological malignancies and is believed to originate from the ovarian surface epithelium. In the present study we investigated the expression of GnRH and its receptor in human ovarian surface epithelial (hOSE) cells and provided novel evidence that GnRH may have antiproliferative effects in this tissue. Using RT-PCR and Southern blot analysis, we cloned the GnRH and GnRH receptor (GnRHR) in hOSE cells. Sequence analysis revealed that GnRH and its receptor have sequences identical to those found in the hypothalamus and pituitary, respectively. To address whether GnRH regulates its own and receptor messenger RNA (mRNA), the cells were treated with different concentrations of the GnRH agonist (D-Ala6)-GnRH. Expression levels of GnRH and its receptor were investigated using quantitative and competitive RT-PCR, respectively. Interestingly, a biphasic effect was observed for the GnRH and GnRHR mRNA levels. High concentrations of the GnRH agonist (10-7 and 10-9 M) decreased GnRH and GnRHR mRNA levels, whereas a low concentration (10-11 M) resulted in up-regulation of GnRH and receptor mRNA levels. Treatment with the GnRH antagonist, antide, prevented the biphasic effects of the GnRH agonist in hOSE cells, confirming the specificity of the response. Furthermore, to investigate the physiological significance, we studied receptor-mediated growth regulatory effects of GnRH in human ovarian surface epithelial cells. The cells were treated with GnRH analogs, and the proliferative index of cells was measured using a [3H]thymidine incorporation assay. (D-Ala6)-GnRH had a direct inhibitory effect on the growth of hOSE cells in a time- and dose-dependent manner. This antiproliferative effect of the GnRH agonist was receptor mediated, as cotreatment of hOSE cells with antide abolished the growth inhibitory effects of the GnRH agonist. The results strongly suggest that GnRH can act as an autocrine/paracrine regulator in hOSE cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPITHELIAL OVARIAN cancer, which accounts for 80–90% of all ovarian cancers, is the most common cause of death from gynecological malignancies and is believed to originate from the ovarian surface epithelium (1). Regardless of its inconspicuous appearance, it has been demonstrated that the ovarian surface epithelium (OSE) plays an important role in normal ovarian physiology. OSE cells participate in the ovulation process as they remodel the ovarian cortex and secrete proteolytic enzymes (2, 3). Furthermore, these cells are involved in the repair process that follows follicular rupture at ovulation as they proliferate and migrate over the ovulatory site and secrete extracellular matrix (3). Considering the integral role of OSE cells in normal ovarian physiology, understanding their growth regulation appears to be very important. Intraovarian regulators such as insulin-like growth factors, transforming growth factors (TGFs), epidermal growth factor, and GnRH act through autocrine/paracrine mechanisms to modulate ovarian functions and growth of ovarian cells (4).

In addition to its well documented role in the regulation of gonadotropin synthesis and secretion, GnRH has direct effects on the gonads. In the ovary, GnRH modulates both basal and gonadotropin-stimulated steroidogenesis (5, 6) and induces transcription of several genes involved in follicular maturation and ovulation (7, 8). Furthermore, GnRH and its synthetic analogs have direct growth inhibitory effects on hormone-sensitive tumors, including carcinomas of the ovary, breast, endometrium, and prostate (9, 10, 11, 12, 13). In view of role of GnRH in modulating ovarian functions, GnRH may be implicated in the functions of OSE cell physiology. However, the role of GnRH in OSE cells has not been reported.

The presence of an autocrine/paracrine regulatory system based on GnRH in normal and malignant reproductive tissues suggests that GnRH and its receptor may be regulated in these extrapituitary tissues similarly to those in the hypothalamus and pituitary. In the hypothalamus, numerous hormones regulate the synthesis and release of GnRH either directly, involving GnRH neurons, or indirectly, involving neurons that communicate with GnRH neurons (14). GnRH has been shown to regulate its own synthesis in a biphasic manner in immortalized GT1–7 neurons (15). It is well documented that the number of GnRH receptors is both up- and down-regulated by its homologous ligand in the pituitary of many mammalian species (16, 17). Stimulation with physiological concentrations of GnRH in vitro results in a biphasic pattern of changes in receptor numbers. Initially, a down-regulation of receptors is associated with a desensitization of the gonadotropes to GnRH, followed by up-regulation (5, 18, 19).

In the present study, to examine the potential role of GnRH as an autocrine regulator in human OSE (hOSE), we first investigated the expression and homologous regulation of GnRH and its receptor gene. Furthermore, to investigate the physiological significance, we studied the direct receptor-mediated growth regulatory effects of GnRH on hOSE cells. Elucidation of the regulation and function of GnRH and its receptor in the ovarian surface epithelium will contribute to a better understanding of normal ovarian physiology and the role of OSE in ovarian carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatments
hOSE cells were scraped from the ovarian surface during laparoscopies for nonmalignant disorders and were cultured as described previously (3) in medium 199:MCDB 105 (Life Technologies, Inc., Burlington, Canada) containing 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2-95% air and passaged with 0.06% trypsin (1:250)/0.01% EDTA in Mg2+/Ca2+-free HBSS when confluent. The human ovarian epithelial carcinoma cell line, OVCAR-3 (provided by Dr. T.C. Hamilton, Fox Chase Cancer Center, Philadelphia, PA), was cultured in medium 199 supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Primary ovarian tumors (n = 3) were obtained and cultured as described previously (20). Briefly, the minced tissues were dissociated in medium 199 containing 4 mg/ml collagenase type III (Roche Molecular Biochemicals, Laval, Canada) and 0.1 mg/ml bovine pancreatic deoxyribonuclease I (Roche Molecular Biochemicals) for 1 h in a humidified atmosphere of 5% CO2-95% air. The cells were passed through a fine mesh and centrifuged on Ficoll-Paque (Pharmacia Biotech, Morgan, Canada; 1000 x g) to remove red blood cells. To obtain pure cancer cells and remove fibroblasts and mesothelial cells, cell suspensions were placed on 100-mm plastic culture dishes in 10 ml medium 199 supplemented with 20% FBS for 24 h. The supernatant containing unattached tumor cells was collected and subsequently placed on 100-mm culture dishes. The tumor cells were maintained in the presence of medium 199 supplemented with 20% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2-95% air. To study the homologous regulation of GnRH and GnRH receptor (GnRHR) messenger RNA (mRNA) by a GnRH agonist, 2 x 105 hOSE cells (n = 3; passage 2) were plated onto 35-mm culture dishes in 2 ml medium 199:MCDB 105 supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. After a preincubation of 48 h, the cells were treated with (D-Ala6)-GnRH (Sigma-Aldrich Corp., Oakville, Canada) at concentrations of 10-7, 10-9, and 10-11 M for 24 h. To confirm the specificity of the GnRH agonist, the cells were treated with different concentrations of (D-Ala6)-GnRH (10-11, 10-9, or 10-7 M) plus the GnRH antagonist (antide; 10-9 or 10-7 M) for 24 h. Control cultures were treated with vehicle.

RT-PCR amplification of GnRH and GnRHR mRNA
Total RNA was prepared from cultured cells using the RNaid kit (Bio/Can Scientific, Mississauga, Canada) according to the manufacturer’s suggested procedure. The RNA concentration was measured based on absorbance at 260 nm, and its integrity was confirmed by agarose-formaldehyde gel electrophoresis. Total RNA (2.5 µg) was reverse transcribed into first strand complementary DNA (cDNA; Pharmacia Biotech, Morgan, Canada), following the manufacturer’s procedure. To clone GnRH mRNA, one set of primers was designed based on the published sequence of human hypothalamic GnRH (21). Primers for GnRH were: sense, 5'-ATTCTACTGACTTGGTGCGTG-3' (F1); and antisense, 5'-GGAATATGTGCAACTTGGTGT-3' (R1). One set of primers for GnRHR was designed to clone GnRHR mRNA based on the human pituitary GnRHR cDNA sequence (22). Primers for GnRHR were: sense, 5'-AATATGGCAAACAGTGCCTCT-3' (P48–2F); and antisense, 5'-GGATATTTTTCTCTGTGATTG-3' (P48R). The cDNA was amplified in a 50-µl PCR reaction containing 2.5 U Taq polymerase (Life Technologies, Inc.) and its buffer, 1.5 mM MgCl2, 2 mM deoxy-NTP, and 50 pmol specific primers. PCR amplification was carried out for 33 cycles, with denaturing for 1 min at 94 C, annealing for 35 sec at 50–60 C depending on the primers employed, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C. Primers for ß-actin were designed based on published sequences (23), as previously described (24). PCR for ß-actin was performed to rule out the possibility of RNA degradation and was used to control the variation in mRNA concentration in the RT reaction, as previously described (24). Amplified PCR products were subjected to Southern blot analysis. Ten microliters of PCR products were fractionated on a 1.5% agarose gel, stained with ethidium bromide. The PCR products were transferred to a nylon membrane and hybridized with digoxigenin-labeled cDNA probe for human GnRH, GnRHR, and ß-actin (24, 25, 26) following the manufacturer’s recommended procedure (Roche Molecular Biochemicals). After washing, the membranes were exposed to Kodak Omat x-ray film (Eastman Kodak Co., Rochester, NY). PCR products isolated from gel were cloned into pCRII vector using the TA Cloning Kit and were sequenced by the dideoxy nucleotide chain termination method using the T7 DNA polymerase sequencing kit (Pharmacia Biotech, Morgan, Canada).

Construction of the native (target) and mutant (competitive) cDNA for GnRHR
Using internal primers for human GnRHR, a 347-bp fragment of native GnRHR-cDNA (the target) was obtained from PCR amplification of a 760-bp human pituitary cDNA as a template (25). The primers employed were: sense, 5'-GTATGCTGGAGAGTTACTCTGCA-3' (P44F); and antisense, 5'-GGATGATGAAGAGGCAGCTGAAG-3' (P45R). The PCR product was visualized by agarose gel electrophoresis stained with ethidium bromide and was confirmed by Southern blot analysis. The PCR product was extracted from the gel, purified with an agarose gel extraction kit (QIAGEN, Hilden, Germany), and cloned into pCRII cloning vector (Invitrogen, San Diego, CA). Sequence analysis was performed to further confirm the identity of the expected sequence and amplified cDNA. After sequence confirmation, the cloned fragment was subcloned into pBSKII (Stratagene, La Jolla, CA) at SacI and XhoI sites for cloning purposes. To generate a mutant (the competitor) cDNA, the subcloned fragment was digested with HindIII and StyI and subsequently self-ligated. This step resulted in a cDNA fragment of 247 bp with a 120-bp deletion compared with the target cDNA, but with identical primer amplification sites (P44F and P45R).

Quantification of GnRH and GnRHR mRNA
To compare different expression levels for GnRH and GnRHR mRNA, quantitative and competitive PCRs were performed, respectively. PCR amplification for GnRH was carried out with denaturing for 1 min at 94 C, annealing for 35 sec at 53 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C for 26 cycles. For GnRHR, 2 µl of the first cDNA were coamplified with 0.002 pg competitor (mutant GnRHR) cDNA. The PCR for GnRHR was carried out in a 50-µl PCR reaction containing 2.5 U Taq polymerase, its buffer, 1.5 mM MgCl2, 2 mM deoxy-NTP, and 50 pmol specific primers (P44F and P45R), with denaturing for 1 min at 94 C, annealing for 35 sec at 60 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C for 33 cycles. Amplified PCR products were quantified using a computerized visual light densitometer (model 620, Bio-Rad Laboratories, Inc., Richmond, CA) after Southern blot analysis.

[3H]thymidine incorporation assay
To investigate the role of GnRH in growth regulation of hOSE cells, a [3H]thymidine incorporation assay was performed as previously described (27). hOSE cells (n = 3; passage 2) were plated in 24-well plates at 2 x 104 cells/well in 0.5 ml medium 199:MCDB 105 supplemented with 5% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. After a preincubation period of 24 h, the cells were incubated with medium containing 1 µCi [3H]thymidine (5.0 Ci/mmol; Amersham Pharmacia Biotech, Oakville, Canada), collected after 24 h, and served as day 0 controls. On the day of treatment, the cells were treated with different concentrations (10-11, 10-9, or 10-7 M) of the GnRH agonist, (D-Ala6)-GnRH for 2, 4, and 6 days. To block the effect of the GnRH agonist, the cells were treated with the (D-Ala6)-GnRH (10-7 M; GnRHa), antide (10-7 M), and (D-Ala6)-GnRH plus antide at equimolar concentration for 2, 4, and 6 days. Control cultures were treated with vehicle. Before the day of collection, the cells were incubated with medium containing the hormone and 1 µCi [3H]thymidine. After 24 h incubation, the culture medium was removed, and the cells were washed three times with PBS and precipitated with 0.5 ml 10% trichloroacetic acid for 15 min at 4 C. After a methanol wash, the precipitate was solubilized in 0.5 ml 1 N sodium hydroxide, and the incorporated radioactivity was measured in a 1217 Rackbeta liquid scintillation counter (LKB Wallac, Inc., Turku, Finland). Each experiment was repeated three times with triplicate samples.

Data analysis
GnRH mRNA levels were expressed as the ratio of GnRH to ß-actin. The amount of GnRHR transcript was calculated from the ratio of the target to competitive cDNA. Expression levels of GnRH and GnRHR mRNA are expressed as the percent change from the control value. Data are shown as the means of three individual experiments with duplicate samples and are presented as the mean ± SD. In the proliferation study, values are expressed as the percentage of growth compared with the control value and are the mean ± SD of three individual experiments with triplicate samples. The data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of GnRH and GnRHR mRNA in hOSE cells
To investigate the expression of GnRH mRNA in hOSE cells, two primers derived from human hypothalamic GnRH cDNA were designed (Fig. 1AGo), and RT-PCR was performed. Using primers F1 and R1, a 380-bp DNA fragment was obtained from the hOSE cells, a primary culture of ovarian carcinoma, and the OVCAR-3 cell line and were validated as GnRH by hybridization with a specific probe for GnRH cDNA (Fig. 1BGo). To investigate the expression of GnRHR mRNA, one set of primers was designed based on the human pituitary GnRHR cDNA sequence (Fig. 2AGo). As shown in Fig. 2BGo, the predicted PCR products were obtained in hOSE cells, primary culture of ovarian carcinoma, and the OVCAR-3 cell line and were confirmed as GnRHR by hybridization with a GnRHR cDNA probe. The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed and detected in negative controls (without template and without reverse transcriptase in the RT reaction) by ethidium bromide staining and Southern blot analysis, respectively (Figs. 1BGo and 2BGo). In addition, the authenticity of the PCR products was further confirmed, because no PCR products were obtained from human embryonic kidney (HEK-293) cDNA (data not shown). Sequence analysis revealed that GnRH and its receptor have sequences identical to those found in the hypothalamus and pituitary, respectively (data not shown).



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  		Figure 1. Detection of GnRH mRNA by RT-PCR amplification. The locations of the primers (F1 and R1) employed are indicated (A). Primer F1 is located in the second exon and corresponds to a specific region of the signal peptide; primer R1 is complementary to a segment a of 3'-untranslated region that is encoded by exon 4. First strand cDNAs from the hOSE cells (OSE), primary culture of ovarian carcinoma (PCO), and the OVCAR-3 cell line (OV-3) were amplified using one set of PCR primers derived from human hypothalamic GnRH cDNA (21 ). The expected products were observed on an ethidium bromide-stained gel (B, top panel). The PCR products were transferred onto a nylon membrane and hybridized with a digoxigenin-labeled 380-bp hGnRH cDNA probe (B, bottom panel). The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed or detected in negative controls (without template [Tm(-)] and without reverse transcriptase [RT(-)] in the RT reaction) by ethidium bromide staining and Southern blot analysis. The PCR products were gel purified, cloned, and sequenced. Sequence analysis revealed that GnRH mRNA from the hOSE cells, primary culture of ovarian carcinoma, and the OVCAR-3 cell line had a nucleotide sequence identical to that found in the hypothalamus (data not shown).

 


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  		Figure 2. Detection of GnRHR mRNA by RT-PCR amplification. The locations of the primers employed (P48–2F and P48R) are indicated (A). The primers are located in different exons to clone the complete coding region of the hGnRHR gene. First strand cDNAs from hOSE cells (OSE), primary culture of ovarian carcinoma (PCO), and the OVCAR-3 cell line (OV-3) were amplified using the primers derived from human GnRHR cDNA (22 ). The expected products were observed on an ethidium bromide-stained gel (B, top panel). The PCR products were transferred onto a nylon membrane and hybridized with a digoxigenin-labeled 364-bp hGnRHR cDNA probe (B, bottom panel). The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed or detected in negative controls (without template [Tm(-)] and without reverse transcriptase [RT(-)] in the RT reaction) by ethidium bromide staining and Southern blot analysis. The PCR products were gel purified, cloned, and sequenced. Sequence analysis revealed that the complete coding region of GnRHR mRNA from the hOSE cells, primary culture of ovarian carcinoma, and OVCAR-3 cell line had a nucleotide sequence identical to that found in the human pituitary gland (data not shown).

 
Validation of PCR for GnRH and GnRHR transcript
To determine the conditions under which PCR amplification for GnRH and ß-actin mRNA were in the logarithmic phase, 2.5 µg total RNA were reverse transcribed, and aliquots (2 µl) were amplified using different numbers of cycles. PCR for ß-actin was amplified to rule out the possibility of RNA degradation and was used to control the variation in mRNA concentrations in the RT reaction. A linear relationship between PCR products and amplification cycles was observed in both GnRH (Fig. 3Go) and ß-actin mRNA (data not shown). Twenty-six cycles for GnRH and 18 cycles for ß-actin were employed for quantification. For competitive PCR for GnRHR, mutant GnRHR cDNA was generated (Fig. 4AGo). The standard curve for GnRHR was constructed by coamplification of a fixed amount of competitive cDNA (mutant GnRHR) with adding serial dilutions of the target cDNA (native GnRHR). The amounts of target and competitive cDNA added to each PCR are shown in Fig. 4BGo. As increasing amounts of target cDNA was added, decreased amplification of competitive cDNA was observed. When plotted, a linear relationship was found between the native and mutant GnRHR cDNA (Fig. 4BGo). To titrate the amount of competitor, a fixed amount of first strand cDNA from hOSE cells (2 µl from 2.5 µg RNA) was coamplified with serial dilutions of competitive cDNA. Increasing the amount of mutant cDNA resulted in decreased amplification of the native GnRHR from the sample cDNA. A similar degree of amplification was observed when 0.002 pg mutant cDNA was added (Fig. 4CGo). This concentration was employed for competitive PCR for GnRHR transcript.



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  		Figure 3. Validation of quantitative RT-PCR for GnRH. hOSE cells were cultured in 35-mm culture dishes at 2 x 105 cells in 2 ml medium 199:MCDB 105 supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Total RNA was isolated and reverse transcribed, and aliquots were amplified using different number of PCR cycles as described in Materials and Methods. A linear relationship was observed between PCR products and amplification cycles when plotted.

 


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  		Figure 4. Construction of native (target) and mutant (competitive) cDNA and validation of competitive RT-PCR for GnRHR transcript. Using internal primers (P44F and P45R) for hGnRHR, a 347-bp fragment of native GnRHR-cDNA (the target) was obtained by PCR amplification from a 760-bp human pituitary cDNA as a template (25 ). The cloned fragment was subcloned into pBSKII (Stratagene) at SacI and XhoI sites for cloning purposes. To generate mutant (the competitor) cDNA, the subcloned fragment was digested with HindIII and StyI and subsequently self-ligated (A). This step resulted in a cDNA fragment of 247 bp with 120-bp deletion compared with the target cDNA, but with the identical primer amplification sites (P44F and P45R). The standard curve for GnRHR was constructed by a coamplification of a fixed amount of competitive cDNA (mutant GnRHR) and a serial dilution of the target cDNA (native GnRHR). As increasing amounts of target cDNA was added, decreased amplification of competitive cDNA was observed. A linear relationship between the native and mutant GnRHR cDNAs was observed when plotted (B). To titrate the amount of competitive cDNA for competitive PCR, a fixed amount of first strand cDNA from hOSE cells (2 µl from 2.5 µg RNA) was coamplified with serial dilutions of competitive cDNA. Increasing the amount of mutant cDNA resulted in decreased amplification of native GnRHR from the sample cDNA. A similar degree of amplification was observed when 0.002 pg mutant cDNA was added (C).

 
Homologous regulation of GnRH and GnRHR mRNA
Treatment with the GnRH agonist induced a biphasic regulation pattern for GnRH and GnRHR mRNA levels. High concentrations of (D-Ala6)-GnRH (10-7 and 10-9 M) decreased GnRH and GnRHR mRNA levels, whereas a low concentration (10-11 M) resulted in up-regulation of GnRH and its receptor (Fig. 5Go, A and B). To confirm the specificity of the biphasic effect by the GnRH agonist, the cells were treated with different concentrations of the GnRH agonist together with the GnRH antagonist, antide. Treatment with antide abolished the biphasic response in the hOSE cells (Fig. 5BGo).



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  		Figure 5. Effect of GnRH on GnRH mRNA (A) and GnRHR mRNA (B) in hOSE cells. hOSE cells were cultured in 35-mm culture dishes at 2 x 105 cells in 2 ml medium 199: MCDB 105 supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. After a preincubation period of 48 h, the cells were treated with different concentrations of (D-Ala6)-GnRH for 24 h. To confirm the specificity of the GnRH agonist, the cells were cotreated with different concentrations of (D-Ala6)-GnRH and the GnRH antagonist, antide. The cells were incubated with medium containing (D-Ala6)-GnRH (10-11 M) plus antide (10-9 M), (D-Ala6)-GnRH (10-9 M) plus antide (10-7 M), (D-Ala6)-GnRH (10-7 M) plus antide (10-7 M) for 24 h. Control cultures were treated with vehicle. GnRH and GnRHR mRNA levels were measured by quantitative and competitive RT-PCR, as described in Materials and Methods, respectively. Treatment with the GnRH agonist induced a biphasic regulation pattern for GnRH and GnRHR mRNA levels. Cotreatment with antide abolished the biphasic response in the hOSE cells. Data are shown as the means of three individual experiments with duplicate samples and are presented as the mean ± SD. a, P < 0.05 vs. control; b, P < 0.05 vs. 10-11 M (D-Ala6)-GnRH.

 
[3H]thymidine incorporation assay
To evaluate the functional role of GnRH in hOSE, the cells were treated with different concentrations (10-11, 10-9, or 10-7 M) of the GnRH agonist, (D-Ala6)-GnRH for 2, 4, and 6 days, and thymidine incorporation assays were performed. As shown in Fig. 6AGo, (D-Ala6)-GnRH inhibited the growth of hOSE cells in a dose-dependent manner. A significant inhibition of proliferation was detected as early as the second day of treatment at concentrations of 10-7 and 10-9 M. At a lower concentration (10-11 M), reduction of cell growth on day 2 was insignificant. The antiproliferative effect of (D-Ala6)-GnRH was also time dependent as the growth inhibitory effect increased with time of treatment. Inhibition of growth continued through to the fourth day and was further evident on the sixth day of treatment. On day 6, a high concentration of (D-Ala6)-GnRH (10-7 M) had reduced cell proliferation to 68% of the control value, and a low concentration of 10-11 M reduced growth to 76% of the control level. To block the effect of the GnRH agonist, the cells were treated with (D-Ala6)-GnRH (10-7 M; GnRHa), antide (10-7 M), and (D-Ala6)-GnRH plus antide at equimolar concentrations for 2, 4, and 6 days. The antiproliferative effect of the GnRH agonist appeared to be receptor mediated, as cotreatment with antide abolished the effects of the agonist (Fig. 6BGo).



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  		Figure 6. Effect of (D-Ala6)-GnRH and cotreatment of antide on growth of hOSE cells. hOSE cells were plated in 24-well plates at 2 x 104 cells/well in 0.5 ml medium 199:MCDB 105 supplemented with 5% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. After a preincubation period of 24 h, the cells were incubated with medium containing 1 µCi [3H]thymidine (5.0 Ci/mmol; Amersham Pharmacia Biotech), collected after 24 h, and served as day 0 controls. On the day of treatments, the cells were treated with different concentrations of (D-Ala6)-GnRH (10-11, 10-9, or 10-7 M) of the GnRH agonist, (D-Ala6)-GnRH for 2, 4, and 6 days. To block the effect of the GnRH agonist, the cells were treated with (D-Ala6)-GnRH (10-7 M; GnRHa), antide (10-7 M), and (D-Ala6)-GnRH plus antide at equimolar concentration for 2, 4, and 6 days. Control cultures were treated with vehicle. Before the day of collection, the cells were incubated with medium containing the hormone and 1 µCi [3H]thymidine. After 24 h incubation, the cells were collected, and the amount of labeled DNA was measured using the [3H]thymidine incorporation assay as described in Materials and Methods. (D-Ala6)-GnRH inhibited the growth of hOSE cells in a time- and dose-dependent manner. A significant reduction in growth vs. control was found on day 2 for (D-Ala6)-GnRH of 10-7 and 10-9 M and on days 4 and 6 for all concentrations (A). Cotreatment with antide abolished the antiproliferative effects of the (D-Ala6)-GnRH (B). Data are shown as the means of three individual experiments with triplicate samples and are presented as the mean ± SD. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Even though GnRH is primarily known for its role in the regulation of gonadotropin secretion from the pituitary, it is also thought to be an autocrine/paracrine regulator of ovarian functions. The presence of GnRH and its binding sites in the granulosa-luteal cells suggests that GnRH exerts its actions in an autocrine/paracrine manner to modulate both basal and gonadotropin-stimulated steroidogenesis, folliculogenesis, and luteolysis (6, 26). The present study demonstrates for the first time that GnRH and GnRHR mRNA are expressed in hOSE cells. In addition, GnRH and its receptor are homologously regulated in a biphasic pattern. Functionally, a GnRH analog has a direct receptor-mediated growth inhibitory effect on hOSE cells. The ovarian surface epithelium is a simple mesothelium covering of the ovary and is believed to be the source of most human ovarian carcinomas (28, 29). It has been demonstrated that this epithelium plays an important role in ovarian functions such as ovulation and the subsequent repair process. A diverse range of regulatory molecules are known to be involved in these critical processes of normal ovarian function (4). Several growth factors and cytokines have been shown to be localized and produced by OSE cells as well as granulosa or thecal cells (4, 30, 31, 32). In particular, OSE-derived regulatory factors are involved in growth stimulation or inhibition of OSE cells. For example, TGF{alpha} stimulates the growth of OSE cells, whereas TGFß inhibits cell growth (31, 32). In this study, we demonstrated that GnRH might be a novel regulatory agent of OSE cells in the human. Using RT-PCR and Southern blot analysis, we demonstrated that hOSE cells express GnRH and GnRHR mRNA. Sequence analysis indicated that GnRH and GnRHR in hOSE cells have a nucleotide sequence identical to that of GnRH and the receptor found in the hypothalamus and pituitary, respectively. Furthermore, the presence of GnRH and its receptor in hOSE cells as well as in primary culture of ovarian carcinomas and the established ovarian cancer cell line, OVCAR-3, indicate that the local regulatory system based on GnRH in hOSE cells is a normal component of the cells, not one that is newly acquired in the course of neoplastic transformation.

One of the interesting findings of the present study is the demonstration that GnRH gene expression is regulated by GnRH itself in normal epithelial cells as in the hypothalamus. It has been demonstrated that GnRH regulates its own synthesis and release through an ultrashort loop feedback mechanism in the rat hypothalamus (33, 34, 35). In addition, GnRH has been shown to exert biphasic effects on GnRH secretion from immortalized and normal hypothalamic neurons depending on the concentration and duration of treatment (15, 36). As well, it has been documented that GnRH-binding sites are both up- and down-regulated by GnRH in the pituitary of various species (5, 17). In general, low doses or pulsatile treatment of GnRH up-regulate its receptor, whereas high doses or continuous treatment down-regulate the receptor numbers. Changes in the GnRHR mRNA level have been explained as at least part of the mechanisms underlying up- and down-regulation of GnRHR receptor numbers. Pulsatile treatment with 10 nM GnRH induced a decrease in GnRHR mRNA levels in rat pituitary cells (37), whereas continuous treatment with the same concentration of GnRH for 48 h decreased levels of the receptor mRNA in cultured sheep pituitary cells (38). Recently, a biphasic regulation pattern of GnRHR mRNA levels has been reported in human ovarian granulosa-luteal cells (26). In the present study, a low dose of GnRH agonist (10 pM) increased GnRHR mRNA levels. In contrast, higher doses (1 and 100 nM) of the GnRH agonist induced a statistically significant decrease in GnRHR mRNA levels. This regulation appears to be receptor mediated, as cotreatment with a competitive GnRH antagonist, antide (39), blocked the biphasic effect of GnRH. Our results suggest that similar to the pituitary GnRHR, alterations in GnRHR mRNA levels are involved in the regulation of responsiveness to GnRH in hOSE cells. Taken together, our studies strongly indicate the presence of an autocrine/paracrine regulatory system based on GnRH in normal ovarian epithelial cells.

GnRH has been suggested to regulate cell growth and proliferation through its receptor in extrapituitary tissues. GnRH analogs have been used and proven to be efficient in treating GnRHR-bearing tumors, including carcinomas of the ovary, breast, and endometrium (9, 10, 11, 12, 13). Using thymidine incorporation assays, we demonstrated in the present study that a GnRH analog inhibits growth of the normal ovarian surface epithelial cells in a time- and dose-dependent manner. This growth inhibitory effect of the GnRH agonist appears to be receptor mediated, such that cotreatment with antide abolished the effect of the GnRH agonist. The exact mechanism of the growth inhibitory effects of GnRH analogs in hOSE cells at the receptor level is unclear. The putative endogenous ligand may stimulate proliferation of the cells through the receptor, which might be down-regulated by continuous treatment with a potent GnRH agonist. The findings in this study that continuous treatment with the GnRH agonist, which is thought to induce receptor down-regulation, inhibits cell growth, and that this effect was abolished by cotreatment with a specific antagonist uphold this view. Alternatively, the receptor might mediate direct antiproliferative effects of GnRH analogs. This idea is supported by the finding in this study that the agonist, not the antagonistic analog, which does not trigger receptor activation, induces growth inhibition of the cells. Recently, it has been suggested that the well established GnRHR signaling mechanism via activation of phospholipase C and protein kinase C is probably not involved in GnRH effects in tumor cells (40). Rather, GnRH binding in cancer cells could activate a downstream phosphotyrosine phosphatase in GnRHR-bearing tumors, thereby counteracting the effects of growth factors that function through tyrosine kinase (41, 42). It has been reported that analogs of GnRH reverse the growth stimulatory effect of epidermal growth factor-{alpha} and insulin-like growth factor in cancer cells, including carcinoma of the ovary (43, 44, 45). Interference by GnRH analogs with growth factor receptors may activate intracellular signaling pathways that partially account for the growth inhibitory effects of GnRH agonist seen in hOSE cells. At the moment, it is not known whether the GnRH analog in hOSE cells exerts its antiproliferative effect at the cellular level. It has been demonstrated that GnRH analogs reduce cell proliferation by increasing the portion of cells in the resting phase, G0-G1 (46), or induce cell death or apoptosis in the ovarian cells (47, 48). Further studies will be warranted to elucidate the exact mechanisms of the antiproliferative effect of GnRH analogs in hOSE cells.

In summary, we have demonstrated for the first time that both GnRH and GnRHR mRNAs are expressed in hOSE cells. The levels of GnRH and GnRHR gene expression are autoregulated in a biphasic manner by the GnRH agonist in a dose-related manner. Furthermore, we demonstrated that a GnRH analog has a direct growth inhibitory effect that is mediated through the GnRHR in hOSE cells. Our findings strongly suggest that GnRH is an integral part of the intraovarian regulatory complexes that modulate the physiology of normal ovarian surface epithelium and may have a role in ovarian carcinogenesis.


   Acknowledgments
 
We express our gratitude to Clara Salamanca for providing hOSE cells, and to Drs. T. Ehlen, J. Pike, M. Bertrand, and D. Miller for providing primary ovarian tumors.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada. Back

Received July 30, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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