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Effect of the gonadotropin-releasing hormone antagonist ganirelix on cyclic adenosine monophosphate accumulation of human granulosa-lutein cells
FERTILITY AND STERILITY威 VOL. 74, NO. 5, NOVEMBER 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Effect of the gonadotropin-releasing hormone antagonist ganirelix on cyclic adenosine monophosphate accumulation of human granulosa-lutein cells L. Cem Demirel, M.D.,b Ju¨rgen M. Weiss, M.D.,a Stephan Polack, Cihat U¨nlu¨, M.D.,b Klaus Diedrich, M.D., Ph.D.,a and Olaf Ortmann, M.D., Ph.D.a Medical University of Lu¨beck, Lu¨beck, Germany
Received November 29, 1999; revised and accepted May 19, 2000. Supported by a grant from the Scientific and Technical Research Council of Turkey, Ankara, Turkey. Supported by a grant from the Medical University of Lu¨beck. Presented at the 15th Annual Meeting of the European Society of Human Reproduction and Embryology, Tours, France, June 26 –30, 1999. Reprint requests: Olaf Ortmann, M.D., Ph.D., Medical University of Lu¨ebeck, Ratzeburger Allee 160, 23538 Lu¨ebeck, Germany (FAX: ⫹49 451 500 2139; E-mail: ortmann @medinf.mu-luebeck.de). a Department of Obstetrics and Gynecology, Medical University of Lu¨beck, Lu¨beck, Germany. b Department of Obstetrics and Gynecology, Medical University of Ankara, Ankara, Turkey. 0015-0282/00/$20.00 PII S0015-0282(00)01552-1
Objective: To evaluate whether the GnRH antagonist ganirelix exerts an effect on cyclic adenosine monophosphate (cAMP) production of human granulosa-lutein (GL) cells in vitro. Design: In vitro cell culture study. Setting: Research laboratory of a university hospital. Patient(s): Mural GL and cumulus cells were obtained from 15 patients on whom controlled ovarian hyperstimulation was being performed for intracytoplasmic sperm injection treatment. Intervention(s): Mural GL and cumulus cells were cultured for 48 hours with and without 1 nM ganirelix or triptorelin. For the last 6 hours, the cells were either exposed to 1–5 IU hCG or left unstimulated. Main Outcome Measure(s): At the end of the culturing period, the intracellular and extracellular cAMP accumulations were measured by an 125I-scintillation proximity assay. Result(s): hCG induced dose-dependent increases in total cAMP accumulation. Stimulation with 1 IU/mL hCG resulted in 9-fold and 13-fold increases, and 5 IU/mL hCG resulted in 19-fold and 14-fold increases in total cAMP release from cumulus and mural GL cells, respectively. On the other hand, treatments with 1 nM GnRH antagonist ganirelix and 1 nM GnRH agonist triptorelin did not exert any significant changes on the basal and hCG-stimulated cAMP accumulation of mural GL cells and cumulus cells as compared with controls. Conclusion(s): Ganirelix does not influence basal and hCG-stimulated cAMP accumulation of human GL cells in vitro. cAMP is apparently not involved in the mechanism of action of GnRH analogs in human ovary. (Fertil Steril威 2000;74:1001–7. ©2000 by American Society for Reproductive Medicine.) Key Words: Ganirelix, cAMP, hCG, granulosa cells, cumulus cells
The presence of the GnRH receptor and messenger RNA (mRNA) transcripts of GnRH have been documented by reverse transcription-polymerase chain reaction (RT-PCR) in human granulosa-lutein (GL) cells (1). Furthermore after cloning of the complementary DNA (cDNA) for GnRH receptor (GnRH-R) (2), it became possible to demonstrate the presence of GnRH-R mRNA transcripts in human GL cells as well (1, 3). However the concentration of endogenous GnRH in the systemic circulation is too low to interact with these receptors. Additionally, the level of GnRH-R mRNA is about 200-fold lower in the ovary than in the pituitary (3). Therefore, the expression of GnRH and GnRH-R in the ovary implicates an
autocrine-paracrine role for GnRH. The GnRH agonistic and antagonistic analogs used clinically in reproductive medicine, on the other hand, may interfere with this system in the human ovary because their concentrations are far higher. However, to date there is no consensus on the ovarian actions of GnRH analogs, and the mechanisms of their action with respect to intracellular signaling in extrapituitary tissues are also unclear. With the emerging use of GnRH antagonists in controlled ovarian hyperstimulation (COH) for IVF (4, 5), their possible actions on the ovary should be examined. The occasional observation that the use of GnRH antagonists in lower dose ranges during COH results in a 1001
more pronounced increase in serum estradiol (E2) values (6), seems to implicate direct effects of these compounds on gonadal steroidogenesis. Besides that, there are reports about the reduction of aromatase activity of GL cells from GnRH antagonist treated patients (7). Our previous data have shown that human GL cells treated with the GnRH antagonists cetrorelix and ganirelix (NV Organon, Oss, The Netherlands) in vitro for up to 96 hours did not reveal differences in E2 and progesterone (P) production compared to GnRH agonist triptorelin treated and control groups (8). But the lack of any action on steroidogenesis does not exclude ovarian actions of these compounds in general. It might be possible that other functions of GL cells are influenced by these decapeptides. In this regard we investigated whether GnRH analogs and in particular the GnRH antagonist ganirelix had an impact on basal and gonadotropin-stimulated cyclic adenosine monophosphate (cAMP) response, which plays a key role in signal transduction mechanisms of various peptides regulating the cellular events of granulosa cells in vitro.
MATERIALS AND METHODS Cell Collection and Preparation GL cells were obtained by ovarian puncture during COH for intracytoplasmic sperm injection (ICSI) treatment because of male factor infertility. Institutional review board approval was obtained by the ethics committee at the Medical University of Lu¨beck for the use of GL cells. A group of 15 women with normal ovulatory cycles and a mean (⫾ SEM) age of 33.1 ⫾ 0.9 were enrolled into the study. Ovarian stimulation was commenced with hMG (Menogon; Ferring, Kiel, Germany) 14 days after the midluteal administration of triptorelin depot preparation (3.75 mg of Decapeptyl; Ferring). When there were at least two follicles measuring 18 mm in diameter, 10,000 IU of hCG (Choragon; Ferring) were administered and follicular puncture was performed 36 hours later. After harvesting the cumulus oocyte complex for ICSI and centrifuging the follicular aspirates at 175g for 10 minutes, the cell pellet was washed with PBS (Seromed-Biochrom KG, Berlin, Germany) without Ca2⫹ and Mg2⫹; then resuspended and incubated in 3 mL Ham’s F10 (GIBCO, Life Technologies, Karlsruhe, Germany) medium supplemented with 20 IU/mL hyaluronidase at 5% CO2 and 37°C for 15 minutes. Once overlayed on 5 mL Ficoll (Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 312g for 20 minutes, the GL cells were aspirated from the interphase and resuspended in Hanks M-199 medium (GIBCO, Life Technologies) supplemented with 10% fetal bovine serum, 100 IU/mL of penicillin (Sigma, St. Louis, MO), 100 g/mL of streptomycin (Sigma), and 1.4 mg/mL of sodium bicarbonate. The viability of cells, assessed by trypan blue dye exclusion (Sigma), was consistently above 80%. As for the preparation of cumulus cells (CCs), harvested 1002 Demirel et al.
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cumulus-oocyte complexes were first treated with 0.1% hyaluronidase in HEPES-buffered medium for 30 seconds. The separation of CC from the oocytes was further facilitated by repeated pipetting with a Pasteur pipette. The denuded oocytes were spared for ICSI. After the CC were recovered by centrifugation, they were resuspended in culture medium.
Experimental Design Both mural GL and CC were plated onto 24-well culture dishes at a density of 100,000 cells in 1 mL medium per well in triplicate. After an overnight incubation in 5% CO2 at 37°C, the incubation medium was exchanged with Hanks M-199 medium containing 100 IU/mL of penicillin, 100 g/mL of streptomycin, 1.4 ng/mL of sodium bicarbonate, and 3 mg/mL of bovine serum albumin (Sigma). A further 48 hours of culturing of the cells was performed either with or without the addition of 1 nM ganirelix (Org 37462, batch G, NV Organon, Oss, The Netherlands) or triptorelin (Ferring). During the last 6 hours of this culturing period, the cells were stimulated with 1–5 IU of hCG with the GnRH analogs still present. The overnight incubation enabled the cells both to attach to the bottom of the culture plates and to recover from any immediate effect of in vivo exposure to the GnRH agonistic analog triptorelin and hMG during COH. The intracellular and extracellular cAMP accumulations of the cultured cells were determined by an 125I scintillation proximity assay (Amersham Pharmacia, Buckinghamshire, England). This assay is based on the competition between unlabeled cAMP and a fixed quantity of 125I-labeled cAMP for a limited number of binding sites on a cAMP-specific antibody. Cells in each well were lysed by vigorous successive pipetting following the addition of 20 L of 10% dodecyltrimethyl ammonium bromide in assay buffer. A microscopic examination using trypan blue dye confirmed the complete lysis of all of the cell populations before the cAMP-measurements. The extracted cAMP was then processed with 125I scintillation proximity radioimmunoassay using a  scintillation counter. The cAMP content of the samples could be extrapolated from a standard curve previously generated by dilution of known cAMP standards. Total cAMP measurements after 125I scintillation proximity radioimmunoassay were expressed in pmol/mL. Because of the occasional wide variation in terms of absolute cAMP values (pmol/mL) among different individual experiments, data were expressed as a percentage of basal release. This variation is from the heterogeneity of granulosa cells from follicular aspirates (9). Data were expressed as the mean (⫾ SEM) percentage of the basal release from triplicates. Each experiment was carried out with cells from a single patient, and in none of the experiments were the cells pooled from different subjects. For each patient included in the study, enough cells could be obtained to proceed with the design of the experiment. Vol. 74, No. 5, November 2000
Statistical Analysis All data are expressed as means (⫾ SEM). The KruskalWallis test, as the nonparametric equivalent of one-way analysis of variance, was used to make comparisons between group mean values. For all comparisons, P⬍.05 was considered statistically significant.
RESULTS
FIGURE 1 Total cAMP production in (A) mural granulosa-lutein and (B) cumulus cell cultures at a density of 100,000 cells per well after stimulation with 1–5 IU hCG for the last 6 of a 72-hour culturing period. The data are expressed as the mean (⫾ SEM) percentage of the basal value from six experiments performed in triplicate. (䊐 ⫽ basal; ■ ⫽ stimulation with 1 IU hCG; ⫽ stimulation with 5 IU hCG.
Patient Characteristics The cell yield from individual patients ranged between 1 million to 4.3 million cells for cumulus and 3.5 million to 27.0 million cells for mural GL cell populations. The mean (⫾ SEM) serum E2 concentration on the day of hCG injection was 1968 ⫾ 186 pg/mL, and the mean (⫾ SEM) length of ovarian stimulation was 13 ⫾ 0.7 days (range 9 to 16 days) for the study group.
Effects of HCG on cAMP Production Stimulation with hCG at various concentrations (1–5 IU) resulted in a 13- to 14-fold increase in total cAMP accumulation of mural GL cells cultured in vitro as compared to basal values. Similarly, CCs cultured in vitro responded to 1 IU of hCG stimulation with a 9-fold increase and to 5 IU of hCG with a 19-fold increase in total cAMP accumulation as compared to basal levels (Fig. 1).
Effects of Ganirelix and Triptorelin on cAMP Production As compared to the control treatment group, treating the cells in vitro for 48 hours with 1 nM of antagonistic GnRH analog ganirelix or agonistic GnRH analog triptorelin did not reveal any differences in their cAMP accumulation, regardless of whether the cells were stimulated with 5 IU of hCG (Fig. 2). Figure 2A depicts the data derived from a representative experiment. The lack of any effect of GnRH antagonist ganirelix on the cAMP accumulation of both mural GL and CCs was also demonstrated in the presence of different stimulation concentrations of hCG (Fig. 3). Because mural GL and CCs are two distinct subpopulation of somatic follicular cells in terms of steroidogenetic capabilities and hormonal receptor expressions, the effect of ganirelix treatment on hCG-induced cAMP accumulation was studied on both cell types with differing concentrations of hCG. Though there was a trend toward a decreased increment in cAMP accumulation of ganirelix-treated cells when stimulated with 1 or 5 IU of hCG as compared to cells that were not exposed to ganirelix in both mural GL and CC cultures, this did not reach statistical significance.
DISCUSSION So far the clinical trials have documented the safety and effectiveness of GnRH antagonists within the context of COH (4, 5). But the information available on their actions on FERTILITY & STERILITY威
Demirel. Effect of ganirelix on granulosa cells. Fertil Steril 2000.
the human ovary is limited and conflicting. The direct impact of clinically used GnRH analogs on the ovarian GnRH system, which is thought to be autocrine and paracrine in nature, has been a matter of debate for years. Although no consensus has been reached, a number of inhibitory and stimulatory effects have been attributed to direct in vivo and in vitro actions of GnRH analogs in the ovary. Among these, ovarian steroidogenesis, aromatase activity, LH-FSH receptor expression, follicular development, and adenylate cyclase activity were reported to be inhibited; steroidogenesis, prostaglandin synthesis, meiotic maturation of oocytes, ovulation, apoptosis/atresia within the follicle, and CC dispersion were reported to be stimulated (10 –20). 1003
FIGURE 2 Total cAMP production in mural granulosa cell cultures treated in vitro for 48 hours with 1 nM ganirelix or 1 nM triptorelin after stimulation with 5 IU of hGC for the last 6 hours of the culturing period. (A) Data derived from a single experiment (䊐 ⫽ no stimulation; ⫽ stimulation with 5 IU of hCG). (B) Basal total cAMP accumulation, and (C) hCG-stimulated total cAMP production (䊐 ⫽ control group; ■ 1 nM ganirelix-treated group; and ⫽ 1 nM triptorelin-treated group). The data are expressed as the mean (⫾ SEM) percentage of the control value from seven experiments performed in triplicate. P ⬎.05 between groups (Kruskal-Wallis test).
Demirel. Effect of ganirelix on granulosa cells. Fertil Steril 2000.
For instance, in a study by Hsueh et al. (21), a direct inhibitory effect of GnRH on FSH induction of LH receptor and aromatase activity in rat granulosa cells was demonstrated. Minaretzis et al. (7) reported that the aromatase activity of GL cells in GnRH antagonist Nal-Glu treated IVF patients was reduced during the first 6 hours of in vitro culturing as compared to agonist treated patients. During the dosagefinding studies for cetrorelix, another GnRH antagonist, to suppress endogenous LH surges in IVF cycles, a more pronounced increase in serum E2 values was observed when the antagonist was used in a 0.5 mg/day dosage, compared to higher dosage ranges (6). These results may point out a direct inhibitory effect of the antagonist on GL cell aromatase activity. An in vitro study by Spona et al. (22) found this inhibitory effect. A direct inhibitory effect of a GnRH antagonist on corpus 1004 Demirel et al.
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luteum has also been postulated in pregnant rats (23). The need for luteal support after the use of GnRH antagonists, which seems to point to an inhibitory effect of the antagonist on steroidogenesis, still needs clarification. Recently, de Jong et al. (24) reported on the use of the GnRH antagonist ganirelix to prevent the occurrence of severe ovarian hyperstimulation syndrome (OHSS). Ganirelix was able to decrease serum E2 levels quickly and effectively at a high dose level (2 mg/day) in a patient with imminent ovarian hyperstimulation syndrome. This effect may be merely because of the suppression on the pituitary level, but a direct ovarian effect cannot be excluded. Our previous in vitro studies have not shown any actions of GnRH antagonists on steroid production of GL cells (8). This result was confirmed by experiments in which we performed in vivo and in vitro treatments with ganirelix or Vol. 74, No. 5, November 2000
FIGURE 3 Total cAMP accumulation in cells treated with 1 nM of GnRH antagonist ganirelix. (A) Mural granulosa and (B) cumulus cell cultures after stimulation with different concentrations of hCG for the last 6 hours of their 48-hour culturing period (䊐 ⫽ basal; ⫽ basal plus 1 nM of ganirelix; 1 IU of hCG; ■ ⫽ 1 IU of hCG plus 1 nM of ganirelix ; z ⫽ 5 IU of hCG; d ⫽ 5 IU hCG plus 1 nM of ganirelix). The data are expressed as the mean (⫾ SEM) percentage of the basal value from five experiments performed in triplicate. P ⬎.05 within groups.
Demirel. Effect of ganirelix on granulosa cells. Fertil Steril 2000.
cetrorelix. Similarly Lin et al. (25) were unable to find a difference in basal P and E2 production of GL cells obtained from patients undergoing IVF with the GnRH agonist buserelin and GnRH antagonist cetrorelix during the first 48 hours of culturing in vitro after recovery. GnRH may share some common pathways with LH in its mode of action in the follicle because GnRH and its analogs induce a number of stimulatory responses normally elicited by LH such as resumption of oocyte meiosis, dispersion of CCs and secretion of mucoid material, and induction of ovulation in hypophysectomized rats (12–15). It is well known that gonadotropins act on the GL cells via cAMPmediated postreceptor signaling (13, 26, 27). cAMP, as an intracellular second messenger, is also involved in a number of processes in the ovary such as steroidogenesis. Therefore, cAMP plays a key role for many of the intracellular events that may be influenced directly by GnRH analogs. Earlier studies have suggested that the suppression of FERTILITY & STERILITY威
gonadotropin-induced cAMP production is the mechanism by which GnRH inhibits steroidogenesis in the ovary (10). In cultured porcine and rat granulosa cells, GnRH and its agonists were shown to inhibit FSH- and LH-induced cAMP accumulation and steroidogenesis (28, 29). In a study by Ranta et al., a GnRH agonist was shown to inhibit FSH- and LH-dependent adenylate cyclase activity by a mechanism involving the loss of binding sites for FSH in diethylstilbestrol-primed hypophysectomized rats (10). The main in vivo effect of the GnRH agonist upon rat ovarian function was through the impairment of adenylate cyclase responses from a decrease in FSH receptor content. Our in vitro data on human GL cells again fails to comply with this in vivo effect of GnRH agonists on rat ovaries. GnRH analogs did not have any influence on the hCGstimulated cAMP accumulation of GL cells in vitro. This may be attributed to the relatively higher expression of GnRH-R in the rat ovary (30). These findings are contradictory to our data showing no action of the GnRH analogs on basal or hCG-stimulated cAMP production of GL cells and may arise from the fact that the action of GnRH on granulosa cells may be species specific, as the availability of specific binding sites for GnRH in the gonadal compartments has been shown to vary between species (30). Furthermore, in a study by Brus et al. (31), GnRH-R in human GL cells was found to be present predominantly after an LH surge, which casts doubt about the action of GnRH analogs in the ovary before this time. Here, we studied both mural GL and CC, which represent two distinct subpopulations of somatic follicular cells. They differ in their steroidogenetic capabilities (32), level of LHreceptor expressions (33) and a variety of other cellular functions (34, 35). Although LH-receptor expression is a more prominent feature of mural GL cells (35), the ability of CCs to respond to hCG in terms of cAMP accumulation in our study proves that CCs in culture establish adequate LHreceptor expression within 72 hours of their separation from the oocyte, to yield cAMP in response to hCG stimulation. In rat experiments, GnRH was shown to activate the Ca2⫹ and phospholipid-dependent protein kinase C activity in granulosa cells (36, 37). The [Ca2⫹]intracellular increases significantly in human granulosa cells with GnRH stimulation as well (38). In this respect, GnRH differs from gonadotropins that mediate their ovarian effects through the cAMPdependent protein kinase A pathway (13). In rat granulosa cells, stimulation with GnRH analogs was found to be distinct from that by LH. After GnRH stimulation, no changes in cAMP production could be detected (13, 39), even though GnRH analogs and LH/hCG seem to elicit similar responses in the ovarian follicle. Nevertheless, some posttranslational modifications of the GnRH-R may occur in extrapituitary tissues. In ovarian and endometrial carcinoma cell lines, GnRH-R activation does not lead to hydrolysis of phosphoinositides by phospholipase 1005
C as in pituitary gonadotrophs (40). Therefore, in the human ovary alternative functional forms of the GnRH-R may also exist. Previously, it has been claimed that the mechanism of GnRH action in cultured rat granulosa cells involved lowering of intracellular cAMP levels by inhibiting adenylate cyclase activity (29, 41). Thus, the mechanism of GnRH action with respect to intracellular signaling in human ovary still needs further clarification. Hyperstimulation of intracellular cAMP levels before a follicle reaches full maturity may initiate atresia (42), a phenomenon that might be induced by GnRH analogs as well. High cAMP within the cell also down-regulates some of the genes induced by FSH, including p450 aromatase (42). Therefore, it is important to note that the kinetics of cAMP production by human GL cells are not affected by either GnRH antagonists or agonists. In conclusion, our study revealed that the GnRH antagonist ganirelix does not exert any effect on basal and hCGstimulated cAMP accumulation of human mural GL and CCs in vitro.
Acknowledgments The authors thank Prof. Dr. H. Coelingh Bennink (Organon, Oss, Netherlands) for the donation of ganirelix compound.
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Effect of the gonadotropin-releasing hormone antagonist ganirelix on cyclic adenosine monophosphate accumulation of human granulosa-lutein cells L. Cem Demirel, M.D.,b Ju¨rgen M. Weiss, M.D.,a Stephan Polack, Cihat U¨nlu¨, M.D.,b Klaus Diedrich, M.D., Ph.D.,a and Olaf Ortmann, M.D., Ph.D.a Medical University of Lu¨beck, Lu¨beck, Germany
Received November 29, 1999; revised and accepted May 19, 2000. Supported by a grant from the Scientific and Technical Research Council of Turkey, Ankara, Turkey. Supported by a grant from the Medical University of Lu¨beck. Presented at the 15th Annual Meeting of the European Society of Human Reproduction and Embryology, Tours, France, June 26 –30, 1999. Reprint requests: Olaf Ortmann, M.D., Ph.D., Medical University of Lu¨ebeck, Ratzeburger Allee 160, 23538 Lu¨ebeck, Germany (FAX: ⫹49 451 500 2139; E-mail: ortmann @medinf.mu-luebeck.de). a Department of Obstetrics and Gynecology, Medical University of Lu¨beck, Lu¨beck, Germany. b Department of Obstetrics and Gynecology, Medical University of Ankara, Ankara, Turkey. 0015-0282/00/$20.00 PII S0015-0282(00)01552-1
Objective: To evaluate whether the GnRH antagonist ganirelix exerts an effect on cyclic adenosine monophosphate (cAMP) production of human granulosa-lutein (GL) cells in vitro. Design: In vitro cell culture study. Setting: Research laboratory of a university hospital. Patient(s): Mural GL and cumulus cells were obtained from 15 patients on whom controlled ovarian hyperstimulation was being performed for intracytoplasmic sperm injection treatment. Intervention(s): Mural GL and cumulus cells were cultured for 48 hours with and without 1 nM ganirelix or triptorelin. For the last 6 hours, the cells were either exposed to 1–5 IU hCG or left unstimulated. Main Outcome Measure(s): At the end of the culturing period, the intracellular and extracellular cAMP accumulations were measured by an 125I-scintillation proximity assay. Result(s): hCG induced dose-dependent increases in total cAMP accumulation. Stimulation with 1 IU/mL hCG resulted in 9-fold and 13-fold increases, and 5 IU/mL hCG resulted in 19-fold and 14-fold increases in total cAMP release from cumulus and mural GL cells, respectively. On the other hand, treatments with 1 nM GnRH antagonist ganirelix and 1 nM GnRH agonist triptorelin did not exert any significant changes on the basal and hCG-stimulated cAMP accumulation of mural GL cells and cumulus cells as compared with controls. Conclusion(s): Ganirelix does not influence basal and hCG-stimulated cAMP accumulation of human GL cells in vitro. cAMP is apparently not involved in the mechanism of action of GnRH analogs in human ovary. (Fertil Steril威 2000;74:1001–7. ©2000 by American Society for Reproductive Medicine.) Key Words: Ganirelix, cAMP, hCG, granulosa cells, cumulus cells
The presence of the GnRH receptor and messenger RNA (mRNA) transcripts of GnRH have been documented by reverse transcription-polymerase chain reaction (RT-PCR) in human granulosa-lutein (GL) cells (1). Furthermore after cloning of the complementary DNA (cDNA) for GnRH receptor (GnRH-R) (2), it became possible to demonstrate the presence of GnRH-R mRNA transcripts in human GL cells as well (1, 3). However the concentration of endogenous GnRH in the systemic circulation is too low to interact with these receptors. Additionally, the level of GnRH-R mRNA is about 200-fold lower in the ovary than in the pituitary (3). Therefore, the expression of GnRH and GnRH-R in the ovary implicates an
autocrine-paracrine role for GnRH. The GnRH agonistic and antagonistic analogs used clinically in reproductive medicine, on the other hand, may interfere with this system in the human ovary because their concentrations are far higher. However, to date there is no consensus on the ovarian actions of GnRH analogs, and the mechanisms of their action with respect to intracellular signaling in extrapituitary tissues are also unclear. With the emerging use of GnRH antagonists in controlled ovarian hyperstimulation (COH) for IVF (4, 5), their possible actions on the ovary should be examined. The occasional observation that the use of GnRH antagonists in lower dose ranges during COH results in a 1001
more pronounced increase in serum estradiol (E2) values (6), seems to implicate direct effects of these compounds on gonadal steroidogenesis. Besides that, there are reports about the reduction of aromatase activity of GL cells from GnRH antagonist treated patients (7). Our previous data have shown that human GL cells treated with the GnRH antagonists cetrorelix and ganirelix (NV Organon, Oss, The Netherlands) in vitro for up to 96 hours did not reveal differences in E2 and progesterone (P) production compared to GnRH agonist triptorelin treated and control groups (8). But the lack of any action on steroidogenesis does not exclude ovarian actions of these compounds in general. It might be possible that other functions of GL cells are influenced by these decapeptides. In this regard we investigated whether GnRH analogs and in particular the GnRH antagonist ganirelix had an impact on basal and gonadotropin-stimulated cyclic adenosine monophosphate (cAMP) response, which plays a key role in signal transduction mechanisms of various peptides regulating the cellular events of granulosa cells in vitro.
MATERIALS AND METHODS Cell Collection and Preparation GL cells were obtained by ovarian puncture during COH for intracytoplasmic sperm injection (ICSI) treatment because of male factor infertility. Institutional review board approval was obtained by the ethics committee at the Medical University of Lu¨beck for the use of GL cells. A group of 15 women with normal ovulatory cycles and a mean (⫾ SEM) age of 33.1 ⫾ 0.9 were enrolled into the study. Ovarian stimulation was commenced with hMG (Menogon; Ferring, Kiel, Germany) 14 days after the midluteal administration of triptorelin depot preparation (3.75 mg of Decapeptyl; Ferring). When there were at least two follicles measuring 18 mm in diameter, 10,000 IU of hCG (Choragon; Ferring) were administered and follicular puncture was performed 36 hours later. After harvesting the cumulus oocyte complex for ICSI and centrifuging the follicular aspirates at 175g for 10 minutes, the cell pellet was washed with PBS (Seromed-Biochrom KG, Berlin, Germany) without Ca2⫹ and Mg2⫹; then resuspended and incubated in 3 mL Ham’s F10 (GIBCO, Life Technologies, Karlsruhe, Germany) medium supplemented with 20 IU/mL hyaluronidase at 5% CO2 and 37°C for 15 minutes. Once overlayed on 5 mL Ficoll (Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 312g for 20 minutes, the GL cells were aspirated from the interphase and resuspended in Hanks M-199 medium (GIBCO, Life Technologies) supplemented with 10% fetal bovine serum, 100 IU/mL of penicillin (Sigma, St. Louis, MO), 100 g/mL of streptomycin (Sigma), and 1.4 mg/mL of sodium bicarbonate. The viability of cells, assessed by trypan blue dye exclusion (Sigma), was consistently above 80%. As for the preparation of cumulus cells (CCs), harvested 1002 Demirel et al.
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cumulus-oocyte complexes were first treated with 0.1% hyaluronidase in HEPES-buffered medium for 30 seconds. The separation of CC from the oocytes was further facilitated by repeated pipetting with a Pasteur pipette. The denuded oocytes were spared for ICSI. After the CC were recovered by centrifugation, they were resuspended in culture medium.
Experimental Design Both mural GL and CC were plated onto 24-well culture dishes at a density of 100,000 cells in 1 mL medium per well in triplicate. After an overnight incubation in 5% CO2 at 37°C, the incubation medium was exchanged with Hanks M-199 medium containing 100 IU/mL of penicillin, 100 g/mL of streptomycin, 1.4 ng/mL of sodium bicarbonate, and 3 mg/mL of bovine serum albumin (Sigma). A further 48 hours of culturing of the cells was performed either with or without the addition of 1 nM ganirelix (Org 37462, batch G, NV Organon, Oss, The Netherlands) or triptorelin (Ferring). During the last 6 hours of this culturing period, the cells were stimulated with 1–5 IU of hCG with the GnRH analogs still present. The overnight incubation enabled the cells both to attach to the bottom of the culture plates and to recover from any immediate effect of in vivo exposure to the GnRH agonistic analog triptorelin and hMG during COH. The intracellular and extracellular cAMP accumulations of the cultured cells were determined by an 125I scintillation proximity assay (Amersham Pharmacia, Buckinghamshire, England). This assay is based on the competition between unlabeled cAMP and a fixed quantity of 125I-labeled cAMP for a limited number of binding sites on a cAMP-specific antibody. Cells in each well were lysed by vigorous successive pipetting following the addition of 20 L of 10% dodecyltrimethyl ammonium bromide in assay buffer. A microscopic examination using trypan blue dye confirmed the complete lysis of all of the cell populations before the cAMP-measurements. The extracted cAMP was then processed with 125I scintillation proximity radioimmunoassay using a  scintillation counter. The cAMP content of the samples could be extrapolated from a standard curve previously generated by dilution of known cAMP standards. Total cAMP measurements after 125I scintillation proximity radioimmunoassay were expressed in pmol/mL. Because of the occasional wide variation in terms of absolute cAMP values (pmol/mL) among different individual experiments, data were expressed as a percentage of basal release. This variation is from the heterogeneity of granulosa cells from follicular aspirates (9). Data were expressed as the mean (⫾ SEM) percentage of the basal release from triplicates. Each experiment was carried out with cells from a single patient, and in none of the experiments were the cells pooled from different subjects. For each patient included in the study, enough cells could be obtained to proceed with the design of the experiment. Vol. 74, No. 5, November 2000
Statistical Analysis All data are expressed as means (⫾ SEM). The KruskalWallis test, as the nonparametric equivalent of one-way analysis of variance, was used to make comparisons between group mean values. For all comparisons, P⬍.05 was considered statistically significant.
RESULTS
FIGURE 1 Total cAMP production in (A) mural granulosa-lutein and (B) cumulus cell cultures at a density of 100,000 cells per well after stimulation with 1–5 IU hCG for the last 6 of a 72-hour culturing period. The data are expressed as the mean (⫾ SEM) percentage of the basal value from six experiments performed in triplicate. (䊐 ⫽ basal; ■ ⫽ stimulation with 1 IU hCG; ⫽ stimulation with 5 IU hCG.
Patient Characteristics The cell yield from individual patients ranged between 1 million to 4.3 million cells for cumulus and 3.5 million to 27.0 million cells for mural GL cell populations. The mean (⫾ SEM) serum E2 concentration on the day of hCG injection was 1968 ⫾ 186 pg/mL, and the mean (⫾ SEM) length of ovarian stimulation was 13 ⫾ 0.7 days (range 9 to 16 days) for the study group.
Effects of HCG on cAMP Production Stimulation with hCG at various concentrations (1–5 IU) resulted in a 13- to 14-fold increase in total cAMP accumulation of mural GL cells cultured in vitro as compared to basal values. Similarly, CCs cultured in vitro responded to 1 IU of hCG stimulation with a 9-fold increase and to 5 IU of hCG with a 19-fold increase in total cAMP accumulation as compared to basal levels (Fig. 1).
Effects of Ganirelix and Triptorelin on cAMP Production As compared to the control treatment group, treating the cells in vitro for 48 hours with 1 nM of antagonistic GnRH analog ganirelix or agonistic GnRH analog triptorelin did not reveal any differences in their cAMP accumulation, regardless of whether the cells were stimulated with 5 IU of hCG (Fig. 2). Figure 2A depicts the data derived from a representative experiment. The lack of any effect of GnRH antagonist ganirelix on the cAMP accumulation of both mural GL and CCs was also demonstrated in the presence of different stimulation concentrations of hCG (Fig. 3). Because mural GL and CCs are two distinct subpopulation of somatic follicular cells in terms of steroidogenetic capabilities and hormonal receptor expressions, the effect of ganirelix treatment on hCG-induced cAMP accumulation was studied on both cell types with differing concentrations of hCG. Though there was a trend toward a decreased increment in cAMP accumulation of ganirelix-treated cells when stimulated with 1 or 5 IU of hCG as compared to cells that were not exposed to ganirelix in both mural GL and CC cultures, this did not reach statistical significance.
DISCUSSION So far the clinical trials have documented the safety and effectiveness of GnRH antagonists within the context of COH (4, 5). But the information available on their actions on FERTILITY & STERILITY威
Demirel. Effect of ganirelix on granulosa cells. Fertil Steril 2000.
the human ovary is limited and conflicting. The direct impact of clinically used GnRH analogs on the ovarian GnRH system, which is thought to be autocrine and paracrine in nature, has been a matter of debate for years. Although no consensus has been reached, a number of inhibitory and stimulatory effects have been attributed to direct in vivo and in vitro actions of GnRH analogs in the ovary. Among these, ovarian steroidogenesis, aromatase activity, LH-FSH receptor expression, follicular development, and adenylate cyclase activity were reported to be inhibited; steroidogenesis, prostaglandin synthesis, meiotic maturation of oocytes, ovulation, apoptosis/atresia within the follicle, and CC dispersion were reported to be stimulated (10 –20). 1003
FIGURE 2 Total cAMP production in mural granulosa cell cultures treated in vitro for 48 hours with 1 nM ganirelix or 1 nM triptorelin after stimulation with 5 IU of hGC for the last 6 hours of the culturing period. (A) Data derived from a single experiment (䊐 ⫽ no stimulation; ⫽ stimulation with 5 IU of hCG). (B) Basal total cAMP accumulation, and (C) hCG-stimulated total cAMP production (䊐 ⫽ control group; ■ 1 nM ganirelix-treated group; and ⫽ 1 nM triptorelin-treated group). The data are expressed as the mean (⫾ SEM) percentage of the control value from seven experiments performed in triplicate. P ⬎.05 between groups (Kruskal-Wallis test).
Demirel. Effect of ganirelix on granulosa cells. Fertil Steril 2000.
For instance, in a study by Hsueh et al. (21), a direct inhibitory effect of GnRH on FSH induction of LH receptor and aromatase activity in rat granulosa cells was demonstrated. Minaretzis et al. (7) reported that the aromatase activity of GL cells in GnRH antagonist Nal-Glu treated IVF patients was reduced during the first 6 hours of in vitro culturing as compared to agonist treated patients. During the dosagefinding studies for cetrorelix, another GnRH antagonist, to suppress endogenous LH surges in IVF cycles, a more pronounced increase in serum E2 values was observed when the antagonist was used in a 0.5 mg/day dosage, compared to higher dosage ranges (6). These results may point out a direct inhibitory effect of the antagonist on GL cell aromatase activity. An in vitro study by Spona et al. (22) found this inhibitory effect. A direct inhibitory effect of a GnRH antagonist on corpus 1004 Demirel et al.
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luteum has also been postulated in pregnant rats (23). The need for luteal support after the use of GnRH antagonists, which seems to point to an inhibitory effect of the antagonist on steroidogenesis, still needs clarification. Recently, de Jong et al. (24) reported on the use of the GnRH antagonist ganirelix to prevent the occurrence of severe ovarian hyperstimulation syndrome (OHSS). Ganirelix was able to decrease serum E2 levels quickly and effectively at a high dose level (2 mg/day) in a patient with imminent ovarian hyperstimulation syndrome. This effect may be merely because of the suppression on the pituitary level, but a direct ovarian effect cannot be excluded. Our previous in vitro studies have not shown any actions of GnRH antagonists on steroid production of GL cells (8). This result was confirmed by experiments in which we performed in vivo and in vitro treatments with ganirelix or Vol. 74, No. 5, November 2000
FIGURE 3 Total cAMP accumulation in cells treated with 1 nM of GnRH antagonist ganirelix. (A) Mural granulosa and (B) cumulus cell cultures after stimulation with different concentrations of hCG for the last 6 hours of their 48-hour culturing period (䊐 ⫽ basal; ⫽ basal plus 1 nM of ganirelix; 1 IU of hCG; ■ ⫽ 1 IU of hCG plus 1 nM of ganirelix ; z ⫽ 5 IU of hCG; d ⫽ 5 IU hCG plus 1 nM of ganirelix). The data are expressed as the mean (⫾ SEM) percentage of the basal value from five experiments performed in triplicate. P ⬎.05 within groups.
Demirel. Effect of ganirelix on granulosa cells. Fertil Steril 2000.
cetrorelix. Similarly Lin et al. (25) were unable to find a difference in basal P and E2 production of GL cells obtained from patients undergoing IVF with the GnRH agonist buserelin and GnRH antagonist cetrorelix during the first 48 hours of culturing in vitro after recovery. GnRH may share some common pathways with LH in its mode of action in the follicle because GnRH and its analogs induce a number of stimulatory responses normally elicited by LH such as resumption of oocyte meiosis, dispersion of CCs and secretion of mucoid material, and induction of ovulation in hypophysectomized rats (12–15). It is well known that gonadotropins act on the GL cells via cAMPmediated postreceptor signaling (13, 26, 27). cAMP, as an intracellular second messenger, is also involved in a number of processes in the ovary such as steroidogenesis. Therefore, cAMP plays a key role for many of the intracellular events that may be influenced directly by GnRH analogs. Earlier studies have suggested that the suppression of FERTILITY & STERILITY威
gonadotropin-induced cAMP production is the mechanism by which GnRH inhibits steroidogenesis in the ovary (10). In cultured porcine and rat granulosa cells, GnRH and its agonists were shown to inhibit FSH- and LH-induced cAMP accumulation and steroidogenesis (28, 29). In a study by Ranta et al., a GnRH agonist was shown to inhibit FSH- and LH-dependent adenylate cyclase activity by a mechanism involving the loss of binding sites for FSH in diethylstilbestrol-primed hypophysectomized rats (10). The main in vivo effect of the GnRH agonist upon rat ovarian function was through the impairment of adenylate cyclase responses from a decrease in FSH receptor content. Our in vitro data on human GL cells again fails to comply with this in vivo effect of GnRH agonists on rat ovaries. GnRH analogs did not have any influence on the hCGstimulated cAMP accumulation of GL cells in vitro. This may be attributed to the relatively higher expression of GnRH-R in the rat ovary (30). These findings are contradictory to our data showing no action of the GnRH analogs on basal or hCG-stimulated cAMP production of GL cells and may arise from the fact that the action of GnRH on granulosa cells may be species specific, as the availability of specific binding sites for GnRH in the gonadal compartments has been shown to vary between species (30). Furthermore, in a study by Brus et al. (31), GnRH-R in human GL cells was found to be present predominantly after an LH surge, which casts doubt about the action of GnRH analogs in the ovary before this time. Here, we studied both mural GL and CC, which represent two distinct subpopulations of somatic follicular cells. They differ in their steroidogenetic capabilities (32), level of LHreceptor expressions (33) and a variety of other cellular functions (34, 35). Although LH-receptor expression is a more prominent feature of mural GL cells (35), the ability of CCs to respond to hCG in terms of cAMP accumulation in our study proves that CCs in culture establish adequate LHreceptor expression within 72 hours of their separation from the oocyte, to yield cAMP in response to hCG stimulation. In rat experiments, GnRH was shown to activate the Ca2⫹ and phospholipid-dependent protein kinase C activity in granulosa cells (36, 37). The [Ca2⫹]intracellular increases significantly in human granulosa cells with GnRH stimulation as well (38). In this respect, GnRH differs from gonadotropins that mediate their ovarian effects through the cAMPdependent protein kinase A pathway (13). In rat granulosa cells, stimulation with GnRH analogs was found to be distinct from that by LH. After GnRH stimulation, no changes in cAMP production could be detected (13, 39), even though GnRH analogs and LH/hCG seem to elicit similar responses in the ovarian follicle. Nevertheless, some posttranslational modifications of the GnRH-R may occur in extrapituitary tissues. In ovarian and endometrial carcinoma cell lines, GnRH-R activation does not lead to hydrolysis of phosphoinositides by phospholipase 1005
C as in pituitary gonadotrophs (40). Therefore, in the human ovary alternative functional forms of the GnRH-R may also exist. Previously, it has been claimed that the mechanism of GnRH action in cultured rat granulosa cells involved lowering of intracellular cAMP levels by inhibiting adenylate cyclase activity (29, 41). Thus, the mechanism of GnRH action with respect to intracellular signaling in human ovary still needs further clarification. Hyperstimulation of intracellular cAMP levels before a follicle reaches full maturity may initiate atresia (42), a phenomenon that might be induced by GnRH analogs as well. High cAMP within the cell also down-regulates some of the genes induced by FSH, including p450 aromatase (42). Therefore, it is important to note that the kinetics of cAMP production by human GL cells are not affected by either GnRH antagonists or agonists. In conclusion, our study revealed that the GnRH antagonist ganirelix does not exert any effect on basal and hCGstimulated cAMP accumulation of human mural GL and CCs in vitro.
Acknowledgments The authors thank Prof. Dr. H. Coelingh Bennink (Organon, Oss, Netherlands) for the donation of ganirelix compound.
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39. Hillensjo T, LeMaire WJ. Gonadotropin releasing hormone agonists stimulate meiotic maturation of follicle-enclosed rat oocytes in vitro. Nature 1980;287:145– 6. 40. Emons G, Ortmann O, Schulz KD, Schally AV. Growth inhibitory actions of analogues of luteinizing hormone releasing hormone on tumor cells. Trends Endocrinol Metab 1997;8:355– 62. 41. Knecht M, Catt KJ. Gonadotropin releasing hormone: regulation of adenosine 3, 5—monophosphate in ovarian granulosa cells. Science 1981;214:1346 – 8. 42. Hillier SG. Roles of androgens in ovarian folliculogenesis. In: Filicori M, ed. The role of luteinizing hormone in folliculogenesis and ovulation induction. Bologna: Monduzzi Editore, 1999:69 –78.
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