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Endocrinology Vol. 138, No. 9 3638-3644
Copyright © 1997 by The Endocrine Society

ARTICLES

Induction of Cyclooxygenase-2 and Prostaglandin F2{alpha} Receptor Expression by Interleukin-1ß in Cultured Human Granulosa-Luteal Cells1

Kirsi Narko, Olli Ritvos and Ari Ristimäki

Departments of Obstetrics and Gynecology and Clinical Chemistry (K.N., A.R.) and the Haartman Institute, Department of Bacteriology and Immunology (O.R., A.R), University of Helsinki, Helsinki, Finland

Address all correspondence and requests for reprints to: Dr. Ari Ristimäki, Research Laboratory, Department of Obstetrics and Gynecology, University of Helsinki, Haartmaninkatu 2, SF-00290 Helsinki, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostanoids are important regulators of ovarian function, especially during ovulation and luteolysis. Cyclooxygenase (Cox) is the rate-limiting enzyme in conversion of arachidonic acid to prostanoids. We have examined the expression and regulation of the inducible Cox isoform (Cox-2) and of the receptor for PGF2{alpha} (FP) in human granulosa cells obtained from women undergoing oocyte retrieval for in vitro fertilization. Freshly isolated granulosa cells express Cox-2 and FP receptor messenger RNAs (mRNAs). FP receptor mRNA is also expressed in cultured human granulosa-luteal (GL) cells, but Cox-2 transcripts are expressed only upon induction. Interleukin-1ß (IL-1ß) elevated Cox-2 mRNA steady state levels in a concentration-dependent manner, and kinetic studies showed that Cox-2 mRNA levels were already induced at the 2 h point and returned to the basal level after incubation for 24 h. The protein synthesis inhibitor, cycloheximide, induced Cox-2 mRNA expression and potentiated the effect of IL-1ß. Degradation of Cox-2 mRNA was inhibited by IL-1ß, which suggests regulation at the posttranscriptional level. IL-1ß also induced the expression of Cox-2 protein, as detected by immunofluorescence staining using Cox-2-specific polyclonal antibodies. Further, IL-1ß-induced synthesis of prostanoids was blocked by a Cox-2-specific inhibitor, NS-398. In addition, hCG induced Cox-2 mRNA expression and potentiated the effect of IL-1ß. However, in contrast to the rapid and transient effect of IL-1ß on Cox-2 mRNA, the effect of hCG followed slower kinetics. We have previously shown that hCG induces expression of human FP receptor mRNA in cultured human GL cells. We now show that IL-1ß induces FP receptor mRNA in a time- and concentration-dependent manner. Our data suggest that Cox-2 and FP receptor are coexpressed in freshly isolated human granulosa cells and that their expression is up-regulated by IL-1ß and hCG in cultured human GL cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYCLOOXYGENASE (Cox) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids (1). Two Cox isoforms exist, Cox-1 and Cox-2. Cox-1 is expressed constitutively and ubiquitously, and it is thought to be responsible for the synthesis of prostanoids necessary for physiological functions, such as platelet aggregation, vasodilatation in the kidney, and cytoprotection of the stomach. In contrast, Cox-2 is usually low under basal conditions, but it is highly induced by tumor promoters, growth factors, and proinflammatory cytokines in several cell types in vitro, and its expression is elevated in chronic inflammatory diseases and in colon tumors in vivo (1, 2). Gonadotropins have also been shown to modulate the expression of Cox-2 in rat and bovine preovulatory follicles (3, 4, 5, 6). In the rat and bovine ovary, Cox-2 is expressed in the granulosa cells, and it has been suggested to be responsible for the increased synthesis of prostanoids during ovulation, which leads to increased blood flow, vascular permeability, and protease production and eventually to the rupture of the follicular wall (4). This is supported by the antiovulatory effect of nonsteroidal antiinflammatory drugs, such as aspirin and indomethacin, in several species (7, 8, 9, 10), including rhesus monkeys (11) and humans (12). Further, targeted deletion of the Cox-2 gene has been shown to reduce the frequency of ovulation in mice (13), whereas Cox-1 knock-out mice had normal ovarian function (14). However, it is not known which Cox isoforms are expressed in the human ovary.

Most prostanoids exert their actions through specific cell surface receptors, and the response to prostanoids could potentially be modulated by regulation of expression of the respective receptor. PGF2{alpha} is a prostanoid that triggers regression of corpus luteum (15). It acts via the PGF2{alpha} receptor (FP), which is a putative seven-transmembrane domain receptor that signals through G proteins that activate phospholipase C, which leads to formation of inositol trisphosphate and a subsequent increase in cytosolic free calcium (16, 17). Human FP receptor has been cloned from uterine (16) and placental tissues (17). We have recently shown that the expression of FP receptor is induced by hCG and down-regulated by phorbol 12-myristate 13-acetate (PMA) in human granulosa-luteal (GL) cells (18). Further, the expression of FP receptor is induced by LH and decreased by PMA and PGF2{alpha} in ovine corpus luteum (19) and is induced by hCG and forskolin in cultured bovine granulosa cells (6).

Interleukin-1 (IL-1) is an important mediator of inflammation (20). Several lines of evidence suggest that IL-1 may also have a regulatory role in the ovary: 1) IL-1ß is synthesized in ovarian stroma by macrophages (21); 2) IL-1-like activity is present in human (22) and porcine (23) follicular fluid; and 3) synthesis and expression of IL-1ß, its receptor, and receptor antagonist have been localized in human ovarian granulosa cells (24). Finally, IL-1ß induces ovulation in perfused rat (25, 26) and rabbit (27) ovaries. The proovulatory effect of IL-1ß may be facilitated by prostanoids, as IL-1ß has been shown to stimulate PG production in rat whole ovarian dispersates (28) and preovulatory follicles (22) and in cultured human granulosa cells (29, 30).

The objective of the present study was to examine the expression of Cox enzymes in freshly isolated human granulosa cells and to investigate whether Cox-2 and FP receptor expression can be regulated by IL-1ß in cultured human GL cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Human granulosa cells were obtained from women undergoing hormone treatment for in vitro fertilization. Approval for the study was obtained from the committee of ethics of the Department of Obstetrics and Gynecology, University of Helsinki. The granulosa cells from three or four patients were pooled, dispersed with hyaluronidase (Sigma Chemical Co., St. Louis, MO), and separated from red blood cells by centrifugation through Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells were seeded in 35-mm six-well dishes (Costar, Cambridge, MA) at a density of 2–5 x 105 cells/well. The cells were maintained in DMEM supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics (Life Technologies, Grand Island, NY). The cells were grown at 37 C in 5% CO2, and the culture medium was changed every other day. All experiments were conducted in DMEM containing 2.5% FCS with or without human recombinant IL-1ß (0.1–10 ng/ml) obtained from R&D Systems (Abingdon, UK) or hCG (30–100 ng/ml) from the National Hormone and Pituitary Program (Rockville, MD). 8-Bromo-cAMP (1 nM), cycloheximide (10 µg/ml), and actinomycin D (10 µg/ml) were purchased from Sigma, and NS-398 (15 µM) was obtained from Cayman Chemical Co. (Ann Arbor, MI).

RNA isolation and Northern and dot blot hybridization analyses
Cytoplasmic RNA was isolated from cultured granulosa cells using the Nonidet P-40 lysis procedure (31) or TriZol reagent (Life Technologies). Total RNA from freshly isolated granulosa cells was extracted using the guanidine isothiocyanate-cesium chloride method (32). For Northern blot analysis, 10–20 µg RNA were first denatured in 1 M glyoxal, 50% dimethylsulfoxide, and 10 mM phosphate buffer at 50 C for 60 min and then electrophoresed through 1.2% agarose gel. The RNA was transferred to Hybond-N nylon membranes (Amersham International, Aylesbury, UK). For dot blots, 1–2 µg RNA were denatured in 7.5% formaldehyde and 6 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) at 60 C for 30 min and then spotted onto nylon membranes using a 96-well Minifold device (Schleicher and Schuell, Dassel, Germany). All blots were baked for 1 h at 80 C and cross-linked under UV light for 6 min (Reprostar II UV, Camag, Muttenz, Switzerland). Human Cox-1, Cox-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNAs (cDNAs) (33) were labeled using [{alpha}-32P]deoxy-CTP (DuPont-New England Nuclear, Boston, MA) and the Prime-a-Gene kit (Promega, Madison, WI). Linear amplification labeling was performed as previously described (18, 34) to obtain a single stranded human FP receptor cDNA probe. Probes were purified with nick columns (Pharmacia) and used at 1 x 106 cpm/ml. Hybridizations were performed at 42 C for 16 h in solution containing 50% formamide, 6 x SSC, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA, 100 µg/ml herring sperm DNA, 100 µg/ml yeast RNA, and 0.5% SDS. Membranes were washed three times at 50 C for 15 min each time in 0.1 x SSC and 0.1% SDS. Dot blots were quantitated using a Fujifilm IP-Reader Bio-Imaging Analyzer BAS 1500 (Fuji Photo Co., Tokyo, Japan) and MacBas software supplied by the manufacturer. Northern blots were visualized by autoradiography.

Reverse transcriptase-PCR (RT-PCR)
Total RNA (1 µg) was converted to cDNA and PCR amplified with Cox-1, Cox-2, and GAPDH sense and antisense primers as described previously (33). Cox-1 and Cox-2 were amplified for 35 cycles, and GAPDH was amplified for 30 cycles of denaturation at 94 C for 1 min, annealing at 60 C for 1 min, and extension at 72 C for 1 min. Amplified products were electrophoresed through 1% agarose gel and visualized by ethidium bromide staining.

Assay of 6-keto-PGF1{alpha} and measurement of DNA content of cell cultures
GL cells were incubated with arachidonic acid (10 µM; Sigma) for 15 min. Conversion of arachidonic acid to prostacyclin (PGI2) was evaluated by measuring the production of its stable hydrolysis product, 6-keto-PGF1{alpha}, from cell culture medium by RIA, employing a specific antibody (35) and a tritiated 6-keto-PFG1{alpha} (Amersham). DNA concentrations in cell cultures were measured using a slightly modified method originally described by Rymaszewski et al. (36). Briefly, the cells were lysed in alkaline EDTA and neutralized with KH2PO4, followed by the addition of fluorochrome bisbenzimidazole (Hoechst 33258, Pharmacia) and measurement of fluorescence by DyNA Quant 200, as suggested by the manufacturer (Hoefer Pharmacia Biotech, San Francisco, CA).

Immunofluorescence staining
For immunofluorescence, GL cells were grown on chamber slides (Nunc, Naperville, IL) for 6 days and then treated with or without IL-1ß (10 ng/ml) for 4 h. The cells were fixed with ice-cold acetone for 10 min, and the chambers were washed three times for 10 min each time in PBS (HyClone, Northumberland, UK). Nonspecific binding of antibody was blocked with 1% BSA in PBS for 15 min. The samples were incubated with human Cox-2 polyclonal antibodies (Cayman Chemical Co.) at room temperature for 30 min and then washed with PBS three times for 10 min each time. The secondary antibody, fluorescein isothiocyanate-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), was added and incubated for 30 min, and the samples were then washed with PBS three times for 10 min each time. As a negative control, the cells received the same treatment with the exception of the primary antibody.

Statistical analysis
Statistical significance for a single comparison was calculated using Student’s t test. For multiple comparison, the t test was used only if one-way ANOVA indicated a significant difference. All results are shown as the mean ± SEM, and P < 0.05 was selected as the statistically significant value. All experiments were repeated at least three times, and each experiment consisted of three separate observations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cox-2 messenger RNA (mRNA) is expressed in human granulosa cells
Freshly isolated human granulosa cells expressed Cox-2 mRNA, but the signal was undetectable or very weak in cultured GL cells, as detected by Northern blot hybridization (Fig. 1AGo) or RT-PCR (Fig. 1BGo). In Northern blot analysis, 4.6- and 2.8-kb transcripts were detected. The transcript sizes are consistent with our earlier results obtained in human lung fibroblasts, and they are likely to represent alternatively polyadenylated Cox-2 isoforms (37). Cox-1 mRNA was detected in uncultured and cultured cells by RT-PCR (Fig. 1BGo), but not by Northern blot analysis. FP receptor mRNA was detected by Northern blot hybridization in both freshly isolated cells and cultured GL cells (Fig. 1AGo).



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  		Figure 1. Detection of Cox-2 and FP receptor mRNA from human granulosa cells. A, Cytoplasmic RNA from two separate pools of freshly isolated human granulosa cells (lanes 1 and 2) and from one pool of cultured (lane 3) human granulosa cells was analyzed by Northern blot hybridization using Cox-2 and FP receptor probes and the probe for GAPDH as a loading control. The 4.6- and 2.8-kilobase Cox-2 transcripts are depicted by arrows. B, mRNA was isolated from three individual pools of human granulosa cells before culture (lanes 1–3) and after 6 days in culture (lanes 4–6). Cytoplasmic RNA was reverse transcribed and PCR amplified with primers for human Cox-1, Cox-2, and GAPDH (see Materials and Methods for details).

 
IL-1ß induces Cox-2 mRNA expression in cultured human GL cells
To investigate the effect of IL-1ß on Cox-2 mRNA expression, GL cells were incubated with IL-1ß (10 ng/ml) for 2–48 h. Kinetic studies showed that Cox-2 mRNA levels were already induced at the 2 h point and had returned to the basal level after incubation for 24 h (Fig. 2AGo). As shown in Fig. 2BGo, the effect of IL-1ß was concentration dependent. To examine whether the cell culture stage has an effect on the induction of Cox-2 mRNA, GL cells were treated with IL-1ß (10 ng/ml) or hCG (30 ng/ml) for 8 h at days 2, 5, and 7 after initiation of the cultures. As shown in Fig. 3AGo, IL-1ß induced Cox-2 mRNA at all stages of culture. This was in contrast to the results obtained with hCG, which did not initially (on day 2) have an effect on Cox-2 mRNA expression. Interestingly, hCG potentiated the effect of IL-1ß on Cox-2 expression (Fig. 3BGo). Incubation of GL cells for 2 h with cycloheximide, an inhibitor of protein synthesis, resulted in an approximately 4-fold induction of Cox-2 mRNA steady state levels. Cycloheximide also potentiated the effects of IL-1ß, hCG, and 8-bromo-cAMP (Fig. 4Go). IL-1ß did not modulate Cox-1 mRNA expression, as detected by RT-PCR (not shown).



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  		Figure 2. Time- and concentration-dependent effect of IL-1ß on Cox-2 mRNA expression. A, Human GL cells were cultured for 4 days and incubated with or without IL-1ß (10 ng/ml) for 2–48 h. The mRNAs were analyzed by Northern blot hybridization. B, Human GL cells were cultured for 4 days and incubated for 2 h with different concentrations (0–10 ng/ml) of IL-1ß. The values of dot blot analyses are shown as the ratios of Cox-2 mRNA and GAPDH mRNA (x1000) calculated from arbitrary densitometric units (mean ± SEM of three observations). Asterisks indicate significant (P < 0.05) differences between IL-1ß-treated cultures and the control.

 


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  		Figure 3. Effects of IL-1ß and hCG on Cox-2 mRNA expression at different stages of culture. A, Human GL cells were cultured for 2, 5, and 7 days. The cultures were incubated with or without IL-1ß (10 ng/ml) or hCG (30 ng/ml) for 8 h. B, Human GL cells were cultured for 5 days and incubated with or without IL-1ß (10 ng/ml), hCG (30 ng/ml), or IL-1ß and hCG in combination for 8 h. Cytoplasmic RNA was analyzed by dot blot hybridization. The values shown represent the ratio of Cox-2 mRNA and GAPDH mRNA calculated from arbitrary densitometric units (mean ± SEM of three observations). Asterisks indicate significant (P < 0.05) differences between treated cultures and controls.

 


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  		Figure 4. Effect of cycloheximide on Cox-2 mRNA expression. Human GL cells were cultured for 6 days and incubated with hCG (30 ng/ml), 8-bromo-cAMP (1 mM), and IL-1ß (10 ng/ml) alone or in combination with cycloheximide (10 µg/ml) for 2 h. Cytoplasmic RNA was analyzed by dot blot hybridization. The values represent the ratio of Cox-2 mRNA and GAPDH mRNA (x1000) calculated from arbitrary densitometric units (mean ± SEM of three observations). Asterisks indicate a significant (P < 0.05) difference between treated cultures and the control.

 
Effect of IL-1ß on degradation of Cox-2 transcripts
To investigate whether IL-1ß has an effect on the stability of Cox-2 mRNA, GL cells were first incubated with cycloheximide for 2 h, after which the cells were washed, and actinomycin D was added to inhibit any further transcription. Cox-2 message decayed with a half-life of approximately 1.7 h (Fig. 5AGo), and IL-1ß was able to inhibit degradation of the Cox-2 transcript (Fig. 5BGo).



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  		Figure 5. Effect of IL-1ß on degradation of Cox-2 mRNA. A, Human GL cells were cultured for 6 days, preincubated with cycloheximide for 2 h, and then incubated with actinomycin D (10 µg/ml) for 0–4 h. Cytoplasmic RNA was analyzed by dot blot hybridization. Cox-2 mRNA values were first normalized by GAPDH mRNA, and the values shown are the mRNA remaining (mean ± SEM of three observations) compared with that at the 0 h point. B, Human GL cells were preincubated with cycloheximide for 2 h and then incubated with actinomycin D (control) alone or with IL-1ß for 4 h. Dot blot analyses were performed as described in A. Asterisks indicate a significant (P < 0.05) difference between treated cultures and the control.

 
IL-1ß induces Cox-2 protein expression and enzyme activity
We next examined expression of Cox-2 protein by immunofluorescence in cultured GL cells. Cox-2 was present in IL-1ß-treated GL cells, but not in untreated cells (Fig. 6Go). We also examined the effect of a Cox-2-specific inhibitor, NS-398, on IL-1ß-induced prostanoid production in GL cells by measuring the concentrations of 6-keto-PGF1{alpha}, a stable metabolite of prostacyclin. IL-1ß increased 6-keto-PGF1{alpha} concentrations in the cell culture medium approximately 2-fold (Fig. 7AGo), and this induction was blocked by NS-398 (Fig. 7BGo).



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  		Figure 6. Immunofluorescence staining of Cox-2 in IL-1ß-treated human GL cells. Human GL cells were subjected to immunofluorescence staining using Cox-2 polyclonal antibodies and fluorescein isothiocyanate-conjugated goat antirabbit IgG as described in Materials and Methods. A, Nontreated cells stained with Cox-2 antibody. B, IL-1ß-treated (10 ng/ml for 4 h) GL cells stained with Cox-2 antibody. C, IL-1ß-treated cells without primary antibody. Magnification, x400.

 


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  		Figure 7. Effects of IL-1ß and NS-398 on prostanoid production in human GL cells. A, Human GL cells were cultured for 6 days and incubated with or without IL-1ß (10 ng/ml) for 6 h. Conversion of exogenous arachidonic acid to PGI2 was evaluated by measuring 6-keto-PGF1{alpha} by RIA. The values represent the ratio of 6-keto-PGF1{alpha} concentrations to DNA concentrations (mean ± SEM of nine observations). B, The cells were cultured for 6 days and treated with or without IL-1ß (10 ng/ml) and NS-398 (15 µM). The values represent the ratio of 6-keto-PGF1{alpha} to DNA content of the cultures (mean ± SEM of three observations). Asterisks indicate a significant (P < 0.05) difference between the IL-1ß-treated cultures and controls and between IL-1ß-treated cultures and IL-1ß- and NS-398-treated cultures.

 
Kinetics of hCG-induced Cox-2 mRNA expression
Induction of Cox-2 mRNA by hCG was slow and sustained. Increased Cox-2 mRNA levels were detected after incubation for 8 h, and transcript levels remained elevated for at least 72 h (Fig. 8AGo). 8-Bromo-cAMP induced Cox-2 expression with similar kinetics as hCG (data not shown).



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  		Figure 8. Time-dependent effect of hCG on Cox-2 mRNA expression. Human GL cells were cultured for 4 days and treated with or without hCG (100ng/ml) for 2–72 h. The mRNAs were analyzed by Northern blot hybridization.

 
IL-1ß induces FP receptor mRNA
IL-1ß induced steady state levels of FP receptor mRNA. The induction peaked at 24 h (Fig. 9AGo) and was concentration dependent (Fig. 9BGo). IL-1ß together with hCG resulted in 2-fold induction compared with that produced by IL-1ß or hCG alone (Fig. 9BGo).



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  		Figure 9. Effect of IL-1ß on FP receptor mRNA expression. A, Human GL cells were cultured for 6 days and incubated with or without IL-1ß (10 ng/ml) for 2–48 h. B, Human GL cells were cultured for 6 days and incubated with or without IL-1ß (0–10 ng/ml), hCG (30 ng/ml), or IL-1ß and hCG in combination for 24 h. Cytoplasmic RNA was analyzed by Northern blot hybridization.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we show that Cox-2 mRNAs are expressed in freshly isolated human granulosa cells and that Cox-2 transcripts and protein are expressed after induction by IL-1ß in human GL cells. Using RT-PCR we detected Cox-1 message, but the expression was not regulated by IL-1ß. However, Cox-1 mRNA was not detected by Northern blot analysis, indicating that only low levels of Cox-1 mRNA are present in isolated human granulosa cells and in GL cell cultures. Cox-1 is constitutively expressed in thecal cells in the rat ovary (38), but the cellular origin of Cox-1 expression in human follicular aspirates was not studied further.

Wong et al. (39) and Sirois et al. (40) previously reported that IL-1ß does not induce the expression of endogenous Cox-2 or activity of a transfected Cox-2 promoter construct in rat granulosa cells. However, induction of Cox-2 mRNA has been detected by RT-PCR in rat preovulatory granulosa cells (41). Further, IL-1ß has been shown to stimulate synthesis of prostanoids in rat whole ovarian dispersates (28) and preovulatory follicles (21), and the synthesis of PGE2 and PGF2{alpha} has been shown to be stimulated by IL-1ß in cultured human granulosa cells (29, 30). In our experiments IL-1ß treatment resulted in a rapid and transient induction of Cox-2 mRNA in cultured human GL cells. Expression of Cox-2 protein was also stimulated by IL-1ß in these cells. In addition, IL-1ß-induced conversion of arachidonic acid to a prostanoid, which was inhibited by the Cox-2-specific inhibitor, NS-398. This suggests that elevated expression of Cox-2 enzyme is important in IL-1ß-induced prostanoid production in human ovarian granulosa cells.

Inhibition of protein synthesis induces expression of Cox-2 mRNA in several cell types (33, 42). However, cycloheximide did not induce Cox-2 mRNA expression in rat granulosa cells (43), and it decreased the induction of Cox-2 mRNA stimulated by gonadotropins (44). In the present study cycloheximide induced the expression of Cox-2 mRNA and potentiated the effects of IL-1ß, hCG, and 8-bromo-cAMP, indicating that induction of Cox-2 mRNA expression is not dependent on protein synthesis in human GL cells. The mechanism by which cycloheximide induces Cox-2 mRNA is not known, but it is possible that it inhibits the synthesis of proteins responsible for the rapid degradation of Cox-2 transcripts. IL-1{alpha} and IL-1ß have been previously shown to induce Cox-2 mRNA in fibroblasts and endothelial cells by inducing transcription and preventing degradation of the Cox-2 mRNA (33, 37). IL-1ß also stabilizes Cox-2 message in mesangial cells (45). Induction of Cox-2 mRNA by IL-1ß in human GL cells seems to depend at least partially on posttranscriptional stabilization of Cox-2 transcript.

Cox-2 mRNA and protein have been shown to be rapidly and transiently induced by ovulatory concentrations of gonadotropins via cAMP-dependent protein kinase A (PKA) pathway (3, 43, 46). In rat granulosa cells Cox-2 mRNA levels peak after 4 h and disappear after incubation for 6 h. Induction of Cox-2 by hCG in bovine granulosa cells from preovulatory follicles follows slower kinetics and is sustained (5, 6), which was also the case in our experiments with human GL cells. We detected similar kinetics in induction of Cox-2 mRNA with 8-bromo-cAMP as with hCG, suggesting that Cox-2 is also induced via PKA pathway in human GL cells. Cox-2 is also induced by other signaling pathways, including protein kinase C (PKC) and tyrosine kinases, in rat granulosa cells (47). Involvement of the PKC pathway in the induction of Cox-2 in human granulosa cells is supported by the findings that PMA increases prostacyclin production (48) and that PMA up-regulates Cox-2 mRNA levels in human GL cells (our unpublished observation). In our study, Cox-2 expression was stimulated by hCG only when the GL cells were cultured for more than 2 days. A similar lag period in responsiveness of other parameters to hCG has been observed in previous studies using this cell culture model (18, 34). This may reflect the down-regulation of LH/hCG receptors or their signaling pathways in response to hormone treatment received by the granulosa cell donors. Interestingly, simultaneous administration of hCG and IL-1ß resulted in synergistic induction of Cox-2 mRNA in GL cells.

Our group has previously shown that the FP receptor is induced after incubation for 24–48 h with hCG in human GL cells (18). We now show that IL-1ß induces FP receptor mRNA expression in GL cells. Interestingly, the induction followed slower kinetics than that of Cox-2, suggesting that the mechanism of induction may be different. However, as FP receptor Cox-2 mRNAs were induced with similar kinetics by hCG, these transcripts may be coregulated via the PKA pathway in human GL cells.

Our data show that IL-1ß-induced production of prostanoids is dependent on Cox-2 expression in cultured human GL cells. We also show that FP receptor mRNA is regulated by IL-1ß in human GL cells. To better define the biological significance of the present findings, additional studies are in progress to further characterize the role of prostanoid-forming enzymes and PG receptors in the human ovary.


   Acknowledgments
 
We thank Prof. Olavi Ylikorkala and Dr. Ulf-Håkan Stenman for their support. We also thank Ritva Javanainen, Kaija Antila, and Marjatta Vallas for their excellent technical assistance. The personnel of the Felicitas In Vitro Fertilization Clinic, the Finnish Population Council In Vitro Fertilization Laboratory, and the In Vitro Fertilization Unit at the Departments of Obstetrics and Gynecology, University of Helsinki, are kindly acknowledged for their collaboration.


   Footnotes
 
1 This work was supported by the Academy of Finland, Helsinki University Central Hospital research funds, and the Novo Nordisk Foundation. Back

Received March 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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