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Endocrinology Vol. 138, No. 1 162-168
Copyright © 1997 by The Endocrine Society

ARTICLES

Induction of Guanosine Triphosphate-Cyclohydrolase by Follicle-Stimulating Hormone Enhances Interleukin-1ß-Stimulated Nitric Oxide Synthase Activity in Granulosa Cells1

Carlos Tabraue, Raquel Diaz Peñate, German Gallardo, Inmaculada Hernandez, José Quintana, Felix Lopez Blanco, Juan Gonzalez Reyes, Luisa F. Fanjul and Carlos M. Ruiz de Galarreta

Department of Biochemistry Molecular Biology and Physiology, University of Las Palmas School of Medicine, Las Palmas, Spain

Address all correspondence and requests for reprints to: Dr. Carlos M. Ruiz de Galarreta, Department of Biochemistry Molecular Biology and Physiology, University of Las Palmas School of Medicine, Las Palmas 35016, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In cultured granulosa cells, interleukin-1ß (IL-1ß) induced a time-dependent (16–72 h) and dose-related (0.3–30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [3H]arginine to [3H]citrulline and NO2- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ml) or the cell-permeant cAMP analog (Bu)2cAMP (0.5 mM) markedly enhanced IL-1ß-induced NO generation in cultured granulosa cells. The effect of IL-1ß on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-{gamma}, or tumor necrosis factor-{alpha}, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [3H]citrulline formation or NO2- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1ß-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1ß and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1ß-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH4 biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH4 is essential for full iNOS biosynthetic capacity in IL-1ß-stimulated granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DIFFUSIBLE free radical nitric oxide (NO) is a versatile signaling molecule produced during the enzymatic conversion of L-arginine to L-citrulline by a family of nitric oxide synthases (NOS), exhibiting distinct patterns of tissue distribution and expression (reviewed in Refs. 1–5). All NOS gene products are extraordinarily well regulated enzymes that require molecular oxygen, NADPH, FAD, FMN, and tetrahydrobiopterin (BH4) for full catalytic activity (3, 4, 5). In addition, two constitutively expressed categories first characterized in neurons and vascular endothelium are calcium-calmodulin-dependent enzymes, whereas in a variety of immunocompetent and nonimmunocompetent cells, NO generation occurs through a functionally distinct inducible (iNOS) isoform that is Ca2+/calmodulin independent (1, 2, 3, 4, 5).

The expression of iNOS is up-regulated in macrophages and other cell types after exposure to bacterial lipopolysaccharide (LPS), interferon-{gamma} (IFN{gamma}), tumor necrosis factor-{alpha} (TNF{alpha}), interleukin-1ß (IL-1ß), and other agents (1, 2). Once iNOS is induced, NO is generated for prolonged periods and mediates an intriguing variety of physiological responses, including tissue injury, cell death, and inflammation (1, 2, 3, 4, 5).

Taking into account that in the female gonad, IL-1ß triggers a variety of biological responses and probably mediates the inflammatory-like reaction of ovulation (for a review, see Ref.6), the physiological role of NO in the ovary has been recently explored (7, 8, 9, 10, 11, 12). An in vivo role for NO in the ovary was initially suggested by the reported ability of NOS inhibitors to suppress ovulation in CG-hCG-primed immature rats (8) and additionally supported by the demonstration that human granulosa-luteal cells express an endothelial-type constitutively expressed NOS enzyme (9). Efforts aimed at identifying an intraovarian regulatory mechanism for NO biosynthesis also revealed that IL-1ß induced NOS activity in primary cultures of rat ovarian dispersates (7, 10, 11). In addition, NO generated in response to IL-1ß or hCG has been recently shown to promote survival and prevent apoptosis in cultured rat preovulatory follicles (12). Although collectively these observations reinforce the concept that IL-1ß plays a key role as an intraovarian regulator of NOS activity (7, 9, 10, 11, 12), the mechanism(s) implicated in the regulation of NOS catalytic activity in the ovary remains unknown.

In the present study we show that IL-1ß induced iNOS and GTP-cyclohydrolase (GTPCH) messenger RNA (mRNA) in cultured granulosa cells. As mRNA levels of GTPCH, which represents the rate-limiting step in tetrahydrobiopterin (BH4) biosynthesis (3, 4, 5), are induced by FSH and IL-1ß in a synergistic fashion, the results presented herein strongly suggest that gonadotropin-induced cofactor biosynthesis is a critical event for cytokine-induced intraovarian NO production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and hormones
The FSH used (NIDDK oFSH-S16) was donated by the National Hormone and Pituitary Agency (Baltimore, MD). The NOS inhibitor NG-methyl-L-arginine (L-NMA), the inactive analog NG-methyl-D-arginine, 2,4-diamino-6-hydroxy-pyrimidine (DAHP), diethylstilbestrol (DES), (Bu)2cAMP, LPS (from Escherichia coli), sulfanilamide, naphthylethylenediamine dihydrochloride, and 3-(4,5-dimethyl-thiazolyl-2)2,5-diphenyltetrazolium bromide were obtained from Sigma Chemical Co. (St. Louis, MO). Human recombinant IL-1ß, TNF{alpha}, IFN{gamma}, the naturally occurring IL-1ß receptor antagonist protein (IL-1RA), and 2-(2-aminoethyl)-2-thiopseudourea (AETU), a selective inhibitor of iNOS, were obtained from Calbiochem (Barcelona, Spain). Labeled [6-3H]arginine (35 Ci/mmol) was purified before use by ion exchange chromatography on Dowex Na+-AG50WX-8 (Bio Rad, Madrid, Spain) columns as recommended by the manufacturer (DuPont-New England Nuclear, Bad Homburg, Germany). Sepiapterin (SEP) was from Dr. Schricks Laboratories (Jona, Switzerland), and digoxigenin-11-2'deoxy (d)-UTP (DIG-dUTP; 1–093-088) and the positively charged nylon membranes (1–209-299) were obtained from Boehringer Mannheim (Madrid, Spain). Polydeoxythymidine primers and deoxynucleotide triphosphates (dNTP) were purchased from Pharmacia (Barcelona, Spain). The ribonuclease inhibitor RNAsin (N211), AMV reverse transcriptase (M519), and Taq DNA polymerase were purchased from Promega (Madison, WI). Culture media (normal or arginine-free McCoy’s 5a) and other tissue culture reagents were obtained from Life Technologies (Grand Island, NY).

Cell culture procedures
Granulosa cells were obtained from the ovaries of immature (21- to 23-day-old) Sprague-Dawley rats (Lettica, Barcelona, Spain) implanted for 5 days with SILASTIC brand capsules (Dow Corning, Midland, MI) containing 25 mg DES. After this period, the ovaries were aseptically removed and cleaned of fat and connective tissue, and granulosa cells were harvested into McCoy’s 5a medium by repeated puncturing of the follicles with sterile 25-gauge needles (13). Cells were washed twice by centrifugation (250 x g; 3 min) resuspended in fresh culture medium supplemented with 2 mM glutamine and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), and incubated (3–4 h at 37 C) in a humidified 95% air-5% CO2 atmosphere. After this period, the floating nonadherent granulosa cells were carefully collected, and an equal number of viable cells was inoculated into Costar 24-well tissue culture cluster plates (Becton Dickinson, Oxnard, CA). All experimental agents were freshly diluted in sterile culture medium and added in 50-µl aliquots, with control incubations receiving the same volume of medium and a similar final concentration of diluent.

Radiometric assay of NOS
As only a reduced number of granulosa cells can be obtained from the ovaries of immature DES-treated rats (2–3 x 106 cells/gland), NOS enzyme activity was routinely determined in intact cells as the rate of [3H]citrulline formed during the enzymatic oxidation of [3H]arginine substrate by previously described methods (14). Briefly, cell monolayers were rinsed twice with arginine-free McCoy’s 5a medium, allowed to equilibrate at 37 C (5 min) in 0.3 ml fresh medium, and reactions were initiated by adding freshly purified [3H]arginine (final concentration, 1.6 µM; {approx}2 x 105 dpm/nmol). Aliquots of the supernatants (25 µl) were removed at 30-min intervals (to ensure linearity) and transferred to capped disposable chromatography columns containing 0.5 ml stop buffer (20 mM HEPES and 2 mM EDTA, pH 5.5) and 0.5 ml preequilibrated Na+-AG50WX-8 as previously described (14). After a 10-min incubation, the columns were uncapped, [14C]citrulline (~2000 dpm) was added to each column to calculate recovery, and the eluate was gently forced out with compressed air and collected directly into 20-ml polypropylene vials. After an additional wash with 0.5 ml buffer, the radioactivity associated with [3H]citrulline in the combined eluates was determined by liquid scintillation counting. Confirmation that the reaction was specifically mediated by NOS was obtained by adding the NOS inhibitor L-NMA (2.5 mM), which markedly decreased cellular [3H]citrulline production during the incubation period used in this study (results not shown). Under these assay conditions, reactions were linear for up to 3 h, and radioactivity in blank incubations established in the absence of cells was negligible (<50 dpm/well). Many cell types can regenerate arginine from citrulline by a pathway comprising the cytosolic urea cycle enzymes arginosuccinate synthase and arginosuccinate lyase (5). Nevertheless, when granulosa cells were cultured for 2 days with IL-1ß alone or with FSH and thereafter incubated for up to 5 h in arginine-free medium supplemented with [3H]- or [14C]citrulline, no labeled arginine was detected in culture medium or cell lysates using the column chromatography method described above (results not shown).

With the technical approach described, it is possible to study NOS enzyme activity in minute biological samples such as those represented by this study of rat granulosa cells, and as determined in preliminary experiments, similar results for basal and ligand-induced iNOS activities were obtained in the supernatants of cell lysates (107 cells/culture) assayed by previously validated methods (15).

Nitrite assay
After the periods indicated in each experiment, media were collected for NO2- determination (16). Suitable aliquots (100 µl) of culture medium were mixed with an equal volume of Griess reagent (a 1:1 mixture of 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4) and incubated for 10 min at room temperature (16), and concentrations were determined at 550 nm in an EL-312 Microplate reader (Biotek Instruments, Winooski, VT). Sterile-filtered standard solutions of sodium nitrite (0–50 µM) were freshly prepared in culture medium and incubated in parallel with each experiment, and comparable results were obtained using a nitrate reductase assay kit provided by Alexis Biochemicals (Laufelfingen, Switzerland).

Analysis of mRNA levels for granulosa cell iNOS and GTPCH using reverse transcriptase-PCR (RT-PCR)
Total granulosa cell RNA was extracted from duplicate 35-mm dishes for each treatment using a modified guanidinium isothiocyanate method (RNAzol-B, Cinna/Biotex, Houston TX), and samples were stored frozen (-70 C) in diethylpyrocarbamate-treated water until used. After drying and optical density determination, reverse transcription was performed by standard protocols adapted for RT-PCR analysis of different mRNA species in minute quantities of rat granulosa cells (17, 18, 19). Briefly, equal amounts of RNA (1 µg) were incubated for 75 min at 42 C in 20 µl (final volume) of 1 x PCR buffer (10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, and 0.1% Triton X-100, pH 9), 500 ng of polydeoxythymidine primers, 1 mM dNTP, 5 U AMV reverse transcriptase, and 20 U RNAsin ribonuclease inhibitor.

After an initial denaturation step (94 C for 3 min), amplification of each complementary DNA (cDNA; 125 ng total RNA input) was performed in 25 µl of 1 x PCR buffer containing (final concentrations) 2.5 µM DIG-dUTP, 0.625 U Taq DNA polymerase, and 125 ng (10–15 pmol each) of the appropriate gene-specific synthetic primers (17, 20, 21, 22).

Due to the high sensitivity of the chemiluminescence detection method and to ensure linearity with respect to the amount of RNA added (0–250 ng), the original PCR procedures were optimized for each primer set by reducing the number of amplification cycles of the published thermal profiles (17, 20, 21, 22). Under these conditions, spurious background signals were reduced to undetected levels, and the rates of amplification were exponential for up to 28 cycles (iNOS), 24 cycles (GTPCH), or 18 cycles for L19, which was used as an internal amplification control (results not shown).

The amplification step for iNOS was run (27 cycles) with a thermal profile of 94 C for 1 min (denaturation), 58 C for 2 min (annealing), and 72 C for 2 min (elongation) with specific primers (forward, 5'CTGCAGGTCTTTGACGCTCGG-3'; reverse, 5'-GTGGAACACAGGGGTGATGCT-3') to generate a PCR product of 807 bp previously shown to be 96% identical to murine macrophage iNOS (20, 22). Amplification of GTPCH (22 cycles) was performed using the same thermal schedule and primers (forward, 5'-GGATACCAGGAGACCATCTCA-3'; reverse, 5'-TAGCATGGTGCTAGTGACAGT-3') previously shown to generate a 372-bp product 100% identical to rat liver GTPCH (21, 22). To ensure that equal amounts of reverse transcribed RNA were added to the PCR reaction, the constitutively expressed ribosomal L19 protein was amplified for 16 cycles, and RNA samples with no L19 band were excluded from further investigation (17, 18, 19).

The amplified products were resolved by 1.8% agarose gel electrophoresis and transferred to positively charged nylon membranes, and chemiluminescent detection was performed with a commercially available DIG luminescent detection kit following the instructions of the manufacturer (Boehringer Mannheim). The membranes were exposed to Polaroid 667 instant film (St. Albans, UK), and the intensities of the signals were quantified with a video densitometer linked to an image computer analysis system (Pharmacia-Biotech, Uppsala, Sweden).

Statistical analysis
Results are expressed as the mean ± SEM from triplicate or quadruplicate cultures, and the experiments were repeated at least three times. The minimal effective doses and ED50 were determined with a software program based on a four-parameter logistic equation as previously described (23). Statistical differences were examined using ANOVA and, as indicated, Student’s t test for comparison of the means. P < 0.5 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of NOS activity in IL-1ß- and FSH-treated granulosa cells
As shown in Fig. 1Go, addition of IL-1ß (10 ng/ml) stimulated citrulline formation (upper panel) and total NO2- accumulation (lower panel) in a time-dependent and dose-related manner (insets in upper and lower panels). A significant increase in NOS activity was observed in cells stimulated with 10 ng/ml IL-1ß for 18 h (P < 0.05) or after treatment with lower doses of the cytokine (1 ng/ml) for 2 days (insets in upper and lower panels). Although FSH (200 ng/ml) by itself did not affect citrulline or NO2- formation, the time-dependent effect of IL-1ß on granulosa cell NOS activity was further enhanced (P < 0.05) by simultaneous treatment with the gonadotropin and was specifically abrogated by the NOS inhibitor L-NMA (2.5 mM). In a similar manner, treatment with the cell-permeant (Bu)2cAMP (0.5 mM) significantly enhanced citrulline formation and total NO2- accumulation in cultured granulosa cells (insets in upper and lower panels).



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  		Figure 1. Effects of IL-1ß and FSH on granulosa cell NOS activity. Granulosa cells (~5 x 105 cells/well) were treated for the periods indicated with IL-1ß (10 ng/ml) alone ({circ}) or in combination with 200 ng/ml FSH ({square}). Groups of cells were cultured for 72 h in the absence or presence of FSH and served as controls. Total NOS activity in intact cells, representing the rate of citrulline formation from [3H]arginine substrate (upper panel), and NO2- accumulation in culture medium (lower panel) were also determined in the presence (2.5 mM) of the NOS inhibitor L-NMA (closed symbols). The inset (upper and lower panels) shows the effect of 48-h stimulation with increasing concentrations (0.3–30 ng/ml) of IL-1ß ({circ}), 0.5 mM (Bu)2cAMP alone (open bars), or (Bu)2cAMP in combination with 10 ng/ml IL-1ß (hatched bars). Similar results (mean ± SEM) were obtained in three additional experiments.

 
The specific activity of NOS, as determined by [3H]arginine conversion to [3H]citrulline, reflecting the biosynthetic capacity of granulosa cells at the time of measurement, increased continuously, and maximal activity was observed 48 h later. In contrast, accumulation of NO2-, the stable end product of NO, represents the summation of enzyme activity throughout the entire period studied, and a significant increase in medium nitrite accumulation was observed at 72 h compared with medium NO2- levels at 48 h (P < 0.05). Regardless of treatment, no changes in cell viability, as determined by the release of the cytoplasmic lactic dehydrogenase to the culture medium (24), mitochondrial reduction of 3-(4,5-dimethyl-thiazolyl-2)2,5-diphenyltetrazolium bromide to formazan dye (25), or protein content determined by the standard bichinchoninic acid method (Pierce Chemical Co., Madrid, Spain), were observed (results not shown).

Specificity of IL-1ß-induced NOS activity in cultured granulosa cells
As NOS can be induced by bacterial endotoxin (LPS) and other proinflammatory mediators in macrophages (1, 2, 3, 4, 5) and a variety of nonimmunocompetent cells, e.g. hepatocytes (26), vascular smooth muscle cells (22, 27, 28, 29), thyrocytes (30), and pancreatic cells (31), we next tested whether LPS and other well known inducers of the L-arginine-NO pathway (TNF{alpha} and IFN{gamma}) were also able to increase NOS activity in our culture system (Table 1Go). It is of interest to note that NOS activation was evident only in granulosa cells treated for 48 h with IL-1ß, and addition of the IL-1RA (32) partially reversed this effect. In the same experiments, low doses (10 µM) of AETU, a specific inhibitor of iNOS (33), abrogated cytokine-induced NO generation with the same order of potency as higher doses (2.5 mM) of L-NMA, a well known and widely used inhibitor of all NOS isoforms (for a review, see Ref.33). In contrast, addition of different doses of LPS, TNF{alpha}, or IFN{gamma} did not affect citrulline formation or NO2- accumulation in either untreated or FSH-stimulated cells.


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  		Table 1. Lack of effect of LPS, TNF{alpha}, and IFN{gamma} on granulosa cell NOS activity

 
Effects of IL-1ß and FSH on iNOS and GTPCH mRNA levels in cultured granulosa cells
As a lag period of several hours required for IL-1ß to augment NOS activity and a sustained production of NO2- for several days are characteristics of an iNOS-type action (1, 2, 3, 4, 5), we next analyzed iNOS mRNA transcripts by RT-PCR (20, 22). Moreover, taking into account that catalytic conversion of L-arginine to L-citrulline by iNOS is dependent on an adequate supply of BH4 (2, 3), we also investigated mRNA expression of GTPCH (21, 22), the rate-limiting enzyme in the de novo pathway for tetrahydrobiopterin biosynthesis (3).

Under the standard amplification conditions described, the iNOS mRNA signal was not detectable in reverse transcribed RNA samples prepared from control or FSH-stimulated granulosa cells (Fig. 2Go). In contrast, iNOS transcripts were augmented in IL-1ß-treated cells in a time-dependent manner; a small increase was observed as early as 12 h after cytokine stimulation. A similar increase in IL-1ß-stimulated iNOS transcripts was observed in FSH-treated cells, and a small increase in iNOS signal was consistently observed at 48 h, compared with levels in cells challenged with cytokine alone. In contrast, GTPCH mRNA levels rose after treatment with IL-1ß or FSH in a time-dependent fashion, and after 48 h, a more than additive effect was observed when cells were simultaneously stimulated with both agents. Regardless of treatment, mRNA levels of the constitutively expressed ribosomal L19 protein remained unchanged (17).



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  		Figure 2. RT-PCR analysis of granulosa cell iNOS and GTPCH-I expression in cultured granulosa cells. Cells were incubated for 2 days in the absence or presence of FSH (200 ng/ml), IL-1ß (30 ng/ml), or their combination; thereafter, RNA (from duplicate 35-mm dishes per treatment) was extracted and reverse transcribed into cDNA, and samples were subjected to PCR amplification in the presence of DIG-11-UTP as described in Materials and Methods. The fractionated PCR products of 807 bp (iNOS), 372 bp (GTPCH), and 194 bp (L19) were transferred to nylon membranes by positive pressure blotting, and chemiluminescent detection was performed with a commercially available kit. Identical results were obtained in three additional experiments.

 
As treatment with IL-1RA partially abrogated cytokine-induced NOS-specific activity (Table 1Go), the effect of the receptor antagonist on iNOS and GTPCH mRNA transcripts was also investigated (Fig. 3Go). The addition of IL-1RA significantly reduced iNOS and GTPCH signals in cells treated with IL-1ß alone or in combination with FSH. As the effect of FSH on GTPCH mRNA and cytokine-induced iNOS transcripts was also observed in granulosa cells stimulated with (Bu)2cAMP (results not shown), an adequate cAMP-mediated supply of BH4 could explain the ability of FSH or the cell-permeant analog to enhance IL-1ß-stimulated NOS catalytic activity in granulosa cells (Fig. 1Go).



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  		Figure 3. Effects of IL-1RA on iNOS and GTPCH gene expression in cultured granulosa cells. Cells were incubated for 2 days in the absence or presence of FSH (200 ng/ml) with and without IL-1ß (30 ng/ml) alone or in the presence of IL-1RA (2.5 µg/ml). Upper panel, Equal amounts of RNA (1 µg) per treatment were reverse transcribed into cDNA, and aliquots were amplified with primers derived from the known sequences of iNOS, GTPCH, and L19 as described in Materials and Methods. The fractionated products were transferred to positively charged nylon membranes and detected with a commercially available chemiluminescent detection kit following the instructions of the manufacturer. Similar results were obtained in two other experiments.

 
BH4-dependent iNOS activity in cultured granulosa cells
Based on the above-mentioned observations, the requirement of BH4 for granulosa cell NO production was further investigated. As shown in Fig. 4Go, treatment with SEP (100 µM), a precursor of the alternative dihydrofolate reductase-dependent pathway for BH4 biosynthesis in many cell types (3), enhanced IL-1ß-induced citrulline formation and nitrite accumulation to levels similar to those observed in FSH-treated cells. In addition, treatment with DAHP (3 mM) to inhibit GTPCH (34) significantly attenuated cytokine-induced iNOS activity, and the SEP precursor fully reversed this effect in both untreated and FSH-stimulated cells. However, when methotrexate (10 µM), which prevents salvage of SEP to BH4 (3, 4), was present, cytokine-induced [3H]citrulline formation and nitrite accumulation were no longer restored by SEP (results not shown).



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  		Figure 4. Effects of SEP and the GTPCH inhibitor on IL-1ß-induced NOS activity. Granulosa cells (~106 cells/well) were cultured in the absence (left panel) or presence of 200 ng/ml FSH (right panel) with IL-1ß (30 ng/ml), SEP (100 µM), the GTPCH inhibitor DHAP (3 mM), and combinations thereof. After 2 days in culture, the catalytic conversion of [3H]arginine to [3H]citrulline (upper panel) and nitrite accumulation in the culture medium (lower panel) were determined by the specific methods described in Materials and Methods. Results (mean ± SEM) show an experiment representative of three others, each performed with four culture wells per condition. Groups with different letters (a–d) are significantly different (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The first piece of evidence provided by this study concerns the demonstration that FSH or the gonadotropin second messenger analog (Bu)2cAMP enhanced IL-1ß-induced granulosa cell NOS catalytic activity. The effect on the enzymatic oxidation of [3H]arginine and nitrite accumulation in the culture medium was abrogated by the IL-1RA (32), the specific iNOS inhibitor AETU (33), or the competitive inhibitor L-NMA (34).

The results presented herein contrast with data obtained with whole ovarian dispersates or cocultures of Percoll-purified granulosa and thecal cells (7, 10, 11), which suggest a heterologous cell-cell contact as a prerequisite for intraovarian IL-1ß-induced NOS activity. Although the reason for these differential responses is presently unclear, they may be related to the distinct preparations and doses of IL-1ß used in this study and/or the presence of specific subpopulations of granulosa cells within the ovary that are lost during the enzymatic dispersion or the purification procedure (35). This later possibility may also explain why in the dispersed ovarian cell model, IL-1ß-induced NO generation was reported to mediate the morphogenic/cytotoxic actions of the cytokine in one study (7), whereas no such a relationship could be established in the other reports (10, 11). Although interaction of granulosa and thecal cells may be important in regulating IL-1ß-induced NOS activity (7, 10, 11) and cytotoxicity (7, 36), at the doses and time periods used in this study neither IL-1ß alone nor in combination with other agents adversely affected granulosa cell viability and/or attachment to the culture dishes (results not shown).

Although an overriding concern using granulosa cells or whole ovarian dispersates is the possibility of contaminating macrophages in the cultures (6, 37), it is important to note that IL-1ß alone has not been demonstrated to induce NO synthesis by the macrophage or other cells of myeloid lineage (1, 2, 5). In addition, as LPS and the other likely candidates (TNF{alpha} and IFN{gamma}) for iNOS induction in other cell types of nonhematopoietic origin (20, 26, 30) did not affect NOS activity in either untreated or FSH-stimulated cultures, the present results support the concept that intraovarian NO is generated at least in part by the granulosa cell.

In an effort to evaluate the specificity of IL-1ß and FSH-induced granulosa cell iNOS activity, we also performed RT-PCR analysis of iNOS and GTPCH mRNA transcripts under a variety of experimental conditions. The IL-1ß-induced delayed (16–18 h), but sustained (48–72 h), iNOS specific activity was accompanied by a time-dependent increase in IL-1ß-induced iNOS and GTPCH mRNA levels. Although FSH alone did not affect iNOS mRNA levels, the GTPCH signal was gradually augmented (12–48 h) in gonadotropin-treated cells, and a more than additive effect was observed when cells were simultaneously stimulated with FSH and IL-1ß. In addition, a small increase in iNOS mRNA levels was consistently observed in cells stimulated with FSH and IL-1ß compared with cells challenged for 48 h with the cytokine alone. Although these results do not address the exact mechanism(s) by which FSH and IL-1ß regulate NOS specific activity, they support the idea that regulation of granulosa cell function implicates a complex network of gonadotropins and other intraovarian regulatory factors acting through different types of receptors and second messenger systems (reviewed in Refs. 38–40). In this context, it has been recently shown that cAMP-inducing agents enhanced mRNA expression of IL-1ß and its type I receptor (pp80 IL-1ß receptor) in human granulosa cells (41), thus raising the interesting possibility that a similar mechanism may account at least in part for the observed effect of FSH on cytokine-induced iNOS and GTPCH mRNA signals.

In its totally reduced form, biopteryn plays a critical role in NO production by all NOS categories (3, 4), and several lines of evidence support the concept that the primary source of intracellular BH4 derives from GTP via the de novo GTPCH-regulated and DAHP-sensitive biosynthetic pathway (3, 5, 42). Taking into account that induction of GTPCH correlates with elevated intracellular concentrations of BH4 (5, 42), results presented herein suggest that FSH-induced mRNA levels for GTPCH may contribute to the efficiency of granulosa cell NO production by providing adequate levels of this essential cofactor (2, 20).

As BH4 functions catalytically but is not recycled during the enzymatic reaction (4, 5), this possibility seems reasonable and was further confirmed by the demonstration that 1) administration of SEP, a substrate for BH4 synthesis via the dihydrofolate reductase-dependent pterin salvage pathway, augmented IL-1ß-induced [3H]citrulline and nitrite accumulation to levels similar to those observed in cells simultaneously treated with FSH; and 2) inhibition of GTPCH by DAHP was completely reversed by SEP.

Although stimulation with cAMP-inducing agents has been previously shown to induce and/or enhance LPS- or cytokine-mediated GTPCH expression (22, 29, 43, 44), to our knowledge this is the first report demonstrating GTPCH mRNA induction after ligand activation of a membrane "serpentine receptor" coupled to adenylyl cyclase.

Although gonadotropins play an important role in the complex process of follicular maturation and ovulation (38, 39, 40, 45, 46), it is of interest to note that different biochemical events associated with the ovulatory cascade appear to be promoted and/or modulated by IL-1ß (6, 37). Although we have been able to show an IL-1ß- and FSH-regulated NOS system in cultured granulosa cells, the ultimate molecular and cellular targets for cytokine-induced NO generation and/or the functional contribution of the free radical as an intraovarian physiological regulator remain to be answered. In this regard, although ip injection or local administration into the ovarian sac of NOS inhibitors has been shown to effectively suppress hCG-induced ovulation in immature rats (8), a clear-cut correlation between the inflammatory-like process of ovulation (45, 46) and intraovarian NO generation in vivo has not been established.

Recently, NO generated in response to IL-1ß or hCG has been reported to play an important role as an intrafollicular survival factor, suppressing cell death by spontaneous apoptosis in cultured rat preovulatory follicles (12). As FSH receptors are present exclusively in granulosa cells (38, 39, 40), which are the major cell type undergoing apoptotic DNA fragmentation in the atretic follicle (reviewed in Ref.46), it is tempting to speculate that FSH-induced GTPCH mRNA expression during the phase of follicular growth and maturation and the subsequent increase in BH4 levels could represent a physiologically relevant mechanism for the synthesis of large quantities of NO in mature follicles exposed to IL-1ß and other ovulatory signals.

In such a scenario, the results presented herein suggest that the coordinate induction of GTPCH and iNOS represents two arms of a common pathway required for full granulosa cell NO generation. In turn, the diffusible free NO radical, acting through autocrine and/or paracrine mechanisms, may play an important role in preventing the process of follicular atresia (47).


   Footnotes
 
1 This work was supported by Grants DGYCIT PB 95/0131 (to L.F.F.) and DGYCIT PM 95/0130 (to C.M.R.G.). Back

Received March 26, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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