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Biology of Reproduction 60, 946-953 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Glucocorticoid Receptor-Mediated Post-Ceramide Inhibition of the Interleukin-1ß-Dependent Induction of Ovarian Prostaglandin Endoperoxide Synthase-2 in Rats1

Minoru Irahara3,a, Motomu Ando4,a, Jean Siroisb, Juichiro Saito5,a, and Eli Y. Adashi2,a

a Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, Maryland 21201 b Centre de Recherche en Reproduction Animale, University of Montreal, Quebec, Canada J2S 7C6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovulation may constitute a cyclic, inflammatory-like process, wherein interleukin (IL)-1 induction and increased biosynthesis of prostanoids may feature prominently. In excess, glucocorticoids, potent anti-inflammatory agents, may exert an antiovulatory effect. This paper addresses the possibility that the antiovulatory action of glucocorticoids may be partly due to interference with ovarian prostanoid biosynthesis. Specifically, we examined the effect of treatment with dexamethasone, a synthetic glucocorticoid, on the IL-1-induced expression and activity of ovarian prostaglandin endoperoxide synthase (PGS)-2, the inducible variety of the rate-limiting enzyme in the prostaglandin cascade. Treatment of cultured whole ovarian dispersates from immature rats with dexamethasone for 48 h produced a significant decrease (98.9% inhibition) in the IL-1-supported expression of PGS-2 transcripts. Comparably marked inhibition was also noted for the corresponding immunoreactive protein. The dexamethasone effect was not limited to the IL-1-mediated induction of PGS-2 transcripts, comparable suppression being noted for the IL-1-mediated up-regulation of ovarian transcripts corresponding to IL-1ß, the IL-1 receptor antagonist, and the type I IL-1 receptor. The order of potency of the glucocorticoids studied was dexamethasone > prednisolone = cortisol. Dexamethasone proved equally effective in suppressing the induction of PGS-2 transcripts by congeners of the sphingomyelin-ceramide cycle (e.g., C-2 ceramide, sphingomyelinase, and sphingosine). The dexamethasone effect proved glucocorticoid-specific, as synthetic agonists representative of the progestin (R-5020), androgen (R-1881), and estrogen (diethylstilbestrol) steroid series proved to be without effect. Cotreatment with RU-486 resulted in reversal of the ability of dexamethasone to suppress PGS-2 activity or expression. Taken together, these observations suggest that dexamethasone is capable of glucocorticoid receptor-mediated/post-ceramide suppression of IL-1-supported ovarian PGS-2 transcript, protein, and activity. These findings are compatible with the view that the chronic anovulatory state associated with adrenal hyperactivity or glucocorticoid excess may be due in part to inhibition of ovarian prostaglandin biosynthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The notion that ovulation may constitute a cyclic inflammatory-like process, originally postulated by Espey [1], has been the subject of intense interest. Central to the hypothesis is the recognition that prostanoids, established mediators of inflammation, play an obligatory role in the ovulatory process [2–5]. Although the biosynthesis of prostanoids constitutes a complex process, there is little doubt as to the central importance of the initial rate-limiting event wherein arachidonic acid substrate is processed by the enzyme prostaglandin endoperoxide synthase (PGS).

The notion that ovulation may constitute a cyclic inflammatory-like process is supported by a growing body of direct and indirect evidence implicating intraovarian interleukin (IL)-1ß, an established mediator of inflammation, in the ovulatory process [6–16]. First, the ex vivo provision of IL-1ß has been shown to bring about ovulation and to synergize with LH in this regard [6, 7]. Second, the addition of an IL-1 receptor antagonist has been shown to attenuate LH-supported ovulation under both ex vivo [8] and in vivo [9] circumstances. Third, some components of the intraovarian IL-1 system (e.g., IL-1ß and the type I IL-1 receptor) appear to be expressed in vivo mostly during a narrow periovulatory window [10–13]. Fourth, IL-1ß has been shown to induce a host of ovulation-associated phenomena in vitro such as the stimulation of hyaluronic acid biosynthesis [14], the induction of collagenase activity [15], and the activation of nitric oxide synthase activity [16].

Glucocorticoids, anti-inflammatory principles, may when in excess exert an antiovulatory effect [17–24]. This paper addresses the possibility that the antiovulatory action of glucocorticoids may be due, in part, to interference with prostanoid biosynthesis. Specifically, we examined the effect of treatment with dexamethasone on the IL-1-stimulated expression and activity of ovarian PGS-2, the inducible PGS isoform. Our findings reveal that dexamethasone is capable of inhibiting IL-1-supported ovarian PGS activity, an effect due, in part, to a decrease in PGS-2 transcript and protein accumulation. We have previously made similar observations as they relate to the activity and expression of phospholipase A2, whose role in mobilizing arachidonic acid substrate is well established (Kol et al., personal communication). Taken together, these observations are compatible with the view that the antiovulatory action of glucocorticoids [17–24] may be due, in part, to suppression of ovarian prostaglandin biosynthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Immature Sprague-Dawley female rats from Zivic-Miller Laboratories (Zelienople, PA) were killed by CO2 asphyxiation on Day 25 of life. The project was approved by the Institutional Animal Care and Use Committee.

Hormones and Reagents

Recombinant human IL-1ß (2 x 107 U/mg) was generously provided by Drs. Errol B. De Souza and C.E. Newton, DuPont-Merck Pharmaceutical Co. (Wilmington, DE). Dexamethasone (DEX), diethylstilbestrol (DES), cortisol, prednisolone, tamoxifen, flutamide, spironolactone, prostaglandin E2 (PGE2), sphingomyelinase, and sphingosine were from Sigma Chemical Co. (St. Louis, MO). C2-ceramide was from BioMol (Plymouth Meeting, PA). R-5020, R-1881, and RU-486 were generously provided by Roussel-UCLAF (Romaineville, France).

McCoy's 5a medium (serum free), penicillin-streptomycin solution, L-glutamine, trypan blue stain, and BSA were from Gibco-BRL Life Technologies (Grand Island, NY). Collagenase (clostridium histolyticum; CLS type I; 144 U/mg) was from Worthington Biochemical Corp. (Freehold, NJ). DNase (bovine pancreas), diethyldithiocarbamic acid (DEDTC), n-octyl ß-D-glucopyranoside (octyl glucoside), and RNase A were from Sigma. T7 and SP6 RNA polymerases, pGEM7Zf, and other molecular biology grade reagents were from Promega (Madison, WI). Nitrocellulose filters (0.45 µm) were from Schleicher & Schuell (Keene, NH). LC rainbow molecular weight markers were from Amersham (Arlington Heights, IL). [32P]UTP was from New England Nuclear (Boston, MA). Nitrocellulose filters (0.45 µm) were from Schleicher & Schuell, 125I-protein A from ICN Biochemicals, Inc. (Costa Mesa, CA).

Tissue Culture Procedures

Whole ovarian dispersates were prepared and cultured under serum-free conditions as previously described [25].

Nucleic Acid Probes

The rat PGS-2 cDNA [26] was generously provided in Bluescript vector by Drs. Daniel Hwang and Shuenn S. Liou of Pennington Biochemical Research Center, Louisiana State University (Baton Rouge, LA). A 385-nt XbaI EcoRI fragment of the original PGS-2 cDNA was subcloned into a pGEM7Zf vector. T7-driven transcription of the HindIII-linearized construct yielded a 328-nucleotide (nt) riboprobe that upon hybridization was projected to generate a 297-nt protected fragment.

The rat IL-1ß cDNA was generously provided in pGEM2 vector by Dr. Alan Shaw of the Glaxo Institute for Molecular Biology (Geneva, Switzerland). T7-driven transcription of the EcoRI-linearized construct yielded a 272-nt riboprobe that upon hybridization was projected to generate a 222-nt protected fragment [10].

The rat type I IL-1 receptor cDNA was generated in this laboratory by reverse transcription-polymerase chain reaction as previously described [13]. T7-driven transcription of the BamHI-linearized construct yielded a 374-nt riboprobe that upon hybridization was projected to generate a 307-nt protected fragment.

The rat IL-1 receptor antagonist (IL-1RA) cDNA was generated in this laboratory by reverse transcription-polymerase chain reaction (Kol et al., unpublished communication). T7-driven transcription of the HindIII-linearized construct yielded a 300-nt riboprobe that upon hybridization was projected to generate a 267-nt protected fragment.

The ribosomal protein large (RPL19) probe was generated and employed as previously described [13].

RNA Extraction

RNA of cultured cells was extracted with RNAZOL-B (Tel Test, Friendswood, TX) according to the manufacturer's protocol.

RNase Protection Assay

Linearized DNA templates were transcribed with the appropriate RNA polymerase to specific activities of 800 Ci/mmol [{alpha}-32P]UTP (PGS-2, IL-1ß, type I IL-1 receptor, IL-1RA) or 160 Ci/mmol [{alpha}-32P]UTP (RPL19). The riboprobes were gel purified as described previously [27] in an effort to eliminate transcribed products shorter than the full-length probes. The assay was performed as previously described [28]. Gels were exposed to XAR film (Eastman Kodak, Rochester, NY) for varying lengths of time with intensifying screens. To generate quantitative data, gels were also exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA). The resultant digitized data were analyzed with ImageQuant Software (Molecular Dynamics). The hormonally independent RPL19 mRNA signal was used to normalize the PGS-2, IL-1ß, type I IL-1 receptor, and IL-1RA mRNA data for possible variation in RNA loads. Specifically, the net protected signal (respective background subtracted) to net RPL19 ratio was calculated for each sample and gene of interest.

RIA of PGE2

The RIA for PGE2 was carried out as previously described [29].

Immune Western Blot Analysis [30]

To prepare solubilized cell extracts, ovarian cells were homogenized in TED buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1 mM DEDTC) containing 2 mM octyl glucoside and centrifuged at 30 000 x g for 1 h at 4°C. The crude pellets (membranes, nuclei, mitochondria) were sonicated (8 sec/cycle, 3 cycles) in TED sonication buffer (20 mM Tris, pH 8.0, 50 mM EDTA, 0.1 mM DEDTC) containing 45 mM octyl glucoside. The sonicates were centrifuged at 16 000 x g for 15 min at 4°C. The recovered supernatant (solubilized cell extract) was stored at -70°C until electrophoretic analyses were performed. Protein concentration was determined by the method of Bradford (Bio-Rad protein assay; Richmond, CA).

Proteins present in cell extracts were resolved by one-dimensional SDS-PAGE and electrophoretically transferred to nitrocellulose filters as previously described [30–32]. Filters were incubated with an affinity-purified PGS-2-directed antibody (#9181 antibody) diluted 1:25 in TBS (10 mM Tris-buffered saline, pH 7.5) containing 2% nonfat dry milk. 125I-Protein A (1 x 106 cpm/ml TBS-2% milk) was used to visualize immunopositive proteins. Filters were washed three times (20 min/wash) in TBS-0.05% Tween and exposed to film at -70°C.

Data Analysis

Except as noted, each experiment was replicated three times. Data points are presented as mean ± SE, and statistical significance (Fisher's protected least-significance difference) was determined by ANOVA and Student's t-test. Statistical values were calculated using Statview 512+ for MacIntosh (Brain Power, Inc., Calabasas, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Treatment with Dexamethasone onIL-1ß-Induced PGS-2, IL-1ß, Type I IL-1 Receptor, orIL-1RA Gene Expression by Cultured WholeOvarian Dispersates

To examine a possible in vitro effect of dexamethasone on IL-1ß-induced PGS-2 gene expression, whole ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without dexamethasone (10-7 M). As shown in Figure 1, treatment with dexamethasone led to significant (p < 0.01) inhibition (98.9%) of IL-1ß-induced PGS-2 gene expression. A comparable inhibitory effect was noted for the IL-1ß-induced expression of ovarian IL-1ß, type I IL-1 receptor, and IL-1RA (99%, 52.5%, and 72.8% inhibition, respectively). Treatment with dexamethasone also appeared to significantly suppress basal IL-1RA expression by approximately 80%.



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  		FIG. 1. Effects of treatment with dexamethasone on IL-1ß-induced PGS-2, IL-1ß, type I IL-1 receptor, or IL-1RA gene expression by cultured whole ovarian dispersates. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were cultured for 48 h under serum-free conditions in the absence or presence of IL-1ß (10 ng/ml), with or without dexamethasone (10-7 M). Total cellular RNA was extracted and subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2, IL-1ß, type I IL-1 receptor, IL-1RA, and RPL19. The intensity of the signals was quantified as described. Each panel depicts (in bar graph form) the mean ± SE of 3 experiments. In each individual experiment data were normalized relative to the IL-1ß value.

Inhibitory Effect of Treatment with Dexamethasone on IL-1ß-Induced PGS-2 Protein Synthesis by Cultured Whole Ovarian Dispersates

To examine a possible in vitro effect of dexamethasone on IL-1ß-induced PGS-2 protein synthesis, whole ovarian dispersates from immature rats were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without dexamethasone (10-7 M). As was the case for PGS-2 transcripts (Fig. 1), a marked inhibitory effect was noted in assessment of the IL-1-dependent content of immunoreactive PGS-2 (Fig. 2) as determined by immune Western blot analysis carried out with the PGS-2-directed antibody #9181. In addition to the 72-kDa holoenzyme, a 59-kDa proteolytic fragment was noted, in keeping with previous observations [31, 32].



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  		FIG. 2. Inhibitory effect of treatment with dexamethasone on IL-1ß-induced protein synthesis by cultured whole ovarian dispersates. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without dexamethasone (10-7 M). The immunoreactive content of PGS-2 (antibody #9181) was determined by immune Western blot analysis (50 µg protein per lane). In addition to the 72-kDa holoenzyme, a 59-kDa proteolytic fragment was noted, in keeping with previous observations [36, 37].

Inhibitory Effect of Dexamethasone on IL-1ß-Induced Ovarian PGS-2 Expression and Activity: Dose Requirements

To further characterize the inhibitory effect of dexamethasone on ovarian IL-1ß-induced PGS-2 gene expression, we examined its dose requirements. Whole ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without increasing concentrations (10-9-10-6 M) of dexamethasone. As shown in Figure 3 (left panel), treatment with dexamethasone produced dose-dependent decrements in PGS-2 gene expression, the first significant (p < 0.05) decrease being detected at the 10-8 M dose level (approximate ED50 = 7 x 10-9 M). Comparable observations were made for the accumulation of media PGE2 (Fig. 3; right panel), the first significant (p < 0.01) decrease being noted at the 10-9 M dose level.



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  		FIG. 3. Inhibitory effect of dexamethasone on IL-1ß-induced ovarian PGS-2 expression and activity: dose requirements. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without dexamethasone (10-9-10-6M). Total RNA was extracted and subjected to a solution hybridization RNase protection assay with antisense riboprobes corresponding to rat PGS-2 and RPL19. The intensity of the signals was quantified as described. In each individual experiment, data were normalized relative to the IL-1ß value. Additionally, the medium content of PGE2 was measured by RIA. The upper left and right panels depict the mean ± SE of 3 experiments. The lower left panels depict representative autoradiographs.

Inhibitory Effect of Dexamethasone on IL-1ß-Induced Ovarian PGS-2 Expression: Time Dependence

To further characterize the inhibitory effect of dexamethasone on IL-1ß-induced ovarian PGS-2 gene expression, we examined its time dependence. Whole ovarian dispersates were cultured for the duration indicated (up to 48 h) in the absence or presence of IL-1ß (10 ng/ml), with or without dexamethasone (10-7 M). As shown in Figure 4, treatment with dexamethasone produced significant (p < 0.01) inhibition (97.8%) of PGS-2 gene expression as early as 24 h into the culture period. Equally significant (p < 0.01) suppression (93.8%) was noted at the 48-h time point. A modest but distinct and measurable signal was noted for controls.



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  		FIG. 4. Inhibitory effect of dexamethasone on IL-1ß-induced ovarian PGS-2 expression: time dependence. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were cultured for the duration indicated (up to 48 h) in the absence or presence of IL-1ß (10 ng/ml), with or without dexamethasone (10-6 M). Total RNA was extracted and subjected to a solution hybridization RNase protection assay with antisense riboprobes corresponding to rat PGS-2 and RPL19. The intensity of the signals was quantified as described. The upper panel depicts (in bar graph form) the mean ± SE of 3 experiments. In each individual experiment, data were normalized relative to the 48-h IL-1ß value. The lower panels depict representative autoradiographs.

Inhibitory Effect of Glucocorticoids on IL-1ß-Induced Ovarian PGS-2 Expression: Rank Order of Potency

To begin to evaluate the role of the glucocorticoid receptor in mediating the dexamethasone effect, whole ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), increasing concentrations (10-9-10-6 M) of the indicated glucocorticoid, or combinations thereof. As Figure 5 shows, all the agonists tested produced dose-dependent decrements in the ability of IL-1ß to up-regulate PGS-2 transcripts, the rank order of potency being dexamethasone > prednisolone = cortisol (ED50s of 7 x 10-9 M, 6 x 10-8 M, and 7 x 10-8 M, respectively).



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  		FIG. 5. Inhibitory effect of glucocorticoids on IL-1ß-induced ovarian PGS-2 expression: rank order of potency. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), increasing concentrations (10-9-10-6 M) of the indicated glucocorticoid, or combinations thereof. Total cellular RNA was extracted and subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. The intensity of signals was quantified as described. The upper panel depicts (in bar graph form) the mean ± SE of 3 experiments. In each individual experiment, data were normalized relative to the IL-1ß value. The lower panels depict representative autoradiographs.

Inhibitory Effect of Steroid Class Representatives on IL-1ß-Induced Ovarian PGS-2 Expression: Specificity

To evaluate the specificity of the dexamethasone effect, the impact of other steroid class representatives was also evaluated. Whole ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), increasing concentrations (10-9-10-6 M) of the indicated steroid class representative, or combinations thereof. As shown in Figure 6, synthetic agonists representative of the progestin (R-5020), androgen (R-1881), and estrogen (DES) steroid series were without effect on the ability of IL-1ß to up-regulate ovarian PGS-2 expression.



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  		FIG. 6. Inhibitory effect of steroid class representatives on IL-1ß-induced ovarian PGS-2 expression: specificity. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), increasing concentrations (10-9-10-6M) of the indicated steroid class representative, or combinations thereof. Total cellular RNA was extracted and subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. The intensity of signals was quantified as described. The upper panel depicts the mean ± SE of 3 experiments. In each individual experiment, data were normalized relative to the IL-1ß value. The lower panels depict representative autoradiographs. * = p < 0.05; ** = p < 0.01.

Inhibitory Effect of Dexamethasone on IL-1ß-Induced Ovarian PGS-2 Expression: Reversal by Steroid Receptor Antagonists

To further establish that the ability of dexamethasone to suppress IL-1ß-induced PGS-2 gene expression is glucocorticoid receptor-mediated, whole ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß, IL-1ß plus dexamethasone, the indicated steroid receptor antagonist, or combinations thereof. As shown in Figure 7, cotreatment with RU-486, an established glucocorticoid/progesterone receptor antagonist [33, 34], all but abolished the ability of dexamethasone to inhibit IL-1ß-induced (but not basal) PGS-2 expression. In contrast, the concurrent provision of tamoxifen, flutamide, or spironolactone (established antagonists of the estrogen, androgen, and aldosterone receptors, respectively) was without effect.



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  		FIG. 7. Inhibitory effect of dexamethasone on IL-1ß-induced ovarian PGS-2 expression: reversal by steroid receptor antagonists. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), IL-1ß plus dexamethasone (10-7 M), the indicated steroid receptor antagonist (10-6 M), or combinations thereof. Total cellular RNA was extracted and subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. The intensity of the signals was quantified as described. The top panel depicts (in bar graph form) the mean ± SE of 3 experiments. In each individual experiment, data were normalized relative to the IL-1ß effect. The lower panels depict representative autoradiographs.

Inhibitory Effect of Dexamethasone on IL-1ß-Induced Ovarian PGS-2 Expression: Role of theSphingomyelin-Ceramide Cycle

Given the ability of dexamethasone to suppress IL-1 hormonal action, we undertook to begin localization of the relevant site(s) of inhibition in the IL-1 transduction cascade. Whole ovarian dispersates were initially cultured for 24 h without treatment. Thereafter, the cells were washed and reincubated for up to 2 h in the absence or presence of IL-1ß or the indicated representative of the sphingomyelin cycle, with or without dexamethasone (10-7 M). As Figure 8 shows, treatment with sphingomyelinase (0.3 µ/ml), sphingosine (10 µM), or C2-ceramide (30 µM) resulted in significant increments in the steady-state levels of PGS-2 transcripts (7.7-, 11.7-, and 33.3-fold, respectively). However, the concurrent application of dexamethasone produced significant (p < 0.01) inhibition of the ability of sphingomyelinase, sphingosine, or C2-ceramide to up-regulate PGS-2 expression (82.1%, 71.6%, and 72.2% inhibition, respectively).



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  		FIG. 8. Inhibitory effect of dexamethasone on IL-1ß-induced ovarian PGS-2 expression: role of the sphingomyelin-ceramide cycle. Whole ovarian dispersates (1.5 x 106 viable cells per dish) were initially cultured for 24 h without treatment. Thereafter, the cells were cultured for a further 1 h in the absence or presence of IL-1ß (10 ng/ml; 1 h), sphingomyelinase (SMase; 0.3 µ/ml; 1 h), sphingosine (SPH; 10 µM; 2 h), or C2-ceramide (CER; 30 µM; 2 h), with or without dexamethasone (10-7 M). Total cellular RNA was extracted and subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. The intensity of the signals was quantified as described. The upper panels depict (in bar graph form) the mean ± SE of 3 experiments. In each individual experiment, data were normalized relative to the IL-1ß value. The lower panels depict representative autoradiographs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A growing body of literature supports the proposition that ovulation may constitute an inflammatory-like process [1]. Indeed, ovulation appears to display the key attributes of the classic inflammatory process including heat, swelling, redness, temporary loss of function, and perhaps even pain. Yet another corollary of ovulation is the biosynthesis of prostaglandins, a phenomenon first suggested by Kuehl et al. [35]. This periovulatory gonadotropin-driven event is due, at least in part, to the promotion of PGS expression/activity [36–39]. Indeed, pharmacologic [2–4] or genetic [5] inhibition/elimination of PGS activity has been reproducibly shown to arrest follicular rupture. Although the precise role of prostaglandins in the ovulatory cascade remains uncertain, it is highly likely that prostaglandins may serve as coordinating messengers for a series of ovulation-associated phenomena such as the induction of periovulatory hyperemia [40–42] and the promotion of collagenolysis [43].

Glucocorticoids have been used for decades as clinical tools to suppress both the immune response and the process of inflammation. However, the precise molecular and cellular mechanism(s) underlying the immunosuppressive property of glucocorticoids remains poorly understood. In part, glucocorticoids may markedly decrease cytokine secretion and thus effectively block the activation of the immune system [44]. In this context, the inhibition of nuclear factor kappa B, a regulator of immune system and inflammation genes, has been shown to be targeted for a decrease by glucocorticoids [45, 46].

The present study concerns the possibility that the antiovulatory effect of glucocorticoids is attributable to their ability to inhibit inflammation in general and in the ovary in particular. Specifically, the ability of glucocorticoids to suppress one component of the inflammatory response, i.e., prostaglandin biosynthesis, was assessed. The ability of glucocorticoids to inhibit prostaglandin biosynthesis may be due to the suppression of PGS-2 activity [47]. Glucocorticoids could also exert a direct effect by diminishing the steady-state levels of PGS-2 transcripts [48, 49]. Either way, glucocorticoid-mediated suppression of PGS-2 activity may have a profound effect on the generation of inflammatory mediators. Our present findings establish the ability of dexamethasone to exert a significant inhibitory effect on IL-1-supported ovarian PGS-2 activity (Fig. 3). This phenomenon was associated with a marked decrease in the steady-state levels of IL-1-supported PGS-2 transcripts (Fig. 1 and Figs. 3–8). These findings notwithstanding, note is made of observations exemplified in Figure 3 showing that dexamethasone at the 10-9 M level was able to significantly suppress PGE2 production without any affect on PGS-2 expression. Either 10-9 M dexamethasone can inhibit PGS-2 activity without altering expression at this concentration, or 10-9 M dexamethasone inhibits PGE2 production at a site other than PGS-2.

It is generally agreed that glucocorticoids bind to a nuclear glucocorticoid receptor, a member of the steroid hormone receptor superfamily, which acts as a transcription factor [50]. It must be assumed that the ability of dexamethasone to inhibit ovarian PGS-2 is in fact glucocorticoid receptor mediated. This presumption appears to be supported by the demonstration that the dexamethasone effect is characterized by an expected rank order of potency of dexamethasone > prednisolone = cortisol (Fig. 5). Moreover, treatment with synthetic agonists representative of the progestin (R-5020), androgen (1881), and estrogen (DES) steroid series proved to be without effect (Fig. 6). Finally, cotreatment with RU-486, an established glucocorticoid receptor antagonist [44, 45], all but abolished the ability of dexamethasone to down-regulate IL-1-supported PGS-2 transcripts (Fig. 7).

In view of the rank order of potency described above, consideration might be given to the possible role of the known ovarian isoforms of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). Although dexamethasone may constitute a poor substrate for 11ß-HSD, there is little question that the relative potency of cortisol could be substantially reduced by virtue of metabolism. It is likely that the degree of metabolism of prednisolone by 11ß-HSD is intermediate between that of cortisol and dexamethasone. Consequently, the rank order of potency stated above may reflect not only relative affinities for the glucocorticoid receptor but also relative differences in the extent of metabolism by 11ß-HSD.

The precise site(s) wherein dexamethasone perturbs the IL-1 transduction signal remain uncertain. If nothing else, it is likely that the ability of dexamethasone to suppress IL-1-induced PGS-2 gene expression may be due, if only in part, to the 50% reduction in the steady-state levels of transcripts corresponding to the type I IL-1 receptor (Fig. 1). However, postreceptor elements appear to be involved as well. Indeed, dexamethasone proved effective in suppressing the induction of PGS-2 by congeners of the sphingomyelin-ceramide cycle, whose role in the transduction of the IL-1 signal is the subject of active investigation [51,52]. Specifically (Fig. 8), dexamethasone appeared to attenuate the activity of C2-ceramide, a cell preamble analogue of ceramide and a putative postreceptor second messenger of IL-1. Comparable data were noted for sphingomyelinase, a cell membrane-anchored enzyme and a putative proximal effector capable of degrading cell membrane sphingomyelin into ceramide. The activity of sphingosine, a metabolite of ceramide, was likewise arrested. Taken together, these observations suggest that the ability of dexamethasone to attenuate ovarian IL-1 action may be post-ceramide in location. Studies are currently under way to identify the downstream target(s) of dexamethasone in the IL-1 transduction cascade.

The association between Cushing's syndrome and ovarian dysfunction is well documented [17]. Autopsy of ovarian material revealed the absence of growing follicles and a decreased complement of primordial follicles. Moreover, glucocorticoid excess of an iatrogenic nature was shown to produce ovulatory dysfunction in 7 of 11 normally cycling women [20]. Comparable observations have been reported for various mammalian species in association with the administration of exogenous glucocorticoids [17–24]. Although the precise cellular and molecular mechanism(s) involved remains uncertain, consideration must be given to a hypothalamic/pituitary effect. However, the possibility of a direct ovarian effect cannot be excluded. In this context, previous observations have shown glucocorticoids to exert a variety of cytodifferentiative effects at the level of the rat granulosa cell [53–56]. Given the ability of glucocorticoids to suppress ovarian PGS-2 activity (and by extension prostaglandin biosynthesis), we speculate that these phenomena account, if only in part, for the antiovulatory activity of glucocorticoids. Such observations are supported by the recognition that prostaglandin biosynthesis is obligatory to the ovulatory process [2–5].

In vitro, dexamethasone was active at concentrations as low as 10 nM (Fig. 6), although statistically significant effects were apparent only at the 10 nM dose. Circulating levels of dexamethasone were 2.5 x 10-8 M [57] after ingestion of 0.75–2 mg of the drug. Higher circulating concentrations are likely after use of immunosuppressive doses of dexamethasone.


   ACKNOWLEDGMENTS
 
The authors wish to thank Ms. Carol E. Resnick and Ms. Cornelia T. Szmajda for their invaluable assistance and advice in the preparation of this article.


   FOOTNOTES
 
1 This work was supported in part by NIH grant HD-30288 (E.Y.A.). Back

2 Correspondence and current address: Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, ARUP II, Mail Box #20, Suite 1100, Room 109, 546 Chipeta Way, Salt Lake City, UT 84108. FAX: 801 585 9256; eadashi{at}hsc.utah.edu Back

3 Current address: Department of Obstetrics and Gynecology, University of Tokushima School of Medicine, Tokushima City, Japan. Back

4 Current address: Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Tokyo 181, Japan. Back

5 Current address: Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, UT 84108. Back

Accepted: November 25, 1998.

Received: September 3, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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