Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saito, J.
Right arrow Articles by Adashi, E. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saito, J.
Right arrow Articles by Adashi, E. Y.
Agricola
Right arrow Articles by Saito, J.
Right arrow Articles by Adashi, E. Y.
Biology of Reproduction 65, 1759-1765 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Interleukin 1 Upregulates Ovarian Prostaglandin Endoperoxide Synthase-2 Expression: Evidence for Prostaglandin-Dependent/Ceramide-Independent Transcriptional Stimulation and for Message Stabilization1

Juichiro Saito4,a,c, Motomu Ando3,a,c, Daniel Sussmanb, Hiroaki Negishic, Gretchen Kingc, and Eli Y. Adashi2,a,c

a Divisions of Reproductive Endocrinology and b Human Genetics, Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, Maryland 21201 c Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Huntsman Cancer Institute, Salt Lake City, Utah 84112


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently documented a marked dependence of ovarian prostaglandin endoperoxide synthase (PGS)-2 transcripts, proteins, and activity on interleukin (IL) 1, a putative intermediary in the ovulatory cascade. The purpose of the present study was to characterize the cellular and molecular mechanisms underlying the ability of IL-1ß to upregulate the steady-state levels of ovarian transcripts corresponding to PGS-2. Results of studies designed to enrich or deplete nitric oxide strongly suggest that the stimulatory effect of IL-1ß on ovarian PGS-2 expression is independent of nitric oxide. Utilization of a series of agents designed to simulate or enhance transduction via the sphingomyelin ceramide cycle suggests that the long-term stimulatory effect of IL-1ß on ovarian PGS-2 gene expression is independent of ceramide. In contrast, inhibition of prostaglandin biosynthesis with a series of distinct inhibitors suggests that the ability of IL-1ß to upregulate ovarian PGS-2 transcripts is due, if only in part, to the generation of endogenous prostaglandin estradiol-17ß (E2). Inhibition of protein biosynthesis suggested that the IL-1ß-induced PGS-2 gene expression required de novo protein biosynthesis. Our findings revealed substantial IL-1ß-mediated stabilization of PGS-2 transcripts, as assessed by a threefold increase in the half-life of the message. We have also observed the ability of IL-1ß to upregulate the transcription of PGS-2 promoter constructs subjected to transient transfection into whole-ovarian dispersates (twofold increase as assessed by activation of the luciferase reporter gene). Taken together, these findings suggest that the stimulatory effect of IL-1ß on PGS-2 expression is 1) independent of nitric oxide as well as ceramide, 2) dependent on prostaglandin E2, 3) contingent on de novo protein biosynthesis, and 4) accounted for by both increased transcription and message stabilization. These observations provide indirect support for the hypothesis that IL-1ß, acting in part through PGS-2 (an obligatory ovulatory principal), may constitute a key intermediary in the ovulatory cascade.

cytokines, female reproductive tract, ovary, ovulation, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A growing body of direct and indirect evidence supports the hypothesis that intraovarian interleukin (IL) 1ß may constitute an intermediary in the ovulatory process [1–12]. Specifically, intraovarian IL-1ß has been found to be induced by the midcycle surge [6] and to upregulate key intraovarian components of the ovulatory cascade, such as collagenase activity [11] and proteoglycan biosynthesis [10]. Yet another corollary of ovulation is the biosynthesis of prostaglandins [13, 14], a phenomenon first suggested by Kuehl et al. [15]. This periovulatory, gonadotrophin-driven event is due, at least in part, to the promotion of prostaglandin endoperoxide synthase (PGS) activity [16–18]. Indeed, pharmacologic [19–23] or genetic [24] inhibition/elimination of PGS activity has been reproducibly shown to arrest follicular rupture. In that context, we have disclosed a marked dependence of ovarian PGS-2 transcripts, proteins, and activity on IL-1 [25]. The effects of IL-1 have proved to be relatively specific, contingent on somatic cell-cell cooperation, dependent on dose and time, and mediated by the IL-1 receptor. The ability of IL-1ß to promote ovarian prostaglandin biosynthesis was also been reported by other investigators [26–28].

The purpose of the present study was to characterize the cellular and molecular mechanisms underlying the ability of IL-1ß to upregulate the steady-state levels of ovarian transcripts corresponding to PGS-2.


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

Immature female Sprague-Dawley 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). The Dulbecco modified Eagle medium/F-12 medium, opti-MEM I reduced-serum medium, penicillin-streptomycin solution, L-glutamine, trypan blue stain, and BSA were from Life Technologies (Grand Island, NY). Collagenase (Clostridium histolyticum; CLS type I; 144 U/mg) was from Worthington Biochemical Corp. (Freehold, NJ). The DNase (bovine pancreas), aminoguanidine hemisulfate salt (AG), cycloheximide (CHX), S-nitroso-n-acetyl-penicilamine (SNAP), prostaglandin E2 (PGE2), diclofenac, sphingomyelinase, D-sphingosine, and RNase A were from Sigma Chemical Co. (St. Louis, MO). The C6-ceramide and C8-ceramide were from Biomol Research Labs, Inc. (Plymouth Meeting, PA). The T7 and SP6 RNA polymerases, pGEM7Zf+, and other molecular biology-grade reagents were from Promega (Madison, WI). The [32P]UTP was from New England Nuclear (Boston, MA).

Tissue Culture Procedures

In the context of transfection studies (see Fig. 8), granulosa cells were prepared for culture by repeatedly stabbing the ovaries with a 25-gauge needle. Thereafter, the remaining ovarian shells (i.e., the ovarian residuum after harvesting of granulosa cells) were collagenase-dispersed as previously described [29]. Whole-ovarian dispersates (1.0 x 106 cells/dish) consisted of a 7:3 mixture of granulosa cells and shells [30], which were cultured as previously described [29]. For all other experiments, whole-ovarian material was collagenase-dispersed and cultured as previously described [29]. Briefly, whole-ovarian dispersates (2.5 x 105 or 2 x 104 viable cells) were inoculated onto 35 tissue culture dishes (10- or 16-mm diameter, respectively; Falcon Plastics, Oxnard, CA; Costar, Cambridge, MA) containing 1 ml of McCoy 5a medium (modified without serum) supplemented with insulin (1 µg/ml), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin sulfate (100 µg/ml). Insulin was included in all cultures to potentiate hCG-stimulated steroidogenesis and, thereby, to magnify the inhibitory effects of IL-1. It is estimated that only 20% of the cells in whole-ovarian dispersates are theca-interstitial in origin and, thus, capable of responding to hCG. Highly purified theca-interstitial cells were obtained over a Percoll gradient according the method described by Magoffin and Erickson [31] and then plated (5 x 104 cells/culture) as described above. To assess the relative purity of the latter preparation, equal numbers of highly purified theca-interstitial or granulosa cells were compared for their aromatase activity using the tritiated water assay as described below [29, 32]. Aromatase activity in highly purified theca-interstitial cells was 0.8%–3% that of granulosa cells. This suggests that, in our hands, the highly purified theca-interstitial cells are contaminated with <3% granulosa cells. All experimental agents (diluted in sterile culture media) were added in 50-µl aliquots. Cell cultures were maintained for 96 h at 37°C under a water-saturated atmosphere of 5% (v/v) CO2 and 95% (v/v) air.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 8. IL-1ß-induced PGS-2 gene expression: gene transcription. Collagenase-dispersed ovarian shells or collagenase-dispersed shells (30%) plus isolated granulosa cells (70%) (1.0 x 106 cells/dish) were initially cultured for 24 h in the absence of treatment. Thereafter, the cells were washed and subjected to transient transfection with PGS-2 promoter/luciferase reporter constructs as described in Materials and Methods and treated for 8 h in the absence or presence of IL-1ß. The scale for plasmids pPG891LUC and pXP1 is presented on left and the scale for pSU2 LUC on right

Aromatase Activity

Androstenedione was obtained from Sigma. Ready-solv EP was purchased from Beckman Instruments, Inc. (Fullerton, CA). Chloroform (spectrophotometric grade) was obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ). Carbon decolorizing alkaline Norit-A was purchased from Fisher Scientific Co. (Chemical Manufacturing Division, Fair Lawn, NJ). Dextran T-70 was obtained from Pharmacia Fine Chemicals (Division of Pharmacia, Inc., Piscataway, NJ). The [1ß,2ß-3H]androstenedione (40 Ci/mmol) was obtained from New England Nuclear and used to prepare [1ß-3H]androstenedione. Tracers were checked for purity before use by thin-layer chromatography (ether:hexane, 3:1 [v/v]).

The cells were initially cultured for up to 3 days in an androstenedione-free medium in the absence or presence of FSH, with or without insulin-like growth factor (treatment interval). At the conclusion of this period, the media were discarded, and the cells were washed twice with 2-ml portions of medium and then reincubated for an additional 8-h test interval in an androstenedione (10-7 M, unless indicated otherwise)-supplemented medium, with or without [1ß-3H]androstenedione (0.13 µCi/3.25 cpm; ~100 000 cpm). The 10-7 M androstenedione dose was selected to provide a saturating substrate concentration. At the end of the experiment, the media were collected and stored at -20°C until assayed for 3H20 as described below.

Aromatase activity was assessed from the stereospecific release of tritium from [1ß-3H]androstenedione to produce . Blanks established from identical incubations in the absence of cells were consistently associated with <100 cpm/dish. Collected media were extracted with five volumes of chloroform, and the aqueous phase was further subjected to treatment with an equal volume of dextran (0.25% [w/v])-activated charcoal (2.5% [w/v]) solution for 10 min at 4°C. After centrifugation at 800 x g, the supernatant radioactivity was subjected to liquid scintillation counting (Packard Instrument Co., Anbac Industries, New York, NY) in ready-solv EP for 5 min at 35% efficiency. Aromatase activity was expressed in terms of accumulation (cpm per 8 h of culture).

Nitrite Assays

Nitrite (NO2-) concentrations in conditioned media were determined as previously described [33, 34] using a modification of a colorimetric method adapted to a microtiter plate procedure.

RIA for PGE2

The RIA for PGE2 was carried out as previously described [35].

Nucleic Acid Probes

The rat PGS-2 cDNA [36] was generously provided in Bluescript vector by Drs. Daniel Hwang and Shuenn S. Liou of the Pennington Biochemical Research Center (Louisiana State University, Baton Rouge, LA). A 385 XbaI-EcoRI fragment of the original PGS-2 cDNA was subcloned into a pGEM7Zf+ vector. The T7-driven transcription of the HindIII-linearized construct yielded a 328-nucleotide riboprobe, which on hybridization was projected to generate a 297-nucleotide protected fragment. The RPL19 probe was generated and employed as previously described [37].

RNA Extraction

The RNA of cultured cells and of tissues 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) or 160 Ci/mmol [{alpha}-32P]UTP (RPL19). The riboprobes were gel-purified as previously described [38] in an effort to eliminate transcribed products shorter than the full-length probes. The assay was performed as previously described [39]. Gels were exposed to XAR film (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 Image Quant Software (Molecular Dynamics). The hormonally independent RPL19 mRNA signal was used to normalize the PGS-2 mRNA data for possible variation in RNA loads. Specifically, the ratio of net protected signal (respective background subtracted) to net RPL19 was calculated for each sample and gene of interest.

Transient Transfection Studies

Whole-ovarian dispersates (1.0 x 106 cells/dish) were initially cultured without treatment for 24 h. At the conclusion of this treatment period, the cells were transfected for 8 h with the following constructs: pPGS891LUC, and pSV2LUC [40]. Both constructs were generously provided by Dr. Lee-Ho Wang, University of Texas-Houston (Houston, TX). The pPGS891LUC construct consisted of nucleotides -891 to +9 of the human PGS-2 gene promoter subcloned into a luciferase reporter vector, pXP1. The pSV2LUC construct consisted of the SV40 early promoter coupled to the luciferase reporter gene as described for pPGS891LUC. pXP1, a promoter-less vector, was used as a negative control. The lipid-mediated transfection methodology conformed to that employed by the Trans IT-100 products (PanVera Corporation, Madison, WI). At the conclusion of the transfection period, the cells were thoroughly washed, after which fresh media were added and the cells reincubated for 8 h in the absence or presence of IL-1ß (10 ng/ml). Thereafter, transfected cells were resuspended in cell-culture lysis buffer, and the resultant supernatant used to determine luciferase activity as per the luciferase assay system (Promega). Quantification of light emission was carried out with conventional TD-20e (Turner, Mt. View, CA) and Lumat LB9507 (Wallac, Inc. Gaitherburg, MD) luminometers.

Data Analysis

Except as noted, each experiment was replicated a minimum of three times. Data points are presented as mean ± SEM. Statistical significance (Fisher protected least significant difference) was determined by ANOVA and Student 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
 
IL-1ß-Induced PGS-2 Gene Expression by Cultured Whole-Ovarian Dispersates: Dependence on Nitric Oxide

To determine whether the effect of treatment with IL-1ß on PGS-2 gene expression is contingent on endogenously produced nitric oxide, whole-ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß, with or without AG. As shown in Figure 1, a "nitric oxide vacuum" was without significant effect on either the spontaneous or the IL-1ß-induced expression pattern of PGS-2. The validity of the preceding observations is strengthened by documentation (Fig. 2) of the ability of AG to inhibit IL-1-stimulated nitrite but not PGE2 accumulation. Moreover, a 48-h application of SNAP, an established nitric oxide generator [33], failed to alter the steady-state levels of PGS-2 transcripts (Fig. 3, left and middle panels) in the face of marked increments in nitrite accumulation (Fig. 3, right panel) above and beyond those elicited by IL-1ß. These observations suggest that the stimulatory effect of IL-1ß on ovarian PGS-2 expression is independent of nitric oxide.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 1. IL-1ß-induced PGS-2 gene expression by cultured whole-ovarian dispersates: dependence on nitric oxide. Whole-ovarian dispersates (1.5 x 106 cells/dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without AG (0.4 mM). The resultant RNA samples were subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. The left panel presents the mean ± SEM of three experiments. In each individual experiment, data were normalized relative to the IL-1ß value. The right panel shows a representative autoradiograph, wherein the full-length PGS-2 and RPL19 probes are in italics. Protected fragments are in bold



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 2. IL-1ß-induced ovarian PGS-2 gene expression: dependence on nitric oxide. Whole-ovarian dispersates (1.5 x 106 cells/dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without AG (0.4 mM). Media content of PGE2 (left panel) and nitrite (right panel) were determined as described in Materials and Methods



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 3. IL-1ß-induced ovarian PGS-2 gene expression: dependence on nitric oxide. Whole-ovarian dispersates (1.5 x 106 cells/dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without SNAP (0.1 mM). In some experiments, an additional dose of SNAP (0.1 mM) was added at the 24-h time point (24h/SNAP). The resultant RNA samples were subjected to an RNase protection assays using antisense riboprobes corresponding to rat PGS-2 and RPL19. The left panel shows the mean ± SEM of three experiments. In each individual experiment, data were normalized relative to the effect of IL-1ß. The middle panel shows a representative autoradiograph, wherein the full-length PGS-2 and RPL19 probes are in italics. Protected fragments are in bold. The right panel shows the medium content of nitrite, which was determined as described in Materials and Methods

IL-1ß-Induced Ovarian PGS-2 Gene Expression: Dependence on Ceramide

To determine whether the effect of treatment with IL-1ß on PGS-2 gene expression entails the intermediacy of the sphingomyelin-ceramide cycle, whole-ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß, C6-ceramide, C8-ceramide, sphingomyelinase, or sphingosine. As shown in Figure 4, treatment with ceramide, sphingomyelinase, or sphingosine was without effect on ovarian PGS-2 gene expression as assessed 48 h after application. These observations suggest that the long-term stimulatory effect of IL-1ß on ovarian PGS-2 gene expression is independent of ceramide.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 4. IL-1ß-induced ovarian PGS-2 gene expression: dependence on ceramide. Whole-ovarian dispersates (1.5 x 106 cells/dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), C6-ceramide (100 µM), C8-ceramide (100 µM), sphingomyelinase (SMase; 0.3 U/ml), or sphingosine (Sph; 10 µM). The resultant RNA samples were subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. Shown are the mean ± SEM of three experiments. In each individual experiment, data were normalized relative to the effect of IL-1ß

IL-1ß-Induced PGS-2 Gene Expression: Dependence on Prostaglandin

To determine if the IL-1ß-induced PGS-2 gene expression involves the intermediacy of prostaglandins, whole-ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß, diclofenac, PGE2, or combinations thereof. As shown in Figure 5, treatment with diclofenac resulted in significant (P < 0.001) inhibition (64.8%) of the IL-1ß effect. Moreover, the addition of PGE2 to either diclofenac-untreated or diclofenac-treated cells resulted in significant (P < 0.001) upregulation of IL-1ß-induced PGS-2 gene expression. These observations suggest that the ability of IL-1ß to upregulate ovarian PGS-2 transcripts is due, if only in part, to the generation of endogenous PGE2.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 5. IL-1ß-induced PGS-2 gene expression: dependence on prostaglandin. Whole-ovarian dispersates (1.5 x 106 cell/dish) were cultured for 48 h in the absence or presence of IL-1ß, diclofenac (Diclo), PGE2, or combinations thereof. The resultant samples were subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. The left panel depicts the mean ± SEM of three experiments. Data were normalized relative to the IL-1ß value in each individual experiment. The right panel shows a representative autoradiograph, wherein the protected fragments are in bold

IL-1ß-Induced Ovarian PGS-2 Gene Expression: Dependence on Protein Synthesis

To determine if IL-1ß-induced PGS-2 gene expression is contingent on intact protein biosynthesis, whole-ovarian dispersates were cultured for 48 h in the absence or presence of IL-1ß, with or without increasing concentrations of CHX. As shown in Figure 6 (left and middle panels), treatment with CHX led to complete blockade of the ability of IL-1ß to upregulate PGS-2 transcripts. A comparable effect was noted at the functional level as assessed by PGE2 accumulation (Fig. 6, right panel). Importantly, RPL19 transcripts remained unaffected (Fig. 6, middle panel), thereby arguing against an overt, general toxic effect of CHX on the cellular transcription system. These observations suggest that IL-1ß-induced PGS-2 gene expression requires de novo protein biosynthesis.



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 6. IL-1ß-induced ovarian PGS-2 gene expression: dependence on protein synthesis. Whole-ovarian dispersates (1.5 x 106 cells/dish) were cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml), with or without increasing concentrations of CHX (1–100 ng). The resultant RNA samples were subjected to an RNase protection assay by using antisense riboprobes corresponding to rat PGS-2 and RPL19. The left panel shows the mean ± SEM of three experiments. Data were normalized relative to the peak value in each individual experiment. The middle panel shows a representative autoradiograph, wherein protected fragments are in bold. The right panel depicts the corresponding accumulation of PGE2 in conditioned media

IL-1ß-Induced Ovarian PGS-2 Gene Expression: Message Stability

To determine whether the effect of IL-1ß is due to stabilization of the PGS-2 message, untreated and IL-1ß-pretreated, whole-ovarian dispersates were exposed to actinomycin D, an established inhibitor of transcription. As shown in Figure 7, pretreatment with IL-1ß resulted in significant stabilization of PGS-2 (but not of RPL19) transcripts, as assessed by a threefold prolongation in the half-life of the message. These observations suggest that the ability of IL-1ß to increase the steady-state levels of ovarian PGS-2 transcripts is due, in part, to stabilization of the corresponding transcript.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 7. IL-1ß-induced ovarian PGS-2 gene expression: message stability. Whole-ovarian dispersates (1.5 x 106 cells/dish) were initially cultured for 48 h in the absence or presence of IL-1ß (10 ng/ml). At the conclusion of this incubation period, the media were removed and replaced with fresh media containing actinomycin D (10 µg/ml) for up to 10 h. The resultant RNA samples were subjected to an RNase protection assay using antisense riboprobes corresponding to rat PGS-2 and RPL19. Data represent the mean ± SEM of three experiments and were normalized relative to 0 h

IL-1ß-Induced Ovarian PGS-2 Gene Expression: RNA Synthesis

To determine whether the effect of IL-1ß is due to increased transcription of the PGS-2 gene, whole-ovarian dispersates or collagenase-dispersed ovarian shells were initially cultured for 24 h in the absence of treatment. Thereafter, the cells were subjected to transient transfection with PGS-2 promoter/luciferase reporter constructs, as described in Materials and Methods. The cells so transfected were then treated for 8 h in the absence or presence of IL-1ß (10 ng/ml). As shown in Figure 8, treatment with IL-1ß resulted in a twofold increase (P < 0.005) in transcription, as assessed by activation of the luciferase reporter gene in whole-ovarian dispersates. A more modest, but statistically significant, increase was also noted in collagenase-dispersed ovarian shells. These observations suggest that the ability of IL-1ß to increase the steady-state levels of PGS-2 transcripts is due, in part, to enhanced transcription of the corresponding gene.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This laboratory has previously demonstrated the ability of IL-1 to stimulate the biosynthesis of prostaglandins in cultured whole-ovarian dispersates [35]. We have since documented that this prostaglandin-promoting property of IL-1 is due, in part, to the upregulation of ovarian secretory phospholipase A2 and cytosolic phospholipase A2 [41, 42]. In a more recent communication, we documented that the prostaglandin-promoting property of IL-1 is also due, in large measure, to the induction of PGS-1 and PGS-2 [25]. Consequently, the ability of IL-1 to promote ovarian prostaglandin biosynthesis involves the activation of several enzymatic steps along the biosynthetic cascade, leading to the genesis of prostaglandins.

To better characterize the IL-1 effect, we examined the possible intermediary role of nitric oxide. Although the role of nitric oxide in ovarian physiology remains undefined, its relevance to the ovulatory process has been proposed [43]. Given the proposed role of IL-1 in the ovulatory process [1] and its established ability to activate the inducible variety of ovarian nitric oxide synthase [33, 34, 44, 45], we examined the role of nitric oxide in IL-1 hormonal action. Our findings (Figs. 1–3) reveal that generation of a "nitric oxide vacuum" with AG, an established inhibitor of the inducible variety of ovarian nitric oxide synthase [33, 34, 44, 45], is without significant effect on IL-1 action. Conceivably, the IL-1 effect may then be mediated through activation of the sphingomyelin pathway, the role of which in transduction of the IL-1 signal is under active investigation [46–48]. However, treatment with C6- or C8-ceramide (i.e., cell-permeable analogues of ceramide), sphingomyelinase (i.e., an enzyme capable of degrading cell membrane sphingomyelin to ceramide), or sphingosine (i.e., a metabolite of ceramide) proved to be without effect on the steady-state levels of PGS-2 transcripts (Fig. 4). Consequently, the ability of IL-1 to upregulate PGS-2 transcripts appears to be independent of nitric oxide and ceramide.

To assess the possible involvement of eicosanoids in IL-1-hormonal action, we set out to evaluate the consequences of blockade of PGS activity as well as of replacement with exogenously provided PGE2, the most abundant prostaglandin produced by IL-1ß-treated, whole-ovarian dispersates [35]. Our findings reveal that blockade of IL-1ß-induced prostaglandin biosynthesis with diclofenac results in substantial attenuation of the IL-1ß effect (Fig. 5). Moreover, addition of PGE2 to either diclofenac-untreated or diclofenac-treated cells substantially augments the effect of IL-1ß. These observations suggest that the ability of IL-1ß to upregulate PGS-2 gene expression is due, if only in part, to the activation of endogenous prostaglandin biosynthesis.

Our current observations suggest that blockade of protein biosynthesis results in complete abrogation of IL-1 hormonal action (Fig. 6). Although the nature of the obligatory protein remains unknown, it is tempting to speculate that the synthesis of the type I IL-1 receptor is at play. Clearly, however, numerous, more distal proteins may also be relevant. Future studies will focus on the potential role of IL-1 in upregulating the phospholipase A2-activating protein, the prostaglandin transporter, and/or prostaglandin receptors.

To determine whether the effect of IL-1 is due to stabilization of the PGS-2 message, untreated and IL-1-pretreated, whole-ovarian dispersates were exposed to actinomycin D, an established inhibitor of transcription. Our findings (Fig. 7) reveal that pretreatment with IL-1ß is associated with stabilization of the PGS-2 message, as assessed by a reduction of its half-life. However, our findings also reveal that the ability of IL-1 to upregulate the steady-state levels of ovarian PGS-2 transcripts is due, in part, to enhanced transcription of the corresponding gene. These conclusions (Fig. 8) are based on the ability of IL-1 to increase the activity of a PGS-2 promoter-driven luciferase reporter gene [40] that was transiently transfected into whole-ovarian dispersates. Accordingly, the upregulatory effect of IL-1ß on ovarian PGS-2 transcripts appears to represent a combination of increased transcription and enhanced message stabilization. In this respect, our present findings are in keeping with those reported by Srivastava et al. [49] for the renal mesangial cell [49].


   ACKNOWLEDGMENTS
 
The authors wish to thank Ms. Cornelia T. Szmajda, Ms. Linda Elder, Ms. Andrea Raposa, and Ms. Michelle S. Lewandowski for their invaluable assistance in the preparation of this manuscript. The authors also wish to thank Dr. A. Tsafriri of the Weitzman Institute of Science (Rehovot, Israel) for helpful discussion.


   FOOTNOTES
 
First decision: 22 November 2000.

1 Supported in part by NIH Research Grant HD-30288 (to E.Y.A.). Back

2 Correspondence: Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Huntsman Cancer Institute, 2000 Circle of Hope, Room 5221, Salt Lake City, UT 84112. FAX: 801 585 9256; eadashi{at}hsc.utah.edu Back

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

4 Current address: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa 216, Japan. Back

Accepted: July 19, 2001.

Received: October 18, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ben-Shlomo I, Adashi EY. Interleukin-1 as a mediator in the ovulatory sequence: evidence for a meaningful role of cytokines in ovarian physiology. Curr Sci Ser 1994; 1:187-192
  2. Brannstrom M, Wang L, Norman RJ. Ovulatory effect of interleukin-1 on the perfused rat ovary. Endocrinology 1993; 132:399-404[Abstract]
  3. Takehara Y, Dharmarajan AM, Kaufman G, Wallach EE. Effects of interleukin-1ß on ovulation in the in vitro perfused rabbit ovary. Endocrinology 1994; 134:1788-1793[Abstract]
  4. Peterson CM, Hales HA, Hatasaka HH, Mitchell MD, Rittenhouse L, Jones KP. Interleukin-1ß (IL-1ß) modulates prostaglandin production and the natural IL-1 receptor antagonist inhibits ovulation in the optimally stimulated rat ovarian perfusion model. Endocrinology 1993; 133:2301-2306[Abstract]
  5. Simon C, Tsafriri A, Chun S-Y, Piquette GN, Dang W, Polan ML. Interleukin-1 receptor antagonist suppresses hCG-induced ovulation in the rat. Biol Reprod 1994; 51:662-667[Abstract]
  6. Hurwitz A, Ricciarelli E, Botero L, Rohan RM, Hernandez ER, Adashi EY. Endocrine- and autocrine-mediated regulation of rat ovarian (theca-interstitial) interleukin-1ß gene expression: gonadotropin-dependent preovulatory acquisition. Endocrinology 1992; 129:3427-3429[Abstract]
  7. Hurwitz A, Loukides J, Ricciarelli E. Human intraovarian interleukin-1 (IL-1) system: highly-compartmentalized and hormonally-dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist. J Clin Invest 1992; 89:1746-1754
  8. Simon C, Frances A, Piquette G, Polan ML. Immunohistochemical localization of the interleukin-1 system in the mouse ovary during follicular growth, ovulation, and luteinization. Biol Reprod 1994; 50::449-457[Abstract]
  9. Brannstrom M, Norman RJ, Seamark RF, Robertson SA. Rat ovary produces cytokines during ovulation. Biol Reprod 1994; 50:88-94[Abstract]
  10. Kokia E, Hurwitz A, Ben-Shlomo I, Adashi EY, Yanagishita M. Receptor mediated stimulatory effects of IL-1ß on hyaluronic acid and proteoglycan biosynthesis by cultured rat ovarian cells: role for heterologous cell-cell interactions. Endocrinology 1993; 133:2391-2394[Abstract]
  11. Hurwitz A, Dushnik M, Solomon H, Ben-Chetrit A, Finci-Yeheskel Z, Milwidsky A, Meyer M, Adashi EY, Yagel S. Cytokine-mediated regulation of rat ovarian function: interleukin-1 stimulates the accumulation of 92-kD gelatinase. Endocrinology 1993; 132:2709-2714[Abstract]
  12. Hurwitz A, Finci-Yeheskel Z, Dushnik M, Milwidsky A, Ben-Chetrit A, Yagel S, Adashi EY, Mayer M. Cytokine-mediated regulation of rat ovarian function: interleukin-1 inhibits plasminogen activator activity through the stimulation of plasminogen activator inhibitor-1 (PAI-1). Mol Cell Endocrinol 1994; 101:307-315[CrossRef][Medline]
  13. Tsafriri A, Lindner HR, Zor U, Lamprecht SA. Physiological role of prostaglandins in the induction of ovulation. Prostaglandins 1972; 2::1-10[CrossRef][Medline]
  14. Lindner HR, Zor U, Kohen F, Bauminger S, Amsterdam A, Lahav M, Salomon Y. Significance of prostaglandins in the regulation of cyclic events in the ovary and uterus. Adv Prostaglandin Thromboxane Leukotriene Res 1980; 8:1371-1390
  15. Kuehl FA, Humes JL, Tarnoff J, Cirillo VJ, Ham EA. Prostaglandin receptor site: evidence for an essential role in the action of luteinizing hormone. Science 1970; 169:883-886[Abstract/Free Full Text]
  16. Huslig RL, Malik A, Clark M. Human chorionic gonadotropin stimulation of immunoreactive prostaglandin synthase in the rat ovary. Mol Cell Endocrinol 1987; 50:237-246[CrossRef][Medline]
  17. Hedin L, Gaddy-Kurten G, Kurten R, DeWitt DL, Smith WL, Richards JS. Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation. Endocrinology 1987; 121:722-731[Abstract]
  18. Sirois J. Induction prostaglandin endoperoxide synthase 2 by human chorionic gonadotropin in bovine preovulatory follicles in vivo. Endocrinology 1994; 135:841-848[Abstract]
  19. Tsafriri A, Koch Y, Lindner HR. Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2. Prostaglandins 1973; 3:461-467[CrossRef][Medline]
  20. Diaz-Infante AJ, Wright KH, Wallach EE. Effects of indomethacin and prostaglandin F2{alpha} on ovulation and ovarian contractility in the rabbit. Prostaglandins 1974; 5:567-581[CrossRef][Medline]
  21. Hamada Y, Bronson A, Wright KH, Wallach EE. Ovulation in the perfused rabbit ovary: the influences of prostaglandins and prostaglandin inhibitors. Biol Reprod 1977; 17:58-63[Abstract]
  22. Downey BR, Ainsworth L. Reversal of indomethacin blockade of ovulation in gilts by prostaglandins. Prostaglandins 1980; 19:17-22[CrossRef][Medline]
  23. Espey LL, Norris C, Saphire D. Effects of time and dose of indomethacin on follicular prostaglandins and ovulation in the rabbit. Endocrinology 1986; 119:746-754[Abstract]
  24. Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, Covington MB, Contel NR, Eng VM, Collins RJ, Czerniak PM, Gorry SA, Trzaskos JM. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 1995; 378:406-409[CrossRef][Medline]
  25. Ando M, Kol S, Ruutiainen-Altman K, Sirois J, Payne DW, Rohan RM, Adashi EY. Rat ovarian prostaglandin endoperoxide synthase-1 and 2: periovulatory expression of granulosa cell-based interleukin-1-dependent enzymes. Endocrinology 1998; 139:2501-2508[Abstract/Free Full Text]
  26. Brannstrom M, Wang L, Norman RJ. Effects of cytokines on prostaglandin production and steroidogenesis of incubated preovulatory follicles of the rat. Biol Reprod 1993; 48:165-171[Abstract]
  27. Pitzel L, Jarry H, Wuttke W. Effects and interactions of prostaglandin F2{alpha}, oxytocin, and cytokines on steroidogenesis of porcine luteal cells. Endocrinology 1993; 132:751-756[Abstract]
  28. Townson DH, Pate JL. Regulation of prostaglandin synthesis by interleukin-1 in cultured bovine luteal cells. Biol Reprod 1994; 51:480-485[Abstract]
  29. Hurwitz A, Payne DW, Packman JN, Andreani CL, Resnick CE, Hernandez ER, Adashi EY. Cytokine-mediated regulation of ovarian function: interleukin-1 inhibits gonadotropin-induced androgen biosynthesis. Endocrinology 1991; 129:1250-1256[Abstract]
  30. Erickson GE, Magoffin DA, Dyer CA, Hofeditz C. The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev 1985; 6:371-399[CrossRef][Medline]
  31. Magoffin DA, Erickson GF. Purification of ovarian thecal-interstitial cells by density gradient centrifugation. Endocrinology 1988; 122::2345-2347[Abstract]
  32. Adashi EY, Resnick CE, Brodie AM, Svoboda ME, Van Wyk JJ. Somatomedin-C-mediated potentiation of follicle-stimulating hormone-induced aromatase activity of cultured rat granulosa cells. Endocrinology 1985; 117:2313-2320[Abstract]
  33. Ben-Shlomo I, Adashi EY, Payne DW. The morphogenic/cytotoxic and prostaglandin-stimulating activities of interleukin-1ß in the rat ovary are nitric oxide-independent. J Clin Invest 1994; 94:1463-1469
  34. Ben-Shlomo I, Kokia E, Jackson M, Adashi EY, Payne DW. Interleukin-1ß stimulates nitrate production in the rat ovary: evidence for heterologous cell-cell interaction and for insulin-mediated regulation of the inducible isoform of nitric oxide synthase. Biol Reprod 1994; 51:310-318[Abstract]
  35. Kokia E, Hurwitz A, Ricciarelli E, Tedeschi C, Resnick CE, Mitchell MD, Adashi EY. Interleukin-1 stimulates ovarian prostaglandin biosynthesis: evidence for heterologous contact-independent cell-cell interaction. Endocrinology 1992; 130:3095-3097[Abstract]
  36. Feng L, Sun W, Xia Y, Tang WW, Chanmugam P, Soyoola E, Wilson CB, Hwang D. Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys 1993; 307:361-368[CrossRef][Medline]
  37. Scherzer WJ, Ruutianen-Altman KSL, Putowski LT, Kol S, Adashi EY, Rohan RM. Detection and in vivo hormonal regulation of rat ovarian type I and type II interleukin-1 receptor mRNA: increased expression during the periovulatory period. J Soc Gynecol Invest 1996; 3:131-140[CrossRef][Medline]
  38. Kol S, Ben-Shlomo I, Adashi EY, Rohan RM. Simplified riboprobe purification using translucent straws as gel tubes. Gen Anal Biomol Eng 1996; 12:129-132
  39. Lowe WJ Jr, Roberts CT Jr, Lasky SR, LeRoith D. Differential expression of alternative 5' untranslated regions in mRNA encoding rat insulin-like growth factor 1. Proc Natl Acad Sci U S A 1987; 84::8946-8950[Abstract/Free Full Text]
  40. Tazawa R, Xu X-M, Wu KK, Wang L-H. Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun 1994; 203:190-199[CrossRef][Medline]
  41. Kol S, Ruutiainen-Altman K, Ben-Shlomo I, Payne DW, Ando M, Adashi EY. The rat ovarian phospholipase A2 system: gene expression, cellular localization, activity characterization and IL-1 dependence. Endocrinology 1997; 138:322-331[Abstract/Free Full Text]
  42. Kol S, Ben-Shlomo I, Ando M, Payne DW, Adashi EY. Interleukin-1 stimulates rat ovarian phospholipase A2 (PLA2) expression and activity: upregulation of both secretory and cytosolic PLA2. Endocrinology 1997; 138:314-321[Abstract/Free Full Text]
  43. Shukovsky L, Tsafriri A. The involvement of nitric oxide in the ovulatory process in the rat. Endocrinology 1994; 135:2287-2290[Abstract]
  44. Ellman C, Corbett JA, Misko TP, McDaniel M, Beckerman KP. Nitric oxide mediates interleukin-1-induced cellular cytotoxicity in the rat ovary: a potential role for nitric oxide in the ovulatory process. J Clin Invest 1993; 92:3053-3056
  45. Bonello N, McKie K, Jasper M, Andre L, Ross N, Braybon E, Brannstrom M, Norman RJ. Inhibition of nitric oxide: effects of interleukin-1ß-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat. Biol Reprod 1996; 54:436-445[Abstract]
  46. Hannun YA, Bell RM. Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science 1989; 243:500-507[Abstract/Free Full Text]
  47. Kolesnick R, Golde DW. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 1994; 77:325-328[CrossRef][Medline]
  48. Zhang Y, Kolesnick R. Signaling through the sphingomyelin pathway. Endocrinology 1995; 136:4157-4159[CrossRef][Medline]
  49. Srivastava SK, Tetsuka T, Daphna-Iken D, Morrison AR. IL-1ß stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region. Am J Physiol 1994; 267:F504-F508[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum Reprod UpdateHome page
J. Sirois, K. Sayasith, K. A. Brown, A. E. Stock, N. Bouchard, and M. Dore
Cyclooxygenase-2 and its role in ovulation: a 2004 account
Hum. Reprod. Update, September 1, 2004; 10(5): 373 - 385.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saito, J.
Right arrow Articles by Adashi, E. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saito, J.
Right arrow Articles by Adashi, E. Y.
Agricola
Right arrow Articles by Saito, J.
Right arrow Articles by Adashi, E. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS