Transcriptional and Posttranscriptional Regulation of Intraovarian ...

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society

Vol. 140, No. 8 Printed in U.S.A.

Transcriptional and Posttranscriptional Regulation of Intraovarian Insulin-Like Growth Factor-Binding Proteins by Interleukin-1b (IL-1b): Evidence for IL-1b as an Antiatretic Principal* DIRAN CHAMOUN†, MARCOS D. DEMOURA‡, ELIAHU LEVITAS§, CAROL E. RESNICK, SHARRON E. GARGOSKY, RON G. ROSENFELD, TOMOKO MATSUMOTOi, AND ELI Y. ADASHI¶ Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Maryland School of Medicine (D.C., M.D.D., E.L., C.E.R.), Baltimore, Maryland 21201; and the Department of Pediatrics, University of Oregon Health Sciences Center (S.E.G., R.G.R., T.M.), Portland, Oregon 97201 ABSTRACT Intraovarian interleukin-1 (IL-1), a putative intermediary in the ovulatory cascade, has recently been implicated as an antiatretic agent. Given the reported antigonadotropic and thus atretogenic potential of granulosa cell-derived insulin-like growth factor-binding proteins (IGFBPs), we evaluated the ability of IL-1b to regulate ovarian IGFBP-4 and -5, the IGFBP species elaborated by the rat granulosa cell. Treatment of whole ovarian dispersates of immature rat origin with increasing concentrations of IL-1b for 96 h resulted in substantial and significant time-dependent inhibition of IGFBP-4 and IGFBP-5 transcripts compared with that in untreated controls. The IL-1 effect proved relatively specific in that no significant alterations in IGFBP transcripts were observed in the presence of select ovarian agonists, including transforming growth factor-a, tumor necrosis factor-a, endothelin-1, hepatocyte growth factor, keratinocyte growth factor, or basic fibroblast growth factor. The inhibitory effect of IL-1b on ovarian IGFBP-4 and -5 expression was almost completely reversed in the presence of IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. The addition of actinomycin D to IL-1b-pretreated whole ovarian dispersates produced a pattern of (IGFBP-4 and -5) messenger RNA decay indistinguishable from that noted for the untreated control group. Medium

T

HE EXISTENCE of a complete intraovarian interleukin-1 (IL-1) system replete with ligands (IL-1a and IL-1b), receptors (types I and II), and receptor antagonist (IL-1RA) Received December 8, 1998. Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, 546 Chipeta Way, Salt Lake City, Utah 84108. E-mail: [email protected]. * This work was supported in part by NIH Research Grant HD-30288 (to E.Y.A.). † Current address: Fertility & Reproductive Medicine, 95 Bulldog Boulevard, Suite 204, Melbourne, Florida 32901. ‡ Recipient of scholarship awards from the Fundac¸ao de Amparo a Pesquisa do Estado de Sao Paulo and the National Brazilian Council of Scientific and Technologic Development. Current address: Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeirao Pret, University of Sao Paulo, Ribeirao Pret 14049-900, Brazil. § Recipient of the American Physician Fellowship Award. Current address: Department of Obstetrics and Gynecology, Soroka-Ben Gurion University Medical Center, Beer-Sheva 84101, Israel. i Current address: Fourth Department of Internal Medicine, University of Tokyo, Bunkyo-Ku, Tokyo 112 Japan.

conditioned by IL-1b-treated (but not untreated) whole ovarian dispersates displayed a marked diminution in the relative content of the IGFBP-4 and IGFBP-5 proteins (24- and 28- to 29-kDa proteins, respectively). Medium conditioned by IL-1b-treated (but not untreated) whole ovarian dispersates proteolyzed [125I]IGFBP-5 (but not IGFBP-4) into fragments with apparent molecular masses of 18 and 14 kDa, respectively. In conclusion, our present observations demonstrate the ability of IL-1 to 1) inhibit the steady state levels of transcripts corresponding to IGFBP-4 and -5 in a time-dependent, relatively specific, and receptormediated fashion; 2) suppress the accumulation of the corresponding IGFBP proteins; and 3) stimulate the activity of the IGFBP-5-directed (but not IGFBP-4) endopeptidase, a posttranscriptional phenomenon. Our findings also suggest, by inference, that the IL-1b-mediated inhibition of IGFBP-4 and -5 transcripts is due in part to a decrease in the rate of transcription of the corresponding genes and not to a change in the stability of the relevant messenger RNAs. Consequently, the ability of IL-1 to influence ovarian IGFBP economy appears multifaceted, comprising both transcriptional and posttranscriptional effects. To the extent that IGFBP-4 and -5 constitute atretogenic agents, our present findings support the view that IL-1b may play an antiatretic role in the context of ovarian physiology. (Endocrinology 140: 3488 –3495, 1999)

has been well documented in several species (1– 4). A growing body of evidence suggests that this intraovarian system may play an intermediary role in the ovulatory cascade (5, 6). Recently, Chun et al. suggested that IL-1b is also capable of exerting an antiatretic effect (7), as assessed by the blockade of spontaneous apoptosis experienced in vitro by cultured (gonadotropin-primed) preovulatory ovarian follicles (8). Given the reported antigonadotropic and thus atretogenic activity of granulosa cell-derived insulin-like growth factor (IGF)-binding proteins (IGFBPs) (9 –14), we set out to evaluate a possible regulatory effect of IL-1b on the expression and processing of ovarian IGFBP-4 and -5, the IGFBP species elaborated by the rat granulosa cell.

¶ Current address: Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, 546 Chipeta Way, Mailbox 20, Salt Lake City, Utah 84108.

3488

EVIDENCE FOR IL-1b AS AN ANTIATRETIC PRINCIPAL

3489

Immature (25- to 28-day-old) intact Sprague Dawley female rats, purchased from Zivic-Miller Laboratories, Inc. (Zelienople, PA), were killed by CO2 asphyxiation. All protocols were approved by the institutional animal care and use committee.

isopropanol, followed by freezing for 30 min at 270 C. The RNA pellet was recovered by centrifugation for 15 min at 12,000 3 g at 4 C, washing once with 1 ml 75% ethanol, air-drying, and resuspending in 10 ml diethylpyrocarbonate-treated distilled deionized water. The integrity of the resulting RNA was assessed by visual inspection of the ethidium bromide-stained 28S and 18S ribosomal RNA bands after electrophoresis through a 1.0% agarose-2.2 mol/liter formaldehyde gel.

Hormones and reagents

Northern blot analysis

McCoy’s 5a medium (modified, serum-free), penicillin-streptomycin solution, l-glutamine, and trypan blue stain (0.4%; wt/vol) were obtained from Life Technologies, Inc. (Grand Island, NY). Collagenase (Clostridium histolyticum; CLS type 1; 144 U/mg) was obtained from Worthington Biochemical Corp. (Freehold, NJ). Recombinant human IL-1b (2 3 107 U/mg) was provided by Drs. Errol B. De Souza and C. E. Newton, DuPont-Merck Pharmaceutical Co. (Wilmington, DE). A recombinantly expressed preparation of the naturally occurring human IL-1RA was provided by Dr. Jerome F. Strauss III, University of Pennsylvania (Philadelphia, PA). Basic fibroblast growth factor (bFGF) was generously provided by Dr. Andreas Summer, Synergen, Inc. (Boulder, CO). Tumor necrosis factor-a (TNFa) was a generous gift from Dr. Jennie P. Mather, Genentech, Inc. (South San Francisco, CA). Transforming growth factor-a (TGFa) was obtained from Oncogene Science, Inc. (Uniondale, NY). Endothelin-1 (ET-1) was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Hepatocyte growth factor (HGF) and actinomycin D were obtained from Sigma Chemical Co. (St. Louis, MO). Vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF) were obtained from PeproTech, Inc. (Rocky Hill, NJ).

Glyoxal-denatured RNA was electrophoresed and transferred to nylon membranes (Magna Graph, MSI, Westboro, MA) using 10 3 SSC (standard saline citrate). Prehybridization was carried out at 42 C for 12–24 h in a final buffer composition of 1.5 3 SSPE, 10 3 Denhardt’s solution (0.2% BSA, 0.2% polyvinylpyrrolidone, and 0.2% Ficoll), 50% formamide, 1% SDS, and 67 mg/ml denatured salmon sperm DNA. Membranes were hybridized overnight at 42 C in 15 ml hybridization buffer (same as prehybridization buffer except for the addition of 10% dextran sulfate) with {a-32P}deoxy-CTP-labeled probes (50 mCi) prepared using the random hexanucleotide-primed second strand synthesis method. Membranes were washed sequentially at room temperature with 5 3 SSPE-0.5% SDS, followed by 1 3 SSPE-0.75% SDS, and finally at 65 C with 0.1 3 SSPE-1% SDS. Membranes were stripped by heating to 95 C in 0.2 3 SSC-0.5% SDS before hybridization with another probe. The corresponding gels were autoradiographed as well as exposed to a phosphor screen (Molecular Dynamics, Inc., Sunnyvale, CA) to quantify the extent of hybridization. The resultant digitized data were analyzed with ImageQuant software (Molecular Dynamics, Inc.).

Materials and Methods Animals

Western ligand blot analysis In vitro studies Whole ovarian dispersates of immature rat origin, prepared by sequential collagenase digestion as described by Magoffin and Erickson (15), were cultured for up to 96 h in 35 3 10-mm plastic culture dishes (Falcon Plastics, Oxnard, CA) at a density of 5 3 105 viable cells/dish. The cells were maintained at 37 C under a water-saturated atmosphere of 95% air and 5% CO2 in 1 ml serum-free McCoy’s 5a medium supplemented with 2 mmol/liter l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate. All reagents were dissolved in sterile culture medium. All treatments were added in 50-ml aliquots. At the conclusion of the incubation period, conditioned media were subjected either to Western ligand blot analysis to assess their relative IGFBP content or to a cell-free IGFBP-5 endopeptidase activity assay. The corresponding cellular pellets were subjected to RNA extraction and Northern blot analysis as described below.

Nucleic acid probes The rat IGFBP-4 and -5 complementary DNAs (cDNAs) were provided in pBluescript SK1 by Dr. Shunichi Shimasaki (16, 17), The Whittier Institute for Diabetes and Endocrinology (La Jolla, CA). Membranes were probed with a 444-bp SmaI-HindIII restriction fragment of the rat IGFBP-4 cDNA and a 300-bp SacII-HindIII restriction fragment of the rat IGFBP-5 cDNA. The ribosomal protein large (RPL)19 probe (18) was generated as previously described (4). As RPL19 messenger RNA (mRNA) expression is not hormonally regulated in the rat ovary (19), a 244-bp EcoRI-HindIII restriction fragment of the rat RPL19 cDNA was used to normalize the IGFBP-4 and -5 mRNA data for possible variation in RNA loading. Specifically, the ratio of the net signal of interest (IGFBP-4 or -5) to net RPL19 was calculated for each sample and expressed as the fold difference from the value in the control untreated group.

RNA extraction Total cellular RNA was extracted with RNAzol-B as recommended by the manufacturer (Tel-Test, Friendswood, TX). Briefly, cells grown in 35 3 10-mm culture dishes were lysed at 4 C by 1 ml RNAzol-B solution before transfer to a microfuge tube. Phase separation was achieved by mixing and incubating with chloroform at 4 C for 5 min, and centrifugation at 1200 3 g for 15 min at 4 C. After transferring the upper phase to a second microfuge, RNA was precipitated by the addition of 0.6 ml

Synthetic IGF-II was iodinated by a modification of the chloramine-T technique to specific activities of up to 250 mCi/mg. The iodinated peptide was then purified by gel filtration over a Sephadex G-50 column (1.0 3 120 cm) at 4 C and eluted with 100 mm HEPES buffer, pH 7.4, containing 0.5% BSA, 120 mm NaCl, 1.2 mm MgSO4, 5 mm KCl, 50 mm Na acetate, and 10 mm dextrose. Conditioned media were electrophoresed on 10% SDS-PAGE under nonreducing conditions. The size-fractionated proteins were then electroblotted onto nitrocellulose for 1 h. Thereafter, the filter-immobilized proteins were blocked, incubated overnight at 4 C with 1 3 106 cpm [125I]IGF-II, washed, and visualized by autoradiography according to the method of Hossenlopp et al. (20). Molecular masses were estimated using prestained protein standards.

IGFBP endopeptidase activity assays Recombinant human IGFBP-4 or -5 (1 mg) was iodinated by a modification of the chloramine-T method and purified on a Sep-Pak C18 column (Millipore Corp., Bedford, MA). Approximately 30,000 cpm [125I]recombinant human IGFBP-4 or -5 (in 25 ml 20 mm HEPES, 0.1% BSA, 2.5 mm CaCl2, pH 7.5) were added to 50 ml conditioned medium. The mixture was incubated for up to 6 h at 37 C, and the reaction was stopped by the addition of SDS-sample buffer. The samples were then separated by 15% SDS-PAGE overnight under nonreducing conditions. The gels were dried and exposed to X-Omat film (Eastman Kodak Co., Rochester, NY) for 1–3 days.

Statistical analysis Except as noted, each experiment was replicated a minimum of three times. Data points are presented as the mean 6 se. Statistical significance (Fisher’s protected least significance difference) was determined by ANOVA and Student’s test. Statistical values were calculated using StatView 5121 for Macintosh (Brain Power, Inc., Calabasas, CA).

Results Granulosa cell-derived IGFBP-4 and -5 gene expression: effect of treatment with IL-1b

To examine the effect of treatment with IL-1b on ovarian IGFBP-4 and-5 gene expression, whole ovarian dispersates

3490


Endo • 1999 Vol 140 • No 8

were cultured for 96 h in the absence or presence of increasing concentrations of the cytokine. Treatment with 10 or 50 ng/ml IL-1b produced significant decrements in the relative expression of the IGFBP-4 gene, yielding 86% and 90% inhibition (P 5 0.0001), respectively, compared with untreated controls (Fig. 1). Similarly, provision of increasing concentrations of IL-1b resulted in substantial (.95%) and significant (P 5 0.001) inhibition of IGFBP-5 transcripts compared with untreated controls. IL-1-attenuated IGFBP-4 and -5 gene expression: time requirements

To assess the time requirements of the IL-1b effect on ovarian IGFBP-4 and -5 gene expression, whole ovarian dispersates were cultured for the duration indicated (up to 96 h) in the absence or presence of IL-1b (10 ng/ml). Untreated cultures of whole ovarian dispersates displayed progressive time-dependent increments in the relative expression of IGFBP-4 transcripts (Fig. 2). A 3-fold increase was noted at 48 h (P , 0.05); no additional changes were noted at 72 and 96 h. In contrast, a modest spontaneous increase was noted in the steady state levels of IGFBP-5 transcripts at 24 h, followed by modest inhibition at 48 h (a decrease of 34%

FIG. 2. IL-1-attenuated IGFBP-4 and -5 gene expression: time requirements. Whole ovarian dispersates (5 3 105 viable cells/culture), obtained and maintained as described, were cultured under serumfree conditions for the duration indicated for up to 96 h in the absence or presence of IL-1b (10 ng/ml). At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described in Fig. 1. The intensity of the signals was quantified as described. The line graph represents the mean 6 SE of five experiments normalized relative to the zero time point value.

FIG. 1. Granulosa cell-derived IGFBP-4 and 5 gene expression: effect of treatment with IL-1b. Whole ovarian dispersates (5 3 105 viable cells/culture), obtained and maintained as described, were cultured for 96 h under serum-free conditions in the absence or presence of IL-1b (10 or 50 ng/ml). At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described using 32P-labeled probes corresponding to the rat IGFBP-4 and -5 genes, respectively. The blots were subsequently probed with the rat RPL19 cDNA to normalize for possible variation in RNA loading or transfer. The intensity of RNA bands was quantified as described. The bar graphs (upper panel) represent the mean 6 SE of three experiments normalized relative to control values (no treatment). The lower panel reflects a representative experiment.

relative to that at time zero) and a significant decrease at 72 h (52% relative to that at time zero; P , 0.05). An almost complete recovery of IGFBP-5 transcripts to levels indistinguishable from those noted at time zero was noted at 96 h. Treatment with IL-1b prevented the spontaneous time-dependent increase in the relative expression of IGFBP-4 transcripts [85% (P 5 0.042), 73% (P 5 0.049), and 88% (P 5 0.014) inhibition relative to untreated controls at the 48, 72, and 96 h, respectively]. In contrast, treatment with IL-1b produced a minimal change (relative to untreated controls) in the expression of IGFBP-5 transcripts at 24, 48, and 72 h; a 68% reduction (P , 0.05) was noted at 96 h. These findings indicate that the inhibitory effect of IL-1b on IGFBP-4 and IGFBP-5 transcripts is time dependent. Granulosa cell-derived IGFBP-4 and -5 gene expression: specificity of the IL-1b effect

To examine the specificity of the IL-1b effect on ovarian IGFBP-4 and -5 gene expression, whole ovarian dispersates were cultured for 96 h in the absence or presence of select growth factors and cytokines. Whereas treatment with IL-1b resulted in the expected inhibition of IGFBP-4 transcripts (70%; P 5 0.02), no significant alterations were observed in


3491

the presence of TGFa, TNFa, ET-1, HGF, KGF, or bFGF (not shown). Similarly, whereas IL-1b proved a potent inhibitor of IGFBP-5 expression (90%; P 5 0.005), no significant changes were noted in the presence of the aforementioned agents (Fig. 3). The moderate inhibition of IGFBP-5 transcripts associated with the addition of TNFa, ET-1, or bFGF proved (statistically) nonsignificant. These findings suggest that the inhibitory effect of IL-1b on the relative expression of IGFBP-4 and -5 is relatively specific and not replicated by a series of regulatory agonists, most of which, with the exception of VEGF, have been shown to modulate ovarian cell function (21–26). The inhibitory effect of IL-1b on granulosa cell-derived IGFBP-4 and -5 gene expression is receptor mediated

To assess the possibility that the IL-1b effect is receptor mediated, whole ovarian dispersates were cultured for 96 h in the absence or presence of IL-1b, with or without an IL-1RA, a validated reagent in the context of the rat ovary (27). As shown in Figs. 1–3, treatment with IL-1b resulted in the inhibition of IGFBP-4 and -5 transcripts [decreases of 83% (P 5 0.0001) and 66% (P 5 0.025), respectively]. However, the inhibitory effect of IL-1b on IGFBP-4 and -5 expression was almost completely reversed in the presence of IL-1RA (Fig. 4). These findings suggest that the inhibitory action of IL-1b on IGFBP-4 and -5 transcripts is mediated via a specific IL-1R.

FIG. 4. The inhibitory effect of IL-1b on granulosa cell-derived IGFBP-4 and -5 gene expression is receptor mediated. Whole ovarian dispersates (5 3 105 viable cells/culture), obtained and maintained as described, were cultured under serum-free conditions for up to 96 h in the absence or presence of IL-1b, with or without IL-1RA. At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described in Fig. 1. The intensity of the signals was quantified as described. The bar graphs (upper panel) represent the mean 6 SE of four experiments normalized relative to control values (no treatment). The lower panel reflects a representative experiment.

IL-1b-attenuated IGFBP-4 and -5 gene expression: lack of effect on the stability of the corresponding transcripts

FIG. 3. Granulosa cell-derived IGFBP-5 gene expression: specificity of the IL-1b effect. Whole ovarian dispersates (5 3 105 viable cells/ culture), obtained and maintained as described, were cultured under serum-free conditions for up to 96 h in the absence or presence of 10 ng/ml IL-1b, TGFa, TNFa, ET-1, HGF, VEGF, KGF, or bFGF. At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described in Fig. 1. The intensity of the signals was quantified as described. The bar graphs (upper panel) represent the mean 6 SE of three experiments normalized relative to control (no treatment). The lower panel reflects a representative experiment.

To determine whether the IL-1b effect reflects an increase in the rate of mRNA degradation or a decrease in the rate of gene transcription, whole ovarian dispersates were initially cultured for 48 h in the absence or presence of IL-1b. After the addition of actinomycin D (10 mg/ml), an established inhibitor of transcription, the cells were harvested, and total RNA was extracted at 0, 1, 2, 4, 8, 12, and 24 h. The addition of actinomycin D to untreated whole ovarian dispersates resulted in progressive and time-dependent decrements in the relative expression of IGFBP-4 and -5 transcripts, yielding by 24 h 95% and 87% inhibition, respectively (Fig. 5). The addition of actinomycin D to IL-1b-pretreated whole ovarian dispersates produced a pattern indistinguishable from that noted for the untreated control group (93% and 95% inhibition of IGFBP-4 and -5 transcripts, respectively, at 24 h). Pretreatment with IL-1b was likewise without effect on the half-life of RPL19 transcripts (Fig. 6). These findings suggest, by inference, that the IL-1b-mediated inhibition of IGFBP-4 and -5 transcripts is due in part to a decrease in the rate of transcription of the corresponding genes and not to a change in the stability of the relevant mRNAs.

3492


FIG. 5. IL-1b-attenuated IGFBP-4 and -5 gene expression: lack of effect on the stability of the corresponding transcripts. Whole ovarian dispersates (5 3 105 viable cells/culture), obtained and maintained as described, were cultured under serum-free conditions for 48 h in the absence or presence of IL-1b (10 ng/ml). After the addition of actinomycin D (10 mg/ml), the cells were harvested, and total RNA was extracted at 0, 1, 2, 4, 8, 12, and 24 h and subjected to Northern blot analysis as described in Fig. 1. The intensity of the signals was quantified as described. The line graph (upper panel) represents the mean 6 SE of three experiments normalized relative to the zero time point value. The lower panel reflects a representative experiment.

Accumulation of IGFBPs in media conditioned by cultured whole ovarian dispersates: effect of treatment with IL-1b

To determine whether the IL-1 effect is also manifest at the protein level, conditioned media corresponding to control and IL-1b-treated cells were subjected to Western ligand blot analysis. Media conditioned by IL-1b-treated whole ovarian dispersates displayed a substantial increase in the relative content of the IGFBP-3 protein (Fig. 7). In contrast, similarly treated whole ovarian dispersates displayed a marked inhibitory effect for IGFBP-4 and particularly for IGFBP-5 in four independent experiments. These findings demonstrate the ability of IL-1b to suppress the accumulation of the IGFBP-4 and -5 proteins (in addition to the corresponding transcripts) in media so conditioned. IGFBP endopeptidase activity in media conditioned by cultured whole ovarian dispersates: effect of treatment with IL-1b

To determine whether the IL-1b-mediated decrease in the IGFBP-5 protein content may reflect activation of an IGFBP endopeptidase(s), whole ovarian dispersates were cultured for 96 h in the absence or presence of IL-1b (10 ng/ml). At the conclusion of the culture period, media were collected and subjected to cell-free IGFBP protease assays as described. Media corresponding to untreated control cultures disclosed no evidence of IGFBP-5 proteolytic activity (Fig. 8). In contrast, media from IL-1b-treated cultures proteolyzed [125]IGFBP-5 (but not IGFBP-4; not shown) into fragments with apparent molecular masses of 18 (13) and 14 (23) kDa,

Endo • 1999 Vol 140 • No 8

FIG. 6. IL-1b-attenuated IGFBP-4 and -5 gene expression: lack of effect on the stability of the RPL19 transcripts. Whole ovarian dispersates (5 3 105 viable cells/culture), obtained and maintained as described, were cultured under serum-free conditions for 48 h in the absence or presence of IL-1b (10 ng/ml). After the addition of actinomycin D (10 mg/ml), the cells were harvested, and total RNA was extracted at 0, 1, 2, 4, 8, 12, and 24 h and subjected to Northern blot analysis as described in Fig. 1. The intensity of the signals was quantified as described. The line graph (upper panel) represents the mean 6 SE of three experiments normalized relative to the zero time point value. The lower panel reflects a representative experiment.

respectively. This proteolytic effect of IL-1b was completely reversed in a dose-dependent fashion by the addition of increasing concentrations of IL-1RA, suggesting receptor mediation. These findings suggest that the ability of IL-1b to reduce the IGFBP-5 (but not IGFBP-4) content in media conditioned by whole ovarian dispersates is due in part to a posttranscriptional phenomenon, i.e. the activation of IGFBP-5 endopeptidase activity. Discussion

IL-1, a cytokine implicated as an intermediary in the ovulatory process, has been the subject of intense investigation (5, 6). First, the ex vivo provision of IL-1b has been shown to bring about ovulation and to synergize with LH in this regard (28). Second, the addition of an IL-1RA has been shown to attenuate LH-supported ovulation under both ex vivo (29) and in vivo (30) circumstances. Third, some components of the intraovarian IL-1 system (e.g. IL-1b and the type I IL-1 receptor) appear to be expressed in vivo, mainly during a narrow periovulatory window (1– 4). Fourth, IL-1b has been shown to induce a host of ovulation-associated phenomena in vitro such as the promotion of PG production (31), the stimulation of hyaluronic acid biosynthesis (32), the induction of collagenase activity (33), the modulation of plasminogen activator economy (34 –36), and the activation of nitric oxide synthase activity (37–39).


FIG. 7. IGFBP accumulation in media conditioned by cultured whole ovarian dispersates: effect of treatment with IL-1b. Whole ovarian dispersates (5 3 105 viable cells/culture), obtained and maintained as described, were cultured for 96 h under serum-free conditions in the absence or presence of IL-1b (10 ng/ml). At the conclusion of the incubation period, conditioned medium samples from whole ovarian dispersates were electrophoresed on 10% SDS-PAGE under nonreducing conditions, electroblotted onto nitrocellulose for 1 h, incubated overnight at 4 C with 1 3 106 cpm [125I]IGF-II, and visualized by autoradiography. Molecular masses were estimated using prestained protein standards. Four independent representative experiments are shown.

More recently, several investigators have implicated IL-1 in the context of follicular atresia. Specifically, Chun et al. suggested that IL-1b is also capable of exerting an antiatreic effect (7). Indeed, treatment with IL-1b markedly attenuated the apoptotic degradation of DNA in cultured isolated preovulatory follicles from gonadotropin-primed rats (7). Given the reported antigonadotropic and thus atretogenic activity of granulosa cell-derived IGFBPs (9 –14), we set out to evaluate a possible regulatory effect of IL-1b on the expression and processing of ovarian IGFBP-4 and -5, the IGFBP species elaborated by the rat granulosa cell. Our current observations document a profound inhibitory effect of IL-1 on the expression and posttranscriptional processing of granulosa cell-derived IGFBP-4 and 5. Although the possibility that IGFBP-4 and -5 may be elaborated by cell types other than the granulosa cell must be considered, such a possibility is viewed as less likely given extensive localization studies consisting of molecular probing, in situ hybridization, Western ligand blotting, glycosylation analysis, and immunoprecipitation studies. Marked (in excess of 80%) decrements in IGFBP transcripts have been observed. The effect of IL-1b proved to be time dependent, relatively specific, as well as receptor mediated in that the concurrent presence of IL-1RA markedly attenuated the IL-1b effect. Presumably, only the type I IL-1 receptor was involved, as the type II receptor does not appear to engage in cellular signaling (40). Moreover, transcripts for the type I IL-1 receptor are more abundant than those for the type II receptor in the immature rat ovary (41). Furthermore, our findings suggest, by inference, that the inhibitory effect of IL-1b on the steady state levels of IGFBP-4 and -5 transcripts appears to be due in part to a decrease in the rate of transcription of the corresponding gene and not to a change in the stability of the mRNA (Fig. 5).

3493

FIG. 8. IGFBP-5 endopeptidase activity in media conditioned by cultured whole ovarian dispersates: effect of treatment with IL-1b. Whole ovarian dispersates (5 3 105 viable cells/culture), obtained and maintained as described, were cultured for 96 h under serum-free conditions in the absence or presence of IL-1b (10 ng/ml), with or without increasing concentrations (0.1–5 mg/ml) of IL-1RA. At the conclusion of the incubation period, conditioned medium was subjected to a cell-free IGFBP-5 protease assay. Approximately 30,000 cpm radiolabeled IGFBP-5 were added to 50 ml medium conditioned by whole ovarian dispersates. The mixture was incubated for 6 h at 37 C and separated by 15% SDS-PAGE under nonreducing conditions. The gels were exposed to X-Omat film for 1–3 days. A representative experiment is shown.

The ability of IL-1b to modulate granulosa cell-derived IGFBP expression was not limited to the transcript level. Indeed, IL-1b was shown to suppress the accumulation of the IGFBP-4 and -5 proteins in media so conditioned. Although the IL-1b-mediated decrease in the relative IGFBP content may be due to a decrease in the steady state levels of the corresponding transcripts, a posttranscriptional phenomenon was noted as well. Specifically, treatment with IL-1b resulted in the activation of an IGFBP-5 (but not IGFBP-4) endopeptidase and thus in the enhanced breakdown of the IGFBP-5 protein. Consequently, the ability of IL-1 to influence IGFBP-4 and -5 levels appears multifaceted, comprising both transcriptional and posttranscriptional effects. To the extent that granulosa cell-derived IGFBP-4, and -5 constitute atretogenic agents (9 –14), our present findings support the view that IL-1b may play an antiatretic role in the context of ovarian physiology by virtue of its ability to decrease the intrafollicular content of IGFBP-4 and -5, thereby increasing the bioavailability of unbound IGF-I. In so doing, IL-1 is joining a growing list of ovarian regulators purported to exert an antiatretic effect including FSH, bFGF, EGF, IGF-I, estrogen, and inhibin (14). These observations are compatible with the idea that intrafollicular IGFBPs constitute potent determinants of follicular fate, and that their modulation by putative intraovarian regulators could play a significant role in establishing the outcome of folliculogenesis. The present data reveal a significant up-regulatory effect of IL-1b on IGFBP-3 elaboration. In this respect, these findings contrast with the inhibitory action exerted by IL-1b on IGFBP-4 and -5 expression. As such, these observations dis-

3494


close the complex action plan of IL-1b at the level of ovarian cells. It stands to reason that IGFBP-3, unlike IGFBP-4 and -5, arises from thecal-interstitial as opposed to granulosa cells. This inference is made based on previous localization studies focusing on this IGF-binding species. Our present observations expand the list of regulators of granulosa cell-derived IGFBPs. Indeed, a substantial body of evidence documents the ability of FSH to exert a biphasic A kinase-dependent regulation of granulosa cell-derived IGFBP-4 and -5 (42– 44). In addition, evidence has been presented to support a role for C kinase in the regulation of granulosa cell-derived IGFBPs. In this context, note is made of the role of GnRH (45, 46), and C kinase agonists (45). Most recently, activin has also been implicated as a regulator of granulosa cell-derived IGFBPs (46). Taken together, these observations document the complexity of the regulatory processes responsible for establishing the steady state of granulosa cell-derived IGFBPs and indirectly for establishing the bioavailability of intrafollicular IGF-I. Although the signal transduction cascade involved with the IL-1b effect remains unknown, consideration must be given to the emerging role of the sphingomyelin-ceramide pathway (47, 48). The current state of the art implicates IL-1 in the context of both atresia and ovulation. Given that the ovarian expression IL-1 is particularly striking just before and during ovulation (1–3), it is tempting to speculate that the antiatretic potential of IL-1 is directed primarily at the rapidly growing preovulatory follicles destined for ovulation. According to this view, the early ovarian expression of IL-1 may be designed to ensure follicular selection and indeed dominance as a prelude to subsequent ovulation. Given that ovulation is associated with a wave of atresia affecting nondominant follicles, it may well be that the IL-1 effect is selectively directed at the successful preovulatory cohort, with little or no effect observed in follicles destined for premature demise. Taken together, our present observations demonstrate a profound negative effect of IL-1b on IGFBP-5 economy. This action of IL-1 is multifaceted, comprising both transcriptional and posttranscriptional effects inclusive of the inhibition of gene expression and of a simultaneous activation of an IGFBP-5 endopeptidase(s).

6. 7. 8. 9.

10. 11. 12. 13.

14. 15. 16. 17.

18. 19.

20.

21.

22.

Acknowledgment The authors thank Ms. Cornelia T. Szmajda and Ms. Linda Elder for their invaluable assistance in preparing this manuscript.

References 1. Hurwitz A, Loukides J, Ricciarelli E, Botero L, Katz E, McAllister JM, Garcia JE, Rohan R, Adashi EY, Hernandez ER 1992 The 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 89:1746 –1754 2. Hurwitz A, E Ricciarelli, L Botero, RM Rohan, ER Hernandez, EY Adashi 1991 Endocrine- and autocrine-mediated regulation of rat ovarian (thecainterstitial) interleukin-1b gene expression: gonadotropin-dependent preovulatory acquisition. Endocrinology 129:3427–3429 3. Simon C, Frances A, Piquette G, Polan ML 1994 Immunohistochemical localization of the interleukin-1 system in the mouse ovary during follicular growth, ovulation, and luteinization. Biol Reprod 50:449 – 457 4. Scherzer WJ, Ruutiainen-Altman KSL, Putowski LT, Kol S, Adashi EY, Rohan RM 1996 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 3:131–139 5. Ben-Shlomo I, Hurwitz A, Kokia E, Scherzer WJ, Donesky BW, Rohan RM,

23.

24.

25. 26.

27. 28. 29.

Endo • 1999 Vol 140 • No 8

Payne DW, Adashi EY 1994 Role of cytokines in ovarian physiology: interleukin-1 as a possible centerpiece in the ovulatory sequence. In: Aagarwal B, Puri RK (eds) Human Cytokines: Their Role in Disease and Therapy. Blackwell, Boston, pp 329 –333 Ben-Shlomo I, Adashi EY 1994 Interleukin-1 as a mediator in the ovulatory sequence: evidence for a meaningful role of cytokines in ovarian physiology. Curr Sci Ser 1:187–192 Chun S-Y, Eisenhauer KM, Kubo M, Hsueh AJW 1995 Interleukin-1b suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology 136:3120 –3127 Tilly JL, Hsueh AJ 1993 Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J Cell Physiol 154:519 –526 Putowski LT, Choi DS, Scherzer WJ, Ricciarelli E, Kiefer MC, Mordacq J, Kelly EM, Rohan RM, Adashi EY 1996 Rat ovarian insulin-like growth factor binding protein-4: a hormonally-dependent granulosa-derived antigonadotropin. J Soc Gynecol Invest 4:144 –151 Nakatani A, Shimasaki S, Erickson GF, Ling N 1991 Tissue-specific expression of four insulin-like growth factor-binding proteins (1, 2, 3 and 4) in the rat ovary. Endocrinology 129:1521–1529 Erickson GF, Nakatani A, Ling N, Shimasaki S 1992 Cyclic changes in insulinlike growth factor binding protein-4 messenger ribonucleic acid in the rat ovary. Endocrinology 130:625– 636 Erickson GF, Nakatani A, Ling N, Shimasaki S 1992 Localization of insulinlike growth factor-binding protein-5 messenger ribonucleic acid in rat ovaries during the estrous cycle. Endocrinology 130:1867–1878 Liu XJ, Malkowski M, Guo Y, Erickson GF, Shimasaki S, Ling N 1993 Development of specific antibodies to rat insulin-like growth factor binding proteins (IGFBP-2 to 6): analysis of IGFBP production by rat granulosa cells. Endocrinology 132:1176 –1183 Hsueh AJW, Eisenhauer K, Chun S-Y, Hsu S-Y, Billig H 1996 Gonadal cell apoptosis. Recent Prog Horm Res 51:433– 456 Magoffin DA, Erickson GF 1982 Primary culture of differentiating ovarian androgen-producing cells in defined medium. J Biol Chem 275:4507– 4513 Shimasaki S, Uchiyama F, Shimonaka M, Ling N 1990 Molecular cloning of the cDNAs encoding a novel insulin-like growth factor-binding protein from rat and human. Mol Endocrinol 4:1451–1458 Shimasaki S, Shimonaka M, Zhang HP, Ling N 1991 Identification of five different insulin-like growth factor binding proteins (IGFBPs) from adult rat serum and molecular cloning of a novel IGFBP-5 in the rat and human. J Biol Chem 266:10646 –10653 Chan Y-L, Lin A, McNally J, Peleg D, Meyuhas O, Wool IG 1987 The primary structure of rat ribosomal protein L19. J Biol Chem 257:4507– 4513 Orly J, Rei Z, Greenberg NM, Richards JS 1994 Tyrosine kinase inhibitor AG18 arrests follicle-stimulating-hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. Endocrinology 134:2336 –2346 Hossenlopp P, Seurin D, Sequoia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138 –143 Adashi EY, Resnick CE, Packman JN, Hurwitz Z, Payne DW 1990 Cytokinemediated regulation of ovarian function: tumor necrosis factor a inhibits gonadotropin-supported progesterone accumulation by differentiating and luteinized murine granulosa cells. Am J Obstet Gynecol 162:889 – 899 Andreani CL, Resnick CE, Packman JN, Payne DW, Hurwitz A, Adashi EY 1991 Cytokine-mediated regulation of ovarian function: tumor necrosis factor a inhibits gonadotropin-supported ovarian androgen biosynthesis. J Biol Chem 266:6761– 6766 Tedeschi C, Hazum E, Adashi EY, Payne DW 1992 Endothelin-1 inhibits rat granulosa cell progesterone accumulation via selective modulation of key steroidogenic steps affecting both progesterone formation and degradation. Endocrinology 131:2476 –2479 Hurwitz A, Hernandez ER, Resnick CE, Packman JN, Payne DW, Adashi EY 1990 Basic fibroblast growth factor (bFGF) inhibits gonadotropin-supported ovarian androgen biosynthesis: mechanism(s) and site(s) of action. Endocrinology 126:3089 –3096 Zachow RJ, Weitsman SR, Magoffin DA 1997 Hepatocyte growth factor regulates ovarian theca-interstitial cell differentiation and androgen production. Endocrinology 138:691– 697 Parrott JA, Vigne JL, Chu BZ, Skinner MK 1994 Mesenchymal-epithelial interactions in the ovarian follicle involve keratinocyte and hepatocyte growth factor production by thecal cells and their action on granulosa cells. Endocrinology 135:569 –575 Kokia E, Ben-Shlomo I, Adashi EY 1994 The ovarian action of IL-1 is receptormediated: reversal by a naturally occurring interleukin-1 receptor antagonist. Fertil Steril 63:176 –181 Brannstrom M, Wang L, Norman RJ 1993 Ovulatory effect of interleukin-1 on the perfused rat ovary. Endocrinology 132:399 – 404 Peterson CM, Hales HA, Hatasaka HH, Mitchell MD, Rittenhouse L, Jones KP 1993 Interleukin-1b (IL-1b) modulates prostaglandin production and the


30. 31.

32.

33.

34.

35. 36. 37.

38.

natural IL-1 receptor antagonist inhibits ovulation in the optimally stimulated rat ovarian perfusion model. Endocrinology 133:2301–2306 Simon C, Tsafriri A, Chun S-Y, Piquette GN, Dang W, Polan ML 1994 Interleukin-1 receptor antagonist suppresses hCG-induced ovulation in the rat. Biol Reprod 51:662– 667 Kokia E, Hurwitz A, Ricciarelli E, Tedeschi C, Resnick CE, Mitchell MD, Adashi EY 1992 Interleukin-1 stimulates ovarian prostaglandin biosynthesis: evidence for heterologous contact-independent cell-cell interaction. Endocrinology 130:3095–3097 Kokia E, Hurwitz A, Ben-Shlomo I, Adashi EY, Yanagishita M 1993 Receptor mediated stimulatory effects of IL-1b on hyaluronic acid and proteoglycan biosynthesis by cultured rat ovarian cells: role for heterologous cell-cell interactions. Endocrinology 133:2391–2394 Hurwitz A, Dushnik M, Solomon H, Ben-Chetrit A, Finci-Yeheskel Z, Milwidsky A, Meyer M, Adashi EY, Yagel S 1993 Cytokine-mediated regulation of rat ovarian function: interleukin-1 stimulates the accumulation of 92-kD gelatinase. Endocrinology 132:2709 –2714 Hurwitz A, Finci-Yeheskel Z, Dushnik M, Milwidsky A, Ben-Chetrit A, Yagel S, Adashi EY, Mayer M 1994 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 101:307–315 Bonello NP, Norman RJ, Brannstrom M 1995 Interleukin-1b inhibits luteinizing hormone-induced plasminogen activator activity in rat preovulatory follicles in vitro. Endocrine 3:49 –54 Karakji EG, Tsang BK 1995 Regulation of rat granulosa cell plasminogen activator system: influence of interleukin-1b and ovarian follicular development. Biol Reprod 53:1302–1310 Ellman C, Corbett JA, Misko TP, McDaniel M, Beckerman KP 1993 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 92:3053–3056 Ben-Shlomo I, Kokia E, Jackson M, Adashi EY, Payne DW 1994 Interleukin-1b 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 51:310 –318

3495

39. Bonello N, McKie K, Jasper M, Andre L, Ross N, Braybon E, Brannstrom M, Norman RJ 1996 Inhibition of nitric oxide: effects of interleukin-1b-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat. Biol Reprod 54:436 – 445 40. Sims JE, Gayle MA, Slack JL, Alderson MR, Bird TA, Giri JG, Colotta R, Re F, Mantovani A, Shanesbeck K, Grabstein KH, Dower SK 1993 Interleukin-1 signalling occurs exclusively via the type I receptor. Proc Natl Acad Sci USA 90:6155– 6159 41. Adashi EY, Resnick CE, Hernandez ER, Hurwitz A, Rosenfeld RG 1990 Follicle-stimulating hormone inhibits the constituitive release of insulin-like growth factor binding proteins by cultured rat ovarian granulosa cells. Endocrinology 126:1305–1307 42. Adashi EY, Resnick CE, Hurwitz A, Ricciarelli E, Hernandez ER, Rosenfeld RG 1991 Ovarian granulosa cell-derived insulin-like growth factor binding proteins: a modulatory role of follicle-stimulating hormone. Endocrinology 128:754 –760 43. Adashi EY, Resnick CE, Tedeschi C, Rosenfeld RG 1993 A kinase-mediated regulation of granulosa cell-derived insulin-like growth factor binding proteins (IGFBPs): disparate response sensitivities of distinct IGFBP species. Endocrinology 132:1463–1468 44. Erickson GF, Li D, Sadrkhanloo R, Liu XJ, Shimasaki S, Ling N 1994 Extrapituitary actions of gonadotropin-releasing hormone: stimulation of insulin-like growth factor-binding protein-4 and atresia. Endocrinology 134:1365–1372 45. Onoda N, Li D, Mickey G, Erickson G, Shimasaki S 1995 Gonadotropinreleasing hormone overcomes follicle-stimulating hormone’s inhibition of insulin-like growth factor-5 synthesis and promotion of its degradation in rat granulosa cells. Mol Cell Endocrinol 110:17–25 46. Choi DS, Rohan RM, Adashi EY 1997 Activin-attenuated expression of transcripts encoding granulosa cell-derived insulin-like growth factor binding proteins 4 and 5 in the rat: a putative antiatretic effect. Biol Reprod 56:508 –515 47. Schutze S, Machleidt T, Kronke M 1994 The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. J Leukocyte Biol 56:533–541 48. Kuno K, Matsushima K 1994 The IL-1 receptor signal pathway. J Leukocyte Biol 56:542–547