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Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor the role of which in ovarian angiogenesis has been the subject of increasing interest. It was the objective of this communication to explore the possibility that interleukin (IL)-1 may regulate the in vitro expression of rat ovarian VEGF mRNA, as well as to study the in vivo expression of rat ovarian VEGF transcripts during follicular maturation, ovulation, and corpora lutea formation. Taken together, our findings 1) reaffirm the rat ovary as a site of VEGF expression; 2) document an in vivo increase in VEGF transcripts before ovulation; 3) disclose a marked dependence of VEGF on IL-1 beta; 4) reveal the IL-1 beta effect to be receptor mediated and dose and time dependent and to be shared by at least two growth factors--epidermal growth factor and basic fibroblastic growth factor; and 5) demonstrate a lack of VEGF effect on ovarian progesterone biosynthesis as assessed in cultured isolated granulosa cells. It is tempting to speculate that the up-regulatory effect of IL-1 beta on VEGF transcripts may be relevant to the marked angiogenesis and increased vascular permeability displayed by the hyperemic ovarian Graafian follicle during the terminal stages of follicular development. In this context, VEGF may be joined by other IL-1-dependent angiogenesis promoters such as IL-6 or transforming growth factor beta 1. Thus, IL-1-mediated VEGF induction may constitute one of several end points through which IL-1 may coordinate and perhaps amplify the ovulatory cascade.

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-1beta 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-1beta 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-1beta 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-1beta to upregulate ovarian PGS-2 transcripts is due, if only in part, to the generation of endogenous prostaglandin estradiol-17beta (E(2)). Inhibition of protein biosynthesis suggested that the IL-1beta-induced PGS-2 gene expression required de novo protein biosynthesis. Our findings revealed substantial IL-1beta-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-1beta 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-1beta on PGS-2 expression is 1) independent of nitric oxide as well as ceramide, 2) dependent on prostaglandin E(2), 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-1beta, acting in part through PGS-2 (an obligatory ovulatory principal), may constitute a key intermediary in the ovulatory cascade.

Interleukin (IL)-6, traditionally an IL-1-induced immune regulator, is nevertheless synthesized by a variety of tissues including the ovary. The purposes of this communication were to assess the ovarian expression of IL-6, examine its cyclic variation, and study its regulation by IL-1, a putative intermediary in the ovulatory process. Molecular probing revealed IL-6 transcripts to be most abundant in the thymus, liver, and ovary, which suggests that, in relative terms, untreated immature whole ovarian tissue is a significant site of IL-6 gene expression. Treatment of immature rats with pregnant mare serum gonadotropins resulted in a measurable, statistically significant (P.05) increase in ovarian IL-6 mRNA compared with untreated controls. Isolated and indeed transient increments in the relative abundance of IL-6 transcripts were noted 4 hours after exposure to hCG, a point in time 8 hours from projected follicular rupture, a pattern highly reminiscent of that previously recognized for IL-1 beta transcripts. Treatment of whole ovarian dispersates from immature rats with IL-1 beta for 48 hours resulted in a significant (P.05) increase (11-fold) over control in IL-6 transcripts. The IL-1 effect proved dose dependent; the first significant increase was noted at the 1-ng/mL dose level. Evaluation of the time requirements revealed IL-1 beta to significantly (P.05) up-regulate (4.5-fold) IL-6 transcripts as early as 24 hours into the culture period. Cotreatment with the IL-1 receptor antagonist completely reversed the IL-1 effect, which suggests mediation through a specific IL-1 receptor. Treatment with indomethacin (10 microg/mL), an established inhibitor of prostaglandin biosynthesis, resulted in a significant (P.05) decrease (79%) in the ability of IL-1 beta to up-regulate IL-6 transcripts. Importantly, the addition of prostaglandin E(2) (10 microg/mL) to untreated or indomethacin-treated cells significantly (P.05) augmented the IL-1 beta effect. This suggests a role for eicosanoid signaling in IL-1 beta action. Treatment with aminoguanidine, an established inhibitor of nitric oxide synthase, significantly (P.05) decreased (85%) the IL-1 beta effect. However, the addition of S-nitroso-n-acetyl-penicilamine, an established nitrite generator, failed to reverse the aminoguanidine effect, which suggests that the inhibitor effect of aminoguanidine may be nitrite independent. Treatment with cycloheximide produced dose-dependent inhibition of the ability of IL-1 to up-regulate IL-6 transcripts; the maximal inhibitory effect was 89%. Taken together, these findings (1) reaffirm the rat ovary as a site of IL-6 expression; (2) document an in vivo increase in IL-6 transcripts before ovulation; (3) disclose a marked dependence of IL-6 on IL-1 beta; and (4) reveal the IL-1 beta effect to be dose and time dependent, receptor mediated, contingent upon de novo protein biosynthesis, and eicosanoid dependent but nitric oxide independent. These findings suggest that IL-1 action in the ovary may require the intermediacy of IL-6 in a manner like that encountered in extraovarian sites.

It is hypothesized that the intraovarian interleukin (IL)-1 system plays a prominent role in the regulation of follicular development and ovulation. A central component of the intraovarian IL-1 system is the IL-1 receptor antagonist (IL-1RA), a protein acting as a pure IL-1 receptor antagonist and one for which intracellular (icIL-1RA) and secretory (sIL-1RA) varieties have been described. It was the objective of this study to explore rat ovarian IL-1RA gene expression, to establish the identity and relative abundance of its alternative transcripts, to study its cellular localization, to determine its cyclic variation, and to assess its hormonal regulation. Protected IL-1RA cDNA fragments corresponding to either sIL-1RA or icIL-1RA were barely detectable in untreated whole ovarian tissue of immature rat origin. However, sIL-1RA transcripts reached a maximal value (3.3-fold increase over untreated control values; p0.05) 12 h after hCG administration (time of projected ovulation). In situ hybridization localized IL-1RA to mural, antral, and cumulus granulosa cells. Modestly intense staining was also apparent in oocytes. The basal pattern of sIL-1RA expression by cultured whole ovarian dispersates was characterized by a spontaneous increase to a peak value at 4 h. The early (4 h) sIL-1RA burst proved IL-1-, nitric oxide-, and protein biosynthesis-independent. However, treatment with IL-1beta led to a secondary sIL-1RA peak at 48 h, an effect that was substantially reversed by IL-1RA. This stimulatory effect of IL-1beta on IL-1RA expression proved relatively specific, and nitric oxide independent, but contingent upon de novo protein biosynthesis. The in vitro expression of icIL-1RA was barely detectable. Taken together, these in vivo and in vitro observations 1) document the rat ovary as a site of IL-1RA (sIL-1RAcIL-1RA) expression, 2) localize the relevant transcripts to the granulosa cell, 3) disclose peak expression at the time of ovulation, and 4) establish IL-1 dependence.

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-1beta, the IL-1 receptor antagonist, and the type I IL-1 receptor. The order of potency of the glucocorticoids studied was dexamethasoneprednisolone = 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.

Ovulation may constitute a cyclic, inflammatory-like process, wherein the increased expression of interleukin (IL)-1 and the biosynthesis of prostaglandins may be established corollaries. In this communication we hypothesize that glucocorticoids, potent anti-inflammatory principles, may exert an antiovulatory effect by interfering with ovarian IL-1-driven prostaglandin biosynthesis. To test this hypothesis, we examined the effect of treatment with dexamethasone on the activity of ovarian phospholipase A2 (PLA2), the event-limiting enzyme in prostaglandin biosynthesis, and on the gene expression pattern of secretory and cytosolic PLA2 (sPLA2 and cPLA2, respectively). Whole ovarian dispersates from immature rats were cultured under serum-free conditions for 48 h in the absence or presence of dexamethasone. At the conclusion of this culture period, PLA2 activity was determined in cell sonicates and conditioned media. Parallel probing for sPLA2 and cPLA2 transcripts was also undertaken using a solution hybridization/RNAse protection assay. Treatment of whole ovarian dispersates with dexamethasone produced a significant (P0.005) decrease in basal cellular and extracellular PLA2 activity to 27 and 40% of controls, respectively. A 5-fold decrease in the basal steady state levels of sPLA2 (but not cPLA2) transcripts was also noted. Co-treatment with dexamethasone produced complete inhibition of IL-1-stimulated cPLA2 transcripts but not of IL-1-supported cellular and extracellular PLA2 activity or sPLA2 transcripts. A glucocorticoid receptor antagonist (RU486), blocked the ability of dexamethasone to inhibit basal sPLA2 transcripts and extracellular PLA2 activity. The inhibitory effect of dexamethasone proved glucocorticoid-specific in that aldosterone and 17beta-estradiol were without effect. Taken together, these observations suggest that dexamethasone is capable of inhibiting basal (but not IL-1-supported) ovarian PLA2 activity, a glucocorticoid receptor-mediated effect due, in part, to a decrease in sPLA2 gene expression. Our findings further suggest that sPLA2 and cPLA2 are differentially regulated and that they may well differ in their relative contribution to ovarian prostaglandin biosynthesis in general and to PLA2 activity in particular. To the extent that IL-1 plays a central role in the ovulatory process, these findings argue against the view that the chronic anovulatory state induced by glucocorticoid excess is due, if only in part, to suppression of ovarian IL-1-dependent PLA2 activity.

Interleukin (IL)-1beta is a putative regulator of ovulation and perhaps luteal function. In this work, we examine its actions on the steroidogenic cascade of the rat granulosa cell. Whereas treatment of immature granulosa cells with FSH for 72 h produced substantial increments in the accumulation of progesterone, the addition of IL-1beta produced dose-dependent inhibition of this FSH effect. Pulse labeling of cells with [3H]pregnenolone revealed IL-1beta to effect a decrease in the FSH-supported accumulation of [3H]progesterone while enhancing the accumulation of its proximal metabolite, [3H]20alpha-dihydroprogesterone. IL-1beta was without effect on the activity levels of the progesterone-synthesizing enzymes, even though the corresponding transcripts were elevated. The effect of IL-1beta on some progesterone-degrading enzymes was negligible (5alpha-reductase) or modest (3alpha-hydroxysteroid dehydrogenase). In contrast, IL-1beta markedly stimulated both control and FSH-supported 20alpha-hydroxysteroid dehydrogenase activity (4.8- and 3.3-fold, respectively) and transcripts (16.4- and 7.5-fold, respectively). These data demonstrate an IL-1beta-mediated inhibition of gonadotropin-stimulated steroidogenesis via modulation of specific enzymes, and suggest a role for IL-1beta in mediating the observed decline of these bioactive hormones during ovulation and luteolysis.

This laboratory has previously shown that interleukin-1 (IL-1), a putative intermediary in the ovulatory process, is capable of up-regulating PG biosynthesis by cultured whole ovarian dispersates from immature rats. In part, this phenomenon was attributable to the stimulation of ovarian phospholipase A2 activity. In this communication we examine the possibility that the PG-promoting property of IL-1 is also due to the up-regulation of PG endoperoxide synthase (PGS), the rate-limiting step in prostanoid biosynthesis. The in vivo expression of ovarian PGS-2 transcripts in the course of a simulated estrous cycle rose abruptly to a peak (35-fold increase over the control value; P0.05) 8-12 h after hCG administration (i.e. before or during projected ovulation). PGS-1 transcripts, in turn, were not significantly altered during the periovulatory period. Treatment of cultured whole ovarian dispersates with IL-1beta resulted in dose- and time-dependent up-regulation of PGS-2 transcripts (as well as of immunoreactive PGS-2 protein and PGE2 accumulation), characterized by an ED50 of 2 ng/ml and a maximal (72-fold) increase at 10 ng/ml. Although treatment with IL-1beta also led to an increase in PGS-1 transcripts and immunoreactive PGS-1 protein, the relative magnitude of the effect was markedly reduced compared with that of PGS-2. Cotreatment with an IL-1 receptor antagonist completely reversed the IL-1 effects, thereby suggesting mediation via the IL-1 receptor. The ability of IL-1 to up-regulate PGS-2 transcripts proved relatively specific, in that other cellular regulators (insulin-like growth factor I, activin A, endothelin-1, transforming growth factor-alpha, tumor necrosis factor-alpha, vascular endothelial growth factor, leukemia inhibitor factor, hepatocyte growth factor, or keratinocyte growth factor) were not effective. The optimal IL-1 effect required heterologous contact-dependent coculturing of granulosa and thecal-interstitial cells. Taken together, these observations 1) reaffirm (by molecular probing) the granulosa cell as the primary site of ovarian PGS-1 and PGS-2 expression, 2) document an increase in ovarian PGS-2 transcripts before ovulation, and 3) reveal a marked dependence of ovarian PGS (21) transcripts, proteins, and activity on IL-1. The effects of IL-1 proved relatively specific, contingent upon somatic cell-cell cooperation, dose and time dependent, and IL-1 receptor mediated. These results are compatible with the proposition that the PG-promoting property of IL-1 is due, in large measure, to the activation of ovarian PGS transcription and translation. The ability of IL-1 to up-regulate ovarian PGS, an obligatory component of ovulation, is in keeping with the idea that IL-1 may constitute an intermediary in the ovulatory process.

It has been suggested that ovulation may constitute a cyclic inflammatory-like process and that gonadotropin-inducible intraovarian interleukin (IL)-1, an established mediator of inflammation, may play a central role in this regard. In support of this hypothesis, our group has been able to document the ability of IL-1 to potently stimulate prostaglandin biosynthesis by cultured rat ovarian cells. Herein we explore the possibility that the prostaglandin-stimulating action of IL-1 is due, in part, to the enhanced expression of ovarian secretory phospholipase-A2 (sPLA2). A single sPLA2 transcript of 1.4 kilobases was noted in all extraovarian tissues of immature rat origin subjected to Northern blot analysis. However, only a barely detectable signal was apparent in ovarian tissue. In contrast, the more sensitive RNase protection assay revealed the unequivocal presence of ovarian sPLA2 transcripts. Cellular localization studies by way of in situ hybridization documented sPLA2 transcripts primarily in the granulosa cell of the periovulatory ovary. Molecular probing of untreated cultured whole ovarian dispersates disclosed spontaneous elaboration of sPLA2 transcripts as early as 20 h after the introduction of cells into culture. Treatment of cultured whole ovarian dispersates with IL-1beta for 48 h produced a 1.7-fold increase (over the value in untreated controls) in the relative expression of sPLA2 transcripts (p0.01) along with a 1.7-fold increase in media PLA2 activity (p0.01). A more marked increase was documented for IL-1beta-treated cultured isolated granulosa cells (12.5-fold increase, p0.001). Treatment of whole ovarian dispersates with an IL-1 receptor antagonist (IL-1RA) produced a reduction in the basal expression of sPLA2 transcripts (55% at the 5 microg/ml dose level; p0.01) and PLA2 activity (40%; p0.01), thereby suggesting basal endogenous IL-1-like bioactivity. Treatment of cultured whole ovarian dispersates with either hCG or FSH led to 2.6-fold (p = 0.056) and 3-fold (p = 0.029) increases in the abundance of sPLA2 transcripts, respectively, effects blocked by the concurrent presence of IL-1RA. These observations 1) document the immature rat ovary as a site of sPLA2 gene expression, 2) localize the relevant transcripts to the postovulatory granulosa cell, 3) confirm the presence of functional secreted ovarian PLA2 activity, 4) reveal PLA2 expression to be IL-1- and gonadotropin-dependent, and 5) suggest the existence of endogenous PLA2-stimulating IL-1-like activity. These findings also suggest that the ability of hCG or FSH to up-regulate ovarian sPLA2 transcripts may be due, in part, to the endogenous elaboration of IL-like activity.

A body of information now supports the existence of an ovarian intrafollicular insulin-like growth factor (IGF)-I system concerned with the amplification of FSH action at the level of the rat granulosa cell. In this study we examined the ability of IGF-I to modulate the basal and FSH-supported activity and expression of key steroidogenic enzymes concerned with progesterone generation and metabolism in cultured granulosa cells from immature rats. The provision of IGF-I stimulated FSH-supported (20 ng/ml) accumulation of progesterone in a dose-dependent manner, reaching a plateau at an IGF-I dose of 50 ng/ml. This dose of IGF-I substantially enhanced FSH action over a broad range of FSH concentrations, reaching a maximum at an FSH dose of 20 ng/ml. Pulse labeling of FSH-pretreated cells with [3H]pregnenolone revealed relatively rapid (5 h) transformation to [3H]progesterone and other distal products that was accelerated by the concurrent addition of IGF-I. These changes in progesterone metabolism were associated with IGF-I-mediated enhancement of the activities and expression of key steroidogenic enzymes. Specifically, treatment with IGF-I produced significant augmentation of the FSH-stimulated activities of cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/ isomerase (3 beta-HSD) enzymes (2.4- and 1.8-fold, respectively). Similarly, P450scc and type I 3 beta-HSD transcripts were elevated by FSH in a dose-dependent manner, the concurrent addition of IGF-I further increasing expression (up to an additional 3-fold) in the range of 1-5 ng/ml (but not at the maximally stimulating dose of 20 ng/ml FSH). The addition of IGF-I also increased basal levels of type I 3 beta-HSD transcripts (3.8-fold). IGF-I enhanced FSH-stimulated 20 alpha-HSD activity and transcripts (2.3-fold and 1.8-fold, respectively) and increased the basal levels of 20 alpha-HSD transcripts (3-fold). Basal levels of 5 alpha-reductase were slightly elevated (1.3-fold) by IGF-I, but the FSH-attenuated activity was unchanged. Taken together, these findings suggest that IGF-I enhances the FSH-supported accumulation of progesterone in cultured granulosa cells through up-regulation of the expression and activity of key enzymes in the steroidogenic pathway. The acceleration of progesterone accumulation reflects a newly established steady state, favoring the activities of progesterone-forming over progesterone-metabolizing enzymes.

Given the suggestion that intraovarian insulin-like growth factor (IGF)-binding proteins (IGFBPs) may constitute markers of follicular atresia, we investigated the possibility that activin, a putative antiatretic principle, may modulate granulosa cell-derived IGFBPs. Untreated granulosa cells cultured for 72 h exhibited a progressive increase in the steady-state levels of transcripts corresponding to IGFBP-4 and IGFBP-5 (1.5-fold and 12-fold, respectively). Transcript levels corresponding to IGFBP-5 were consistently higher than their IGFBP-4 counterparts. Treatment with activin-A (50 ng/ml) for 72 h produced significant (p0.05) decrements in the steady-state levels of IGFBP-4 and IGFBP-5 transcripts (46% and 79%, respectively) as compared to controls. Thus, treatment with activin-A appears to be capable of blocking the spontaneous increase in IGFBP-4 and IGFBP-5 transcripts exhibited by untreated cultured granulosa cells. Consistent activin-A-induced decrements were also observed in the accumulation of the IGFBP-5 (but not the IGFBP-4) protein. Dose-response analysis revealed monophasic dose dependence (half maximal inhibitory doses of 16.2 and 7.8 ng/ml for IGFBP-5 and IGFBP-4 transcripts, respectively). The addition of increasing concentrations of the putative activin-binding protein, follistatin, produced dose-dependent reversal of the activin-A effect on IGFBP transcripts (IGFBP-5IGFBP-4). Activin-B was as effective as activin-A in reducing IGFBP-4 transcripts (31% decrement, p0.05) whereas it had little or no effect on IGFBP-5 transcripts (21% decrement, p0.1). No apparent effect was observed for the corresponding proteins. Activin-A action was specific in that treatment with transforming growth factor (TGF)-beta1, inhibin-A, or Müllerian-inhibiting substance (MIS)--all related peptides--failed to produce statistically significant alterations in the steady-state levels of IGFBP-4 and IGFBP-5 transcripts. Taken together, these observations reveal that activin-A exerts a substantial, relatively rapid, follistatin-neutralizable, dose- and time-dependent inhibitory effect on granulosa cell-derived IGFBP transcripts (IGFBP-5IGFBP-4). Other members of the TGFbeta superfamily (e.g., inhibin-A, TGFbeta1, and MIS) were without significant effect on the expression of IGFBP-4 and IGFBP-5. To the extent that the inhibition of IGFBP-4 and IGFBP-5 expression is associated with, and possibly causally related to, the promotion of follicular health, the present observations are in keeping with the proposition that activin may play an antiatretic role in the dynamic process of follicular selection.

Interleukin (IL)-1 beta has been shown to stimulate ovarian prostaglandin biosynthesis. We hypothesized that this effect entails the induction of phospholipase A2 (PLA2). Treatment of cultured whole ovarian dispersates of immature rat origin with IL-1 beta produced significant increases in [3H]arachidonic acid (AA) release and [3H]prostanoid accumulation as well as increases in cellular PLA2 activity and in secretory PLA2 and cytosolic PLA2 transcripts. Cotreatment with IL-1 receptor antagonist reversed IL-mediated (and basal) release of [3H]labeled AA and prostaglandin products, as well as cellular PLA2 activity. Treatment with IL-1 beta also promoted a significant decrease in the cellular content of [3H]phospholipids (apparently phosphatidylethanolamine but not phosphatidylcholine). These observations establish the ovary as a site of IL-1-dependent sPLA2 and cPLA2 gene expression, document the presence of a possible phosphatidylethanolamine-dependent PLA2 activity in cultured whole ovarian dispersates, reveal the up-regulatory, receptor-mediated action of IL-1 beta in this regard and suggest the existence of endogenous PLA2-stimulating/ IL-1-like bioactivity.

We have previously demonstrated that interleukin-1 beta (IL-1 beta), a putative intermediary in the ovulatory process, is a potent stimulator of ovarian PG biosynthesis. In this communication, we examine the possibility that this IL-1 effect reflects in part the induction of arachidonic acid mobilization by phospholipase A2 (PLA2). Molecular probing of whole ovarian material revealed the immature rat ovary to be a site of modest secretory PLA2 (sPLA2) gene expression. However, no change in ovarian sPLA2 gene expression was noted during the periovulatory period. Comparable probing for cytosolic PLA2 (cPLA2) failed to disclose a quantifiable signal. However, in situ hybridization localized both sPLA2 and cPLA2 (sPLA2cPLA2) transcripts to the granulosa cell layer of the ovarian follicle. Treatment of cultured whole ovarian dispersates with IL-1 beta produced significant (P0.01) increments in the steady state levels of transcripts corresponding to both sPLA2 (1.7-fold increase) and cPLA2 (5-fold increase), an effect reversed by an IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. Treatment with cycloheximide, a protein synthesis inhibitor, resulted in significant attenuation of the ability of IL-1 beta to up-regulate sPLA2 and cPLA2 gene expression as well as medium PLA2 activity. Treatment with aminoguanidine, an inhibitor of inducible nitric oxide synthase, led to augmentation of the ability of IL-1 beta to up-regulate sPLA2 and cPLA2 gene expression as well as medium PLA2 activity. Total cellular PLA2 activity proved time, cell density, and calcium dependent, with an optimal pH of 8.0-9.0 and K(m) values in the low micromolar range (2-5 microM). Our observations 1) establish the rat ovary as a site of sPLA2 and cPLA2 gene expression, 2) localize the corresponding transcripts to the granulosa cell layer, and 3) establish IL-1 beta as an up-regulatory agent for ovarian sPLA2 and cPLA2 gene expression as well as for ovarian PLA2 activity. These findings also indicate that the IL-1 effect is 1) receptor mediated, 2) contingent in part upon de novo protein biosynthesis, and 3) inhibited by nitric oxide. These observations support the proposition that PLA2 may be a key component in the IL-1-stimulated biosynthesis of ovarian PGs.

To study the expression, localization, and in vivo hormonal regulation of type I and type II interleukin-1 (IL-1) receptors in the rat ovary.Segments of the cDNAs for rat type I and type II IL-1 receptors were cloned and used as probes in RNase protection assays and in situ hybridization. Tissues obtained from immature rats and hormonally treated rat ovaries were examined.Type I IL-1 receptor (IL-1R(1)) was ubiquitously expressed in rat tissues, including granulosa cells prepared from immature ovaries, whereas type II IL-1 receptor (IL-1R(2)) expression was restricted to macrophages, thymus, and lung. Hypophysectomy and subsequent treatment with FSH and/or diethylstilbestrol did not alter significantly the abundance of IL-1R(1) transcripts in the whole ovary. However, the relative amount of ovarian IL-1R(1) transcripts increased 7.3-fold 6 hours after the administration of hCG to pregnant mare serum gonadotropin-primed immature rats. During this time, IL-1R(1) mRNA was localized primarily in the granulosa cells. The increased expression of IL-1R(1) persisted 24 hours after hCG administration but declined to baseline by 48 hours. Ovarian expression of IL-1R(2) mRNA was observed only before ovulation in amounts that were approximately 70-fold lower than IL-1R(1).The increased intraovarian expression of IL-1R(1) in granulosa cells during the periovulatory period implies that this cell type has a heightened receptivity to IL-1 and provides further indirect evidence that this cytokine is involved in the ovulatory process.

Because of the potential importance of insulin-like growth factor binding protein-4 (IGFBP-4) to ovarian physiology and the obvious limitations imposed by in vivo-exclusive experimental paradigms, we set out to delineate the characteristics and hormonal regulation of granulosa cell-derived IGFBP-4 under in vitro circumstances.Granulosa cells obtained by follicular puncture of the ovaries from diethylstilbestrol-primed intact immature rats were subjected to culture for up to 72 hours. Insulin-like growth factor binding protein-4 mRNA extracted from culture was subjected to Northern blot hybridization. Data normalization was assured by reprobing with the hamster Chinese hamster ovary B (CHOB) cDNA, and the IGFBP-4/CHOB ratio was calculated. Conditioned culture media were subjected to Western ligand blot before and after immunoprecipitation with a rat IGFBP-4-directed polyclonal antiserum (alpha-B104).Immunoprecipitation studies revealed granulosa cell-derived IGFBP-4 to be composed of a major 24-kDa species as well as a relatively minor 27-kDa moiety. Given cultures of untreated granulosa cells from immature estrogen-treated rats, transcripts corresponding to IGFBP-4 displayed an initial temporary decline culminating in a 6-hour nadir (a decrease of 67%; P.05) followed by relatively prompt recovery (within 24 hours) to levels comparable to those noted at the outset of the culture (time 0). However, additional (albeit statistically insignificant) increments were noted at the 48-hour (but not 72-hour) time point. Treatment of granulosa cells with increasing concentration of FSH resulted in decrements of up to 30% (P.05) in the steady-state levels of IGFBP-4 transcripts. A modest, biphasic, time-dependent response was noted for IGFBP-4 transcripts after treatment with high-dose FSH (100 ng/mL), an effect characterized by 24- and 48-hour increments (51% [P.05] and 26% [P = .052] over untrated controls, respectively) and a 72-hour decrement (25%; P = .16). The concurrent provision of the C19 aromatase substrate androstenedione (10(-7) mol/L) to the culture medium from 72 hours enhanced the inhibitory effect of FSH (100 ng/mL) for a maximal decrement in IGFBP-4 transcripts of 49% (P.05). Treatment with insulin-like growth factor (IGF)-I produced limited inhibition (up to 26%) of the steady-state levels of IGFBP-4 transcripts (P.05).Findings indicate the existence of heterogeneously-sized IGFBP-4 species, of which the 27-kDa (as distinct from the 24-kDa) IGFBP-4 moiety constitutes a relatively minor component. The steady-state levels of granulosa cell-derived IGFBP-4 transcripts display relatively limited regulation in response to treatment with either FSH or IGF-I.

Despite the potential importance of insulin-like growth factor binding protein-5 (IGFBP-5) to follicular development, the hormonal regulation of this antigonadotropic IGFBP has not been investigated. Therefore, it was the objective of this study to eludicate the role of gonadotropins and estrogen in the in vivo regulation of IGFBP-5 mRNA expression.Two models of follicular development in immature rats were used. Specifically, rats were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Alternatively, terminal follicular development was induced in intact immature rats by pregnant mare serum gonadotropin (PMSG) and hCG. The IGFBP-5 mRNA in whole ovarian RNA was assayed by Northern blot hybridization. Localization of expression in PMSG and hCG-stimulated ovaries was further assessed by in situ hybridization.Expression of IGFBP-5 mRNA was increased in ovaries from hypophysectomized rats. Treatment with FSH and/or DES did not alter the abundance of this mRNA. Treatment with PMSG induced a transient increase in IGFBP-5 expression that was localized in a subset of alpha-inhibin-negative follicles. At later times after PMSG, IGFBP-5 expression persisted in the surface epithelium but was not detected in large preovulatory follicles. In vitro studies affirmed the antigonadotropic action of IGFBP-5.In vivo expression of IGFBP-5 in the rat ovary is moderated by hormonal treatment both in terms of total expression and follicular localization.

During studies on the regulation of rat ovarian steroidogenic enzymes by interleukin-1 beta (IL-1 beta), we observed substantial metabolism of 25-hydroxycholesterol to two unusual polar products. This unexpected effect was observed both in isolated granulosa cells and in whole ovarian dispersates and was also induced by tumor necrosis factor alpha, but not by insulin-like growth factor I or follicle-stimulating hormone. The effect was dependent on time and the dose of IL-1 beta and was blocked by and IL-1 receptor antagonist. The formation of the polar metabolites was inhibited by ketoconazole and trilostane, but not by aminoglutethimide. Subsequent purification of these novel metabolites and analysis by gas chromatography/ mass spectrometry, NMR, and high performance liquid chromatography revealed them to be related 7 alpha-hydroxylated hydroxycholesterols (cholest-4-ene-7 alpha,25-diol-3-one and cholest-5-ene-3 beta,7 alpha,25-triol). IL-1 beta-stimulated ovarian 7 alpha-hydroxylase activity (3-10 pmol/min/mg of cellular protein) was nearly 4-fold that of control levels using 25-hydroxycholesterol as substrate. Activities at or below control levels were observed when IL-1 beta-treated cell sonicates were boiled or assayed in the presence of NADH (rather than NADPH), indicating that involvement of a nonenzymatic process was unlikely. IL-1 beta-stimulated 7 alpha-hydroxylase activity was inhibited to basal levels by a 10-fold excess of unlabeled 25- or 27-hydroxycholesterol, but not by cholesterol, pregnenolone, progesterone, testosterone, or dehydroepiandrosterone, suggesting that ovarian 7 alpha-hydroxylase is specific for hydroxycholesterols. Furthermore, when IL-1 beta-treated ovarian cultures were incubated with radiolabeled cholesterol or testosterone, no 7 alpha-hydroxylated products were observed. We were also unable to detect any mRNA transcripts for liver cholesterol 7 alpha-hydroxylase in IL-1 beta-stimulated ovarian cultures. This study describes an ovarian hydroxycholesterol 7 alpha-hydroxylase that differs from liver cholesterol 7 alpha-hydroxylase and from other nonhepatic progestin/ androgen 7 alpha-hydroxylases. The novel finding of the regulation of a 7 alpha-hydroxylase by IL-1 beta (and tumor necrosis factor alpha) suggests a unique role for cytokines in the regulation of cholesterol metabolism in the ovary and possibly other tissues.