Izhar Ben-Shlomo

The morphogenic/cytotoxic and prostaglandin-stimulating activities of interleukin-1 beta in the rat ovary are nitric oxide independent.

Nitric oxide (NO) has been implicated as a mediator of physiologic and pathologic cellular injury. Since the cytokine interleukin-1 beta (IL-1 beta) induces nitric oxide synthase (NOS) activity as well as effects morphogenic/cytotoxic changes and increased prostaglandin (PGE2) levels in cultured whole ovarian dispersates, we set out to determine whether these actions are interrelated. Treatment with IL-1 beta resulted in a marked increase in media nitrite and nitrate accumulation, morphological alterations, and increased release of lactate dehydrogenase (LDH) into media. Addition of IL-1 receptor antagonist (RA) eliminated these IL-1 beta effects. In contrast, specific inhibitors of NOS failed to reverse IL-1 beta-induced morphogenic changes or LDH release in spite of complete reduction of media nitrite to control levels. Similarly, treatment with transforming growth factor beta 1, inhibited IL-1 beta-induced nitrite accumulation, but had no effect on the morphologic or cytotoxic endpoints. Moreover, the addition of sodium nitroprusside, an NO generator, resulted in progressive increments in media nitrite content without a corresponding increase in the IL-1 beta-associated morphogenic changes or media LDH content. Furthermore, IL-1-induced PGE2 accumulation remained unaffected by specific NOS inhibition. These observations support the view that NO does not mediate the morphogenic/cytotoxic or inflammatory-like (e.g., PGE2 inducing) properties of IL-1 beta in cultured whole ovarian dispersates. Although the precise role of NO in ovarian physiology remains unknown, it is possible that NO participates in the periovulatory modulation of ovarian blood flow by virtue of its potent vasodilatory activity.

The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process.

This study characterizes the rat ovary as a site of hormonally dependent glucose transporter (Glut) expression, and explores the potential role of interleukin (IL)-1, a putative intermediary in the ovulatory process, in this regard. Molecular probing throughout a simulated estrous cycle revealed a significant surge in ovarian Glut3 (but not Glut1) expression at the time of ovulation. Treatment of cultured whole ovarian dispersates from immature rats with IL-1beta resulted in upregulation of the relative abundance of the Glut1 (4.5-fold) and Glut3 (3.5-fold) proteins as determined by Western blot analysis. Other members of the Glut family (i.e., Gluts 2, 4, and 5) remained undetectable. The ability of IL-1 to upregulate Glut1 and Glut3 transcripts proved time-, dose-, nitric oxide-, and protein biosynthesis-dependent but glucose independent. Other ovarian agonists (i.e., TNF alpha, IGF-I, interferon-gamma, and insulin) were without effect. Taken together, our findings establish the mammalian ovary as a site of cyclically determined Glut1 and Glut3 expression, and disclose the ability of IL-1 to induce the ovarian expression as well as translation of Glut1 and Glut3 (but not of Gluts 2, 4, or 5). Our observations also establish IL-1 as the first known regulator of Glut3, the most efficient Glut known to date. In so doing, IL-1, a putative component of the ovulatory process, may be acting to meet the increased metabolic demands imposed on the growing follicle and the ovulated cumulus-enclosed oocyte.

A Novel Nonhepatic Hydroxycholesterol 7α-Hydroxylase That Is Markedly Stimulated by Interleukin-1β CHARACTERIZATION IN THE IMMATURE RAT OVARY

During studies on the regulation of rat ovarian steroidogenic enzymes by interleukin-1β (IL-1β), 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 α, but not by insulin-like growth factor I or follicle-stimulating hormone. The effect was dependent on time and the dose of IL-1β and was blocked by an 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 7α-hydroxylated hydroxycholesterols (cholest-4-ene-7α,25-diol-3-one and cholest-5-ene-3β,7α,25-triol). IL-1β-stimulated ovarian 7α-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-1β-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-1β-stimulated 7α-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 7α-hydroxylase is specific for hydroxycholesterols. Furthermore, when IL-1β-treated ovarian cultures were incubated with radiolabeled cholesterol or testosterone, no 7α-hydroxylated products were observed. We were also unable to detect any mRNA transcripts for liver cholesterol 7α-hydroxylase in IL-1β-stimulated ovarian cultures. This study describes an ovarian hydroxycholesterol 7α-hydroxylase that differs from liver cholesterol 7α-hydroxylase and from other nonhepatic progestin/androgen 7α-hydroxylases. The novel finding of the regulation of a 7α-hydroxylase by IL-1β (and tumor necrosis factor α) suggests a unique role for cytokines in the regulation of cholesterol metabolism in the ovary and possibly other tissues.

The rat ovarian phospholipase A2 system: Gene expression, cellular localization, activity characterization, and interleukin-1 dependence

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 (sPLA2 > cPLA2) transcripts to the granulosa cell layer of the ovarian follicle. Treatment of cultured whole ovarian dispersates with IL-1 beta produced significant (P < 0.01) 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.

Simplified riboprobe purification using translucent straws as gel tubes

Gel purification of radioactive riboprobes enhances the quality of the ribonuclease protection assay. A simple and effective method for riboprobe purification is described. The method uses acrylamide gels in plastic tubes to achieve electrophoretic separation of the RNA polymerase products.

Interleukin (IL)-1β Increases Glucose Uptake and Induces Glycolysis in Aerobically Cultured Rat Ovarian Cells: Evidence That IL-1β May Mediate the Gonadotropin-Induced Midcycle Metabolic Shift1

This communication explores the possibility that interleukin (IL)-1beta, a putative intermediary in the ovulatory process, may take part in the gonadotropin-driven midcycle diversion of ovarian carbohydrate metabolism toward glycolysis. We examined the effect of treatment with IL-1beta on glucose metabolism in aerobically cultured whole ovarian dispersates from immature rats. Treatment with IL-1beta increased cellular glucose consumption/uptake, stimulated extracellular lactate accumulation and media acidification, and decreased extracellular pyruvate accumulation in a receptor-mediated, time-, dose- and cell density-dependent manner. Endogenous IL-1beta-like bioactivity was shown to mediate the ability of gonadotropins to exert these same metabolic effects. The IL-1beta effect was also (1) apparent over a broad range of glucose concentrations, inclusive of the putative physiological window; (2) relatively specific, because tumor necrosis factor-alpha and insulin were inactive; (3) contingent upon cell-cell cooperation (4) and reliant on de novo protein synthesis. Comparison of the molar ratios of lactate accumulation to glucose consumption in IL-1beta-replete vs. IL-1beta-deplete cultures suggests that IL-beta promotes the conversion of all available glucose to lactate but that other substrates for lactate production may also exist. However, no lactate was generated by cells grown under glucose-free conditions. Taken together, our data suggest that IL-1beta may act as a metabolic hormone in the ovary. Subject to the limitations of the in vitro paradigm, our data also suggest that IL-1beta may mediate the gonadotropin-associated midcycle shift in ovarian carbohydrate metabolism. By converting the somatic ovarian cells into a glucose-consuming glycolytic machinery, IL-1beta may establish glycolysis as the main energy source of the relatively hypoxic preovulatory follicle and the resultant cumulus-oocyte complex. The consequent oxygen sparing may conserve the limited supply of oxygen needed for vital biosynthetic processes such as steroidogenesis. This adaptational response may also provide the glycolytically incompetent oocyte with the obligatory tricarboxylic cycle precursors it depends on to meet the increased energy demands imposed upon it by the resumption of meiosis.

Interleukin-1β Stimulates Ovarian Phospholipase A2 (PLA2) Expression and Activity: Up-Regulation of Both Secretory and Cytosolic PLA21

Interleukin (IL)-1β 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-1β 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-1β 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 u...

Rat Ovarian Interleukin-1α: Interleukin-1-Dependent In Vitro Expression

Evidence exists supporting the possibility that intraovarian interleukin-1 (IL-1) may play an intermediary role in the periovulatory cascade. Although the existence of a mammalian intraovarian IL-1 system has been convincingly demonstrated, most efforts have focused on the possibility that the mammalian ovary is a site of IL-1b production, reception, and action. the objective of this study was to explore the possibility of ovarian IL-1a expression, characterize its pattern of expression by cultured ovarian cells, and study its hormonal regulation. The basal in vitro expression of IL-1a by cultured whole ovarian dispersates from immature rats increased spontaneously, reaching a peak (sixford increase over untreated controls) at 4 h. Treatment with an IL-1 receptor antagonist (IL-1RA), human chorionic gonadorropin, or IL-1b failed to attenuate the initial 4-h burst of IL-1a expression. By contrast, treatment of whole ovarian dispersates with IL-1b for 48 h resulted in significant upregulation of IL-1 a transcripts (60-fold increase). This IL-1b effect was completely blocked by cotreatment with IL-1RA, thereby suggesting mediation via a specific IL-1 receptor. The IL-1b effect proved to be protein biosynthesis and eicosanoid dependent, nitric oxide independent, and relatively specific in that it was not reproduced by a select series of other granulosa cell agonists.