Glucocorticoids suppress basal (but not interleukin-1-supported) ovarian phospholipase A2 activity: evidence for glucocorticoid receptor-mediated regulation.

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 (P < 0.005) 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-1beta inhibits steroidogenic bioactivity in cultured rat ovarian granulosa cells by stimulation of progesterone degradation and inhibition of estrogen formation.

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.

Rat ovarian prostaglandin endoperoxide synthase-1 and -2: periovulatory expression of granulosa cell-based interleukin-1-dependent enzymes.

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; P < 0.05) 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 (2 >> 1) 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.

The mouse intraovarian insulin-like growth factor I system: departures from the rat paradigm.

Although the rat intraovarian insulin-like growth factor I (IGF-I) system is well documented, the increasing availability of null mouse mutants for components of the IGF system necessitates characterization of the mouse model as well. Therefore, we undertook to define the components of the mouse intraovarian IGF-I system and to examine its operational characteristics. The cellular pattern of ovarian gene expression was comparable in the immature rat and mouse for IGF-I and the type I IGF receptor. In both species, IGF-I messenger RNA (mRNA) is selectively expressed by granulosa cells in growing, healthy appearing follicles. Type I IGF receptor mRNA was also concentrated in granulosa cells, but was uniformly expressed in all follicles large and small, healthy and atretic appearing alike. Cellular patterns of IGF-binding protein (IGFBP) gene expression were similar in mouse and rat, except in the case of IGFBP-2. IGFBP-2 mRNA was localized to the mouse granulosa cell, in contrast to its concentration in the rat thecal-interstitial compartment. This difference in IGFBP expression pattern was also noted in cultured mouse and rat granulosa cells. Although immunoreactive IGFBP-4 (24 and 28 kDa) and IGFBP-5 (29 kDa) were shared by both species, the cultured mouse granulosa cell also featured immunoreactive IGFBP-2 (30 kDa). The mouse paradigm further differed from its rat counterpart in that a maximal dose of FSH, previously shown to suppress the elaboration of rat granulosa cell-derived IGFBPs, was without effect. The addition of IGF-I proved stimulatory to the accumulation of the 28- to 29-kDa IGFBPs, as previously reported for the rat. However, IGF-I proved inhibitory to the accumulation of the 24-kDa IGFBP (presumptive nonglycosylated IGFBP-4); no consistent effect was reported for the rat model. Functional comparisons of mouse and rat ovarian cell cultures revealed qualitatively comparable FSH-stimulated steroidogenesis, disposition of radiolabeled pregnenolone, IGF-I-amplified FSH action, and IGFBP-mediated antigonadotropic activity. These findings indicate that the mouse intrafollicular IGF-I system differs from the rat paradigm in both the makeup and regulation of granulosa cell-derived IGFBPs as well as in the intensity and character of the steroidogenic process. Studies employing the mouse model must take into account these important distinctions relative to the more established rat paradigm.

Interleukin (IL)-1beta increases glucose uptake and induces glycolysis in aerobically cultured rat ovarian cells: evidence that IL-1beta may mediate the gonadotropin-induced midcycle metabolic shift.

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.

Insulin-like growth factor-I-mediated amplification of follicle-stimulating hormone-supported progesterone accumulation by cultured rat granulosa cells: enhancement of steroidogenic enzyme activity and expression.

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.

Interleukin-1 beta stimulates ovarian phosphoipase A2 (PLA2) expression and activity: up-regulation of both secretory and cytosolic PLA2.

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.

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.

A novel nonhepatic hydroxycholesterol 7 alpha-hydroxylase that is markedly stimulated by interleukin-1 beta. Characterization in the immature rat ovary.

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.

Rat ovarian granulosa cell as a site of endothelin reception and action: attenuation of gonadotropin-stimulated steroidogenesis via perturbation of the A-kinase signaling pathway.

Endothelins (ETs) are a family of vasoactive peptides that may be involved in granulosa cell (GC) luteinization or follicular maturation. However, the precise role of ET in ovarian physiology remains unknown. We have investigated whether the rat GC is a site of ET reception and have characterized the antigonadotropic effect of ET in cultured GC from immature rats. Two major ET binding species (52 and 30 kDa) were observed after cross-linking of GC membranes with radiolabeled ET-1, although the smaller protein may represent a degradative product. Unlabeled ET-1, ET-2, or ET-3 were equipotent in displacing radiolabeled ET-1 from these putative ET receptors, with EC50s of 0.3-0.7 x 10(-9) M. Similarly, ET-1, ET-2, and ET-3 were equipotent (EC50s of about 10(-10) to 10(-9) M) in inhibiting the FSH-supported accumulation of progesterone. ET-1 (10(-7) M) also inhibited (> 90%) FSH-supported estrogen accumulation. Maximum progesterone inhibition (> 90%) by ET-1 (10(-7) M) was achieved throughout the range of FSH does and cell densities tested and by 48 h or 72 h of culture. ET-1 was not cytotoxic in the dose range tested. Forskolin-stimulated progesterone accumulation was similarly inhibited by ET-1, suggesting that ET-1 inhibits cAMP-mediated (e.g., FSH or forskolin-stimulated) progesterone accumulation. ET-1 inhibited (74%) the FSH-stimulated accumulation of cAMP, suggesting that it acts at sites related to cAMP generation or degradation. In addition, ET-1 inhibited 8-bromo-cAMP-generated progesterone accumulation (60%), suggesting that it also acts at sites distal to cAMP generation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Interleukin-1 beta stimulates nitrite production in the rat ovary: evidence for heterologous cell-cell interaction and for insulin-mediated regulation of the inducible isoform of nitric oxide synthase.

Recent studies suggest that endogenously generated nitric oxide (NO) may mediate the effects of cytokines in a variety of tissues. In an effort to determine whether NO generation mediates any of the intraovarian actions of interleukin-1 beta (IL-1 beta), we have looked for and characterized the accumulation of nitrite by IL-1 beta-treated, cultured whole ovarian dispersates. Application of IL-1 beta significantly enhanced basal nitrite release in a dose-, cell density- and time-dependent manner, the latter characterized by a lag time of about 20 h, suggestive of induction of NO synthase (NOS). Cellular NOS activity was also elevated by IL-1 beta. Sustained nitrite accumulation required continuous application of IL-1 beta. The maximally stimulating dose of IL-1 beta (50 ng/ml) produced a 10-fold increase in nitrite accumulation by 96 h of culture, an effect reduced 23% when cells were cultured in substrate (i.e., arginine)-free media. IL-1 beta-stimulated nitrite accumulation was reduced to control levels by the simultaneous application of an IL-1 beta receptor antagonist, thereby suggesting a specific receptor-mediated effect. Both the control and IL-1 beta-stimulated levels of nitrite accumulation were attenuated in a dose-dependent manner by inhibitors that favor the inducible form of NOS. In contrast, selective inhibitors of the constitutive form of NOS were significantly less potent. No inhibition was noted after application of an inactive stereoisomeric analogue. IL-1 beta-induced nitrite accumulation was shown to require cell-cell interaction between granulosa and theca-interstitial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin.

A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4- sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay (r = 0.991) and over a wide range of viability (15-75%) as determined by ethidium bromide/acridine orange staining (r = 0.995).

Endothelin-1 as a luteinization inhibitor: inhibition of rat granulosa cell progesterone accumulation via selective modulation of key steroidogenic steps affecting both progesterone formation and degradation.

Endothelin (ET)-1, is a 21 amino acid vasoactive peptide subject to regulation by cellular oxygen tension. However, an increasing body of information now suggests that ET-1 is a multifunctional peptidergic regulator the actions of which are not limited to the vascular system. Although ET-1 has been shown to inhibit the gonadotropin-supported accumulation of progesterone by cultured granulosa cells, the precise cellular mechanism(s) involved remain unknown. It was therefore the objective of this study to examine in greater detail the effects of ET-1 on progestin economy in cultured granulosa cells from immature rats. Treatment with ET-1 was inhibitory to the FSH-supported accumulation of progesterone in a dose-dependent manner, an action characterized by a median inhibitory dose of 2 x 10(-11) M and a maximal inhibitory effect of 90%. This inhibitory action of ET-1 was reversible following extensive washing and could not be accounted for by a decrease in the viable cell mass. Evaluation of the activities of progesterone-forming enzymes revealed ET-1 to be a potent (P < 0.01) inhibitor of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase (HSD)/isomerase (76.1 +/- 1.2% and 47.3 +/- 8.6% inhibition, respectively). Cellular radiolabeling with [3H]pregnenolone confirmed an ET-1-induced inhibition of the FSH-supported accumulation of radiolabeled progesterone. However, this effect was concomitant with enhancement of the accumulation of more distal metabolites, i.e. 20 alpha-dihydroprogesterone, 5 alpha-pregnane-3 alpha, 20 alpha-diol, and 5 alpha-pregnane-3 alpha-ol-20-one. Analysis of the FSH-supported activities of the progesterone-degrading enzymes revealed ET-1 as a potent (P < 0.05) stimulator of 20 alpha-HSD and 5 alpha-reductase (3.6 +/- 1.0 and 1.7 +/- 0.3-fold, stimulation respectively). In contrast, no significant changes were observed in 3 alpha-HSD activity. Taken together, our findings demonstrate that the ET-1 induced inhibition of gonadotropin-supported progesterone accumulation constitutes a complex phenomenon wherein ET-1 inhibits the activities of steroidogenic enzymes concerned with progesterone formation while enhancing the activities of enzymes concerned with progesterone degradation. We speculate that ET-1, possibly of intraovarian origin, acts as a luteinization-inhibitor to suppress premature luteinization at a time when continued preovulatory expression of ET-1 (in the intact but not ruptured follicle) may be contingent upon relative intrafollicular hypoxia.

Interleukin-1 is both morphogenic and cytotoxic to cultured rat ovarian cells: obligatory role for heterologous, contact-independent cell-cell interaction.

An increasing body of information now suggests that intraovarian interleukin-1 (IL-1) may play an intermediary role in the ovulatory process. Given that follicular rupture inevitably requires marked tissue remodeling and possibly cell death, we set out to examine the morphogenic potential of IL-1 under in vitro circumstances. Treatment of freshly plated whole ovarian dispersates from immature rats with any one of several batches of IL-1 (10 ng/ml) for up to 96 h produced marked time-dependent morphological alterations, including cellular retraction, rounding, clumping, aggregation, blebbing, swelling, and, ultimately, irreversible detachment. Evidence of (asynchronous) cell death consisted of reduced total cell number, diminished cellular protein content, enhanced cellular release of lactic dehydrogenase, failure to exclude trypan blue, and attenuated reduction of the tetrazolium dye 3-[4,5-dimethylthiazol-2-y]2,5-diphenyltetrazolium bromide to spectrophotometrically detectable formazan. Comparable results were obtained when using established day 4 cultures, arguing against a possible critical action of IL-1 at the time of plating. Dose-response curves revealed IL-1 beta (EC50, 0.2-0.4 ng/ml) to be substantially more potent than IL-1 alpha (EC50, 2.7-2.8 ng/ml). Importantly, the concurrent provision of an IL-1 beta-directed polyclonal antibody yielded complete immunoneutralization of the IL-1 beta effect, arguing against the possible involvement of a non-IL-1 contaminant. An unrelated polyclonal antiserum raised against insulin-like growth factor-I was without effect. IL-1 action proved relatively specific, in that tumor necrosis factor-alpha (10 ng/ml), a putative cytotoxic principle, as well as IL-1-inducible ILs (IL-2 and -6; 100 U/ml) were without effect. Although minimally effective at the level of the isolated granulosa or theca-interstitial cell, IL-1 proved highly potent in heterologous (but not homologous), contact-dependent and independent cocultures of these somatic cell types, strongly suggesting obligatory cell-cell cooperation. These observations further indicate that IL-1 action is indirect and may require the induction of an intermediary soluble principle to serve as the final effector. Taken together, these findings indicate that relatively low concentrations of IL-1 (beta >> alpha), possible of somatic ovarian cell or resident ovarian macrophage origin, are capable of exerting specific dose- and time-dependent (immunoneutralizable) morphogenic as well as cytotoxic effects at the level of ovarian cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Follicle-stimulating hormone inhibits granulosa cell 5 alpha-reductase activity. Possible role of 5 alpha-reductase as a steroidogenic pubertal switch.

In order to elucidate the role of 5 alpha-reductase in the ovarian pubertal transition from 5 alpha-reduced to non-5 alpha-reduced steroids, we examined the characteristics and regulation of granulosa cell (GC) 5 alpha-reductase activity. Maximum activity was observed at 37 degrees C and at a pH of 6.5-8.0. Synthetic 4-aza-3-oxosteroids proved to be potent inhibitors (76% inhibition at 0.1 microM) of ovarian 5 alpha-reductase activity, and 20 alpha-DHP was a better substrate than either progesterone or testosterone (4- or 7-fold higher affinity constants, respectively). The Km (20 alpha-DHP) of the enzyme was 0.50 +/- 0.03 microM and 0.75 +/- 0.20 microM in homogenates of whole ovaries and GC, respectively. 17 beta-Estradiol was a non-competitive inhibitor (KI = 6.97 microM). 5 alpha-Reductase activity was 22-fold (immature) to 68-fold (mature) higher in liver than ovary and 4-fold higher in theca-interstitial shells than in isolated GC. Ovarian 5 alpha-reductase activity decreased markedly with age (greater than 60% inhibition in mature, randomly cycling rats as compared to immature rats). In vivo administration of follicle-stimulating hormone (FSH) to immature rats produced a dose-dependent decrease in GC 5 alpha-reductase activity (36 +/- 1.1% and 46 +/- 5.9% inhibition following 12 micrograms and 24 micrograms FSH, respectively). Similarly, the in vitro provision of FSH (100 ng/ml) to cultured GC from immature rats resulted in (36-59%) inhibition in 5 alpha-reduced steroids. Inasmuch as FSH promotes GC development and the advancement of puberty, its ability to "switch-off" ovarian 5 alpha-reductase activity may enhance the formation of biologically potent (i.e. non-5 alpha-reduced) progestins as well as the availability of aromatizable androgens, in the best interests of pubertal steroidogenesis.

Cytokine-mediated regulation of ovarian function: interleukin-1 inhibits gonadotropin-induced androgen biosynthesis.

Resident ovarian macrophages have long been recognized as potential in situ regulators of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, interleukin-1 (IL-1) has recently been shown to exert profound regulatory effects at the level of the ovarian granulosa cell. In this report, we examine the possibility that the adjacent theca-interstitial (androgen-producing) cell may also be a site of IL-1 reception and action. The basal accumulation of androsterone, the major androgenic steroid synthesized by whole ovarian dispersates from immature rats, in the presence of insulin (1 microgram/ml), increased 8- to 9-fold after treatment with human CG (1 ng/ml). Although IL-1 alpha or IL-1 beta (10 ng/ml) by themselves were without effect on basal androsterone accumulation, both cytokines (IL-1 beta greater than IL-1 alpha) inhibited human CG hormonal action (in the presence of insulin) in a dose-dependent manner, the maximal inhibitory effect being 75%. Similar results were obtained when using highly purified theca-interstitial cells derived from the same animal model suggesting that IL-1-attenuated androgen biosynthesis is due, at least in part, to IL-1 acting directly at the level of the theca-interstitial cells. The IL-1 effect proved relatively specific since all other known interleukins (IL-1, IL-3, IL-4, IL-5, and IL-6) were without effect. Moreover, IL-1 beta action was effectively immunoneutralized when concurrently applied with anti-IL-1 beta (but not nonimmune) sera. Significantly, the antigonadotropic action of IL-1 could not be accounted for by a decrease in the viable cell mass. Tracer studies with radiolabeled steroid substrates suggested that IL-1-attenuated ovarian androsterone accumulation is due, if only in part, to inhibition of transformations catalyzed by (theca-interstitial) 17 alpha-hydroxylase/17:20 lyase, stimulation of theca-interstitial (or granulosa 20 alpha-hydroxysteroid dehydrogenase-mediated conversions, or both. Taken together, these findings indicate that relatively low concentrations of IL-1, possibly originating from somatic ovarian cells or resident ovarian macrophages, are capable of exerting an inhibitory effect upon gonadotropin-supported androgen production. As such, these and previous observations suggest that intraovarian IL-1 may play a dual regulatory role in the developing ovarian follicle by targeting both the granulosa and theca-interstitial cells as its sites of action.

Cytokine-mediated regulation of ovarian function. Tumor necrosis factor alpha inhibits gonadotropin-supported ovarian androgen biosynthesis.

Resident ovarian macrophages have been implicated in the regulation of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, tumor necrosis factor (TNF) alpha, has been shown to attenuate the gonadotropin-dependent differentiation of the somatic ovarian (estrogen-producing) granulosa cell. This study examines the possibility that TNF alpha may also regulate the adjacent androgen-producing theca interstitial cell. The basal accumulation of androsterone (the major androgenic steroid), synthesized by whole ovarian dispersates from immature rats, remained unchanged following treatment with TNF alpha (30 ng/ml) alone. In contrast, concurrent treatment with increasing concentrations of TNF alpha (0.03-30 ng/ml), yielded dose-dependent inhibition of the human chorionic gonadotropin (1 ng/ml)-stimulated accumulation of androsterone. This reversible and immunoneutralizable effect of TNF alpha was characterized by a minimal effective dose of 0.1 ng/ml, a median inhibitory dose of 0.9 ng/ml, a maximal inhibitory effect of 90%, and a minimal time requirement of less than or equal to 48 h. Comparable results were obtained when using highly purified theca interstitial cells, thereby indicating that TNF alpha is capable of exerting a direct inhibitory effect at the level of the ovarian androgen-producing cell. TNF alpha action was not accounted for by alterations in the plated viable cell mass. Instead, treatment with TNF alpha resulted in significant inhibition of the human chorionic gonadotropin-supported accumulation of cAMP, the putative second messenger of gonadotropin hormonal action. TNF alpha action at sites distal to cAMP generation was associated with profound inhibition of the conversion of the [3H]pregnanolone (3 alpha-hydroxy,5 alpha-pregnane-20-one) and [3H]17 alpha-hydroxypregnanolone (3 alpha, 17 alpha-dihydroxy,5 alpha-pregnane-20-one) substrates to androsterone, suggesting stimulation of 20 alpha-hydroxysteroid dehydrogenase activity, inhibition of 17 alpha-hydroxylase/17:20 lyase activity, or both. Taken together, these findings indicate that TNF alpha, acting at relatively low concentrations, is capable of inhibiting gonadotropin-supported ovarian androgen biosynthesis by selectively modulating the activity of relevant key steroidogenic enzymes. As such, these observations suggest that the theca interstitial cell is a site of TNF alpha reception and action and that TNF alpha, possibly of resident ovarian macrophage origin, may partake in the regulation of ovarian androgen production, an effect due in part to inhibition of the activity of the key steroidogenic enzymes 17 alpha-hydroxylase/17:20 lyase.

Evaluation of a small-scale method for the extraction of the K88 antigen from enterotoxigenic Escherichia coli.

A small-scale method for the extraction of the K88 major fimbrial subunit from enterotoxigenic Escherichia coli (ETEC) based on heat extraction has been developed. Variation in the buffer composition, time and temperature of extraction had negligible effect on the subsequent SDS-PAGE profile. There was, however, a correlation between the pH of the extraction buffer and the ensuing amount of K88 released. Reduction of the pH from 7 to 4 reduced by six-fold the amount of K88 released from the cells. We suggest that the relative stability of the K88 fimbriae at acid pH may influence the site of infection by ETEC in the intestine.

Transforming growth factor-beta 1 inhibits ovarian androgen production: gene expression, cellular localization, mechanisms(s), and site(s) of action.

It is the aim of this study to establish ovarian transforming growth factor-beta 1 (TGF beta 1) gene expression, to reevaluate its cellular localization, and to explore potential interactions of this regulatory peptide on ovarian androgen biosynthesis. Northern analysis of whole ovarian polyadenylated RNA revealed a single 2.5-kilobase transcript corresponding to the TGF beta 1 precursor. Immunohistochemical staining localized the protein to the thecal-interstitial (interfollicular) compartment. To explore potential autocrine effects of TGF beta 1, use was made of whole ovarian dispersates from immature rats the differentiation of which was monitored by the acquisition of androgen biosynthetic capacity. The accumulation of androsterone, the major androgenic steroid detectable in this culture system, increased 5.4-fold over baseline in response to treatment with hCG (1 ng/ml). This effect was further optimized (2- to 4-fold) by supplementation with insulin (1 microgram/ml) and insulin-like growth factor-I (50 ng/ml). In the absence of these optimizing supplements, TGF beta 1 (10 ng/ml) was without effect on basal androsterone accumulation, producing distinct, albeit relatively limited (25%), inhibition of hCG hormonal action. In contrast, supplement-mediated optimization of ovarian androgen biosynthesis revealed TGF beta 1 to be a highly potent inhibitor (greater than 80%) of hCG hormonal action. This reversible TGF beta 1 action proved time and dose dependent, with a minimal time requirement of 72 h and a median inhibitory dose of 2.6 ng/ml. TGF beta 1 action was not due to diminution in the viable cell mass or altered cAMP generation and, therefore, most likely involved a site(s) of action distal to or independent of cAMP generation. Cellular radiolabeling studies of TGF beta 1-treated ovarian cells disclosed the accumulation of steroid intermediates proximal to the 17 alpha-hydroxylation step, suggesting TGF beta 1-mediated blockade at the level of the steroidogenic enzyme 17 alpha-hydroxylase/17-20-lyase. Taken together, these observations are in keeping with the view that TGF beta 1, possibly of thecal-interstitial origin, may not only play a positive paracrine role at the level of the adjacent granulosa cell (as previously reported), but may also constitute one of several autocrine signals concerned with the regulation of ovarian androgen economy. As such, these findings reaffirm the polyfunctional nature of TGF beta 1 action, as manifested by its diametrically opposed effects in different ovarian compartments.