Insulin-like growth factor (IGF)-I stimulates IGF-I and type 1 IGF receptor expression in cultured rat granulosa cells: autocrine regulation of the intrafollicular IGF-I system.

A growing body of information documents the existence of a complete rat intrafollicular insulin-like growth factor (IGF)-I system replete with a ligand (IGF-I), a receptor (type 1 IGF receptor) IGF binding proteins (4 and 5), and IGFBP-directed endopeptidases (4 and 5). Previous studies have established the ability of IGF-I to promote the elaboration of granulosa cell-derived IGFBP-5 and to suppress the activity of granulosa cell-derived IGFBP-5-directed endopeptidase. It was the purpose of this article to examine the effects of treatment with IGF-I on the other components of the intrafollicular IGF system, i.e., IGF-I itself and the type 1 IGF-receptor. Granulosa cells, obtained by follicular puncture from 25-d-old estrogen-primed rats were cultured in polystyrene tubes for 72 h under serum-free conditions, in the absence or presence of the indicated agents. At the conclusion of each experiment, media were discarded, and RNA was extracted and subjected to an RNase protection assay. Treatment of cultured rat granulosa cells with IGF-I resulted in a significant 1.8-fold increase in the steady-state levels of IGF-I mRNA. No effect was noted on the total cellular DNA content thereby arguing against the possibility that the relative increase in IGF-I transcripts can be ascribed to a possible treatment-induced increase in cell number in culture. The IGF-I effect was apparent (p < 0.05) at IGF-I doses as low as 1 ng/mL, minimal additional increments being noted thereafter. Treatment with insulin and des (1-3) IGF-I proved equally effective, producing 2.0- and 2.6-fold increases, respectively, thereby suggesting that the IGF-I effect may be mediated via the type 1 IGF receptor. Treatment with IGF-I also resulted in a significant (p < 0.005) increase in type 1 IGF receptor expression (2.3-fold increase), the first significant effect being noted at the 30 ng/mL dose level. Similar results obtained for insulin and des (1-3) IGF-I thereby suggest that the ability of IGF-I to upregulate the expression of its own receptor is probably type 1 IGF receptor-mediated. Taken together, these findings indicate that treatment of estrogen-primed granulosa cells with IGF-I will result in upregulation of the steady-state levels of transcripts corresponding to IGF-I itself and to its type 1 IGF receptor. These observations emphasize the importance of positive autoregulatory phenomena as determinants of the intrafollicular content of IGF-I and its receptor.

Transcriptional and posttranscriptional regulation of intraovarian insulin-like growth factor-binding proteins by interleukin-1beta (IL-1beta): evidence for IL-1beta as an antiatretic principal.

Intraovarian interleukin-1 (IL-1), a putative intermediary in the ovulatory cascade, has recently been implicated as an antiatretic agent. Given the reported antigonadotropic and thus atretogenic potential of granulosa cell-derived insulin-like growth factor-binding proteins (IGFBPs), we evaluated the ability of IL-1beta to regulate ovarian IGFBP-4 and -5, the IGFBP species elaborated by the rat granulosa cell. Treatment of whole ovarian dispersates of immature rat origin with increasing concentrations of IL-1beta for 96 h resulted in substantial and significant time-dependent inhibition of IGFBP-4 and IGFBP-5 transcripts compared with that in untreated controls. The IL-1 effect proved relatively specific in that no significant alterations in IGFBP transcripts were observed in the presence of select ovarian agonists, including transforming growth factor-alpha, tumor necrosis factor-alpha, endothelin-1, hepatocyte growth factor, keratinocyte growth factor, or basic fibroblast growth factor. The inhibitory effect of IL-1beta on ovarian IGFBP-4 and -5 expression was almost completely reversed in the presence of IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. The addition of actinomycin D to IL-1beta-pretreated whole ovarian dispersates produced a pattern of (IGFBP-4 and -5) messenger RNA decay indistinguishable from that noted for the untreated control group. Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates displayed a marked diminution in the relative content of the IGFBP-4 and IGFBP-5 proteins (24- and 28- to 29-kDa proteins, respectively). Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates proteolyzed [125I]IGFBP-5 (but not IGFBP-4) into fragments with apparent molecular masses of 18 and 14 kDa, respectively. In conclusion, our present observations demonstrate the ability of IL-1 to 1) inhibit the steady state levels of transcripts corresponding to IGFBP-4 and -5 in a time-dependent, relatively specific, and receptor-mediated fashion; 2) suppress the accumulation of the corresponding IGFBP proteins; and 3) stimulate the activity of the IGFBP-5-directed (but not IGFBP-4) endopeptidase, a posttranscriptional phenomenon. Our findings also suggest, by inference, that the IL-1beta-mediated inhibition of IGFBP-4 and -5 transcripts is due in part to a decrease in the rate of transcription of the corresponding genes and not to a change in the stability of the relevant messenger RNAs. Consequently, the ability of IL-1 to influence ovarian IGFBP economy appears multifaceted, comprising both transcriptional and posttranscriptional effects. To the extent that IGFBP-4 and -5 constitute atretogenic agents, our present findings support the view that IL-1beta may play an antiatretic role in the context of ovarian physiology.

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.

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.