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.

Periovulatory and interleukin-1 beta-dependent up-regulation of intraovarian vascular endothelial growth factor (VEGF) in the rat: potential role for VEGF in the promotion of periovulatory angiogenesis and vascular permeability.

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.

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.

Defective 3-ketosteroid reductase activity in a human monocyte-like cell line.

The human monocyte-like cell line U937, which is a cholesterol auxotroph, does not grow on mevalonate, squalene, or 4,4-dimethyl cholest-7-en-3 beta-ol. It grows on cholest-7-en-3 beta-ol and converts it to cholesterol. When deprived of an exogenous source of cholesterol, the cells accumulate 4 alpha-methyl-cholest-8-en-3-one. The cell-free extracts of U937 are also devoid of 3-ketoreductase activity. The present studies indicate that the lesion in cholesterol synthesis by these cells is located at 3-ketosteroid reductase, making this the first report of a deficiency of this enzyme. In contrast, another U937 strain (U937-N) synthesizes cholesterol, does not accumulate 4 alpha-methyl-cholest-8-en-3-one, and has 3-ketosteroid reductase activity. The two strains should be valuable in studies of the regulation of cholesterol metabolism and of the role of cholesterol in membrane structure and function.