Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells

In the present studies we examined the regulation of insulin-like growth factor I (IGF-I) expression in porcine granulosa cells in vitro. Using Northern analysis and ribonuclease protection assays with exon-specific probes, we identified the IGF-I messenger RNA (mRNA) transcripts present in these cells under basal and hormone-stimulated conditions. We also assessed changes in secreted IGF-I using Western blots and correlated the change in protein secretion after hormone treatment with changes in mRNA levels. By analogy to the human IGF-I gene and its transcription, two major transcripts of approximately 1 and 7.5 kilobases, seen in freshly isolated granulosa cells and follicle wall and in single passaged granulosa (MDGp1) cells, most likely correspond to IGF-IA. Minor transcripts of 3-4 kilobases, which appeared after FSH or forskolin treatments or in control cells after long exposure of the autoradiographs, were attributed to incompletely processed RNA precursors. Ribonuclease protection assay analysis using probes to detect alternative use of exon 5 or exon 6 indicated that most, if not all, of the transcripts contained only exon 6 sequence (IGF-IA). Both class 1 and class 2 transcripts were identified using exon 1- and exon 2-specific probes, respectively. GH increased steady state levels of IGF-I mRNA 3-fold, FSH increased it approximately 10-fold, and forskolin maximally increased it 12- to 15-fold. Estradiol had no effect alone or in combination with the other treatments. All treatments that increased IGF-I mRNA coordinately increased both class 1 and class 2 transcripts, with the increase in class 1 greater than that in class 2. Multiple forms of IGF-I protein were seen under basal conditions and after hormone treatment. These were identified based on mRNA analysis and biochemical methods as both glycosylated and nonglycosylated IGF-IA prohormone, incompletely processed forms of prohormone, and the mature peptide. Changes in the levels of total protein were similar to the changes in mRNA (GH, 3-fold; FSH and forskolin, 10- to 20-fold). All forms of the protein changed coordinately, suggesting that these hormones had no major effect on the intracellular processing mechanism. IGF-binding protein-3 was able to bind to all IGF-I forms. These data conclusively demonstrate FSH and GH induction of ovarian IGF-I. The porcine granulosa cell culture system used in these studies should be an excellent system for studying the hormonal regulation of IGF-I expression.