REGULATION OF INSULIN-LIKE GROWTH-FACTOR-I BIOSYNTHESIS IN PORCINE GRANULOSA-CELLS

In the present studies we examined the regulation of insulin-like grow th factor I (IGF-I) expression in porcine granulosa cells in vitro. Us ing Northern analysis and ribonuclease protection assays with exon-spe cific 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 co rrelated the change in protein secretion after hormone treatment with changes in mRNA levels. By analogy to the human IGF-I gene and its tra nscription, two major transcripts of similar to 1 and 7.5 kilobases, s een in freshly isolated granulosa cells and follicle wall and in singl e passaged granulosa (MDGp1) cells, most likely correspond to IGF-IA. Minor transcripts of 3-4 kilobases, which appeared after FSH or forsko lin treatments or in control cells after long exposure of the autoradi ographs, were attributed to incompletely processed RNA precursors. Rib onuclease protection assay analysis using probes to detect alternative use of exon 5 or exon 6 indicated that most, if not all, of the trans cripts contained only exon 6 sequence (IGF-IA). Both class 1 and class 2 transcripts were identified using exon 1- and exon 2-specific probe s, respectively. GH increased steady state levels of IGF-I mRNA 3-fold , FSH increased it similar to 10-fold, and forskolin maximally increas ed it 12- to 15-fold. Estradiol had no effect alone or in combination with the other treatments. All treatments that increased IGF-I mRNA co ordinately increased both class 1 and class 2 transcripts, with the in crease in class 1 greater than that in class 2. Multiple forms of IGP- 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, incomplet ely 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 ch anged 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 F SH and GH induction of ovarian IGF-I. The porcine granulosa cell cultu re system used in these studies should be an excellent system for stud ying the hormonal regulation of IGF-I expression.