Gonadotropins and Estradiol Stimulate Immunoreactive Insulin-Like Growth Factor-I Production by Porcine Granulosa Cells in Vitro*

Abstract

Previous studies have established the ovarian granulosa cell as a site of insulin-like growth factor-I (IGF-I) secretion and action, suggesting an autocrine function for this peptide in the ovary. To better understand how this putative autocrine system is regulated and its interface with the classic ovarian trophic hormones FSH, LH, and estradiol (E₂), we have studied the effects of these hormones on the secretion of immunoreactive IGF-I (iIGF-I) by cultured porcine granulosa cells. Immature granulosa cells were cultured under serum-free conditions which were optimized to allow maximal iIGF-I production and hormonal responsivity. Measurements of iIGF-I were made after minimizing the influence of IGF-binding proteins by either acid gel filtration or reverse phase chromatography. Since the two preparative procedures gave roughly comparable results, the more expeditious reverse phase procedure was chosen for most samples. Cycloheximide virtually eliminated measurable iIGF-I in culture, suggesting that the peptide measured was newly synthesized, and degradation of IGF-I by cultured granulosa cells was negligible. Consequently, the medium levels provided an accurate indication of cellular secretion over the collection period. Under optimal culture conditions, iIGF-I was readily measurable and responsive to treatment with ovarian trophic hormones. The iIGF-I levels in several experiments with these hormones were as follows: FSH treatment, 1.58 plusmn; 0.21 times the control value (n = 5 experiments); E₂ treatment, 1.26 plusmn; 0.12 times the control value (n = 5); E₂ plus FSH, 3.12 × 0.31 times the control value (n = 8); LH, 1.33 plusmn; 0.12 times the control value (n = 3); LH plus FSH, 1.78 plusmn; 0.2 times the control value (n = 1). To assess the role of cAMP in the mediation of gonadotropin effects in this system, granulosa cells were treated with a phosphodiesterase inhibitor (methylisobutylxanthine), which resulted in iIGF-I levels 1.61 plusmn; 0.7 times the control level. In the presence of FSH, a further stimulatory effect was demonstrated (3.76 plusmn; 0.29 times control). In addition, the cAMP analog 8-bromo-cAMP dramatically increased iIGF-I levels (6.3 plusmn; 0.72 times control). These data provide the first demonstration that gonadal iIGF-I secretion can be stimulated by the principal hormones involved in trophic regulation of the ovary. As with other gonadotropin-dependent functions of granulosa cells, this effect appears to be mediated by cAMP and enhanced by E₂. This interface between circulating hormones and autocrine systems could provide an important mechanism to amplify the effects of gonadotropic hormones on a local level. (Endocrinology120: 198–207,1987)