Insulin-Like Growth Factor-I Regulates Δ5–3β-Hydroxysteroid Dehydrogenase Activity in Rat Granulosa Cells

Abstract

The objectives of this study were: (1) to examine the role of insulin-like growth factor-I (IGF-I) in the regulation of A5–3s-hydroxysteroid dehydrogenase (3β-HSD) in cultures of rat granulosa cells; and, (2) to determine if IGF-I was secreted by granulosa cells. Granulosa cells were isolated from 25-day-old diethylstilbestrol-primed rats and cultured under serum-free conditions. The 3β-HSD activity was measured by the conversion of [3H]pregnenolone to [3H]progesterone by granulosa cell sonicates under optimal conditions. Cells cultured in the presence of FSH for 48 h had elevated levels of 3β-HSD activity. Both insulin and IGF-I alone stimulated 3β-HSD activity and augmented the actions of FSH. The concentrations of insulin required to stimulate 3s-HSD were higher than physiological levels, whereas the concentrations of IGF-I were consistent with those found in follicular fluid. This suggested that IGF-I may play a physiological role in the regulation of 3β-HSD. Since growth factors usually act on cells in close proximity to their site of production, we looked for biological activity in conditioned medium generated by primary cultures of rat granulosa cells under serum-free conditions. The biological activity of the conditioned medium was similar to that described for IGF-I, in that it augmented the actions of FSH on aromatase activity and also acted alone in the absence of FSH to stimulate 3β-HSD activity. When granulosa cell-secreted proteins were separated by chromatography on a Sephadex G75 column, a peak of IGF-I immunoreactivity was found at 7,000 da corresponding to commercially available 125I-IGF-I passed through the same column. In conclusion, the secretion of IGF-I by rat granulosa cells in culture and its actions on 3β-HSD suggest that the production and accumulation of IGF-I in follicular fluid may be important in the regulation of steroidogenesis during follicular development.