Effect of insulin-like growth factors (IGF), FSH, and leptin on IGF-binding-protein mRNA expression in bovine granulosa and theca cells: quantitative detection by real-time PCR

To determine if insulin-like growth factor (IGF)-1 and -2, FSH, or leptin alter IGF-binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and (or) theca cells, granulosa and theca cells were collected from bovine ovarian follicles, plated for 48 h in 10% FCS and then treated for 24 h in serum-free medium containing various hormone treatments arranged in three different experiments. Amounts of IGFBP-2, -3, -4, and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. Neither 100 ng/ml of IGF-1 nor IGF-2 had an effect ( P > 0.10) on IGFBP-2, -3, -4, or -5 mRNA levels in small-follicle (1–5 mm; Experiment 1) granulosa cells. In large-follicle (>7.9 mm; Experiment 2) granulosa cells, 100 ng/ml of IGF-1 increased ( P < 0.05) IGFBP-2 mRNA levels above controls and 3 ng/ml of IGF-1; 100 ng/ml of IGF-1 also decreased ( P < 0.10) IGFBP-5 mRNA levels compared to 3 ng/ml of IGF-1 or FSH or 100 ng/ml leptin, while 100 ng/ml of IGF-2 had no effect ( P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels (Experiment 2). At the doses tested, leptin and FSH had no effect ( P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels in large-follicle granulosa cells. In theca cells, IGF-2 decreased ( P < 0.05) IGFBP-2 mRNA levels, but had no effect on IGFBP-3 or -4 mRNA expression (Exp. 3); IGF-1 did not affect ( P > 0.10) thecal IGFBP-2, -3 or -4 mRNA levels. In contrast, IGF-1 but not IGF-2 increased ( P < 0.01) thecal IGFBP-5 mRNA levels. Ligand blotting revealed that both IGF-1 and -2 increased IGFBP-2 and -5 (protein) and had no effect on IGFBP-3 (protein), whereas IGF-1 (but not IGF-2) increased IGFBP-4 (protein), suggesting IGFBP-2, -4, and -5 are post-transcriptionally regulated. These results suggest that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by IGF-1 and -2, therefore discretely modulating the amount of bio-available IGFs to these cells depending upon the specific hormonal milieu.