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 front 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 100ng/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 nim: Experiment 1) granulosa cells. In large-follicle (>7.9 mm; Experiment 2) granulosa cells, 100 ng/ml of IGF- I increased (P < 0.05) IGFBP-2 mRNA levels above controls and 3 ng/ml of IGF-1; l00ng/m1 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 100ng/ml of IGF-2 had no effect (P > 0.10) on IGFBP-2, -3, -4, and -5 mRNIA 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 blotting 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- I and -2. therefore discretely modulating the amount of bio-available IGFs to these cells depending upon the specific hormonal milieu. (C) 2004 Elsevier Inc. All rights reserved.