EXPRESSION AND REGULATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-1, PROTEIN-2, PROTEIN-3, AND PROTEIN-4 MESSENGER RIBONUCLEIC-ACIDS IN PURIFIED RAT LEYDIG-CELLS AND THEIR BIOLOGICAL EFFECTS

The actions of insulin-like growth factor-I (IGF-I) are modified by bi nding proteins [IGF-binding proteins (IGFBPs)]. Previously, we reporte d that IGF-I enhances Leydig cell steroidogenesis and that IGF-I mRNA is expressed in Leydig cells. In the present study, we evaluated the e xpression and regulation of IGFBP-1, -2, -3, and -4 in purified rat Le ydig cells and their biological effects.We found that none of the test icular crude interstitial cells, purified Leydig cells, or seminiferou s tubules expressed IGFBP-1 mRNA. This indicated that IGFBP-1 mRNA is not expressed in the testis in detectable amounts. In contrast, large amounts of IGFBP-2 with a size of 1.8 kilobases (kb) were expressed in purified Leydig cells, and lesser amounts in crude interstitial cells . Small amounts of IGFBP-2 mRNA were expressed in seminiferous tubules , but none could be detected in liver. IGFBP-3 mRNA was predominantly expressed in purified Leydig cells, crude interstitial cells, and live r, while appreciable amounts were not found in seminiferous tubules. L iver had the highest amounts of IGFBP-4 mRNA, whereas purified Leydig cells and crude interstitial cells had lesser amounts. We next evaluat ed the pituitary dependency of IGFBP mRNAs in Leydig cells. Purified L eydig cells were isolated from 50-day-old rats 5 days after hypophysec tomy. IGFBP-2, -3, and -4 mRNA levels in Leydig cells decreased 22%, 8 0%, and more than 90%, respectively, after hypophysectomy. In the live r, however, IGFBP-2 mRNA levels increased, and IGFBP-3 mRNA levels dec reased, while IGFBP-4 remained unchanged. As expected, hypophysectomy caused decrements in large (7.0- to 7.5-kb; a 75% reduction) and small (0.8- to 1.2-kb; a 90% reduction) IGF-I mRNA levels in Leydig cells. Hypophysectomy also reduced IGF-I mRNA expression in liver. Finally, t he effects of IGFBP-2, -3, and -4 on Leydig cell testosterone formatio n were investigated. hCG in a concentration of 10 ng/ml increased test osterone formation from 0.6 +/- 0.01 to 27.4 +/0 1.01 ng/10(6) cells.h . In the presence of IGF-I (10 ng/ml), testosterone formation was furt her increased to 58.6 +/- 1.6 ng/10(6) cells.h (P < 0.01). IGFBP-3 (0. 1, 1, and 2.5 pmol/ml) caused a dose-dependent inhibition of IGF-I- pl us hCG-induced testosterone formation. IGFBP-3 in a concentration of 2 .5 pmol/ml completely neutralized the effects of IGF-I on Leydig cell steroidogenesis. IGFBP-4 had a lesser effect, while IGFBP-2 had no eff ect on IGF-I- plus hCG-induced testosterone formation. Our results sug gest that multiple species of IGFBPs are expressed in Leydig cells, an d that IGFBPs modify the effect of IGF-I on Leydig cell function.