Prolactin and antiprolactin receptor antibody inhibit steroidogenesis by purified rat Leydig cells in culture

Abstract

The in vitro effects of ovine PRL (oPRL) on testicular testosterone synthesis were determined using isolated, collagenase-dispersed, adult rat Leydig cells in culture. oPRL (50–1000 ng/ml) had no effect either on basal or on LH (50, 100 or 2000 pg/ml)-stimulated testosterone secretion by Leydig cells in short-term culture (4 h). 125I-oPRL binding studies revealed a single class of high affinity sites ( K a 8.7 nM) with a low capacity ( B max 6.7 fmol/mg protein  ~ 980 sites/Leydig cell). Isolated Leydig cells were further purified on a continuous Percoll gradient and cultured in serum-free medium, at 34°C, in 5% CO 2 and 95% air. After 3 days of culture, the media were collected, the cells washed and then stimulated with hCG (3 ng/ml) for 3 h. oPRL (1–1000 ng/ml) added at plating, caused a log dose-dependent inhibition of testosterone accumulation during the 3-day culture period; the highest and most consistent inhibition (31%) was with 500 ng/ml oPRL. hCG increased the sensitivity to the inhibitory effect of PRL, 10 ng/ml oPRL causing 40% inhibition and 100 ng/ml causing a maximal inhibition of 50%. PRL in fact caused a reduction in the maximal effect (efficacy) of hCG on steroidogenesis, without significantly affecting the ED 50 (sensitivity). The effects of an antiPRL receptor antibody raised by the antiidiotypic route and previously shown to bind to rat testis PRL receptors were tested. The antiPRL receptor IgG (13 μg/ml) mimicked the PRL inhibitory effect and acted synergistically with PRL (100 ng/ml) in inhibiting both testosterone accumulation in 3-day cultured Leydig cells and their subsequent response to hCG. In summary, a clear inhibitory effect of PRL and a synergistic effect of antiPRL receptor antibody were demonstrated on testosterone synthesis by rat Leydig cells in 3-day culture.