Stimulatory effect of LH, PGE2 and progesterone on StAR protein, cytochrome P450 cholesterol side chain cleavage and 3β hydroxysteroid dehydrogenase gene expression in bovine luteal cells

Abstract

The aim of these studies was to investigate the effect of LH, progesterone (P4), PGE, noradrenaline (NA) and a nitric oxide donor, S-nitroso- N-acetylpenicillamine ( S-NAP), on steroid acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 side chain cleavage (P450scc) gene expression and on the synthesis of their protein products. Bovine luteal cells were collected and prepared on days 6–10 of the estrous cycle and preincubated in vitro for 24 h. Thereafter, medium was changed and supplemented with one of six treatments: control medium, LH (100 ng/ml), P4 (10 −5 M), PGE2 (10 −6 M), NA (10 −5 M) or S-NAP (10 −4 M). In Experiment 1, luteal cells (10 6/well) were incubated for 3, 6, 18 and 24 h. After incubation, total RNA was isolated and P4 concentrations in medium was determined. Semiquantitative RT-PCR was used to measure gene expression. In Experiment 2, luteal cells were preincubated for 24 h, then stimulated as in Experiment 1. Total protein was isolated from lysed cells and Western blot analysis was performed using specific antibodies against the StAR, 3β-HSD and cytochrome P450scc proteins. Bands were analyzed by means of KODAK 1D Image Analysis Software. In Experiment 1, LH and PGE2 stimulated secretion of progesterone from luteal cells. Concentrations of mRNA for StAR, 3β-HSD, cytochrome P450scc were increased after 6 h in cells stimulated with LH, PGE2 and P4 ( P < 0.05). Gene expression was not affected by NA. In Experiment 2, LH, P4 and PGE2 induced an increase in the concentration of these three proteins. S-NAP inhibited both concentrations of mRNA and protein for StAR, 3β-HSD, cytochrome P450scc. Therefore, the increase in secretion of P4 induced by LH and PGE2 is associated with increases in StAR, 3β-HSD and cytochrome P450scc gene expression. This genomic response may be mediated in part through a positive effect of P4 on the expression of these genes observed in this experiment.