Corticosterone induces steroidogenic lesion in cultured adult rat leydig cells by reducing the expression of star protein and steroidogenic enzymes

Abstract

The present study was designed to investigate the dose-dependent direct effect of corticosterone on adult rat Leydig cell steroidogenesis in vitro. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34 degrees C in a CO(2) incubator under 95% air and 5% CO(2) using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum-containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400, and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34 degrees C. At the end of exposure period, cells were utilized for assessment of the activities and mRNA expression of steroidogenic enzymes (cytochrome P(450) side chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P(450) aromatase) and steroidogenic acute regulatory protein gene expression. Testosterone and estradiol production were also quantified. Activities of cytochrome P(450) side chain cleavage enzyme, 3beta- and 17beta-hydroxysteroid dehydrogenases were declined significantly in a dose-dependent manner after corticosterone exposure, while their mRNA expression were significantly reduced at higher doses of corticosterone exposure. The activity and mRNA expression of cytochrome P(450) aromatase registered a significant increase at 100 nM dose of corticosterone whereas at 200-800 nM doses both the activity as well as the mRNA levels was significantly reduced below the basal level. StAR protein gene expression was significantly inhibited by higher doses of corticosterone employed. At all doses employed, corticosterone significantly reduced the production of testosterone by Leydig cells, while estradiol level registered a significant increase at 50 and 100 nM doses but at higher doses, it registered a significant decrease when compared to basal level. It is concluded from the present in vitro study that the molecular mechanism by which corticosterone reduces the production of Leydig cell testosterone is by reducing the activities and mRNA expression of steroidogenic enzymes and steroidogenic acute regulatory protein.