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Category:Canadian novellasMultiple growth factors modulate expression of steroidogenic enzymes in fetal rat Leydig cells in vitro.
The present study was undertaken to determine the effect of multiple growth factors on the induction of steroidogenic enzymes in Leydig cells cultured in vitro. Serum-starved Leydig cells from 21-day fetal rats were stimulated by treatment with cholera toxin (1.25 ng/ml), 8-bromo-cAMP (3 mM), or the tumor promoter phorbol 12-myristate 13-acetate (TPA, 50 ng/ml) for 6 h. An enhanced expression of steroidogenic enzymes was observed in all growth factor-treated cells by western blot analysis. These enzymes were identified as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase, and 17,20-lyase. Immunohistochemical studies showed that in cholera toxin-treated cells, 3 beta HSD was expressed in the perinuclear region, and in 8-bromo-cAMP- and TPA-treated cells, 3 beta HSD was distributed throughout the cytoplasm. The steroidogenic response to growth factors was blocked by a combination of cycloheximide (0.1 microgram/ml) and actinomycin D (0.25 microgram/ml). This suggests that synthesis of steroidogenic enzymes occurs by a gene-activated event. We also examined the effect of growth factors on the enzymatic activity of the specific steroidogenic enzymes. Induction of 3 beta HSD activity was the earliest response to the various growth factors. The activity increased by approximately sixfold in cholera toxin-treated cells and by approximately sevenfold in 8-bromo-cAMP- and TPA-treated cells, whereas dexamethasone increased the activity by approximately threefold. The overall order of effectiveness of the growth factors was cholera toxin greater than 8-bromo-cAMP greater than TPA. We conclude that the expression of steroidogenic enzymes is regulated by multiple growth factors in fetal rat Leydig cells in vitro and that the rate-limiting enzyme ac619d1d87