Maintenance and regulation of 17 alpha-hydroxylase expression by bovine thecal cells in primary culture.

Abstract

We have developed and characterized a primary cell culture system to study the regulation of 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) gene expression in bovine thecal cells. Conditions have been established for the dispersal and growth of thecal cells isolated from bovine follicles, which maintain the expression of P45017 alpha for up to 8 days. Bovine theca interna cells were grown to subconfluence and transferred into medium containing forskolin, a stimulator of adenylate cyclase. Levels of P45017 alpha transcripts reached a maximum value after 48 h of stimulation with forskolin. Added progesterone was converted to 17 alpha-hydroxyprogesterone at a rate of 214 pmol/mg protein.h in cells treated with forskolin for 72 h, whereas in control cells, the rate was 9.2 pmol/mg protein.h after 72 h. This was reflected in a 10-fold increase in endogenous androstenedione production by forskolin-stimulated cells. Studies employing various growth factors suggest that transforming growth factor-beta, but not basic fibroblast growth factor, is a potent inhibitor of forskolin-induced 17 alpha-hydroxylase activity and androstenedione production in these cells. We have also characterized this cell culture system with respect to expression of other steroidogenic enzymes. Cholesterol side-chain cleavage cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase transcripts as well as endogenous progesterone accumulation were increased in response to forskolin stimulation. On the other hand, aromatase cytochrome P450 expression was undetectable. The ability to maintain bovine thecal cells, which retain 17 alpha-hydroxylase activity, in culture will provide a model system to study the regulation of expression of the P45017 alpha gene in the bovine ovary.