Regulation of cholesterol side-chain cleavage and 17α-hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture

In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17α-hydroxylase, cholesterol side-chain cleavage, and 3β-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28−32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time-and dose-dependent increase in 17α-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17α-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17α-hydroxyprogesterone and 16α-hydroxyprogesterone. In studies using 17α-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarily, 4−pregnen−20α-ol-one (20α-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolinstimulated theca interna cultures verified that the increase in 17α-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17α-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.