Primary culture of differentiating ovarian androgen-producing cells in defined medium.

A method for maintaining primary cultures of ovarian cells in serum-free medium in which the metabolic functions of the androgen-producing cells can be studied is described. Freshly dispersed cells from ovaries of hypophysectomized immature rats contained interstitial cells with specific, high affinity lZ5I-human chorionic gonadotropin (hCG) binding sites which were functionally coupled to adenylate cyclase but not to steroid synthesis. Culturing the interstitial cells with luteinizing hormone (LH) or hCG, but not follicle-stimulating hormone or prolactin, there was a 200-fold increase in steroid synthesis. Of the total steroid secreted, 96% was androgen, of which 98% was androsterone. The stimulation of androsterone synthesis by LH or hCG was dose-dependent (EDSo = 2 * 0.2 ng/ml for both hormones). A time course study with a saturating dose of LH showed that androsterone production was low at days 0-3, increased rapidly to maximum levels at day 4, and remained maximal through day 10. The LH effect on androsterone synthesis was mimicked in a dose-dependent manner by prostaglandin Ez, cholera toxin, and 8-bromo CAMP. Treatment with actinomycin D reversibly inhibited LH-stimulated androsterone synthesis. Experiments in vivo using hCG induced a similar pattern of androgen response indicating our defined in vitro system has physiological relevance. These in vitro results show that physiological concentrations of LH or hCG specifically induce the differentiation of the ovarian androgen-producing cells in a manner similar to that observed in uiuo. This defined culture system should provide an excellent model for studying the mechanisms and control of ovarian androgen biosynthesis.