It is the objective of the in vitro studies reported herein to further evaluate the role of insulin in the regulation of ovarian androgen biosynthesis, to assess its dose requirements, and to elucidate the cellular mechanism(s) underlying its high dose action. To this end, use was made of recently developed primary culture systems of ovarian androgen-producing cells, the differentiation of which is subject to regulation by gonadotropic and insulinotropic signaling. Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) thecal-interstitial cells from immature rats with insulin (1 microgram/ml) or hCG (1 ng/ml), resulted in 1.5- and 2.6-fold increments in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstan-17-one), the main androgenic steroid identified in culture medium by HPLC. However, combined treatment with both agents unmasked a synergistic interaction resulting in 5.7-fold amplification of hCG action, the increase in androsterone accumulation representing enhanced biosynthesis rather than diminished degradation. Unaccounted for by cellular growth and independent of the cellular density of plating (1 X 10(4)-1 X 10(6) viable cells/culture) or the hCG dose (0.1-10 ng/ml) employed, the insulin effect proved time and dose dependent with a minimal time requirement of 72 h. [125I-TyrA14]Iodoinsulin binding to untreated highly enriched thecal-interstitial cells proved highly specific, saturable, and reversible, displaying a single class (Hill coefficient = 0.93 +/- 0.07) of high affinity (Kd = 1.7 X 10(-10) M), low capacity (4746 +/- 283 sites/cell) binding sites. Treatment with physiological concentrations (10 ng/ml) of insulin produced limited, albeit measureable, down-regulation of the insulin receptor. In contrast, provision of relatively high concentrations (1 microgram/ml) of insulin resulted (despite marked adsorption/degradation) in substantial (greater than 60%) down-regulation of the insulin receptor, but not the type I insulin-like growth factor receptor, the ligand of which has also been shown to amplify hCG-supported androgen biosynthesis. These findings suggest that the thecal-interstitial cell is a site of insulin reception and action, that physiological concentrations of insulin are capable of participating in the regulation of ovarian androgen biosynthesis, and that this effect is probably mediated via high affinity insulin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)