Medium was a 1:1 (v:v) mixture of DMEM and Ham’s F-12 containing 0.12 mM gentamicin and 38.5 mM sodium bicarbonate. Approximately 2 X 105 viable cells in 20-105 jxl of medium were added to Falcon multiwell plates (#3047; Becton Dickinson and Co., Lincoln Park, NJ) containing 1 ml of medium. Cultures were kept at 38.5°C in a 5% C02 atmosphere. To obtain optimal attachment, cells were maintained in the presence of 10% FCS for the first 2 days of culture. After this time, cells were washed twice with 0.5 ml of serum-free medium, and incubations were continued in serum-free medium with or without added hormones unless stated otherwise. Medium was changed every day. For experiments evaluating the effects of hormones on steroid production, hormonal treatments were applied for 2 days (i.e., from Day 2 to 4 of culture), unless stated otherwise. buy ampicillin
Experiment 1 was conducted to evaluate the dose-re-sponse effect of leptin on the action of LH and insulin on thecal cell proliferation and steroidogenesis. Thecal cells were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 2 days with 100 ng/ml of LH in the absence or presence of 100 ng/ml of insulin and leptin (0, 10, 30, 100, and 300 ng/ml). The doses of insulin and LH were selected on the basis of previous studies. Concentrations of leptin in blood of lean and obese humans have been reported to be 2-10 ng/ml and 10-100 ng/ml, respectively.