Experiment 2 was conducted to evaluate whether the effect of leptin or insulin on thecal cell steroidogenesis was due to a change in cell viability. Thecal cells were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 2 days with no additional hormones, 100 ng/ml of insulin, 300 ng/ml of leptin, or both insulin and leptin. The doses of insulin and leptin were based on results from experiment 1. At the end of the 2-day treatment, media were collected and centrifuged at 1600 X g for 5 min to isolate any unattached cells. Attached cells were removed from each well after a 15-min exposure to 0.5 ml of trypsin (0.25% [w:v] in 0.15 M NaCl) at 25DC. Each well was washed with 0.25 ml of 0.15 M NaCl. Cells and washes were combined with cells isolated from media, centrifuged at 1600 X g for 5 min, and resuspended in 200 |xl of serum-free medium. The number of viable cells in each sample was determined using the trypan blue dye exclusion method and a hemocytometer. buy antibiotics online
Experiment 3 was conducted to evaluate whether the inhibitory effect of leptin on insulin-induced thecal cell steroidogenesis was due to leptin inhibiting insulin from binding to its receptors. Thecal cells were cultured for 3 days in 10% FCS, and then medium was removed, cells were washed twice with 0.5 ml of 0.9% NaCl, and a [125I]insulin binding assay was conducted at 22°C as previously described. Briefly, 50 000 disintegrations per minute (d.p.m.) of [125I]insulin was added directly into the 24-well culture plates alone, with unlabeled leptin or unlabeled insulin. Final assay volume was 500 |xl of PBS (1.6% BSA, pH 8.0). At the end of the 2-h incubation period at 22°C, wells were washed with PBS, and cells were solubilized with 1 N NaOH and placed in 12 X 75-mm tubes. Culture wells were washed twice, and these washes were combined with cells and counted in an automated gamma counter (counter efficiency = 75%).