Experiment 4 was conducted to evaluate whether specific binding of 125I-leptin existed on thecal cells. Thecal cells from large follicles were cultured for 3 days in the presence of 10% FCS, and then cells were washed, and 125I-leptin binding assays were conducted directly in the culture wells. Leptin was iodinated using a chloramine-T method as previously described for insulin-like growth factor-I. For experiment 4a, approximately 100 000 d.p.m. of 125I-leptin was incubated for 0-6 h at 25°C in a total assay volume of 400 jjlI (0.25% BSA in PBS, pH = 7.5), whereas for experiment 4b, 50 000-300 000 d.p.m. of 125I-leptin was incubated for 6 h at 25°C in a total assay volume of 400 (jlI. Specific binding was determined as the difference between total binding and nonspecific binding; nonspecific binding was determined using 5 (xg/well of excess unlabeled recombinant mouse leptin.
Determination of Thecal Cell Numbers
Numbers of thecal cells were determined at the termination of experiments using a Coulter counter (Model Zm; Coulter Electronics, Hialeah, FL) as previously described. Briefly, cells were exposed to 0.5 ml of trypsin (0.25% [w:v] in 0.15 M NaCl) for 20 min at 25°C, then scraped from each well, diluted in 0.15 M NaCl, and enumerated. ventolin inhalers
Concentrations of androstenedione in culture medium collected on Day 4 of culture were determined using solid-phase RIA kits (ICN Biomedicals, Costa Mesa, CA) as previously described. Intra- and interassay coefficients of variation were 8% and 16%, respectively. Sensitivity of the androstenedione assay was 5 pg/tube.
Concentrations of progesterone in culture medium collected on Day 4 of culture were determined with an RIA as previously described. Intra- and interassay coefficients of variation were 11% and 18%, respectively. Sensitivity of the progesterone assay was 25 pg/tube.
Experimental data are presented as the least-squares means ± SE of measurements for triplicate culture wells from three or more experiments. Each experiment was performed with different pools of thecal cells collected from 7 to 10 follicles from 4 to 8 cows for each pool. Main effects (e.g., dose) and interactions on dependent variables (i.e., steroid production) were assessed using the general linear models procedure of Statistical Analysis Systems. Each well was a replicate, and each experiment contained three replicates per treatment. When steroid production was expressed as nanograms or picograms/105 cells per 24 h, cell numbers at the termination of the experiment were used for this calculation. Specific differences in steroid production between treatments were determined by Fisher’s protected least-significant difference procedure.