Rats were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the University of Califomia-San Diego (UCSD) Animal Subjects Committee. GC were collected from ovaries of immature DES-primed rats and plated into 12-well dishes (Coming Glass, Coming, NY) that had been serum-coated. Cells (1 X 106 viable cells/ ml) were cultured in serum-free tissue culture medium (McCoy’s 5a medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 |xg/ml streptomycin sulfate, and 100 nM androstenedione). Cells were cultured at 37°C in a humidified atmosphere of 95% air:5% C02 with or without the indicated concentrations of test samples. birth control pills
Western Immunoblot Analysis
Cell-free supematants (conditioned medium) were centrifuged (500 X g for 5 min) and concentrated 15 times using the Ultrafree-MC Filter Unit (10 000 Mr cut-off; Mil-lipore, Bedford, MA). Samples were diluted with reducing buffer (5% (3-mercaptoethanol), heated at 85°C for 3 min, and loaded onto 14% Tris-glycine polyacrylamide gel using the Xcell mini-cell system (Novex, San Diego, CA). Following electrophoresis, performed at a constant voltage of 125 V for 1.5 h, the protein samples were transferred onto a 0.45-|xm nitrocellulose membrane (Bio-Rad, Hercules, С A) for 2 h at 150 mA using the Mini Blot module (Novex). Molecular weights were estimated by comparison with known molecular weight standards (Bio-Rad). Im-munostaining was performed by incubating overnight at 4°C with Inh-a antibody (1:2000 dilution in 1% casein/ TBST [50 mM Tris-HCl, 150 mM NaCl with 0.1% Tween 20]). Membranes were washed three times with TBST and then incubated with peroxidase-conjugated goat anti-rabbit IgG (diluted 1:10 000 in casein/TBST buffer) for 2 h. The membranes were washed and incubated with enhanced chemiluminescence (ECL) detection reagents (DuPont, Wilmington, DE) for 1 min and exposed to a Hyperfilm ECL (Amersham) for 1-3 min.