All samples were run in duplicate. Sensitivity for the FSH assay was 200 pg/ml with inter- and intraassay coefficients of variation of 2.7% and 6.7% respectively. Sensitivity for the LH assay was 86 pg/ml with inter- and intraassay coefficients of variation of 5.3% and 2.5% respectively.
After dosing, ovaries were collected from mice and immediately snap frozen in a dry ice and ethanol bath. Each frozen sample was homogenized in lysis buffer (40 mM Tris-HCl, pH 8.0, 1% NP40, 150 mM NaCl, 2.5 mM EDTA, 20 mM NaF, 20% glycerol) containing a protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany). After homogenization, the amount of protein in each sample was evaluated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Protein lysates (40 |xg/lane) were subjected to Western blot analysis using a 1:200 dilution of polyclonal antibody specific for ERa (Santa Cruz Biotechnology) or a polyclonal antibody specific for ERp (Zymed Laboratories) as the primary antibody. A 1:3000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse polyclonal antibody (Santa Cruz Biotechnology) was used as the secondary antibody. Immune complexes were visualized using an enhanced chemiluminescence (ECL) detection kit (Cell Signaling Technologies, Beverly, MA).