Aromatase

Proteins were extracted from rat ovarian granulosa cells, washed with HBSS and then lysed with RIPA buffer (Sigma-Aldrich, USA) containing protease inhibitors (Roche, Germany) for 30 min. The Bradford assay was used to determine the protein concentration. Equal amounts of total protein (15 μg) were loaded, separated on SDS-PAGE, transferred to a nitrocellulose membrane, and then immunoblotted with the appropriate antibody: ERα (#04-820, Millipore), ERβ (#92731, Millipore), FSHR (#sc13935; Santa Cruz, CA) StAR (#sc25806, Santa Cruz, CA), aromatase (#14245, Santa Cruz, CA), β-actin (13E5, Cell Signaling Technology), GADPH (#sc48116, cell signaling). ECL detection kit (GE, health care) was used to visualize the protein and Quality One software (Bio-Rad) was applied to quantify the intensities.

Total proteins were extracted using ice-cold radioimmunoprecipitation assay lysis buffer (Shenggong, Shanghai, China), supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). Equal amounts of protein were denatured at 100°C for 10 min and then electrophoresed in 10% SDS-polyacrylamide gels. Afterward, blots were wet-transferred to nitrocellulose membranes (Millipore, Billerica, MA). After blocking, blots were incubated with primary antibodies against MT1 (Abcam, Cambridge, United Kingdom), AR, and aromatase (both from Santa Cruz Biotechnology, United States) at 1:600–1:1000 dilutions and then incubated against secondary antibodies. An enhanced chemiluminescent detection system (Millipore) was used to detect bands with peroxidase activity. The same blot was probed with GAPDH (Proteintech, Chicago, United States) as the internal loading control. The bands were visualized using a G-Box iChemi Chemiluminescence Image Capture System (Syngene, Frederick, MD, United States).