4 androstene 3 17 dione

Dexamethasone (internal standard), 4-Androstene-3,17-dione-2,3,4-¹³C₃, Estrone-2,3,4-¹³C₃, 17β-Estradiol-2,3,4-¹³C₃, Testosterone-2,3,4-¹³C₃, Anastrozole, Letrozole, KZ, protease from Bacillus licheniformis (subtilisin), formic acid, acetone, methanol, acetonitrile, hexane, methylene chloride, charcoal activated, Tris, HCl, KH₂PO₄, K₂HPO₄, KCl, NADPH, glycerol, BSA, scintillation cocktail, and Supel™-Select SPE HLB (six mL, 200 mg) columns were provided by Sigma-Aldrich (St. Louis, MO, USA). 1β-³H Androstene-3,17-dione (30 Ci/mmol) was obtained from Perkin-Elmer Life Science (Boston, MA, USA).

For hormone assays, mGCs were cultured in 12-well plates in phenol red-free DMEM/F12 (Hyclone) supplemented with 2 % C-FBS (HyClone) and 2 μM 4-androstene-3, 17-dione (Sigma). To determine the effects of 8-Br-cAMP on mGC function, mGCs were treated with medium alone or with 1 mM 8-Br-cAMP (Sigma) for 24 h or 48 h. To determine the effects of miR-132 on mGCs, the medium was changed 6 h after transfection with miR-132 mimics/inhibitors or the corresponding negative controls, and the cells were cultured for an additional 48 h. To determine the effect of Nurr1 on mGCs, siNurr1 was transfected into cells 24 h prior to the transfection of miR-132 mimics, and the cells were cultured for an additional 24 h or 48 h. Culture medium was collected at the indicated time points, and the concentrations of E₂ and progesterone in the culture medium were determined using the Access Immunoassay System 2 (Beckman Coulter, Brea, CA, Germany), an automated random-access chemiluminescence-based assay. The intra- and interassay coefficients of variation were less than 10 % and 15 %, respectively. Each assay was performed in triplicate, and the experiments were repeated at least three times.