Follicles from each ovary were cultured separately (to get n = 3) for 24 h in DMEM with ITS+ under 5% CO2 in air at 37°C in a Heraeus (Life Scientific, Inc., St. Louis, MO) incubator. Follicles were exposed to either 100 ng/ml ovine FSH (NIH-18; Bethesda, MD), 50 ng/ml ovine LH (NIH-25), 50 ng/ml recombinant murine IGF-I (Mallinckrodt, Phillipsburg, NJ), 50 ng/ml murine EGF (Mallinckrodt), or 10 ng/ml human TGFp1 (R&D Systems, Minneapolis, MN). buy cheap antibiotics
Control culture received no treatment. Follicles were retrieved from the culture and rinsed with ice-cold homogenization buffer (10 mM Tris-HCl, pH 7.0, 0.25 M sucrose, 10% glycerol) supplemented with 1 mM PMSF and 10 ^g/ml each of pepstatin, antipain, soybean trypsin inhibitor, and benzidine-HCl to minimize proteolysis; they were then homogenized in 35 ^l homogenization buffer using a handheld micro glass-glass homogenizer Protein content was determined in 1 ^l of homogenate using a protein assay kit (ISS, Natick, MA). The rest of the homogenate was centrifuged at 26 000 X g for 30 min in an Eppendorf microfuge at 4°C to separate the mitochondrial fraction.