Four hundred milligram of pulverized rat testes were homogenized in 1 ml of 2Â MEB buffer (100 mM Tris-HCl pH 7.5, 20% glycerol, 4 mM DTT, 10 ml/ml protease inhibitor cocktail [Roche]) in a Potter homogenizer, filtered on 50 mm Nitex membrane (Tetko) and centrifuged at 2000g, 48C. The pellet (nuclear fraction) was washed two times with 0.5 ml of 2Â MEB buffer. Nuclei were gently resuspended in 0.5 ml of 2Â MEB (small aliquot was analyzed by DAPI staining), passed through a needle and incubated 30 min on ice with 10 ml of DNase I (Warthington). The supernatant was centrifuged at 10 000g, 48C (mitochondrial fraction). After separation of the mitochondrial fraction, the supernatant was centrifuged at 100 000g in an ultracentrifuge and the supernatant (cytoplasmic fraction) was collected.