Gestational Exposure to Ethane Dimethanesulfonate: MATERIALS AND METHODS(7)
Ex Vivo Testis Parenchyma Incubations
Production of T by testis parenchyma following gestational exposure to EDS was determined by modification of the protocol used by Klinefelter et al. and Parks. Briefly, the right testis of each mouse was removed. The tunica was perforated (PND 24) or removed (PND 82), and the whole testis (PND 24) or 50-mg pieces of testis parenchyma (PND 82) were incubated (34°C) while shaking for 30 min in 1 ml of M199 buffered with 0.71 g/L sodium bicarbonate, 2.21 g/L N-2-hydroxyethyl piperazine-N’-2 ethenesulfonic acid (Hepes; Sigma), 0.1% BSA (Schwarz-Mann, Cleveland, OH), and 25 mg/L soy bean trypsin inhibitor, pH 7.4 (Sigma). After this incubation, medium was removed and replaced with fresh medium, with or without maximal hCG stimulation (100 mIU/ml), for 2 h. Media were frozen at — 80°C until RIA analysis.
The fertility analysis was based on a modification of two different mating protocols. Initially, two untreated virgin female mice (PND 65-75) were cohabited with an individual adult male offspring (PND 7080) that had been prenatally exposed to either 160 mg EDS/kg or 30% DMSO vehicle. Initially, two females were placed in a box with a single male at 1700 h (lights out at 1800 h).