Gestational Exposure to Ethane Dimethanesulfonate: MATERIALS AND METHODS(3)
Fetal whole-body T production in male mice was evaluated from GD 15, when morphological identification of the male gonad could first be achieved, through GD 18 just prior to birth. Dosing was terminated the day prior to cesarean section. Thus, a dam killed on GD 16 was dosed from GD 11 to GD 15. Fetuses were killed with CO2 followed by decapitation. They were weighed, placed in 12 X 75 mm borosilicate disposable glass culture tubes (Fisher Scientific, Pittsburgh, PA) (for fetuses aged GD 11-17) or 13 X 100 mm tubes (for fetuses and neonates aged GD 18 to PND 4), and frozen at — 80°C until steroid extraction.
On the day of birth, PND 1, live pups were counted, weighed, and examined for overt signs of toxicity (e.g., tremors, lethargy, malformations). Anogenital distance (AGD) was measured in all offspring on PND 1 and PND 82 from the base of the genital papilla to the proximal end of the anal opening with calipers calibrated to 0.1 mm: distances of 1.5 mm and longer on PND 1 were designated as male. On PND 4, litters were standardized to four males and four females. Male pups were individually identified with a s.c. injection of Permanent Tattoo Pigment 242 (Aims, Inc., Piscataway, NJ) with a 26-gauge needle and tuberculin syringe on the back of alternating paws. At weaning, PND 21, dams and female offspring were killed via CO2 asphyxiation. Male littermates were housed together. On PND 24, two males per litter were removed for prepubertal necropsy. Remaining males were examined for preputial separation from PND 25 through PND 32 until puberty was attained. Thereafter, mice were housed individually until maturity (PND 70) for a 10-d fertility analysis (PND 70-80) and subsequent necropsy on PND 82.