Gestational Exposure to Ethane Dimethanesulfonate: MATERIALS AND METHODS(4)
Postnatal Necropsy of Male Offspring
On PND 1, one male pup per litter was killed via CO2 followed by decapitation. The internal morphology of the urogenital tract and the positioning of the testes were noted. The right testis was removed and placed in a 5-ml glass scintillation vial containing 5% glutaraldehyde in 0.05 M collidine buffer with 0.1 M sucrose for histological analysis. The left testis was removed, placed in a microcentrifuge tube, and frozen at — 80°C for proteomic analysis.
On PND 24 and 82, male mice were killed via CO2 followed by cervical dislocation. Extravasated blood was collected from the neck region following decapitation and transferred to 1 ml serum separator tubes, placed on ice, and centrifuged (30 min, 3000 X g, 4°C). The serum was decanted and frozen at — 80°C until serum T and LH could be assayed. Postmortem analysis was conducted on two prepubertal males per litter on PND 24 and two sexually mature males per litter on PND 82. Body and organ weights (testes, epididymides, and seminal vesicles trimmed free of fat and fascia) were recorded. The left testis was placed in a 20ml scintillation vial containing 5% glutaraldehyde in 0.05 M collidine buffer with 0.1 M sucrose for histological analysis. The right testis (PND 24) or 50-mg pieces of testis parenchyma (PND 82) were removed and placed into a microcentifuge tube with 1 ml Medium 199 (M199; #4001100, Gibco Laboratories, Grand Island, NY) for ex vivo determination of T production. The right cauda epididymis was removed, placed in a microcentrifuge tube, and frozen at — 80°C for subsequent sperm enumeration.