Gestational Exposure to Ethane Dimethanesulfonate: MATERIALS AND METHODS(6)

METHODS(6)

The sample was decanted into a microcentrifuge tube and stored at 4°C. An aliquot (0.1 ml) was diluted with 0.9 ml of buffer, and sperm were counted using a he-mocytometer slide. Minimally, three hemocytometer chambers were counted per sample.

Radioimmunoassay of T and LH

Testosterone levels from dried perinatal extracts were quantified via RIA using a Coat-a-Count Total T kit according to the manufacturer instructions (Diagnostic Products Corporation, Los Angeles, CA). Dried perinatal extracts were resuspended in 120 |xl of zero standard, vortexed for 30 sec, and a 50-|xl aliquot assayed in duplicate for T. Levels of T in both ex vivo media and blood serum were also quantified using a Coat-a-Count total T kit.

Serum LH concentrations were measured using the method from Chan-drashekar and Bartke. Rat LH standards NIDDK-r-LH-19 and rat LH antibody NIDDK-anti-rLH-S-11 were obtained through the National Hormone and Pituitary Program. Radioactive 125I-rat LH was obtained from Covance Laboratories (Vienna, VA), and IgG antiserum was obtained from ICN Pharmaceuticals (Costa Mesa, CA).

Category: Dimethanesulfonate

Tags: Leydig cells, testis, testosterone, toxicology