Rearing of the animals and isolation of the mammary gland
The Sprague-Dawley rat is highly regarded for studies designed to investigate the effects of endocrine modulation on mammary gland apoptosis and carcinogenesis. Nursing Sprague-Dawley female rats were obtained with litters from Taconic (Germantown, NY). The animals were allowed free access to food and water. The mothers were allowed to nurse their pups for 3 weeks. The pups were taken from the mothers at day 0 (lactating) and the involuting breast tissue was harvested following weaning. The excised tissue was fixed overnight in 4 % paraformaldehyde at 4°C, embedded in ophimum culting temperature, frozen in liquid nitrogen, and cut as 5μ m sections onto poly-L-lysine coated slides from Sigma (St. Louis, MO).
Lysosomal enzyme assays
Acid phosphatase activity was measured using an assay from Sigma according to the manufacturer's instructions. Briefly, mammary glands were homogenized in 0.5 mL 0.9% NaCl and the homogenates were clarified by centrifugation for 5 min at room temperature. The reaction mixture [0.5 mL of p-nitrophenyl phosphate (substrate), 0.5 ml of 90 mM citrate buffer, pH 4.8, and 0.1 mL of homogenate] was incubated for 30 min at room temperature, and the reaction was terminated by the addition of 5 mL of 0.1 N NaOH. In alkali, liberated p-nitrophenol was measured spectrophotometrically at 410 nm.
Cathepsin B was monitored using an enzymatic assay from Sigma according to the manufacturer's instructions. Mammary glands were homogenized in a buffer (pH 6.0) containing 352 mM potassium phoshate, 48 mM sodium phosphate, and 4 mM EDTA and clarified by centrifugation. The substrate was Nα-CBZ-Arg-Arg-7-Amido-4-Methylcoumarin. The increase in fluorescence intensity for liberated 7-Amino-4-Methylcoumarin was recorded at the excitation wavelength of 348 nm and the emission at the wavelength of 440 nm for 5 min.
β-N-acetylhexosaminidase (hexosaminidase) was also assayed according to Sigma's protocol. The glands were homogenized in 0.5 mL 0.9% NaCl (pH 4.8) and the homogenates were clarified by centrifugation for 5 min at room temperature. The substrate used was p-nitrophenyl-N-acetyl-β-glucosaminide. The liberation of p-nitrophenol and N-acetyl-D-glucosamine was measured spectrophotometrically at 410 nm.
Student's t-test was used for the determination of statistical significance. Absorbance values for day 0 and day 4 involuting mammary gland extracts were considered statistically different at P = < 0.05.