The number of nipples at necropsy was compared against the number of areolas/nipples on PCD 35. Male necropsies were completed on PCD 213. Testes and epididymides from all males were preserved in Bouin fixative and placed in 70% ethanol after 24 h. Histopathologic examination of the testes and epididymides was conducted by Experimental Pathology Laboratories, Inc. (Research Triangle Park, NC), where tissues were sectioned at 4-6 |xm and stained using hematoxylin-eosin.
Necropsy of Mature Male Offspring
Female and male pups were necropsied separately. Beginning on PCD 147, males were necropsied in a randomized block design in which one male per litter per treatment block was selected at a time. One male was selected from each of the other treatment blocks before an additional male was selected from a given block. This process was continued until all males in the study were necropsied. Androgen-dependent organ weights were measured in each treatment block until three animals per litter were examined.
The degree of necrosis in dead pups was highly variable. Dead pups were not examined further. On PCD 35, areola/nipple number and location were noted for each (male and female) pup in a blinded fashion. Pups were weaned (females on PCD 43 and males on PCD 44) and housed in unisexual groups of two to three littermates. Pups were placed in cages numbered and arranged in treatment blocks according to the assignment of the mother. Pups were given ear punch identification marks according to the mother’s treatment, and picric acid marks to identify them within a litter.
During subsequent observations, the numbers of delivered pups were noted. Animals showing vaginal bleeding or having pups scattered throughout the cage and uncleaned (blood on the pups and dam) or placentas in the bedding were considered to be in the process of delivery. In addition, signs of dystocia were also noted. To reduce observer effects on parturition the litter and nests were not disturbed, and the dams or pups were not handled.
Dams were dosed by gavage daily from GD 14 to GD 18 (fetal differentiation of androgen-dependent tissues) with PZ in corn oil at doses of 0, 31.25, 62.5, 125, or 250 mg/kg BW in a dose volume of 2.5 ml corn oil/kg BW. Treatment volume was calculated according to each dam’s daily weight. Dams were dosed by gavage using a 1.5-inch X 20-gauge curved feeding needle (7910; Popper and Sons, Lincoln, RI) attached to a 1-ml glass syringe.
Weights of pituitary, brain, ventral prostate, dorsolateral prostate, seminal vesicles (wet and dry), testes, epididymides, levator ani plus bul-bocavernosus muscles, bulbourethral glands, adrenals, liver, and kidneys were also taken. In females, brain and pituitary weights were measured and reproductive tissues were examined for malformations. In the pilot study, the offspring data were analyzed using individual and litter mean values.
Water was tested monthly for Pseudomonas and every 4 mo for a suite of chemicals, including pesticides and heavy metals. The current study was conducted under a protocol that had been approved by the National Health and Environmental Effects Research Laboratory institutional animal care and use committee.
Receptor-bound ligand was separated using 2 ml of ethanol and radioactivity (dpm) was determined using [3H] counting on a Beckman LS5000TD liquid scintillation counter.
Timed-pregnant Sprague-Dawley rats (approximately 90 days of age) were purchased from Charles River Laboratories (Raleigh, NC).
Prostate tissue was homogenized on ice in low-salt TEGD buffer (1.5 mM disodium ethelynediaminetetraacetate, 1.0 mM phenyl-methyl sulfonyl fluoride, 1.0 mM sodium molybdate, 1.0 mM dithiothre-itol, 10 mM Tris, and 10% glycerol) at 10 ml per gram of tissue. Homog-enates were centrifuged (30 000 X g) and supernatants were pooled.
Medium was replaced and cells were dosed with PZ (0.03-30 |xM), DHT (0.1 nM), DHT (0.1 nM) + PZ (0.03-30 |xM), DHT (0.1 nM) + hydroxyflutamide (1 |xM) as an antiandrogen control, or vehicle control (ethanol) and incubated overnight. Cells were washed using Dulbecco PBS (14080-055; Gibco BRL) and lysed using 25 |xl of E1531 lysis buffer (Promega, Madison, WI). Luciferase readings were measured using a Dyn-Ex MLX microtiter plate luminometer.