Pubertal Female 2HM (BPS + ethinyl estradiol challenge)
Here, we have evaluated the effects of perinatal exposures to bisphenol S on the response of the mammary gland and other hormone-sensitive endpoints to a peripubertal estrogen challenge. We specifically focused on measures of pubertal onset in mice including the weight of estrogen-sensitive organs, anogenital distance, timing of vaginal opening, and morphology of the pubertal mammary gland. On pregnancy day 8, dams were randomly assigned to treatment groups. From pregnancy day eight until lactational day 2, dams were orally dosed with BPS or vehicle (Tocopherol Stripped Corn Oil) by gently placing a pipet in the mouth and allowing the mouse to drink the solution. 1 µg oil was administered for every 1 g of body weight. The diluted solutions were designed to deliver 2, 200, or 2000 µg BPS/kg/day. At postnatal day (PND) 21, two females from each litter were selected at random. One pup was assigned to receive vehicle, and one was assigned to receive an estrogen challenge. Each pup was orally dosed via a pipet with either vehicle (Tocopherol Stripped Corn Oil) or 1 µg EE2/kg/day. Dose administration continued for 10 days and was adjusted daily for body weight. On PND31, pups were euthanized via CO2 inhalation. Anogenital distance was measured using calipers, and the uterus was weighed using an analytical balance. The right fourth inguinal mammary gland was dissected from the skin, spread on a glass slide (Fisher Scientific, Pittsburgh, PA) and fixed in neutral buffered formalin (10%) (Fisher Scientific) overnight (standard whole mount preparation). The third pectoral mammary glands were frozen at -80C for RNA extraction. Whole mounts mammary glands from female offspring were imaged using a Zeiss Axio Imager dissection microscope. Using Zen Pro software, the whole mounts were quantitatively analyzed. Specific measurements included the area subtended by the ducts (ductal area), the total number of terminal end buds (TEBs, defined as bulb-shaped structures ≥0.03 mm2), and area of TEBs. Total RNA was extracted from mammary glands of individual mice using Trizol reagent and a BeadBug microtube homogenizer. Total RNA was quantified by UV spectrophotometry. One microgram of RNA from each sample was reverse transcribed to cDNA using reverse transcriptase. The FastStart Universal SYBR Green Master kit was used for qPCR along with 1 μL of cDNA and 300 nM forward and 300 nM reverse primers for each target gene. β-actin was used as a housekeeping gene. Relative quantification was determined using the ΔΔCt method to correct for differences in β-actin.