Ch223191

Embryos from CD-1 mice were taken out of the uterus on embryonic day 16.5 and tissue from each embryo was collected for SRY genotyping for sex determination. The whole pituitary glands were taken out and transferred to 96-well plate with 50μL phosphate buffered saline (PBS) +1% antibiotic/antimycotic solution (anti-anti, Thermo-Fisher). Pituitaries were washed twice with PBS +1% anti-anti and 50 μL of media (phenol red-free DMEM/F12 (Thermo-Fisher), supplemented with 10% charcoal-stripped fetal bovine serum (Sigma-Aldrich) and 1% anti-anti) with different treatments (0.1 μg/mL, 1 μg/mL, 10 μg/mL and 100 μg/mL MEHP/DEHP (Sigma) added to each well. The concentrations of MEHP/DEHP were picked based on previous papers describing mouse ovarian cultures with MEHP/DEHP [30 (link),31 (link)]. Pituitaries were cultured at 37 °C for 72 hours without changing the media. To co-treat the pituitary with MEHP and AhR antagonist- CH223191 (ApexBio), pituitaries were harvested and washed as described above and first treated with the antagonist dissolved in DMSO in the 37 °C incubator for 2 hours, then MEHP was added at the final concentration of 100 μg/mL. Some pituitaries were only treated with the antagonist to eliminate possible effects of the antagonist. The final concentration of the AhR antagonist was at 1μM as instructed by the manufacturer’s product catalog.