Comparison of sheep foetal ovarian development after in vivo and in vitro exposure to DEHP/MEHP, PCBs (101+118) and a mixture of both

Our previous studies of sewage sludge exposures, and the literature, highlight polychlorinated bisphenols (PCB) and diethylhexyl phthalate (DEHP) as biologically active at environmental concentrations. We have demonstrated in sheep that a real-life cocktail of environmental chemicals, containing these chemicals, preferentially accumulate in the fetus and affects the development of the ovary. We therefore investigated the effects of dietary intake of DEHP, the two PCBs and a mixture of both, on sheep female reproductive development in vivo and in vitro. Ovaries were collected from day 140 foetal sheep foetuses exposed in-utero to DEHP and/or PCBs (congeners 101+118) and also control (vehicle). 54 ovaries distributed in four groups were included in this study and were analysed by histology and by transcriptomic analysis with a dedicated sheep microarray. There were no overall significant effects of any of the exposures on fetal ovarian follicle dynamics at day 140 compared with control fetuses. In DEHP-exposed ovaries, a total of 136 genes were differentially expressed between controls and exposed fetuses (FDR 5% and a fold-change >1.5). A greater number of genes were down-regulated (106/136) as compared to those that were up-regulated (30/136). A much smaller number of genes were significantly affected by PCB exposure; only 4 genes were differentially expressed. Surprisingly, the mixture DEHP+PCBs produced fewer changes than DEHP alone: 24 genes were differentially expressed (9 up, 13 down). In parallel, we have developed a long-term (20 days) organotypic culture system to investigate the histological and molecular effects of MEHP (the main metabolite of DEHP) and PCB101 & 118 on early ovarian development. In these culture experiments, MEHP and PCB 101 & 118 at 10-6 M or a combination of both and MEHP and PCB 101-118 at 10-8 M or combination of both were tested. Three stages of ovary development were investigated: meiosis (48-70dpc in sheep), primary follicle formation (90-110dpc) and follicle differentiation (110-130dpc). Very few genes were differentially expressed between controls and PCB, MEHP and mixture exposures. There were no significant differences in morphology or in oocyte or follicle numbers. In conclusion, a larger number of effects of exposure were observed in vivo than in vitro for the same compounds. This also suggests further reasons for caution in assessing developmental consequences of exposure to ECs in vitro.