Aryl hydrocarbon receptor antagonists attenuate the deleterious effects of benzo[a]pyrene on isolated rat follicle development  Michael S. Neal, Anne M. Mulligan Tuttle, Robert F. Casper, Alex Lagunov, Warren G. Foster  Reproductive BioMedicine Online  Volume 21, Issue 1, Pages 100-108 (July 2010) DOI: 10.1016/j.rbmo.2010.03.025 Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 FSH-stimulated growth of preantral isolated rat follicles in vitro. Increasing concentrations of benzo[a]pyrene (black bar) attenuated growth and reached statistical significance at a concentration of 1.5ng/ml. Co-treatment with 5μmol/l resveratrol (grey bar) ameliorated the deleterious effect of benzo[a]pyrene. Results are expressed as mean±SEM of six replicates of 96 follicles per culture. ∗P<0.05. Reproductive BioMedicine Online 2010 21, 100-108DOI: (10.1016/j.rbmo.2010.03.025) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 FSH-stimulated growth of preantral isolated rat follicles in vitro. Increasing concentrations of benzo[a]pyrene (black bar) attenuated growth and reached statistical significance at a concentration of 1.5ng/ml. Co-treatment with 5μmol/l 3′,4′-dimethoxyflavone (grey bar) ameliorated the deleterious effect of benzo[a]pyrene. Results are expressed as mean±SEM of seven replicates of 96 follicles per culture. ∗P<0.05. Reproductive BioMedicine Online 2010 21, 100-108DOI: (10.1016/j.rbmo.2010.03.025) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Representative images of isolated rat follicles after 5days of culture in: (a) follicle culture media (FCM) alone (control); (b) FCM supplemented with 1.5ng/ml benzo[a]pyrene; or (c) FCM supplemented with 1.5ng/ml benzo[a]pyrene co-incubated with an aryl hydrocarbon receptor antagonist (resveratrol). Arrows point to the oocyte surrounded by granulosa cells, which are healthier in appearance and growth in both a and c compared with b. Bars=20μm. Reproductive BioMedicine Online 2010 21, 100-108DOI: (10.1016/j.rbmo.2010.03.025) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Isolated rat follicle cell viability, measured with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, for follicles exposed to varying concentrations of benzo[a]pyrene, alone (black bar), with 5μmol/l resveratrol (white bar) or 5μmol/l 3′,4′-dimethoxyflavone (grey bar). Values are expressed as percentage of controls. Follicles exposed to concentrations of 5.0ng/ml benzo[a]pyrene or greater had significantly lower cell viability compared with controls and follicles co-incubated with resveratrol or 3′,4′-dimethoxyflavone (t-test). ∗P<0.001. Reproductive BioMedicine Online 2010 21, 100-108DOI: (10.1016/j.rbmo.2010.03.025) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Concentrations of oestradiol (pg/ml) in spent media from the isolated follicle culture assay. Oestradiol concentration was inversely related to increasing concentration of benzo[a]pyrene (black bar), decreased in a dose-dependent manner and was significantly lower than controls (∗). Co-treatment with 5μmol/l 3′,4′-dimethoxyflavone (grey bar) resulted in a higher oestradiol concentration but still lower than controls. At a concentration of 50.0ng/ml, there was a significant difference in oestradiol concentration between follicles grown in benzo[a]pyrene alone compared with those co-incubated with 3′,4′-dimethoxyflavone (∗∗). ∗P<0.05; ∗∗P<0.05). Reproductive BioMedicine Online 2010 21, 100-108DOI: (10.1016/j.rbmo.2010.03.025) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Concentrations of anti-Müllerian hormone (AMH) measured by enzyme-linked immunosorbent assay in the spent media of isolated rat follicle cultures exposed to increasing concentrations of benzo[a]pyrene alone (black bar) or co-incubated with 5μmol/l of 3′,4′-dimethoxyflavone (white bar). Values are expressed as percentage of controls. AMH concentrations were significantly lower in follicles exposed to increasing concentrations of benzo[a]pyrene. AMH concentrations were similar in spent media samples from follicles co-cultured with 3′,4′-dimethoxyflavone and were significantly different than their treated counterparts. ∗P<0.001; ∗∗P<0.05. Reproductive BioMedicine Online 2010 21, 100-108DOI: (10.1016/j.rbmo.2010.03.025) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions