Bisphenol A and Related Alkylphenols Exert Nongenomic Estrogenic Actions Through a G Protein-Coupled Estrogen Receptor 1 (Gper)/Epidermal Growth Factor Receptor (Egfr) Pathway to Inhibit Meiotic Maturation of Zebrafish Oocytes

Abstract

Xenobiotic estrogens, such as bisphenol A (BPA), disrupt a wide variety of genomic estrogen actions, but their nongenomic estrogen actions remain poorly understood. We investigated nongenomic estrogenic effects of low concentrations of BPA and three related alkylphenols on the inhibition of zebrafish oocye maturation (OM) mediated through a G protein-coupled estrogen receptor 1 (Gper)-dependent epidermal growth factor receptor (Egfr) pathway. BPA (10-100 nM) treatment for 3 h mimicked the effects of estradiol-17beta (E2) and EGF, decreasing spontaneous maturation of defolliculated zebrafish oocytes, an effect not blocked by coincubation with actinomycin D, but blocked by coincubation with a Gper antibody. BPA displayed relatively high binding affinity (15.8% that of E2) for recombinant zebrafish Gper. The inhibitory effects of BPA were attenuated by inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase with ilomastat. Treatment with an inhibitor of Egfr transactivation, AG1478, and an inhibitor of the mitogen-activated protein kinase (MAPK) 3/1 pathway, U0126, increased spontaneous OM and blocked the inhibitory effects of BPA, E2, and the selective GPER agonist, G-1. Western blot analysis showed that BPA (10-200 nM) mimicked the stimulatory effects of E2 and EGF on Mapk3/1 phosphorylation. Tetrabromobisphenol A, 4-nonylphenol, and tetrachlorobisphenol A (5-100 nM) also inhibited OM, an effect blocked by cotreatment with AG1478, as well as with the GPER antagonist, G-15, and displayed similar binding affinities as BPA to zebrafish Gper. The results suggest that BPA and related alkylphenols disrupt zebrafish OM by a novel nongenomic estrogenic mechanism involving activation of the Gper/Egfr/Mapk3/1 pathway.

Keywords: EGFR; GPER; GPR30; MAPkinase; bisphenol A; endocrine disruptors; meiotic arrest; nonylphenol; oocyte maturation; tetrabromobisphenol A; tetrachorobisphenol A; zebrafish.

FIG. 1

Effects of BPA and EGF on spontaneous maturation of denuded zebrafish oocytes. EGF (100 nM) and BPA (100 nM), and E2 (100 nM) effects on inhibition of OM in an in vitro bioassay (A). BPA effects on OM tested over the concentration range of 5–100 nM (B). BPA, bisphenol-A; DHP, 17,20β-dihydroxy-4-pregen-3-one (dihydroxyprogesterone); EGF, 50 nM EGF; Veh, vehicle ethanol control. Bars denote means ± SEM; n = 6–15. Different letters denote significant differences from each other (P < 0.05, one-way ANOVA and nonparametric Bonferroni test). The entire experiment was repeated three or more times and similar results were obtained each time.

FIG. 2

Mechanism of BPA inhibition of OM. Effects of coincubation with the transcription inhibitor, actinomycin D (A), and the specific Gper antibody (B) on the inhibitory effects of BPA on spontaneous maturation of denuded zebrafish oocytes in the in vitro GVBD bioassay. Anti-GPER, 1:300 GPER antibody; BPA + AD, BPA plus 0.1 μg/ml actinomycin-D; IgG, 1:300 rabbit serum; Veh, vehicle control dimethyl sulfoxide (DMSO). Bars for both graphs denote means ± SEM (n = 7). Different letters denote significant differences from each other (P < 0.05, one-way ANOVA and nonparametric Bonferroni test). All the experiments were repeated three or more times and similar results were obtained each time. Competition curve of BPA binding to cell membranes of HEK293 cells transfected with zebrafish Gper expressed as a percentage of maximum specific [³H]-E2 binding (C). Mean binding results ± SEM (n = 6), plotted from two separate binding assays.

FIG. 3

Role of Egfr and its upstream regulators in BPA inhibition of OM and Mapk3/1 phosphorylation. Effects of cotreatment with the Src kinase inhibitor PP2 (A) and MMP inhibitor ilomastat (B) on the inhibitory effects of BPA on spontaneous maturation of denuded zebrafish oocytes in the in vitro bioassay and BPA phosphorylation of Mapk3/1 (C). Representative Western blot of phosphorylated Mapk3/1 (p-Mapk3/1) and total Mapk3/1 (Mapk3/1) levels after 15-min pretreatment with the inhibitors or vehicle followed by 15-min treatment with BPA or EGF (C). Effects of the Erb1 inhibitor AG1478 (D) on the inhibitory effects of BPA on spontaneous maturation of denuded zebrafish oocytes in the in vitro bioassay and BPA phosphorylation of Mapk3/1 (E). Representative Western blot of p-Mapk3/1 and total Mapk3/1 after 15-min pretreatment with AG1478 or vehicle followed by 15-min treatment with BPA, E2, and EGF (E). Veh, vehicle ethanol control; DHP, 5 nM DHP; E2, 100 nM E2; BPA, 100 nM BPA; PP2, 10 μM PP2; ILO, 10 μM ilomastat; BPA + ILO, 100 nM BPA plus 10 μM ilomastat ; BPA+PP2, 100 nM BPA plus 10 μM PP2; EGF, 50 nM EGF; G1, 100 nM G-1; AG1478 + BPA, 50 μM AG1478 plus 100 nM BPA; G1 + AG1478, 100 nM G-1 + 50 μM AG1478. Error bars denote means ± SEM (n = 8) (A and B); (n = 8) (D). Different letters denote significant differences from each other (P < 0.05, one-way ANOVA and nonparametric Bonferroni test). The GVBD experiments were repeated three or more times and similar results were obtained each time.

FIG. 4

Involvement of Mapk3/1 in BPA inhibition of OM and effects of BPA on cAMP production by denuded oocytes. Effects of MAPK3/1 pathway inhibitor U0126 on the inhibitory effects of BPA on spontaneous maturation of denuded zebrafish oocytes in the in vitro GVBD bioassay (A) and BPA phosphorylation of Mapk3/1 (B). Representative Western blot of p-Mapk3/1 and total Mapk3/1 after 15-min pretreatment with U1026 or vehicle followed by 15-min treatment with BPA, E2, and EGF (B). Veh, vehicle ethanol control; BPA, 100 nM BPA; BPA + U0126, 100 nM BPA plus 50 μM U0126; DHP, 5 nM DHP; E2, 100 nM E2; U0126, 50 μM U0126. Effects of BPA on activation of Mapk3/1 (B and C). Representative Western blot of p-Mapk3/1 and total Mapk3/1 levels after 15-min treatment with 10, 100, and 200 nM BPA (C, top) and relative density of p-Mapk3/1 bands relative to total Mapk3/1 determined from densitometry of Western blots (C, bottom). EGF, 50 nM EGF; Veh, vehicle control ethanol. Effects of BPA on cAMP production by denuded oocytes (D). Cyclic AMP levels were measured after 3-h incubation of oocytes with 100 nM BPA, 100 nM E2, and 100 nM G-1 (G1). Error bars denote means ± SEM (n = 8) (A), (n = 6) (C), (n = 3) (D). Different letters denote significant differences from each other (P < 0.05, one-way ANOVA and nonparametric Bonferroni test). The experiments were repeated three or more times and similar results were obtained each time.

FIG. 5

Effects of alkylphenols structurally related to BPA on inhibition of OM. Effects of tetrabromobisphenol A (TBBPA; A), nonylphenol (NP; B), tetrachlorobisphenol A (TCBPA; C) over the concentration range of 5–100 nM on spontaneous maturation of denuded zebrafish oocytes in the in vitro bioassay. (D) Effects of lower concentrations (1 and 2.5 nM) of NP and TCBPA on OM. Veh, vehicle ethanol control. Error bars denote means ± SEM (n = 6). Different letters denote significant differences from each other (P < 0.05, one-way ANOVA and nonparametric Bonferroni test). The experiments were repeated three or more times and similar results were obtained each time.

FIG. 6

Involvement of Gper and Egfr in inhibitory effects of NP, TCBPA, and TBBPA on OM. Effects of the specific GPER antagonist, G-15, on the inhibitory effects of alkylphenols on spontaneous maturation of denuded zebrafish oocytes in the in vitro bioassay (A). G-15, 100 nM G-15; NP, 100 nM NP; NP + G-15, 100 nM NP plus 100 nM G-15; TBB, 100 nM TBBPA; TBB + G-15, 100 nM TBBPA plus 100 nM G-15; TCB, 100 nM TCBPA; TCB + G-15, 100 nM TCBPA plus 100 nM G-15; Veh, vehicle ethanol control; n = 6. Different letters denote significant differences from each other (P < 0.05, one-way ANOVA and nonparametric Bonferroni test). The experiments were repeated three or more times and similar results were obtained each time. Binding of TBBPA, TCBPA, and NP to zebrafish Gper. Displacement of bound [³H]-E2 by 1 μM TCBPA, 1 μM NP, 1 μM BPA, and 1 μM TBBPA compared to that displaced by 100 nM and 1 μM E2 to recombinant zebrafish Gper on transfected HEK293 cell membranes in a single-point competitive binding assay (B). Values are represented as a percent of the total binding (total, membranes incubated with 4 nM [H³]-E2 alone in the absence of any competitor). Error bars denote means ± SEM (n = 4). Different letters denote treatments significantly different from each other (P < 0.05, one-way ANOVA and Dunnett test). Role of Egfr in TBBPA, TCBPA, and NP inhibition of OM (C). Effects of the EGFR inhibitor, AG1478, on the inhibitory effects of alkylphenols on spontaneous maturation of denudedzebrafish oocytes in the in vitro bioassay. +AG1478, plus 50 μM AG1478; NP, 100 nM NP; TBB, 100 nM TBBPA; TCB, 100 nM TCBPA; Veh, ethanol vehicle control. Error bars denote means ± SEM (n = 8). Asterisks denote significant differences between treatments in the presence or absence of AG1478 (P < 0.05, one-way ANOVA and Student t-test).