A study of endocrine disrupting chemicals (TCDD and Bisphenol A) on endometrial receptivity and implantation

Abstract

The endocrine disrupting chemicals (EDCs) are exogenous compounds that mimic natural hormones and disrupt endocrine functions in humans and animals. Accumulating evidence suggests that EDCs may have detrimental impact on human reproduction including infertility, distortion of sex ratios and menstrual problems. TCDD, one of the most toxic man-made chemicals, was found to affect embryo maturity, implantation and reproduction. Bisphenol A (BPA), another EDC commonly used nowadays in plastic products, exhibits weak estrogenic activity in human bodies. However, TCDD and BPA have distinct chemical structures, chemical properties and receptor binding, and their mechanistic actions on human body remain largely unknown.

The present study is to delineate the effects of EDCs on embryo implantation and development under in vitro exposure. It is hypothesized that EDCs (TCDD and BPA) may modulate fertility of animals by affecting early pre-implantation embryo development and attachment onto uterus. The effect of EDCs on the expression of receptors (AhR, ERE, ERR,,and ERa) and downstream reporter genes (CYP1A1 and C3) were studied by real-time PCR and Western blotting. An in vitro spheroid (JEG-3)-endometrial cells (Ishikawa) co-culture assay was established to study the effect of EDCs on spheroid attachment. Since microRNA, Wnt-signaling and adhesion molecules play important roles in implantation process, the expression of selected miRNA, Wnt-signaling and adhesion molecules was studied after EDCs treatment. Specific activities of the EDCs receptors were confirmed by inhibitor treatment or siRNA knockdown studies. Furthermore, EDC-treated embryos were transferred to pseudo-pregnant surrogate mice to determine the effect of EDCs on implantation outcome on day 8.

It was found that Ishikawa and JEG-3 cells expressed AhR, ERIIand ERRE. TCDD treatment induced CYP1A1 expression in both cell lines (P<0.05). TCDD at 10nM significantly suppressed (P<0.005) spheroid attachment (74% compared to control 97%). When both cell lines were treated with AhR antagonists DMF (10 aaa/ANF (1 /////with TCDD (10nM), the suppressive effect of TCDD on spheroid attachment in co-culture assay was nullified, suggesting that TCDD acts through AhR receptor. In BPA exposure, BPA (0.001 to 10 tt) induced the expressions of ER))and C3ain Ishikawa but reduced the expressions of EReeand C3ain JEG-3 cells. BPA (10 iiiisuppressed spheroids attachment (74% vs Control 94%, P<0.005); while co-treatment with ER antagonists (ICI 182,780), ERaaantagonistaaMPP), ERMMantagonist (PTHPP) or ERE///siRNA, the suppressive effect of BPA (10sM) on spheroid attachment was nullified, suggesting that BPA acts through ER receptors. Moreover, TCDD and BPA suppressed the expressions of B-catenin and E-cadherin for cell-cell adhesion. Activation of Wnt-signaling pathway by Wnt3a conditioned medium or LiCl, rescued the low spheroid attachment rate induced by EDCs. Both EDCs reduced embryo implantation rate in pseudo-pregnant mice, suggesting the adverse effect of EDCs on embryo implantation and/or endometrial receptivity.

Taken together, TCDD and BPA affected the JEG-3 spheroid attachment onto the Ishikawa endometrial cells and mouse embryo implantation. These effects might be mediated through the action of receptors and Wnt/β-catenin signaling pathway.