Effects of endocrine disruptors (TCDD and PFOA) on implantation: an in vitro co-culture study

Abstract

Endocrine disruptors (EDs) are exogenous substances that act like hormones in the endocrine system. They affect human health, reduce fertility, cause reproductive tract abnormalities, and distort sex ratios. 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) and Perfluorooctanoate (PFOA) are EDs that are mainly produced from industrial combustion and used as the surfactant in many daily used products, respectively, that are commonly found in wildlife and humans. TCDD affects the growth and reproductive functions in hamster, and disrupts the morphogenesis of rat preimplantation embryos. PFOA causes early pregnancy loss, compromises postnatal survival, as well as delays growth and development. Yet, how these EDs affect animal fertility and embryo implantation is not fully understood. We hypothesized that EDs affected fertility by suppressing the implantation process through down-regulation of Wnt-signaling pathway that regulates implantation process.

The effects of EDs on implantation was studied using an in vitro spheroidendometrium co-culture model with the trophoblast cell lines (BeWo and JEG-3) and an endometrial carcinoma cell line (RL95-2) to mimic the embryo-endometrial implantation process. The effects of EDs on cell proliferation and expression of their receptors (TCDD: aryl hydrocarbon receptor/AhR; PFOA: peroxisome proliferator-activated receptors/PPARs) were investigated. Their antagonists (AhR: alpha-naphthoflavone/ANF; PPARs: MK886, GSK0660 and GW9662) were used to determine whether the signaling pathways is mediated through receptor binding. Moreover, Wnt-signaling activators (Wnt3a and lithium chloride/LiCl) were used to examine the interaction between the EDs and the Wnt molecules. Mouse blastocyst-endometrial cells co-culture assay was also performed to study the effects of EDs on the development and attachment of the mouse embryos in vitro. Human primary endometrial epithelial and stroma cells were isolated and cultured to investigate the effects of the EDs on the protein expression of integrins, adhesion molecules and receptivity markers.

It was found that AhR and PPARs was present in the three cell lines studied. Moreover, EDs did not affect cell proliferation, viability and the expression of the AhR and PPARs. However, TCDD (1 – 10 nM) and PFOA (10 – 100 μM) significantly reduced the attachment of spheroids onto the RL95-2 monolayer. Addition of AhR antagonist (ANF) and PPARs antagonists (MK886 and GW9662), but not GSK0660 nullified the suppressive effect of EDs on spheroids attachment. Moreover, EDs reduced the expression of Wnt-signaling molecule (β-catenin), while cells treated with Wntsignaling activators (Wnt3a) or glycogen synthase kinase-3β inhibitor (LiCl) stimulated-catenin expression and reversed the suppressive effect of the EDs on spheroid attachment.

TCDD but not PFOA significantly suppressed the attachment of mouse blastocysts onto the endometrial cells; while the invasion of embryos was not affected by both EDs. TCDD induced the expression of miR-133a and miR-199a in the treated mice blastocysts. In the human primary endometrial cultures, EDs suppressed the expression of the adhesion molecules (β-catenin and E-cadherin), integrins (αV and β3), and changed the expression of Mucin 1, Leukemia inhibitory factor and Osteopontin. In conclusion, the present study showed that TCDD and PFOA suppress spheroids (blastocysts surrogate) attachment, affect the expression of adhesive molecules and modulate the Wnt-signaling pathway