Aberrant Hoxa10 gene methylation as a mechanism for endosulfan-induced implantation failures in rats

Abstract

DNA methylation is a well-established epigenetic mechanism controlling gene expression. Environmental chemicals, such as pesticides have been shown to alter DNA methylation. We have previously shown that the insecticide endosulfan impairs female fertility in rats by increasing the rate of preimplantation embryo losses. In this study, we evaluated whether early postnatal exposure to endosulfan affects long-term transcriptional regulation of Homeobox A10 (Hoxa10) gene, which is a key marker of endometrial receptivity. Female rats were neonatally exposed to 6 or 600 μg/kg/day (ENDO6 and ENDO600, respectively) of endosulfan and uterine samples collected on gestational day (GD) 5. Hoxa10 protein and mRNA levels were assessed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), respectively. In silico analysis of enzyme-specific restriction sites and predicted transcription factors were performed to investigate the methylation status of the regulatory regions of Hoxa10 gene by methylation-sensitive restriction enzymes-PCR technique. The expression of the DNA methyltransferases (Dnmts) was also evaluated. ENDO600 showed a decreased uterine Hoxa10 expression at protein and transcript level, while ENDO6 decreased only the level of transcripts, during the receptive stage. In addition, endosulfan increased levels of Dnmt3a and Dnmt3b. Dysregulation of DNA methylation patterns of Hoxa10 regulatory regions was detected in ENDO6- and ENDO600-treated rats. All these results suggest that aberrant DNA methylation in Hoxa10 gene could be an underlining mechanism contributing to explain endosulfan-induced preimplantation losses.