Hypermethylation of Hoxa10 by neonatal endosulfan exposure: an epigenetic mechanism for impaired embryo implantation

MILESI MM; GUERRERO SCHIMPF M; LUQUE EH; VARAYOUD J

Mar del Plata

Congreso; LXI Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2016

Sociedad Argentina de Investigación Clínica (SAIC)

The homeobox gene, Hoxa10, is crucial for uterine development during embryogenesis and for embryo implantation at adulthood. In previous work we demonstrated that neonatal exposure to endosulfan alters uterine Hoxa10 expression in prepubertal rats, and decreases the number of implanted embryos at adulthood. This work investigates the effects of neonatal endosulfan exposure on Hoxa10 uterine expression and DNA methylation status during the pre-implantation period. Newborn female rats were treated by sc. injections every 48 h, from postnatal day 1 (PND1) to PND7, with corn oil (vehicle, Control), 6 µg endosulfan/kg (Endo6, reference dose EPA) or 600 µg endosulfan/kg (Endo600, no observed effect level, EPA). On PND90 females were pregnant and on gestational day 5 (pre-implantation period) uterine samples were collected. The expression of Hoxa10 was determined at protein and mRNA levels by immunohistochemistry and real time RT-PCR, respectively. mRNA relative expression of the DNA methyltransferases (DNMT) 3a and 3b was also evaluated. Upon Hoxa10 gene we searched for CpG islands and restriction sites for the BstUI enzyme, to evaluate the methylation status of its regulatory regions by Methylation-Sensitive Restriction Enzymes-PCR technique (MSRE-PCR). Predicted binding sites for transcription factors were also investigated. Both doses of endosulfan decreased the expression of Hoxa10 mRNA, while only Endo600 down-regulated Hoxa10 at protein level. Endo6 and Endo600 groups showed increased DNA methylation levels in regulatory regions of Hoxa10 gene, that are potentially regulated by critical transcription factors associated with the implantation process. An up-regulation of DNMT3a and DNMT3b was detected in endosulfan-treated rats. Neonatal exposure to endosulfan decreases uterine Hoxa10 expression during the pre-implantation period, via hypermethylation of regulatory regions of the gene. These alterations could account for the endosulfan-induced implantation failures.