Hypermethylation of hoxa10 by neonatal endosulfan exposure: an epigenetic mechanism for impaired embryo implantation.

MARLISE L. GUERRERO SCHIMPF,; MA. MERCEDES MILESI, ; JORGELINA G. VARAYOUD. ; ENRIQUE H. LUQUE,

Mar del Plata, Argentina

Otro; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clinica.; 2016

The homeobox gene, Hoxa10, is crucial for uterinedevelopment during embryogenesis and for embryoimplantation at adulthood. In previous work we demonstratedthat neonatal exposure to endosulfan altersuterine Hoxa10 expression in prepubertal rats, anddecreases the number of implanted embryos at adulthood.This work investigates the effects of neonatalendosulfan exposure on Hoxa10 uterine expressionand DNA methylation status during the pre-implantationperiod. Newborn female rats were treated by s.c. injectionsevery 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 PND90females were pregnant and on gestational day 5 (preimplantationperiod) uterine samples were collected.The expression of Hoxa10 was determined at proteinand mRNA levels by immunohistochemistry and realtime RT-PCR, respectively. mRNA relative expressionof the DNA methyltransferases (DNMT) 3a and 3b wasalso evaluated. Upon Hoxa10 gene we searched for CpGislands and restriction sites for the BstUI enzyme, toevaluate the methylation status of its regulatory regionsby Methylation-Sensitive Restriction Enzymes-PCRtechnique (MSRE-PCR). Predicted binding sites fortranscription factors were also investigated. Both dosesof endosulfan decreased the expression of Hoxa10mRNA, while only Endo600 down-regulated Hoxa10at protein level. Endo6 and Endo600 groups showedincreased DNA methylation levels in regulatory regionsof Hoxa10 gene, that are potentially regulated by criticaltranscription factors associated with the implantationprocess. An up-regulation of DNMT3a and DNMT3b wasdetected in endosulfan-treated rats. Neonatal exposureto endosulfan decreases uterine Hoxa10 expression duringthe pre-implantation period, via hypermethylation ofregulatory regions of the gene. These alterations couldaccount for the endosulfan-induced implantation failures.