Perinatal Programming of Adult Rat Germ Cell Death After Exposure to Xenoestrogens: Role of microRNA miR-29 Family in the Down-Regulation of DNA Methyltransferases and Mcl-1

Different studies have pointed out that developmental exposure to environmental endocrine disruptors can induce long-term testicular germ cell death probably through epigenetic mechanisms. By using a model of early neonatal post-natal day (PND) 1 to 5 exposure of male rats to a xenoestrogen, estradiol benzoate (EB), we investigated the role of microRNA and DNA methyltransferases (DNMT) on the developmental effects of EB on the adult germ cell death process. Neonatal exposure to EB induced adult germ cell apoptosis together with a dose-dependent increase in miR-29a, miR-29b, and miR-29c expression. Increased miR-29 expression resulted in a decrease in DNMT1, DNMT3a, and DNMT3b and antiapoptotic myeloid cell leukemia sequence 1 (Mcl-1) protein levels as shown in 1) germ cells of adult rats exposed neonatally to EB and 2) in spermatogonial GC-1 transfected with miR-29. The DNMT decrease was associated with a concomitant increase in transcript levels of DNA methylation target genes, such as L1td1-1 ORF1 and ORF2, Cdkn2a, and Gstp1, in correlation with their pattern of methylation. Finally, GC-1 cell lines transfection with miR-29a, miR-29b, or miR-29c undergo apoptosis evidenced by Annexin-V expression. Together, the increased miR-29 with a subsequent reduction in DNMT and Mcl-1 protein levels may represent a basis of explanation for the adult expression of the germ cell apoptosis phenotype. These observations suggest that the increased expression of the "apoptomir" miR-29 family represents the upstream mechanism identified until now that is involved in adult germ cell apoptosis induced by a neonatal hormonal disruption.