An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming

Autor(es)

Cantao, Isabelle Hernandez ; Tesser, Renato Borges ; Stumpp, Taiza

• Tipo de documento: Artigo Científico
• Fonte: BIOLOGICAL PROCEDURES ONLINE; v. 19, AUG 3 2017.
• Citações Web of Science: 1

Resumo

Background: Primordial germ cells (PGC) are the precursors of the gametes. During pre-natal development, PGC undergo an epigenetic reprogramming when bulk DNA demethylation occurs and is followed by sex-specific de novo methylation. The de novo methylation and the maintenance of the methylation patterns depend on DNA methyltransferases (DNMTs). PGC reprogramming has been widely studied in mice but not in rats. We have previously shown that the rat might be an interesting model to study germ cell development. In face of the difficulties of getting enough PGC for molecular studies, the aim of this study was to propose an alternative method to study rat PGC DNA methylation. Rat embryos were collected at 14, 15 and 19 days post-coitus (dpc) for the analysis of 5mC, 5hmC, DNMT1, DNMT3a and DNMT3b expression or at 16dpc for treatment 5-Aza-CdR, a DNMT inhibitor, in vitro. Methods: Once collected, the gonads were placed in 24-well plates previously containing 45 mu m pore membrane and medium with or without 5-Aza-CdR. The culture was kept for five days and medium was changed daily. The gonads were either fixed or submitted to RNA extraction. Results: 5mC and DNMTs labelling suggests that PGC are undergoing epigenetic reprogramming around 14/15dpc. The in vitro treatment of rat embryonic gonads with 1 mu M of 5-Aza-CdR lead to a loss of 5mC labelling and to the activation of Pax6 expression in PGC, but not in somatic cells, suggesting that 5-Aza-CdR promoted a PGC-specific global DNA hypomethylation. Conclusions: This study suggests that the protocol used here can be a potential method to study the wide DNA demethylation that takes place during PGC reprogramming. (AU)

• Processo FAPESP: 11/07078-0 - Expressão de proteínas RIWI e retrotransposons no desenvolvimento embrionário das células germinativas em ratos
• Beneficiário: Renato Borges Tesser
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 12/25087-9 - Alteração do período de reprogramação epigenética das células germinativas primordiais de rato e influência de sua alteração na expressão do marcador de pluripotência SSEA1
• Beneficiário: Isabelle Hernandez Cantão
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 12/01024-8 - Expressão de piRNAs, proteína RIWI e de retrotransposons durante o desenvolvimento embrionário da células germinativas em ratos
• Beneficiário: Taiza Stumpp Teixeira
• Modalidade de apoio: Auxílio à Pesquisa - Regular