An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic ReprogrammingReportar como inadecuado




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Biological Procedures Online

, 19:9

First Online: 03 August 2017Received: 11 May 2017Accepted: 14 July 2017

Abstract

BackgroundPrimordial 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.

MethodsOnce collected, the gonads were placed in 24-well plates previously containing 45μm 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.

Results5mC and DNMTs labelling suggests that PGC are undergoing epigenetic reprogramming around 14-15dpc. The in vitro treatment of rat embryonic gonads with 1 μM 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.

ConclusionsThis study suggests that the protocol used here can be a potential method to study the wide DNA demethylation that takes place during PGC reprogramming.

KeywordsRat Primordial germ cell Epigenetic reprogramming  Download fulltext PDF



Autor: Isabelle Hernandez Cantão - Renato Borges Tesser - Taiza Stumpp

Fuente: https://link.springer.com/







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