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

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 patt...

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Autores principales: Cantão, Isabelle Hernandez, Tesser, Renato Borges, Stumpp, Taiza
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541664/
https://www.ncbi.nlm.nih.gov/pubmed/28785173
http://dx.doi.org/10.1186/s12575-017-0058-1
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author Cantão, Isabelle Hernandez
Tesser, Renato Borges
Stumpp, Taiza
author_facet Cantão, Isabelle Hernandez
Tesser, Renato Borges
Stumpp, Taiza
author_sort Cantão, Isabelle Hernandez
collection PubMed
description 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 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. 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 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. 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.
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spelling pubmed-55416642017-08-07 An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming Cantão, Isabelle Hernandez Tesser, Renato Borges Stumpp, Taiza Biol Proced Online Methodology 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 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. 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 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. 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. BioMed Central 2017-08-03 /pmc/articles/PMC5541664/ /pubmed/28785173 http://dx.doi.org/10.1186/s12575-017-0058-1 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology
Cantão, Isabelle Hernandez
Tesser, Renato Borges
Stumpp, Taiza
An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming
title An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming
title_full An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming
title_fullStr An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming
title_full_unstemmed An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming
title_short An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming
title_sort initial investigation of an alternative model to study rat primordial germ cell epigenetic reprogramming
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541664/
https://www.ncbi.nlm.nih.gov/pubmed/28785173
http://dx.doi.org/10.1186/s12575-017-0058-1
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