Double-stranded DNA breaks hidden in the neutralcometassay suggest a role of the sperm nuclearmatrix in DNA integrity maintenance
Ribas-Maynou, Jordi (Universitat Autònoma de Barcelona. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia)
Gawecka, Joanna E. (Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa)
Benet i Català, Jordi (Universitat Autònoma de Barcelona. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia)
Ward, W. Steven (Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa)

Fecha:	2014
Resumen:	We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNAbreaks while vas deferens sperm cannot. Here,wedemonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the brokenDNAends remain attached to the nuclear matrix, causing theDNAto remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
Ayudas:	Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-1107 Instituto de Salud Carlos III PI11/0630
Nota:	Altres ajuts: National Institutes of Health Grant 1R01HD060722
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Lengua:	Anglès
Documento:	Article ; recerca ; Versió acceptada per publicar
Materia:	Neutral Comet assay ; Sperm nuclear matrix ; Sperm DNA breaks
Publicado en:	Molecular Human Reproduction, Vol. 20 Núm. 4 (april 2014) , p. 330-340, ISSN 1460-2407

DOI: 10.1093/molehr/gat090
PMID: 24282283

Postprint 28 p, 1.3 MB

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