title: Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.
abstract: 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 dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to 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.
authors.affiliation: Unitat de Biologia Cellular, Fisiologia i Immunologia, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Ribas-Maynou
authors.forename: J E
authors.initials: JE
authors.lastname: Gawecka
authors.forename: J
authors.initials: J
authors.lastname: Benet
authors.forename: W S
authors.initials: WS
authors.lastname: Ward
mesh_terms: D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects; D020552:Comet Assay / Q000379:methods*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D053938:DNA Fragmentation / Q000187:drug effects*; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D004822:Epididymis / Q000166:cytology / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D017895:Manganese Compounds / Q000494:pharmacology; D051379:Mice; D015530:Nuclear Matrix / Q000737:chemistry / Q000187:drug effects*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013094:Spermatozoa / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*; D014649:Vas Deferens / Q000166:cytology / Q000187:drug effects
chemical_list: D002712:Chlorides; D002843:Chromatin; D017895:Manganese Compounds; D012967:Sodium Dodecyl Sulfate; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D002122:Calcium Chloride; C025340:manganese chloride; D004229:Dithiothreitol
keywords: neutral Comet assay; sperm DNA breaks; sperm nuclear matrix