Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis

Abstract

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains approximately 2.5-fold higher density of background DNA breaks with 3'-OH ends, and also approximately 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei.

The DNA integrity of cryopreserved spermatozoa separated for use in assisted reproductive technology is unaffected by the type of cryoprotectant used but is related to the DNA integrity of the fresh separated preparation

“…Many different cryoprotective media have been developed in an effort to eliminate or minimize the effects of cryopreservation on human spermatozoa (9,32), and studies have evaluated and compared different media in terms of the relative recovery of normal, motile spermatozoa (2,3,18,20). In this study, the aim was to compare a larger number of cryoprotective media and evaluate them in terms of their ability to preserve the DNA integrity of the spermatozoa to be used for treatment in ART, and while this is not the first study of its kind to date, it is the largest to the best of our knowledge.…”

Influence of obesity on sperm DNA fragmentation index and outcomes of IVF programs

“…Визуализация встроенных фрагмен-тов возможна с помощью люминисцентного микро-скопа и проточной цитометрии. При NT (ник-трансляции, In-Situ Nick Translation assay) к 3'-ги-дроксильному концу ДНК присоединяются радио-активные нуклеотидные остатки [31][32][33].…”

“…Полученные «кометы» окрашивают флюорес-центным красителем. Чем выше уровень фрагмен-тации, тем большее количество ДНК при электро-форезе будет располагаться в зоне хвоста кометы [31][32][33][34].…”

The comet assay in male reproductive toxicology

“…Under alkaline conditions, nicks in the DNA seem to provide a starting point for DNA unwinding by transforming the breaks into single strands (Rydberg 1975;Vazquez-Gundin et al 2000). Published data suggest that sperm nuclei compared to leucocytes contain more than double the amount of SSB (Muriel et al 2004). More importantly, sperm chromatin is ninefold more enriched in singlestranded segments -potentially prone to becoming ALS.…”

“…More importantly, sperm chromatin is ninefold more enriched in singlestranded segments -potentially prone to becoming ALS. Due to the higher degree of compaction, those partially denatured sections maybe the result of elevated torsional stress of DNA loops (Muriel et al 2004). Impressively, mammalian sperm chromatin is six times more highly compacted than metaphase chromosomes, although it seems to be organised very specifically (Ward and Coffey 1991).…”