Mol. Hum. Reprod. Advance Access originally published online on January 29, 2004
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Molecular Human Reproduction, Vol. 10, No. 3, pp. 203-209, 2004
© European Society of Human Reproduction and Embryology 2004
Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis
1Sección de Genética y Unidad de Investigación, Hospital ‘Teresa Herrera’, Complejo Hospitalario Universitario Juan Canalejo, 15006 La Coruña, 2Unidad de la Mujer, La Coruña and 3Unidad de Genética, Facultad de Biología, Universidad Autónoma de Madrid, Spain
4 To whom correspondence should be addressed at: Sección de Genética, Hospital ‘Teresa Herrera’, Complejo Hospitalario Universitario Juan Canalejo, 15006 La Coruña, Spain. e-mail: JLFernandez{at}canalejo.org
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 
2.5-fold higher density of background DNA breaks with 3'-OH ends, and also 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.
Key words: Key words: alkali-labile sites/alkaline DNA unwinding/chromatin structure/DBD–FISH/sperm DNA
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