Distribution of DNA damage in the sperm nucleus: A study of zebrafish as a model of histone-packaged chromatin.

Reproductive defects can occur when the integrity of the male gamete genome is affected. Sperm chromatin is not homogeneous, having relaxed regions which are more accessible to the transcription machinery in the embryo, and thought to be specially sensitive to DNA damage. The level of damage in specific genes located in these sensitive regions could represent an early biomarker of damage. Our objective is to test the hypothesis that these more relaxed regions show greater susceptibility to damage in zebrafish, a species lacking protamines and whose sperm chromatin is compacted with histones. After sperm UV irradiation, treatment with H₂O₂ and cryopreservation, global chromatin fragmentation was evaluated using the TUNEL assay, and the number of lesions per 10 Kb in specific genes (hoxa3a, hoxb5b, sox2, accessible for early transcription and rDNA 18S and rDNA 28S) was quantified by using a qPCR approach. Additionally, oxidative damage within the sperm nucleus and the potential colocalization of this injury with histone H3 and TOPO IIα+β were located by using immunofluorescence. UV irradiation produced the highest degree of fragmentation (p = 0.041) and the highest number of lesions per 10 Kb in all the genes, but no differences were observed in sensitivity to damage in the studied genes (ranging from 14.93 to 8.03 lesions per 10 Kb in hoxb5b and 28S, respectively). In contrast, H₂O₂ and cryopreservation caused varying levels of damage in the analyzed genes which was not related to their accessibility, ranging from 0.00 to 1.65 lesions per 10 Kb in 28S and hoxb5b, respectively, after H₂O₂ treatment, and from 0.073 to 5.51 in 28S and sox2, respectively, after cryopreservation. Immunodetection near oxidative lesions also revealed different spatial patterns depending on the treatments used, these being mostly homogeneous with UV irradiation or cryopreservation, and peripherally located around the nucleus after H₂O₂ treatment. Oxidative lesions did not colocalize with histone H3 or TOPO IIα+β, thus demonstrating that the relaxed DNA regions associated with these proteins were not more vulnerable to oxidative damage. Results suggest that accessibility of each agent to the nucleus could be the main factor responsible for the distribution of sperm DNA damage rather than the organization of the chromatin. Lesions in these genes important to early embryo development assayed in this study cannot be used as biomarkers of global DNA damage.

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