The nucleohistone compartment in relation to sperm HALO formation, DNA damage and DNA sequence analysis

The presence of DNA damage in mature sperm may be associated with poor chromatin structure due to abnormal protamination. Interestingly, however, approximately 5-15% of the DNA in the human sperm nucleus remains bound to histones. In this study, oxidative stress was used to induce DNA damage in mature sperm aimed at investigating the integrity of sperm chromatin structure. The extent of the damage was assessed by acridine orange, alkaline comet and halo assay (HalospermTM). Experiments were designed to probe the relationship between sperm DNA fragmentation as revealed by halo dynamics and the DNA sequences that constitute the nuclear halo structure, and so provide a more robust link between the halo assay as a discriminator of high-quality sperm and paternal genes that may be disrupted in damaged sperm.
Differential Density Gradient Centrifugation (DDGC) was used to resolve human spermatozoa into 90% percoll solution (high density) and 45% percoll solution (low- density) fractions. DNA damage was induced by exposure to H2O2 at two different concentrations (100 and 300 μM) for fixed times. Acridine orange, alkaline comet and halo assay were used conventionally to measure the extent of DNA fragmentation in peroxide-treated cells. In a variant of the halo assay aimed at investigating the differences between protamine and histone-bound DNA, human sperm nuclei were treated with either low or high ionic strength salt solutions to generate nuclear halos. Halos produced from control (undamaged) sperm by HalospermTM or by salt extraction were treated in suspension with restriction
enzymes to release halo-DNA, which was analysed by Next Generation Sequencing (NGS).
Results of acridine orange, alkaline comet and halo assay revealed that pellets of DDGC processed sperm were far more resistant to H2O2 treatment compared with interface sperm. The efficacy of halo formation as an indicator of DNA damage was shown by the high percentage of strong halos generated by HalospermTM and salt extraction methods from sperm isolated from the pelleted sperm compared with interface sperm. Analysis of NGS data of halos generated by HalospermTM and by low or high salt extraction of nuclear proteins suggests that approximately 2000 genes, many of developmental significance are significantly ‘over-represented’ in nuclear halos compared with residual (nucleoid) DNA. Moreover, the data suggests that halo-DNA was originally associated with the histone compartment of sperm chromatin.
The nuclear halo can indicate the level of DNA fragmentation in sperm, and the sequence composition of halos suggest that such fragmentation could compromise important paternally-derived DNA sequences that the oocyte may be unable to repair.