Fig 1.
Simplified schematic detailing the modified experimental methods for DNA extraction and fragmentation from sperm cells.
Human Donor spermatozoa samples, isolated via Percoll density gradient centrifugation and cleaned of leucocytes with Dynabeads, were pooled together in a single sample (0). Four identical samples with identical volume and cell numbers (1, 4, 9 and 14) were obtained from pooled sperm sample (0). DNA from sperm cells in method 1 samples (1) was collected after cell lysis (2) and DNA isolation via phenol-chloroform extraction (3) prior to comparative analysis. Samples assessed by method 2 (Sonication) (4) were subjected to cell lysis (5) followed by DNA isolation by phenol-chloroform extraction (6) and shearing by sonication (7) before comparative analysis. Samples assessed by method 3 (MNase after lysis) (9) were initially subjected to cell lysis (10) then DNA isolation by phenol-chloroform extraction (11) and DNA fragmentation by MNase digestion (12) followed by an additional DNA isolation step (13). Samples assessed by method 4 (MNase before lysis) (14) were treated with Triton X-100 and DTT prior to MNase digestion (15), sperm cells were then lysed (16) and DNA isolated via phenol-chloroform extraction (17). DNA fragment size was assessed by DNA gel electrophoresis and the amount of DNA recovered by each method was quantified with the Quant-iT PicoGreen dsDNA Kit and DNA oxidation was measured following Southern transfer and immunoblotting (18).
Fig 2.
Simplified schematic detailing the modified DNA immunoprecipitation technique to isolate oxidised DNA fragments.
Human Donor spermatozoa samples, isolated via Percoll density gradient centrifugation and cleaned of leucocytes with Dynabeads, were pooled together in a single sample (0). Each pooled sample was divided into two identical samples with equal volume and cell numbers (1, 2) before being processed by method 2 (sonication) (4, 6–8) or by method 4 (MNase before cell lysis) (3, 5, 9). The genomic DNA extracted and prepared following these methods was divided into 3 samples, the input control samples (10, 15), the antibody treated (11, 13) and the no-antibody negative controls (12, 14). DNA fragments in each sample was ran on a DNA gel and quantified with the Quant-iT PicoGreen dsDNA Kit (22).
Fig 3.
Bioinformatics pipeline framework.
Mock sequencing data (1) was initially processed by Bowtie (2) to align sequences against the human reference genome. Sequences un-aligned by Bowtie (5) were submitted to Bowtie 2 (6) to resolve sequence ambiguity and increase the number of successfully mapped sequences (10). The RepeatMasker software was used to analyse sequence composition of the entire dataset (3), the mapped (12) and un-mapped sequences (13). A constructed algorithm (16) was then used to remove sequences classified as background noise in MoDIP negative controls. The remaining sequences (17) were then grouped into clusters of oxidised sequences by another newly developed algorithm (18). Cluster information (19) was imported into statistical analysis software (20) to determine the significant relevance of this data.
Fig 4.
Comparative assessment of DNA fragmentation and DNA yield generated by different experimental methods.
a) Resolution of extracted DNA via gel electrophoresis showed successful fragmentation was achieved using each experimental method. Method 2 and 4 accurately generated DNA fragments in target range of 200–2,000 bp in size. L1: 100bp DNA Ladder. L2-3: DNA fragments obtained following method 2 (sonication). L4-5: DNA fragments obtained following method 3 (MNase after cell lysis). L6-7: DNA fragments obtained following method 4 (MNase before cell lysis). L8-9: DNA extracted from samples using control method 1. b) The amount of DNA recovered from sperm cells was significantly influenced by the extraction protocol utilized (One-way ANOVA, p = 0.034, n = 6). Method 4 (MNase before cell lysis) achieved the highest DNA yield, isolating an equivalent amount of DNA to that of controls. Method 3 (MNase before cell lysis) generated a significantly lower DNA yield (Tukey’s HDS test, p = 0.02) and performed the worst among compared methods. * p<0.05.
Fig 5.
Levels of DNA oxidation induced by different methodological approaches of DNA extraction.
a) Detection of oxidised DNA was performed following Southern transfer and sequential immunoblotting with anti-8OHdG and HRP-conjugated anti-mouse antibodies. The DNA extracted via experimental method 3 displayed the highest levels of oxidative damage. L1: 100bp DNA Ladder. L2-3: Oxidised DNA extracted via method 2 (sonication). L4-5: Oxidised DNA generated by method 3 (MNase after cell lysis). L6-7: Oxidised DNA induced by method 4 (MNase before cell lysis). L8-9: Oxidised DNA extracted using method 1. b) The amount of residual DNA oxidation was significantly affected by experimental methods used to extract and fragment sperm DNA (Kruskal-Wallis test, p = 0.04, n = 6). Multiple comparison testing revealed a significant increase in DNA oxidation (p<0.05, n = 6) in samples obtained using methods 2 (sonication) and 4 (MNase after cell lysis). *p<0.05.
Fig 6.
Quantification of DNA from human spermatozoa before and after modified immunoprecipitation to extract oxidised DNA.
a). Resolution of isolated DNA via electrophoresis illustrated successful fragmentation was achieved using both experimental methods before and after MoDIP. Further, both methods extracted similar amounts of DNA with an equivalent size range before MoDIP. Following immunoprecipitation, method 2 (sonication) isolated comparatively more oxidised DNA. L1: 100bp DNA Ladder. L2: DNA fragments extracted using method 4 (MNase before cell lysis). L3: DNA fragments obtained following method 2 (sonication). L4: Empty Lane. L5: Oxidised DNA extracted following MoDIP and method 4 (MNase before cell lysis). L6: Oxidised DNA extracted following MoDIP and method 2 (sonication). b) The amount of DNA recovered from human spermatozoa before MoDIP was not significantly different between samples fragmented via sonication or MNase digestion. c) Significant increase in the amount of oxidised DNA extracted from sperm cells following MoDIP in samples fragmented via sonication (Mann-Whitney U test, p = 0.02, n = 4). *p<0.05.