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Novel Bioinformatic Analyses of Somatic Cell Contamination in Sperm Samples

ABSTRACT: Novel Bioinformatic Analyses of Somatic Cell Contamination in Sperm Samples

PROVIDER: PRJNA1035130 | ENA |

REPOSITORIES: ENA

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Novel Bioinformatic Analyses of Somatic Cell Contamination in Sperm Samples

Project description:To establish contamination profiles, the sperm donors with normal sperm counts were analyzed using an Infinium HumanMethylation450 array. Somatic cell lysis, sperm isolation, DNA extraction, and bisulfite conversion were performed as described by Aston et al. The bisulfite converted sperm DNA was hybridized to Illumina Infinium HumanMethylation450K microarrays at the University of Utah and run as recommended by the manufacturer (Bibikova et al. 2011). Unpaired blood samples were extracted using Qiagen's DNeasy Blood and Tissue kit and bisulfite converted using Zymo's EZ DNA Methylation kit. All procedures were performed according to the instructions of the manufacturer. Four permutations were run on each sample, including pure blood, half blood and half sperm by DNA concentration, half blood and half sperm by cell count, and pure sperm (n = 16). Concentration was normalized using a spectrophotometer. A Makler cell counting chamber was used to count white blood cells and sperm, which were then normalized in a 1:1 ratio.

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Project description:Mammalian sperm chromatin carries epigenetic information with the potential to influence offspring phenotype, making its faithful characterization essential. It has been suggested that cauda sperm preparations are contaminated by somatic chromatin, that this contamination dominates genome-wide profiles, and that valid results require pretreatment with somatic cell lysis buffer (SCLB), DNase I, and dithiothreitol. Here we show that properly purified cauda sperm contain no detectable somatic cells or cell-free DNA and that this pretreatment disrupts sperm chromatin organization. SCLB permeabilizes the sperm nucleus, allowing DNase I to fragment the sperm genome in situ, while DTT treatment causes chromatin to leak out of the nucleus. Using ATAC-see, we further demonstrate that Tn5 transposase can access intact protamine-condensed sperm chromatin without DTT, refuting the premise that profiles from untreated sperm reflect contamination. Pretreatment therefore damages the chromatin it claims to purify, and published profiles of untreated cauda sperm are valid and require no systematic re-examination.

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Project description: Not available

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In human sperm, H4K20me3 is abundantly preserved in retrotransposons, satellite repeats and olfactory genes, and maintains herewith the somatic-like pattern

Project description:In human sperm, preserved histones evading histone-to-protamine replacement were observed in certain genes and gene promoters, but also in distal intergenic and repetitive DNA regions. The substantiality of the latter and its putative biological role are still a subject of hot debate. To shed more light on this issue we analyzed H4K20me3, a histone mark regulating heterochromatic and repetitive DNA in somatic cells, which was recently detected in human sperm. Our immunohistochemical and western blot analyses revealed the presence of H4K20me3 in male germ cells at every stage of spermatogenesis and in mature sperms, respectively. By ChIP-sequencing of the motile sperm fractions from three biological replicates we found 4.56% of the sperm genome to be occupied by H4K20me3. By comparing the genome-wide binding sites of H4K20me3 in sperm cells and somatic cells (K562) we found correspondences in 77% of respective peaks. The majority of binding sites (70%) were detected in distal intergenic and intron regions. Intriguingly, H4K20me3 enrichments could be observed in both somatic and sperm cells within satellite repeats and retrotransposons, particularly in long interspersed nuclear repeats (LINEs) and retrotransposons containing long terminal repeats (LTR-retrotransposons). Broad cluster arrangements and strong enrichments in olfactory receptor genes were also characteristic for H4K20me3. This is the first time H4K20me3 is characterized at the genome-wide level in human sperm and compared to somatic cells. Our results reveal that H4K20me3 constitutes the majority of histones preserved in matured human sperm and maintains a somatic-like distribution pattern.

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