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Epigenetics of bovine semen: tools to DNA methylation analysis
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Edité par CCSD ; Cost Office -
Spermatogenesis is a long process requiring several epigenetic modifications to obtain a stably packed chromatin. Recent studies suggest that stochastic and environmental disturbances in this genome-wide epigenetic reprogramming may lead to epigenetic aberrations of semen quality, fertility and embryo development (Boissonnas et al., 2013). Establishing reference values and profiles of spermatozoa DNA methylation provides the opportunity to identify biological disturbances in epigenetic marks, as well as protective measures. Here, we present tools to monitor sperm DNA methylation at different scales and to identify individual and temporal variations in the methylation contents and patterns. The global quantification of DNA methylation amount (MeDNA) estimated by LUminometric Methylation Assay, demonstrated a species specificity (bovine, porcine, ovine, human and murine semen). In bovine, the MeDNA wasn’t higher in semen than in somatic tissues as well as between fresh and cryopreserved semen. However, breed-dependent variations of semen MeDNA were found. To identify methylated regions in bovine semen, we used immunoprecipitation of methylated DNA followed by hybridization on a bovine promoter microarray (MeDIP-chip). The microarray targets the upstream region (-2000/+1360bp) of 21416 genes (UMD3.1 assembly). After normalization of the data, enriched probes were identified using ChIPmix (Martin-Magniette et al., 2008). Genomic DNA from sperm (4 fertile bulls), liver (19 females) and fibroblasts (3 females) were analyzed. Differentially Methylated Regions between tissues (T-DMRs) were identified using Spatstat R package. T-DMRs in sperm were enriched for genes involved in the spliceosome (DAVID analysis). Most of theseT-DMRs were demethylated in sperm. We developed successful and relevant tools to analyze the methylation statue of bovine semen. Now we could analyze the environmental impact on the sperm epigenetic profile according to the spermogram and fertility.
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