Stability Of The Cytosine Methylome During Post-Testicular Sperm Maturation In Mouse

Author summaryIn mammals, after completing development in the testis, sperm continue to mature as they transit a long convoluted tube known as the epididymis. This process entails extensive molecular remodeling, with changes to the lipid composition of the sperm membrane, gain of key proteins, and a dramatic overhaul of sperm RNA contents. Here, to explore whether cytosine methylation patterns might also be edited during this process, we characterized cytosine methylation genome-wide at seven stages of sperm development in the testis and epididymis. Overall, we find remarkably stable methylation patterns throughout, supporting the classical view of mammalian cytosine methylation dynamics. In addition, while our data initially suggested a major methylation remodeling event in the caput epididymis and vas deferens, followup studies revealed that the aberrant methylation signature resulted from contamination by cell-free DNA. Curiously, this DNA was associated with citrullinated histones, suggesting that it might be generated by a cell death process akin to NETosis. While it will be interesting to determine whether epididymal cell-free DNA serves any biological function, the more immediate implication of our results is technical, as cell-free DNA could contaminate studies of the sperm epigenome, and so should be eliminated to improve the robustness of sperm methylation measurements.Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.