DNA methylation analysis for target regions in swim-up sperm and HRCS.

Majority of histones are replaced by protamine during spermatogenesis, but small amount of histones are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important how histones are distributed on sperm genome. We have found that the swim-up sperm used in many studies contains about 10% of immature sperm which do not yet complete the histone-to-protamine replacement. We have developed the novel method to purify the histone replacement-completed sperm (HRCS), and to completely solubilize nucleosomes from cross-linked HRCS without MNase digestion. Here, we analyzed H3 binding profiles in total sperm and HRCS by Chromatin IP – sequencing (ChIP-seq) analysis, and DNA methylation profiles in swim-up sperm and HRCS by target bisulfite sequencing (TGSB). Examination of DNA methylation status in 2 fractions of sperm samples.