New insight into the function of sperm retained histones.

We previously demonstrated that the nuclear form of Glutathione Peroxidase 4 (nGPx4) has a peculiar distribution in sperm head and is required for proper paternal chromatin decondensation at fertilization. While protamines are the major component responsible for sperm chromatin packaging, a small amount of histones is also retained, the relevant role of which has been recently highlighted in influencing early embryo development. We hypothesized that paternal histone modifications are implicated in the process of sperm chromatin disassembly in the zygote, addressing this issue in nGPx4 KO mice as experimental model. We first assessed the presence of acetylated histones in cauda epididymal sperm from WT mouse and by immunofluorescence we were then able to detect hyperacetylated histone H4 in mature sperm partially decondensed by treatment with glutathione and heparin. Western blot analysis of hyperacetylated histone H4 and histone H3 acetylated at K9 and K14 showed significant higher amounts of modified histones in nGPx4 KO sperm compared to WT sperm. When WT sperm chromatin status was analyzed in zona pellucida free oocytes fertilized in vitro in the presence of trichostatin (TSA), a faster decondensation kinetics was observed compared to untreated sperm. Having a higher content of acetylated histones, nGPx4 KO sperm did not show any decondensation following TSA treatment. In addition the analysis of DNA methylation pattern of the paternally imprinted gene Igf2/H19 showed a significant hypomethylation in KO sperm compared to WT ones. These findings reveal a link between acetylated histones retained in sperm and paternal chromatin remodeling after fertilization