Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome

Abstract

Background Paternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit. Results Using proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation. Conclusions These data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Epigenetic markers in the embryonal germ cell development and spermatogenesis

“…The increasing number of data indicates changes of epigenetic markers of sperm DNA also during the maturation in epididymis (and its particular parts), and thus puts attention also on the epigenetic role of this organ, leading to the need of revision of the statement that the histone PTMs are completed before the release of the sperm from the male gonad [ 21 , 217 , 223 ]. So, in the context of male reproduction there is a strong need to deep evaluation of the epididymal processes in the epigenetic manner, particularly in the light of the known rich epididymal microenvironment and its already described influence both: on sperm maturation (motility, fertilization capability), as well as on the rate of sperm DNA damage [ 21 , 202 , 217 – 220 , 223 ]. Thus, the link between proper epimarks and sperm chromatin integrity perhaps will possess part of its principles also from the epididymal side.…”

Alcohol induced increases in sperm Histone H3 lysine 4 trimethylation correlate with increased placental CTCF occupancy and altered developmental programming

“…We acid extracted histones using methods previously described 73 . Briefly, we resuspended frozen sperm pellets in Nuclei Isolation Buffer-250 with 0.3% NP-40 (15 mM Tris–HCl (pH 7.5), 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM Sucrose, 1 mM DTT, 10 mM sodium butyrate, and 1:100 Halt protease inhibitor (Cat# PI78437, Thermo Fisher Scientific, Pittsburgh, PA, USA)) and rotated them at 4 °C for 30 min.…”

Nucleosomes in mammalian sperm: conveying paternal epigenetic inheritance or subject to reprogramming between generations?

“…While predominantly located at transcriptionally active promoters in round spermatids [9] , H4ac levels are globally upregulated upon spermatid elongation 10 , 11 , 12 . Subsequently, H4ac dramatically decreases concomitantly with histone eviction, resulting in only ~2–5% of H4 peptides acetylated at residues K5, K8, or K12 in mature sperm [13] . In vitro, protamines displace hyperacetylated nucleosomes more efficiently than unmodified ones [14] .…”