During spermiogenesis, nucleosome-based DNA packaging is replaced by
protamine toroids to generate the highly compacted nucleus of the mature
sperm of many species including human, mouse and bovine; however, the
sperm from these species also retain some nucleosomes. It has been
suggested that the histones of sperm nucleosomes may play important roles in
embryo development. In the fruit-fly, Drosophila melanogaster it had been
thought that nucleosomes and their component histones are completely
replaced by protamine, and that no residual histones are retained. In this study,
three experimental models, (Drosophila melanogaster, Mus musculus and Bos
taurus) were used to investigate i) the presence of histone protein and/or its
modifications in mature sperm, ii) paternal DNA packaging by histone, and iii)
the possible epigenetic roles of sperm histones in embryogenesis.
In this study, the presence of all the canonical histones of nucleosomal
chromatin (H2A, H2B, H3 and H4) was confirmed in the mature sperm of
Drosophila melanogaster by microscopic, biochemical and immunoprecipitation
analysis. Evidence is also provided for the presence of post-translational
modifications (acetylation and methylation) of the histones from fly, bovine and
murine sperm. The molecular landscape of histones and histone modification
(H3K27me3, H3K36me3 and H3K9me3) was investigated using ChiP-chip on a
Drosophila-based, 2.4 million probe whole genome tiling array. Data analysis
revealed that histones (and by extension, nucleosomes) are preferentially
positioned within exons rather than introns and that they also mark intron/exon
boundaries and gene promoters. However, nucleosomes are depleted
upstream of Transcription Start Sites (TSS). Sperm chromatin also contains
modified histones associated with gene regulation including repressive
(H3K9me3, H3K27me3) and permissive (H3K36me3) marks.
Gene ontology analysis showed that the H3K27me3 and H3K36me3 are
associated with gene sequences carrying distinctive embryological
developmental process terms. Moreover, the in silico analysis indicated that the
early zygote expresses genes are marked by H3K36me3 in sperm chromatin,
while the late zygote expresses genes that are marked by H3K9me3 instead.
The same sequences are depleted of the H3K36me3 modification in sperm
chromatin. These data suggest that modified sperm histones might play
epigenetic roles in post fertilisation events such as formation and functioning of
the male pronucleus and in early and late embryo development.