We recently reported evidence for nucleosome and developmental gene sequence
enrichment in chromatin released by salt-extracted nuclei (1, 2). A large
halo in the sperm chromatin dispersion test corresponds with low levels of
DNA fragmentation and hence good sperm quality (3). We sought to link these
ndings by high resolution sequencing of halo DNA.
Dispersion halos were generated by treating human sperm nuclei with NaCl
solutions (4). Halos and remaining nucleoids were recovered and processed for
DNA sequencing. The mapped read depth and coverage of genomic regions by
sequences from halos and the ‘insoluble’ nucleoid core were compared. Halo
and nucleoid DNAs were also uorescently labelled for FISH on salt-extracted
sperm nuclei. Protein extracts recovered during halo formation were analysed
Sperm DNA fragmentation is a major concern for embryo viability but nothing
known about the fragmented sequences or how they may compromise the sperm.
Quantitative sequence analysis of low-salt
fractions revealed a signi cant enrichment of genomic features, with CpG
islands (>9x) > 5’UTRs (>4x) > CDS (>2x). Intronic sequences
were depleted (x2). Enrichments were reduced in high salt halos but showed
similar hierarchical arrangements. Genes enriched in the halo fraction are
involved in development, cell-cell interactions and DNA replication.
FISH-halo hybridisation signals localised almost exclusively to the periphery
of sperm nuclei while nucleoid signals localised to both peripheral and
internal regions. Histones, but not protamines, were detected in low-salt
We propose a model where CpG islands and the 5’UTRs and exons of many
developmentally important genes are located close to the nuclear envelope in
sperm nuclei. Our data also suggest that at least some of this chromatin is
nucleosomal. Sperm DNA fragmentation as assessed by halo formation could
include vulnerable sequences essential for normal reproductive function.