Environmental Toxin Exposure and Its Effects on Sperm Chromatin Epigenetic Marks
DOI:
https://doi.org/10.5281/zenodo.22756536Keywords:
sperm chromatin, protamine-histone exchange, endocrine-disrupting chemicals, DEHP, DNA methylation, paternal epigenetic inheritanceAbstract
Mature mammalian sperm retain a small but functionally important fraction of nucleosomal histones at
developmental and imprinted loci after the genome-wide histone-to-protamine exchange of spermiogenesis, and
disruption of this retained chromatin landscape has been proposed as a route by which paternal environmental
exposures influence offspring phenotype. Here we exposed adult male mice to the phthalate plasticiser
di(2-ethylhexyl) phthalate (DEHP) at an environmentally relevant dose throughout one full 35-day spermatogenic
cycle and profiled sperm chromatin composition using sperm chromatin structure assay (SCSA), chromomycin A3
and aniline blue staining of protamine deficiency, whole-genome bisulfite sequencing and CUT&RUN for retained
H3K4me3 and H3K27me3. DEHP-exposed males showed a 2.4-fold increase in the protamine-deficient sperm
fraction by chromomycin A3 staining and a significantly elevated DNA fragmentation index by SCSA relative to
vehicle-treated controls, without a significant change in overall sperm concentration or motility. Whole-genome
bisulfite sequencing identified 1,362 differentially methylated regions in DEHP-exposed sperm, enriched at CpG
islands overlapping developmental transcription factor genes and imprinted loci, with a directional bias toward
hypomethylation. CUT&RUN profiling of retained histone marks showed that H3K4me3 domains at a defined set of
developmental gene promoters were preferentially depleted in DEHP-exposed sperm, while H3K27me3 domains
were comparatively unaffected, indicating a mark-specific rather than global disruption of the retained nucleosome
landscape. RNA-sequencing of two-cell and blastocyst-stage embryos sired by DEHP-exposed males, using
unexposed oocytes, revealed differential expression of genes overlapping the sperm hypomethylated and
H3K4me3-depleted promoter set, with effects most pronounced at the two-cell stage and substantially attenuated
by the blastocyst stage, supporting a mechanistic route for paternal chemical exposure to influence offspring
molecular phenotype through sperm chromatin rather than through sperm count or motility alone.
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