Drug-Induced Genetic and Epigenetic Marks in Sperm Affect Zygotic Development.

Abstract

Exposure of male rats to a male mediated developmental toxicant and anticancer alkylating agent, cyclophosphamide (CPA), can result in increased pre- and post-implantation loss, and malformations in the offspring. CPA induces a stage specific stress response in male germ cells. The DNA damage induced by CPA is germ cell phase specific, with the greatest damage occurring during sperm chromatin remodeling. Drug induced insults to sperm chromatin may affect the function of the paternal genome in the zygote. Zygotes sired by drug-treated males display advanced developmental progression. In 1- and 2-cell embryos sired by CPA-treated males, the relative abundance of candidate gene transcripts is elevated. We hypothesized that paternal CPA exposure disrupts epigenetic programming and DNA damage recognition/repair in the early embryo. To test this hypothesis, we examined the temporal patterns of histone H4 acetylation at lysine 5, cytosine methylation at position 5, histone H2AX phosphorylation, and poly(ADP-ribose) polymerase-1 immunostaining in zygotes sired by CPA-exposed and control males. H4 was significantly hyperacetylated in both the male and female pronuclei in embryos sired by CPA-treated males, beginning in G1 and lasting into S-phase; in later stages of zygotic development, the extent of H4 acetylation did not differ. DNA methylation reprogramming was also dysregulated. In control zygotes, the male pronuclei underwent a gradual genome-wide demethylation, while the female pronuclei remained hypermethylated; in contrast, the male pronuclei in zygotes fertilized by CPA-exposed sperm were dramatically hypomethylated. Phosphorylated histone H2AX was increased in a biphasic manner in the paternal genome, while poly(ADP-ribose) polymerase-1 was markedly elevated in both the paternal and maternal genomes in zygotes fertilized by CPA-treated males. Collectively, these studies demonstrate that paternal drug exposure affects the epigenetic programming and DNA damage responses of not only the male, but also the female pronucleus, demonstrating the existence of pronuclear cross talk during zygotic development. Deregulation of epigenetic programming in the zygote may contribute to heritable instabilities later in development. Supported by CIHR