| Paternal exposure to therapeutic drugs or environmental chemicals can alter the genomic integrity of male germ cells, and consequently, be a source of detrimental effects on embryo development. Preconceptional exposure of male rats to the anticancer alkylating agent, cyclophosphamide, resulted in increased embryo loss, malformations and behavioral deficits in the offspring; these drug effects can be transmissible to subsequent generations. The goal of the studies presented in this thesis is to gain an understanding of the manner by which altered spermatozoal genomic integrity, acquired during preconceptional exposure to a DNA damaging agent, may lead to developmental instabilities in the early preimplantation embryo. Spermatozoa must be functional in order to effectively transmit genetic information to the oocyte. Motility is one component contributing to the ability of the spermatozoon to achieve proximity to and penetration of the oocyte for fertilization. Chronic cyclophosphamide exposure had little effect on the kinematic parameters constituting spermatozoal motion patterns. Drug-exposed spermatozoa are motile, and may thus be at an increased risk of transmitting an altered genomic complement to the embryo. An intact paternal genome is essential for normal embryogenesis. Using the rat sperm Y-4 fluorescence in situ hybridization assay, we determined that 9 weeks of chronic cyclophosphamide treatment, but not 6 weeks, significantly increased the frequency of numerical chromosomal abnormalities in epididymal spermatozoa. Epigenetic marks, characterized by various posttranslational modifications, also play an intricate role in chromatin dynamics during spermatogenesis and embryogenesis. Early post-fertilization, histone H4 acetylation and DNA methylation were dysregulated in both parental pronuclei in zygotes sired by cyclophosphamide-treated males. Intriguingly, posttranslational modifications important for DNA damage recognition are rapidly activated following fertilization by drug-exposed spermatozoa. Phosphorylated histone H2AX was dramatically increased in a biphasic manner in the paternal genome; Interestingly, poly(ADP-ribose) polymerase-1 was markedly elevated in both the paternal and maternal genome in zygotes fertilized by cyclophosphamide-treated males. Collectively, these studies demonstrate that male germ cell damage acquired during spermatogenesis can be transmitted to the embryo leading to aberrant epigenetic reprogramming and a dramatic induction of distinct modifications involved in DNA damage recognition in the 1-cell embryo. Importantly, we have highlighted the involvement of pronuclear cross talk as a unique phenomenon for both epigenetic reprogramming and DNA damage response in the zygote. |
