Paternal exposure to cyclophosphamide alters cell-cell contacts and activation of embryonic transcription in the preimplantation rat embryo

Abstract

Paternal exposure to chronic low doses of cyclophosphamide, an anticancer agent, results in aberrant embryonic development of the progeny. We hypothesized that paternal exposure to cyclophosphamide disturbs zygotic gene activity regulating proper progression through preimplantation development and that this disturbance results in improper cell-cell interactions. To test this hypothesis, we analyzed cell-cell interactions and the expression of cytoskeletal elements in preimplantation embryos sired by male rats gavaged with saline or 6 mg kg-1 day-1 cyclophosphamide for 5 wk. Embryos from control litters had 4-12 cells on Day 2 of gestation; cell- cell contacts were observed consistently. Embryos from litters sired by cyclophosphamide-treated males were frequently abnormal and had lower cell numbers and decreased cell-cell contacts. Steady state concentrations of the mRNAs for cell adhesion molecules (cadherins and connexin 43) and structural proteins (β-actin, collagen, and vimentin) were low in two- and four-cell control embryos; expression increased dramatically by the eight-cell stage. In contrast, embryos sired by cyclophosphamide-treated males displayed the highest expression of most trancripts at the two-cell stage. In parallel with the mRNA profiles, E-cadherin immmunoreactivity was nearly absent in two-cell control embryos and was strong by the eight-cell stage; immunoreactivity in embryos sired by drug-treated fathers was strong at the two-cell stage but absent at later stages. Thus, drug exposure of the paternal genome led to dysregulated expression of structural elements and decreased cell interactions during preimplantation embryonic development.