107. RESPONSES OF THE PREIMPLANTATION EMBRYO TO DEFINED GENOTOXIC STRESSES

Abstract

Genotoxic cellular stressors induce damage to the structure of DNA. This potentially compromises the genetic integrity of the cell. Such damage is particularly dangerous in the early embryo since transmission of defects can affect most cells in the body. Canonical responses of cells to such stress include delaying or blocking mitosis to allow DNA repair to occur or induction of cell death (apoptosis) to ensure that damage is not propagated. To date there have been only limited studies of the response of the preimplantation embryo to genotoxic stress. Ultra violet (UV) irradiation typically induces single strand DNA breaks while drugs such as the chemotherapeutic cisplatin commonly induce double strand breaks. ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and rad3 related) are checkpoint kinase that mediate early responses of the cell to DNA damage. ATM primarily responds to DNA double strand breaks while ATR typically responds to single strand breaks and stalled replication forks. However, ATM and ATR may have partially overlapping and complementary functions. Both kinases can exert P53 dependent and independent responses. ATR protein was detected by immunofluorescence in all preimplantation stages up to the morulae stage. ATM protein was detected in oocytes and all stages. Cisplatin or UV-irradiation at the 2-cell stage caused increased nuclear staining of both ATM and ATR. UV-irradiation of 2-cell embryos induced irreversible, ATR-dependent, P53-independent cell-cycle block without apoptosis. Cisplatin allowed cell-cycle progression with progressive ATR and ATM-dependent, P53-independent apoptosis over subsequent cell-cycles. UV-irradiation of morula caused an ATR and ATM-dependent, P53-independent block of blastocyst formation while the block caused by cisplatin was ATR and ATM-dependent and at least partially P53-dependent. The results demonstrate complexity and maturation of the cellular responses of the embryo to defined genotoxic stressors and help to define the nature of embryopathy under these circumstances.