Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos

Abstract

In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (H2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring H2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of H2AX foci (606.1 ± 103.2) and greater area of H2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of H2AX foci or area were detected among the treatments. H2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods forin vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.