The Incidence of DNA Double-Strand Breaks Is Higher in Late-Cleaving and Less Developmentally Competent Porcine Embryos.

Abstract

Studies in different species, including human, mice, bovine, and swine, demonstrated that early-cleaving embryos have higher capacity to develop to the blastocyst stage and produce better quality embryos with superior capacity to establish pregnancy than late-cleaving embryos. It has also been shown that experimentally induced DNA damage delays embryo cleavage kinetics and reduces blastocyst formation. To gain additional insights into the effects of genome damage on embryo cleavage kinetics and development, the present study compared the occurrence of DNA double-strand breaks (DSBs) with the expression profile of genes involved in DNA repair and cell cycle control between early- and late-cleaving embryos. Porcine oocytes matured in vitro were activated, and then early-cleaving (before 24 h) and late-cleaving (between 24 and 48 h) embryos were identified and cultured separately. Developing embryos, on Days 3, 5, and 7, were used to evaluate the total cell number and presence of DSBs (by counting the number of immunofluorescent foci for phosphorylated histone H2A.x [H2AX139ph] and RAD51 proteins) and to quantify transcripts of genes involved in DNA repair and cell cycle control by quantitative RT-PCR. Early-cleaving embryos had fewer DSBs, lower transcript levels for genes encoding DNA repair and cell cycle checkpoint proteins, and more cells than late-cleaving embryos. Interestingly, at the blastocyst stage, embryos that developed from early- and late-cleaving groups had similar number of DSBs as well as transcript levels of genes induced by DNA damage. This indicates that only embryos with less DNA damage and/or superior capacity for DNA repair are able to progress to the blastocyst stage. Collectively, findings in this study revealed a negative correlation between the occurrence of DSBs and embryo cleavage kinetics and embryo developmental capacity to the blastocyst stage.