The Incidence of DNA Double-Strand Breaks Is Higher in Late-Cleaving and Less Developmentally Competent Porcine Embryos1

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.

CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development

“…Therefore, we hypothesized that CDK2 knockdown-induced cell cycle arrest may be caused by increased accumulation of DNA damage during early embryonic development. Indeed, by analyzing the number of γH2AX fluorescent foci [37,38], we demonstrated that CDK2 knockdown embryos have more DNA DSBs than control embryos on days 3 and 5 of development. In response to DNA damage, cell cycle checkpoints (G1, S, G2/M) are activated, stopping cell cycle progression to allow time for repair, thereby preventing transmission of damaged or incompletely replicated chromosomes [3].…”

Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos

“…Cleavage stage embryos have few DNA breaks and low transcription levels for DNA repair and cell cycle checkpoint proteins23. These peculiarities in cleavage stage development can influence embryonic response to genetic insults7.…”

Double‐strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos

“…It has been reported that early developing embryos can respond to DSBs by inducing the phosphorylation of histone H2A.x and activating checkpoint proteins, Chek1 and Chek2, which block the cell cycle to prevent the segregation of DSBs in dividing cells (5, 14, 31, 32). Moreover, we have observed that early developing embryos can respond to DNA damage by up‐regulating the expression of genes that are involved in either the HR or NHEJ pathways (2, 4); however, it has not been determined if both pathways are required for DSB repair in early embryos before the first cell lineage specification. In pluripotent embryonic stem cells, which are derived from blastocysts after the first cell lineage specification, HR is the predominant pathway for DSB repair (33).…”