Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)–p53–p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.
Supplemental Table S1. Primers used for real-time reverse transcription-PCR
Supplemental Figure S1: Messenger RNA expression of RAD51 during porcine embryonic development.
Supplemental Figure S2: (A) Quantification of cell numbers in control and B02 treatment blastocyst. (B) RAD51 mRNA was significantly decreased after dsRNA injection. (C) Zygotes were cultured to develop into two-cell embryos. Developmental rates were determined at 16, 20, 24, and 28 h post activation. (D) The rate of development of embryos treated with B02 over seven days in culture. The developmental stages of embryos were affirmed according to blastomere number. Average numbers of embryos are shown at different stages. *P < 0.05. **P < 0.01; ***P < 0.001. The experiment was performed in triplicate and the data are expressed as the mean ± SEM. The number of oocytes analyzed is specified in brackets.
Supplemental Figure S3: (A) Control and RAD51 knockdown embryos obtained at the six-cell stages and stained with a γH2AX antibody. (B) Quantification of γH2AX foci number per nuclei in Control and B02-treated 6cell-embryos. Scale bar = 20 μm (white), 5 µm (yellow). ***P < 0.001. The experiment was performed in triplicate and the data are expressed as the mean ± SEM. The number of oocytes analyzed is specified in brackets.
Supplemental Figure S4: Quantification of ratio of ICM and TE in the control and B02-treated groups (refer to Figure 6B). The regions defined by dotted lines are as indicated in the picture. *P < 0.05. The experiment was performed in triplicate and the data are expressed as the mean ± SEM. The number of oocytes analyzed is specified in brackets.