Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development

in Reproduction
Correspondence should be addressed to N-H Kim; Email: nhkim@chungbuk.ac.kr

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.

Downloadable materials

  • 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.

 

    Society for Reproduction and Fertility

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    Localization and expression patterns of RAD51 in porcine early embryos. (A) Localization of RAD51 at various stages in early embryos was investigated by immunostaining with an anti-RAD51 antibody and Hoechst nuclear stain. Scale bar: 20 µm (white), 5 µm (yellow). (B) Quantitative RAD51 foci number per nuclei in one-, two-, four- and eight-cell embryos, as well as morula and blastocysts. (C) H2AX was activated by phosphorylation (γH2AX) at sites of DNA damage and served as a marker of DNA DSBs. RAD51 aggregated as foci in the nucleus and colocalized with γH2AX in 2-cell embryos. Red, RAD51; green, γH2AX; blue, DNA. Scale bar = 20 μm (white), 5 µm (yellow).

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    Effects of RAD51 inhibition on the early porcine parthenogenetic embryo development. (A) Blastocyst rates of porcine embryos that were untreated (control) or exposed to 0.10, 25, or 50 μm RAD51 inhibitor B02. (B) RAD51 foci were decreased after B02 (25 μm) treatment. Scale bar = 20 μm (white), 5 µm (yellow). (C) Quantitative RAD51 foci number per nuclei in control and B02-treated 4cell-embryos. (D) Morphology of day-7 embryos derived from zygotes treated with B02, indicating that the blastocyst development rates and cell numbers per blastocyst were lower than in the control group. Blue, DNA; DIC, differential interference contrast. (E and F) Knockdown of endogenous RAD51 protein expression after RAD51 dsRNA injection was verified by immunofluorescent staining analysis. Bar = 5 µm. (G) Effects of RAD51 knockdown on rate of blastocyst over 7 days in culture. (H) Morphology of embryos derived from zygotes treated with B02 (50 μm). For embryos that were treated with B02, two nuclei or no nuclei were found in each blastomere. Blue, DNA; DIC, differential interference contrast. Scale bar = 20 μm. (I) Control and B02-treated embryos are shown at different stages derived from 12, 24, 60 and 84 h post activation. The developmental stages of embryos were affirmed according to blastomere number. Scale bar = 40 μm. (J) Two-cell embryos were collected at 24 h and cultured into the four-cell stage. Developmental rates were determined at 24, 28, 32 and 36 h post activation. *P < 0.05; **P < 0.01; ***P < 0.001. The experiment was performed in triplicate and the data are expressed as the mean ± s.e.m. The number of oocytes analyzed is specified in brackets.

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    DNA damage accumulates in B02-treated four-cell embryos. (A) Representative images of control and B02-treated embryos obtained at the two- and four-cell stages and stained with a γH2AX antibody. The size and number of γH2AX foci increased in B02-treated four-cell embryos. Scale bar = 20 μm (white), 5 µm (yellow). (B) Quantification of γH2AX foci number per nuclei in control and B02-treated two-cell and four-cell embryos. (C) Control and RAD51-knockdown embryos obtained at the four-cell stages and stained with a γH2AX antibody. (D) Quantification of γH2AX foci number per nuclei in control and RAD51-knockdown four-cell embryos. (E) Control and B02-treated four-cell embryos were stained with γH2AX and 53BP1 antibodies. For the negative control, the anti-53BP1 primary antibody was omitted. Scale bar = 20 μm (white), 5 µm (yellow). (F) Quantification of the 53BP1 intensity in four-cell embryos. (G) Levels of Brca1, Mre11a, Brca2, Prkdc and Xrcc6 mRNA were measured in the B02-treated four-cell embryos using qRT-PCR. *P < 0.05; **P < 0.01; ***P < 0.001. The experiment was performed in triplicate, and the data are expressed as the mean ± s.e.m. The number of oocytes analyzed is specified in brackets.

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    Checkpoints are activated in B02-treated four-cell embryos. (A) Control and B02-treated embryos obtained at the four-cell stage and stained with an ATM-p antibody. Scale bar = 20 μm. (B) Control and B02-treated embryos obtained at the four-cell stage and stained with a p53 antibody. For the negative control, the anti-p53 primary antibody was omitted. Scale bar = 20 μm (white), 5 µm (yellow). (C) Control and B02-treated embryos obtained at the four-cell stage and stained with a p21 antibody. For the negative control, the anti-p21 primary antibody was omitted. Scale bar = 20 μm (white), 5 µm (yellow). (D) Western blot analysis showing activation/expression of p53 and p21 at the four-cell stage after B02 treatment; β-actin served as a loading control. (E) Relative intensity of p53 in Western blot was assessed by densitometry. (F) Relative intensity of p21 in Western blot was assessed by densitometry. ***P < 0.001. The experiment was performed in triplicate and the data are expressed as the mean ± s.e.m.

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    RAD51 inhibition increased intracellular ROS levels and abnormal mitochondrial distribution. (A) ROS staining in B02-treated and untreated blastocyst. Scale bar = 120 μm. (B) Mitochondrial distribution in RAD51 inhibitor-treated and untreated control oocytes as detected by immunostaining. (C) Red fluorescence corresponds to activated mitochondria and green fluorescence corresponds to less-activated mitochondria. Scale bar = 120 μm. (D) ROS levels in B02-treated and control blastocyst. (E) The rate of abnormal mitochondria distribution is increased after B02 treatment. (F) Mitochondrial membrane potential was measured as the ratio of red fluorescence to total fluorescence. **P < 0.01; ***P < 0.001. The experiment was performed in triplicate and the data are expressed as the mean ± s.e.m. The number of oocytes analyzed is specified in brackets.

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    Effects of B02 on apoptosis and pluripotency gene expression. (A) TUNEL-positive cells were detected in B02-treated and untreated blastocyst. Scale bar = 20 μm. (B) SOX2 protein detection in B02-treated and untreated blastocyst. Scale bar = 20 μm. (C) OCT4 protein detection in B02-treated and untreated blastocyst. Scale bar = 20 μm. (D) Quantification of the apoptotic rate (number of apoptotic cells/number of total cells) in B02-treated and untreated blastocyst. (E) Quantification of SOX2 signal in B02-treated and untreated blastocyst. (F) Quantification of OCT4 signal in B02-treated and -untreated blastocyst. **P < 0.01; ***P < 0.001. The experiment was performed in triplicate, and the data are expressed as the mean ± s.e.m. The number of oocytes analyzed is specified in brackets.

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