Figure 4. SIRT6 knockdown in oocytes induces DNA damage and apoptosis of early embryos. (A) Relative telomere length in two-cell embryos is expressed as a T/S ratio determined by quantitative RT-PCR analysis. Data are expressed as mean percentage ± SD of three independent experiments (n=125 for control; n=128 for SIRT6-KD). (B) Representative images of young and SIRT6-KD two-cell embryos stained with antibodies against TRF1 (red) and γH2AX (green), and co-stained with Hoechst 33342 for chromosomes (blue). Scale bars, 25 µm. (C) Quantification of DNA damage-induced foci (TIFs) from (B). TIFs were detected by co-localization of TRF1 and γ-H2AX, and cells with at least 3 TIFs were scored (n=35 for control; n=32 for SIRT6-KD). Data represent averages of 3–10 fields. (D) TUNEL analysis of control and SIRT6-KD embryos. Embryos were labeled with Hoechst 33342 (blue) for DNA and by TUNEL for fragmented DNA (red). Arrowheads point to the apoptotic cells in blastocysts. (E) Quantification of control and SIRT6-KD blastocysts with TUNEL positive nuclei (n=82 for control; n=75 for SIRT6-KD). Statistical analyses were performed with Student’s t-test. *P<0.05, ** P<0.01, ***P<0.001.