Research Paper Volume 11, Issue 7 pp 1965—1976

SIRT6 participates in the quality control of aged oocytes via modulating telomere function

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.