Fig 2: Effects of Sirt6 knockdown on the lysine acetylation of histones in mouse oocytes.Control and Sirt6-MO oocytes at GV and metaphase II stages were immunostained with an array of antibodies specifically against different acetylated histones (green), and co-stained with PI for chromosomes (red). Representative confocal images of (A) acetylated H3K9 (H3K9ac), (C) acetylated H3K56 (H3K56ac), (E) acetylated H3K14 (H3K14ac), (G) acetylated H4K12 (H4K12ac), and (I) acetylated H4K16 (H4K16ac) in control and Sirt6-MO oocytes. (B,D,F,H,J) Quantification of the data shown in panel (A,C,E,G,I), respectively. At least 35 oocytes for each group were analyzed, and the experiments were repeated 3 times. Error bars indicate ± SD. *p < 0.05 vs. controls.

Fig 3: Effects of Sirt6 knockdown on spindle assembly and chromosome alignment in oocyte meiosis.(A) Control and Sirt6-MO oocytes were stained with a-tubulin antibody to visualize spindle (green) and counterstained with PI to visualize chromosomes (red). Control oocytes present a bipolar barrel-shaped spindle and well-aligned chromosomes on the metaphase equator (a), whereas spindle defects (arrows) and chromosomes misalignment (arrowheads) were frequently observed in Sirt6-MO oocytes (b–d). Representative confocal sections are shown. Scale bar, 25 µm. (B) Quantification of control and Sirt6-MO oocytes with abnormal spindle and chromosomes. (C) Quantitative analysis of spindle length in control and Sirt6-MO oocytes. Data are expressed as mean ± SD from 3 independent experiments in which at least 100 oocytes were analyzed. *p < 0.05 vs. controls.

Fig 4: Increased incidence of aneuploidy in oocytes depleted of Sirt6.(A) Chromosome spread of control and Sirt6-MO MII oocytes. Chromosomes were stained with Hoechst 33342 (blue) and kinetochores were labeled with CREST (purple). Representative confocal images indicate (a) control oocytes with a normal haploid complement of 20 chromosomes, (b–c) Sirt6-MO oocytes with 21 chromosomes and premature separation of sister chromatids (yellow arrowhead). (B) Histogram showing the incidence of aneuploidy in control and Sirt6-MO oocytes. 50 control oocytes and 52 Sirt6-MO oocytes were analyzed respectively. Error bars indicate ± SD. *p < 0.05 vs. controls.

Fig 5: Sirt6 knockdown disrupts kinetochore-microtubule attachments in meiotic oocytes.(A) Control and Sirt6-MO oocytes were stained with anti-Tubulin antibody for microtubules (green), CREST for kinetochores (purple), and Hoechst 33342 for chromosomes (blue). Representative images are shown. (B) Magnified views for the kinetochore-microtubule attachments in the oocytes shown in (A). Chromosome 1 and 2 represent amphitelic attachment, chromosome 3 and 4 represent merotelic attachment, chromosome 5 and 6 represent monotelic attachment, chromosome 7 and 8 represent loss attachment, and chromosome 9 represents mixed/undefined attachment. (C) Quantitative analysis of K-MT attachments in oocytes as indicated. Kinetochores in regions where fibers were not easily visualized were not included in the analysis. 12 control oocytes and 10 Sirt6-MO oocytes were analyzed respectively. Scale bars, 10 µm. *p < 0.05 vs. controls.