Sirt6 depletion causes spindle defects and chromosome misalignment during meiosis of mouse oocyte

Abstract

Sirt6, a member of the sirtuin family of NAD-dependent protein deacetylases, has been implicated in multiple biological processes. However, the roles of Sirt6 in meiosis have not been addressed. In the present study, by employing knockdown analysis in mouse oocytes, we evaluated the effects of Sirt6 on meiotic apparatus. We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes. Consistent with this observation, incidence of aneuploidy is also markedly increased in Sirt6-depleted oocytes. Furthermore, confocal scanning showed that kinetochore-microtubule interaction, an important mechanism controlling chromosome segregation, is severely impaired in metaphase oocytes following Sirt6 knockdown. Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function. Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus.

Figure 1. Cellular localization of Sirt6 during meiosis.

Mouse oocytes at GV, pre-metaphase I, and metaphase II stages were immunolabeled with Sirt6 antibody (green) and counterstained with PI for nuclear staining (red). Arrowheads indicate Sirt6 signal. Scale bar, 25 μm.

Figure 2. Effects of Sirt6 knockdown on oocyte maturation.

Fully grown oocytes microinjected with Sirt6-MO were cultured in medium with milrinone for 20 hours to repress mRNA translation and then matured in vitro. A sham MO was injected as control. (A) The efficiency of Sirt6-MO was verified by Western blot. Band intensity was calculated using Image J software, and the ratio of Sirt6/actin expression was normalized. (B,C) Quantitative analysis of GVBD and Pb1 extrusion in control (n = 130) and Sirt6-MO (n = 125) oocytes. Bars represent means ± SD of results obtained in 3 independent experiments. *p <0 .05 vs. controls.

Figure 3. Effects of Sirt6 knockdown on spindle assembly and chromosome alignment in oocyte meiosis.

(A) Control and Sirt6-MO oocytes were stained with α-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.

Figure 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.

Figure 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.

Figure 6. 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.