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. 2018;17(21-22):2436-2446.
doi: 10.1080/15384101.2018.1542894. Epub 2018 Nov 14.

Survivin regulates chromosome segregation by modulating the phosphorylation of Aurora B during porcine oocyte meiosis

Li Chen  1   2 , Tailang Yin  3 , Zheng-Wen Nie  1   2 , Tao Wang  1   2 , Ying-Ying Gao  1   2 , Shu-Yuan Yin  1   2 , Li-Jun Huo  2 , Xia Zhang  1   4 , Jing Yang  3 , Yi-Liang Miao  1   2   4
Affiliations

Survivin regulates chromosome segregation by modulating the phosphorylation of Aurora B during porcine oocyte meiosis

Li Chen et al. Cell Cycle. 2018.

Abstract

SURVIVIN is an essential chromosomal passenger complex (CPC) subunit and participates in cell division. In this study, we used porcine oocyte as a model to investigate the roles of Survivin during porcine oocyte maturation. Survivin was highly expressed in germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages oocytes, mainly localized in the GV at GV stage and on the chromosomes after GVBD. We have used RNA interference to specifically deplete Survivin in oocytes during in vitro maturation (IVM). Immunofluorescence assay showed that Survivin-depleted oocytes failed to produce polar body in meiosisⅠ (failed to complete cytokinesis), and they were arrested in metaphaseⅠwith misaligned chromosomes. The homologous chromosomes in Survivin-depleted oocytes could not be separated normally. Moreover, both the phosphorylation levels of Aurora B and the mRNA level of Mad2L1 related to spindle assembly checkpoint (SAC) was decreased in Survivin-depleted oocytes, which thus inhibited the degradation of Cyclin B1 (CCNB1) to complete meiosis. Taken together, we conclude that Survivin is an important mediator of centromere and midbody docking of Aurora-B as well as its activity and regulates SAC and MPF activity during meiosis in porcine oocytes.

Keywords: Survivin; chromosomes; metaphase I; oocyte; pig; spindle assembly checkpoint.

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Figures

Figure 1.
Figure 1.
Expression and subcellular localization of Survivin during porcine oocytes meiotic maturation. (a) Relative expression of Survivin mRNA in GV, GVBD, MI and MII stages; (b) Subcellular localization of Survivin during porcine oocytes maturation. N-Ctr: negative control; Bar = 25 µm. (c) The protein level of Survivin in in GV, GVBD, MI and MII stages. Proteins derived from a total of 300 oocytes were loaded for each sample.
Figure 2.
Figure 2.
Survivin depletion led to oocytes arrested at the MI stage and impaired chromosome alignment. (a) Representative DIC images of oocytes and oocyte maturation percentage in the control and Survivin knockdown groups. Bar = 25 μm. ** P < 0.01 (b) Relative expression of Survivin mRNA in the control and Survivin knockdown groups after 24 h and 68 h siRNA injection. * P < 0.05, ** P < 0.01 (c) Expression of SURVIVIN protein in the siRNA-injected oocytes and relative levels of SURVIVIN protein between control and Survivin knockdown groups. Porcine COCs were injected with siRNA and incubated with dbcAMP for 20 h, followed by Western blotting. (d) Images of SURVIVIN (red) and chromosome (blue) in control and Survivin knockdown groups. Oocytes were stained with Survivin antibody to visualize the protein and co-stained with DAPI for chromosomes. Bar = 25 μm.
Figure 3.
Figure 3.
Survivin depletion led to chromosome misaligned and decrease of phosphorylated Auroa B. (a) Images of Spindle morphologies (green) and chromosome (blue) in control and Survivin knockdown groups. Oocytes were stained with α-tubulin-FITC antibody to visualize the spindles and co-stained with DAPI for chromosomes. Bar = 10 μm. (b) The percentage of spindle/chromosome defects in oocytes from the control and Survivin knockdown groups. (c) The protein level of Survivin, Aurora B, phosphorylated Aurora B and Mad2 examined by western blot in the control and Survivin knockdown groups in MI stage. And the relative level of each protein analysis was showed in (d). * P < 0.05, ** P < 0.01. (e) The protein level of Survivin, Aurora B, and phosphorylated Aurora B examined by western blot in the control and Survivin overexpression groups in MI stage. And the relative level of each protein analysis was showed in (f). * P < 0.05, ** P < 0.01.
Figure 4.
Figure 4.
Survivin depletion inhibited Cyclin B1 degradation in oocytes at MI-AI. (a) Images of cyclin B1-GFP (green) in the control and Survivin knockdown groups. Cyclin B1 was immunolabled with GFP and examined by confocal microscopy. Bar = 25 μm (b) The average fluorescence intensity of Cyclin B1-GFP in the control and Survivin knockdown groups. (c) Relative expression of SAC or APC/C associated genes which were Cdc25b, Cdc20, Cdk1, Mad2L1 and Mad2L2. * P < 0.05. (d) Schematic illustrating function of Survivin during porcine oocytes maturation.
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Grants and funding

This research was supported by the Natural Science Foundation of Hubei Province (Grant# 2018CFA015); National Key Research and Development Program of China; Stem Cell and Translational Research (Grant# 2016YFA0100203); National Project for Breeding of Transgenic Pig (2016ZX08006-002).

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