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Aging and disease    2018, Vol. 9 Issue (4) : 623-633     DOI: 10.14336/AD.2017.0901
Orginal Article |
Involvement of Kif4a in Spindle Formation and Chromosome Segregation in Mouse Oocytes
Tang Feng, Pan Meng-Hao, Lu Yujie, Wan Xiang, Zhang Yu, Sun Shao-Chen*
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
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Abstract  

Kif4a, a member of the kinesin superfamily, has been reported to participate in a series of cellular processes such as chromosome condensation and cytokinesis during mitosis. However, the roles of KIF4a in meiosis are still unknown. In present study we found that the Kif4a protein expression decreased in maternal aged mouse oocytes. We then explored the roles of Kif4a in mouse oocyte meiosis by knockdown analysis. Kif4a was enriched at the spindle during mouse oocyte maturation. By specific knock down of the Kif4a using morpholino microinjection, we found that the disruption of Kif4a caused the failure of polar body extrusion. Further analysis indicated that Kif4a might affect the spindle morphology and chromosome alignment in the mouse oocytes, and this might be due to the regulation of tubulin acetylation. Moreover, our results showed that an increased proportion of aneuploidy in the Kif4a knock down oocytes, and this might be due to the loss of kinetochore-microtubule attachment. Taken together, these results suggested that Kif4a possibly regulated mouse oocyte meiosis through its effects on the spindle organization and accurate chromosome segregation, and the loss of Kif4a might be related with aneuploidy of aging oocytes.

Keywords Kif4a      oocyte      meiosis      aneuploidy      aging     
Corresponding Authors: Sun Shao-Chen   
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These authors contributed equally to this work.

Issue Date: 01 August 2018
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Tang Feng
Pan Meng-Hao
Lu Yujie
Wan Xiang
Zhang Yu
Sun Shao-Chen
Cite this article:   
Tang Feng,Pan Meng-Hao,Lu Yujie, et al. Involvement of Kif4a in Spindle Formation and Chromosome Segregation in Mouse Oocytes[J]. Aging and disease, 2018, 9(4): 623-633.
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http://www.aginganddisease.org/EN/10.14336/AD.2017.0901     OR     http://www.aginganddisease.org/EN/Y2018/V9/I4/623
Figure 1.  Kif4a expression decreases in mouse oocytes with maternal aging

(A) Kif4a expression examined by western blotting. Three independent experiments were performed. (B) Relative intensities of the Kif4a protein bands in young and aged oocytes. Kif4a expression was significantly decreased in aged oocytes (Kif4a:tubulin, 1.0 vs. 0.223 ± 0.053; *p < 0.05). (C) Representative images of young and aged oocytes stained with an anti-Kif4a antibody. Green, Kif4a; blue, DNA. Scale bars, 20 μm. (D) Percentages of young and aged oocytes with abnormal Kif4a signals. Data are presented as mean percentage (± SEM) of at least three independent experiments. The asterisk denotes p < 0.05.

Figure 2.  Localization of Kif4a in mouse oocytes

(A) Oocytes at different stages were immunolabeled with an anti-Kif4a antibody (red) and counterstained with DAPI to visualize DNA (blue). Kif4a was primarily accumulated around chromosomes after GVBD. Kif4a was accumulated at the meiotic spindle region at both the MI and MII stages. Kif4a localized at the midbody at the ATI stage. (B) Double staining of MI oocytes with an anti-Kif4a antibody (red) and an anti-α-tubulin antibody (green). Oocytes were counterstained with DAPI to visualize DNA (blue). Kif4a mainly localized on the meiotic spindle. (C) Subcellular localization of Kif4a after taxol treatment during mouse oocyte meiotic maturation. Green, α-tubulin; red, Kif4a. (D) Subcellular localization of Kif4a after nocodazole treatment during mouse oocyte meiotic maturation. Green, α-tubulin; red, Kif4a. Scale bars, 20 μm.

Figure 3.  Effects of Kif4a KD on oocyte maturation

(A) KD of endogenous Kif4a protein expression after Kif4a morpholino injection was verified by western blot analysis. Three independent experiments were performed. (B) Phase-contrast images of control morpholino-injected and Kif4a-KD oocytes after culture for 12 h. Black arrowheads indicate oocytes that failed to extrude a polar body. Red arrowheads indicate oocytes with an apparently large polar body. Scale bar, 100 μm. (C) The rate of pbI in control and Kif4a-KD oocytes. (D) The rate of pbI in control and antibody-injected oocytes. Data are presented as mean percentage (±SEM) of at least three independent experiments. The asterisk denotes p < 0.05.

Figure 4.  Kif4a KD causes spindle defects and chromosome misalignment during oocyte meiosis

(A) Control morpholino-injected and Kif4a-KD oocytes were stained with an anti-α-tubulin antibody to visualize the spindle (green) and counterstained with DAPI to visualize chromosomes (blue). Control MI oocytes had a typical barrel-shape spindle and well-aligned chromosomes at the metaphase plate. Meanwhile, Kif4a-KD oocytes had several spindle defects, such as malformed spindles, elongated spindles, and inflated spindles. Scale bars, 20 μm. Three independent experiments were performed. (B) Percentages of control and Kif4a-KD oocytes with aberrant spindles. (C) Percentages of control and Kif4a-KD oocytes with misaligned chromosomes. Data are presented as mean percentage (±SEM) of at least three independent experiments. The asterisk denotes p < 0.05. (D) Endogenous ac-tubulin protein expression after Kif4a morpholino injection was verified by western blot analysis. Three independent experiments were performed. (E) Control and Kif4a-KD oocytes were stained with an anti-ac-α-tubulin antibody (red) to visualize ac-microtubules and with DAPI to visualize chromosomes. The ac-α-tubulin signal was significantly decreased in Kif4a-KD oocytes. Three independent experiments were performed. (F) Quantification of the data showed that the ac-tubulin fluorescence signal intensity was decreased in Kif4a-KD oocytes. At least 30 oocytes were analyzed for each group. The asterisk denotes p < 0.05.

Figure 5.  Kif4a KD induces aneuploidy and affects kinetochore-microtubule attachment in mouse oocytes

(A) Chromosome spreads of control and Kif4a-KD MII oocytes. Representative images of euploid control oocytes and aneuploid Kif4a-KD oocytes are shown. (B) Percentages of aneuploid control and Kif4a-KD oocytes. Scale bars, 20 μm. p < 0.05. Data are presented as mean percentage (±SEM) of at least three independent experiments. (C) MI oocytes were subjected to cold treatment to depolymerize unstable microtubules that were not attached to kinetochores. Control and Kif4a-KD oocytes were stained with an anti-α-tubulin antibody to visualize the spindle (green) and with Crest to visualize kinetochores. Oocytes were counterstained with DAPI to visualize chromosomes (blue). Yellow arrowheads indicate the absence of kinetochores or the unattached kinetochore. (D) Percentages of control and Kif4a-KD oocytes with kinetochore-microtubule attachment defects. At least 30 oocytes were analyzed for each group. Data are presented as mean percentage (±SEM) of at least three independent experiments. The asterisk denotes p < 0.05.

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