Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

Abstract

KIFC3 is a member of Kinesin-14 family motor proteins, which play a variety of roles such as centrosome cohesion, cytokinesis, vesicles transportation and cell proliferation in mitosis. Here, we investigated the functional roles of KIFC3 in meiosis. Our findings demonstrated that KIFC3 exhibited expression and localization at centromeres during metaphase I, followed by translocation to the midbody at telophase I throughout mouse oocyte meiosis. Disruption of KIFC3 activity resulted in defective polar body extrusion. We observed aberrant meiotic spindles and misaligned chromosomes, accompanied by the loss of kinetochore-microtubule attachment, which might be due to the failed recruitment of BubR1/Bub3. Coimmunoprecipitation data revealed that KIFC3 plays a crucial role in maintaining the acetylated tubulin level mediated by Sirt2, thereby influencing microtubule stability. Additionally, our findings demonstrated an interaction between KIFC3 and PRC1 in regulating midbody formation during telophase I, which is involved in cytokinesis regulation. Collectively, these results underscore the essential contribution of KIFC3 to spindle assembly and cytokinesis during mouse oocyte meiosis.

Keywords: Cytokinesis; KIFC3; Meiosis; Oocyte; Spindle.

Fig. 1

KIFC3 is essential for mouse oocyte meiotic maturation (A) Oocytes were immunolabeled with anti-α-tubulin (green) and anti-KIFC3 antibody (red), and Hoechst 33,342 was used to label DNA (blue). Bar = 20 μm. (B) Co-localization of KifC3 with Crest at MI and MII stages. Oocytes cultured to 8 h (MI) and 12 h (MII) were stained for KIFC3 (green), Crest (red), and DNA (blue). Bar = 10 μm. (C) Immunoblotting assays for the expression level of KIFC3 at different stages (GV, GVBD, MI, and MII) during mouse oocyte maturation. (D) The typical picture of control oocytes and KIFC3-antibody injection oocytes. Bar = 50 μm. (E) Quantitative analysis of GVBD rate and PB1 extrusion rate in control and KIFC3-antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05

Fig. 2

KIFC3 regulates spindle formation and chromosome alignment in mouse oocyte meiosis (A) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-α-tubulin (green) and counterstained with Hoechst 33,342 to visualize the chromosomes (blue). Bar = 20 μm. (B) The rate of abnormal spindle morphology and misaligned chromosome after KIFC3-antibody injection at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05. (C) Thickness of the spindle middle plate. C indicates the maximal span of chromosomes; S indicates the maximal spindle length. Bar = 10 μm. The scattergram shows the C: S ratios for control and KIFC3-antibody injection oocytes at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. ***P < 0.001. (D) Immunofluorescence analysis of γ-tubulin (red) and α-tubulin (green) in control and KIFC3-antibody injection oocytes. Bar = 10 μm. The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal γ-tubulin. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05. (E) Immunofluorescence analysis of p-Aurka (red) and α-tubulin (green) in control and KIFC3-antibody injection oocytes. Bar = 10 μm. The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal p-Aurka. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05

Fig. 3

KIFC3 regulates kinetochore-microtubule attachment in mouse oocytes (A) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-α-tubulin (green), anti-CREST (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 10 μm. (B) The rate of abnormal kinetochore-microtubule attachment after KIFC3-antibody injection at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05. (C) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-H3S10ph (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 5 μm. (D) The scattergram shows the relative fluorescence intensity of H3S10ph signals in control and KIFC3-antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. ***P < 0.001. (E) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-BubR1 (green) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 5 μm. (F) The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal BubR1. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05. (G) Co-IP was performed with an anti-KIFC3 antibody. The immunoblots of protein precipitates were probed with an anti-Bub3 antibody. (H) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-Bub3 (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 5 μm. (I) The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal Bub3. Graph shows means ± sem of results obtained in 3 independent experiments. **P < 0.01

Fig. 4

KIFC3 regulates stability of spindle microtubules (A) Control and KIFC3-antibody injection oocytes after cold treatment at the MI stage were stained with anti-α-tubulin (green) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 20 μm. (B) The rate of abnormal spindle morphology after cold treatment of KIFC3 antibody injection and control oocytes at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05. (C) Control and KIFC3-antibody injection oocytes after Nocodazole treatment at the MI stage were stained with anti-α-tubulin (green) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 20 μm. (D) The relative area of spindle after nocodazole treatment of KIFC3 antibody injection and control oocytes at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05. (E) Co-IP was performed with an anti-KIFC3 antibody. The immunoblots of protein precipitates were probed with anti-Sitr2 and anti- acetylated tubulin antibody. (F) Immunofluorescence analysis of acetylated tubulin (red) and α-tubulin (green) in control and KIFC3 antibody injection oocytes. Bar = 10 μm. (G) The histogram shows the relative fluorescence intensity of acetylated tubulin signals in control and KIFC3 antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. **P < 0.01. (H) Immunofluorescence analysis of Sirt2 (red) and α-tubulin (green) in control and KIFC3 antibody injection oocytes. Bar = 20 μm. (I) The histogram shows the relative fluorescence intensity of Sirt2 signals in control and KIFC3 antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. *P < 0.05

Fig. 5

KIFC3 affects midbody formation and cytokinesis in mouse oocytes (A) Spindle morphology at the ATI stage and MII stage oocytes. Green, α-tubulin; blue, DNA. Repeat the experiment 4 times. Bar = 20 μm. (B) Co-IP was performed with an anti-KIFC3 antibody. The immunoblots of protein precipitates were probed with anti-PRC1 antibody. (C) Control and KIFC3-antibody injection oocytes at the ATI stage were stained with anti-α-tubulin (green), anti-PRC1 (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Repeat the experiment 3 times. Bar = 20 μm. (D) Diagram of the roles of KIFC3 during mouse oocyte meiotic maturation. KIFC3 regulated tubulin acetylation to stabilize the microtubules for spindle assembly and kinetochore-microtubules attachment, and KIFC3 also interacted with PRC1 for cytokinesis during mouse oocyte meiosis