Beclin-1 knockdown shows abscission failure but not autophagy defect during oocyte meiotic maturation

Abstract

Cytokinesis is the final step in cell division that results in the separation of a parent cell into daughter cells. Unlike somatic cells that undergo symmetric division, meiotic division is highly asymmetric, allowing the preservation of maternal resources for embryo development. Beclin-1/BECN1, the mammalian homolog of yeast Atg6, is a key molecule of autophagy. As part of a class III phosphatidylinositol 3-kinase (PI3K-III) complex, BECN1 initiates autophagosome formation by coordinating membrane trafficking. However, emerging evidence suggests that BECN1 regulates chromosome segregation and cytokinesis during mitosis. Thus, we investigated the function of BECN1 during oocyte meiotic maturation. BECN1 was widely distributed during meiotic maturation forming small vesicles. Interestingly, BECN1 is also detected at the midbody ring during cytokinesis. Depletion of BECN1 impaired the cytokinetic abscission, perturbing the recruitment of ZFYVE26 at the midbody. Similar phenotypes were observed when PI3K-III activity was inhibited. However, inhibition of autophagy by depleting Atg14L did not disturb meiotic maturation. Therefore, our results not only demonstrate that BECN1 as a PI3K-III component is essential for cytokinesis, but also suggest that BECN1 is not associated with autophagy pathway in mouse oocytes.

Keywords: Beclin-1; autophagy; cytokinetic abscission; meiosis; oocyte.

Figure 1.

Expression and subcellular localization of BECN1 during meiotic maturation (A) Oocytes at GV, GVBD, MI, and MII stages were collected and subjected to immunoblot analysis with anti-BECN1 antibody. β-actin was used as a loading control. Each lane contains 50 oocytes. Normalized expression of BECN1 was quantified and expressed as the mean ± SEM from 3 independent experiments. (B) Immunostaining of BECN1 during oocyte meiotic maturation. Each stage of oocytes were fixed and stained with anti-BECN1 antibody. Oocytes at MI stage were fixed and stained with normal rabbit IgG as a negative control. DNA and spindle were stained with DAPI and anti-tubulin antibody, respectively. Representative images are shown from 3 independent experiments with at least 30 oocytes. Bar, 10 μm.

Figure 2.

Knockdown of BECN1 impairs cytokinesis (A, B) GV oocytes injected with indicated dsRNAs were cultured for 24 hours in the presence of IBMX. Knockdown of BECN1 was confirmed by either RT-PCR (A) or immunoblot analysis (B). GAPDH and β-actin were used as a loading control for RT-PCR and immunoblot, respectively. Quantification of the BECN1 level is shown above the representative images. The data are expressed as the mean ± SEM from 3 independent experiments. *p < 0.05; ***p < 0.0001. (C) BECN1 knockdown oocytes were cultured in IBMX-free medium for 13 hours and polar body extrusion (PBE) was scored to assess meiotic maturation. Representative images from at least 3 independent experiments are shown. Bar, 100 μm. The data are expressed as the mean ± SEM. The number of oocytes analyzed was shown above the bar. ns, not significant. (D) BECN1 knockdown oocytes were fixed and stained with anti-tubulin and anti-centromere antibodies (ACA) with DAPI for DNA staining. Representative images from 3 independent experiments are shown. Bar, 10 μm. Arrows indicate the misaligned and lagging chromosomes. The chromosome misalignment and cytokinesis failure were quantified and shown in right panel of images. The data are expressed as the mean ± SEM from 3 independent experiments. The number of oocytes analyzed was shown above the bar. *p < 0.05.

Figure 3.

BECN1 is not associated with autophagy during meiotic maturation (A) Oocytes injected with mRNAs encoding BECN1 tagged with V5 were co-stained with anti- MAP1LC3B/LC3 antibody. Note that BECN1 and MAP1LC3B/LC3 are not colocalized. Bar, 10 μm. (B) Oocytes at the MI stage (8 hours after IBMX release) were cultured with either 100 nM Baf A1 or 2 mM 3-MA for 6 hours and polar body extrusion (PBE) was scored. Data are the mean ± SEM from 3 independent experiments with the indicated number of oocytes. The representative images are shown in the right panel of the bar graph. Bar, 100 μm. (C) Oocytes treated with either Baf A1 or 3-MA were stained with DAPI and anti-tubulin antibody to visualize DNA and spindle, respectively. Bar, 10 μm. Quantification of cytokinesis failure is shown in the right panel of the images. Data are the mean ± SEM from 3 independent experiments with the indicated number of oocytes. *p < 0.05; ns, not significant.

Figure 4.

Cytokinesis failure at abscission is mediated by Pik3c3/Vps34, but not by Atg14L (A) GV oocytes injected with indicated dsRNAs were cultured for 24 hours in the presence of IBMX. Knockdown of Atg14L and Pik3c3/Vps34 was confirmed by RT-PCR analysis. GAPDH was used as a loading control. The normalized expression level of Atg14L and Pik3c3/Vps34 is shown in the right panel of the representative images. The data are expressed as the mean ± SEM from 2 independent experiments. (B) Oocytes depleting Atg14L or Pik3c3/Vps34 were cultured in IBMX-free medium for 13 hours and polar body extrusion (PBE) was scored. The data are expressed as the mean ± SEM from 3 independent experiments. The number of oocytes analyzed is shown above the bar. (C) Oocytes depleting Atg14L or Pik3c3/Vps34 were fixed and stained with anti-tubulin antibody and DAPI for spindle and DNA staining, respectively. Representative images from 3 independent experiments are shown. Bar, 10 μm. Cytokinesis failure was quantified and is shown in the right panel of images. Data are the mean ± SEM from 3 independent experiments with the indicated number of oocytes. **p < 0.05; ns, not significant.

Figure 5.

BECN1 is required to localize ZFYVE26 to the midbody (A) BECN1 knockdown oocytes were fixed and stained with anti-BECN1 antibody. Non-injected oocytes at cytokinesis were used as a control. DNA and spindle were stained with DAPI and anti-tubulin antibody, respectively. Bar, 10 μm. (B) BECN1 knockdown oocytes were fixed and stained with anti-ZFYVE26 antibody. DNA and spindle were stained with DAPI and anti-tubulin antibody, respectively. Bar, 10 μm. (C, D) Oocytes injected with dsRNA targeting Zfyve26 (dsZFYVE) were cultured for 24 hours in the presence of IBMX. Knockdown of ZFYVE26 was confirmed by either RT-PCR (C) or immunoblot analysis (D). GAPDH and β-actin were used as a loading control for RT-PCR and immunoblot, respectively. Normalized expression level of ZFYVE26 is shown above the representative images. The data are expressed as the mean ± SEM from 2 independent experiments. *p < 0.05; **p < 0.001. (E, F) Oocytes depleting ZFYVE26 were fixed and stained with anti-tubulin antibody with DAPI for DNA staining. The representative images from at least 3 independent experiments are shown. Bar, 10 μm. (F) Cytokinesis failure was quantified. Data are the mean ± SEM from 3 independent experiments with the indicated number of oocytes. *p < 0.05. (G) Oocytes depleting ZFYVE26 were fixed at cytokinesis and stained with anti-BECN1 antibody. DNA and spindle were stained with DAPI and anti-tubulin antibody, respectively. Bar, 10 μm.