Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes

Abstract

Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes.

Fig 1. Subcellular localization of Axin-1 during meiotic maturation in mouse oocytes.

Oocytes at various stages were stained with an antibody against Axin-1 (red) and each was counterstained with DAPI to visualize DNA (blue). Key: GV, oocytes at germinal vesicle stage; GVBD, oocytes at germinal vesicle breakdown; Pro MI, oocytes at the first prometaphase stage; MI, oocytes at the first metaphase stage; MII, oocytes at the second metaphase stage. Oocytes in the negative control group were incubated with rabbit IgG. Scale bar = 20 μm.

Fig 2. Disruption of Axin-1 function adversely affected spindle assembly and chromosome alignment in MI oocytes.

(A) Axin1 mRNA level after siRNA microinjection. (B) Axin-1 protein expression level after siRNA microinjection. (C) Oocytes microinjected with Axin1 or control siRNAs were collected after 8.5 h of culture in fresh M2 medium. In the Axin1-specific siRNA-injected group, the oocytes exhibited various morphologically defective spindles and misaligned chromosomes. However, when supplemented with LiCl, the spindles morphology were rescued. (D) Percentages of oocytes with abnormal spindles in the Axin1 siRNA-injected group (n = 92) and control group (n = 94) and LiCl group (n = 89). Data are presented as the mean ± SEM. Different superscripts indicate significant differences (P < 0.05). (E) Percentages of oocytes with misaligned chromosomes in the Axin1 siRNA-injected group (n = 92) and control group (n = 90). Data are presented as the mean ± SEM. Superscripts indicate statistically significant differences (P < 0.05).

Fig 3. Dissociation of γ-tubulin and Nek9 from spindle poles in Axin1-knockdown oocytes at the MI stage.

(A) Oocytes microinjected with Axin1 or control siRNAs were incubated in M2 for 8.5 h, and immunostained for α-tubulin (green), and γ-tubulin (red). DNA is stained with DAPI (blue). (B) In the Axin1 siRNA or control siRNA microinjection group, oocytes were cultured for 8.5 h, and then immunostained for α-tubulin (green) and Nek9 (red). DNA is stained with DAPI (blue). Scale bar = 20 μm.

Fig 4. Axin-1 depletion decreased GVBD, PB1 extrusion and activated SAC.

(A) The rate of GVBD in different group. (B) Percentages of first polar body (PB1) extrusion in the Axin1 siRNA microinjection and control group. (C) Detection of BubR1 (red) in oocytes in the control and Axin1 siRNA groups. DNA is stained with DAPI (blue). Scale bar = 20 μm. (D) The rates of pronuclear formation are shown in different groups. Data are presented as the mean ± SEM of three independent experiments. Different superscript letters indicate significant differences at P < 0.05.