Aurora kinase B modulates chromosome alignment in mouse oocytes

Abstract

The elevated incidence of aneuploidy in human oocytes warrants study of the molecular mechanisms regulating proper chromosome segregation. The Aurora kinases are a well-conserved family of serine/threonine kinases that are involved in proper chromosome segregation during mitosis and meiosis. Here we report the expression and localization of all three Aurora kinase homologs, AURKA, AURKB, and AURKC, during meiotic maturation of mouse oocytes. AURKA, the most abundantly expressed homolog, localizes to the spindle poles during meiosis I (MI) and meiosis II (MII), whereas AURKB is concentrated at kinetochores, specifically at metaphase of MI (Met I). The germ cell-specific homolog, AURKC, is found along the entire length of chromosomes during both meiotic divisions. Maturing oocytes in the presence of the small molecule pan-Aurora kinase inhibitor, ZM447439 results in defects in meiotic progression and chromosome alignment at both Met I and Met II. Over-expression of AURKB, but not AURKA or AURKC, rescues the chromosome alignment defect suggesting that AURKB is the primary Aurora kinase responsible for regulating chromosome dynamics during meiosis in mouse oocytes.

Figure 1

Relative mRNA levels of Aurka, Aurkb, and Aurkc in GV-intact oocytes and MII-arrested eggs. RNA was isolated from GV-intact oocytes and MII-arrested eggs from sexually mature mice. Following reverse transcription, mRNA levels of Aurka, Aurkb, and Aurkc were determined using quantitative RT-PCR and were normalized against Prkaca. Data are shown as mean ± SEM from three independent experiments.

Figure 2

Immunocytochemical detection of AURKA during meiotic maturation. A: GV-intact oocytes were collected from sexually mature mice and matured in vitro for 0 hr (GV), 3 hr (GVBD), 7 hr (Met I), 10 hr (Telo I), or 16 hr (Met II) prior to fixation in cold methanol and staining with anti-AURKA and anti-γ-tubulin (TUBG1) antibodies. DNA was visualized with DAPI. Merged images show AURKA in red, γ-tubulin in green, and DNA in blue. The arrows point to spots where AURKA and γ-tubulin co-localized. The asterisk indicates the midbody. B: GV-intact oocytes were microinjected with Aurka-eGfp mRNA, held for 1 hr in maturation medium containing milrinone, and matured in vitro for 7 hr (Met I) prior to fixation in 3.7% paraformaldehyde. Experiments were repeated at least three times with approximately 20 oocytes per stage.

Figure 3

AURKB-eGFP expression during meiotic maturation. A: GV-intact oocytes were microinjected with Aurkb-eGfp mRNA, held for 1 hr in maturation medium containing milrinone, and matured in vitro for 0 hr (GV), 3 hr (GVBD), 7 hr (Met I), 9 hr (Ana I), 10 hr (Telo I), or 16 hr (Met II) prior to fixation in cold methanol. DNA was visualized with DAPI. Merged images show AURKB-eGFP in green and DNA in blue. In the GVBD panel, a region containing one chromosome was zoomed in to highlight the association of AURKB and DNA. B,C: GV-intact oocytes were microinjected with Aurkb-eGfp mRNA, held for 1 hr in maturation medium containing milrinone, and matured in vitro for 7 hr (MI) prior to fixation in 3.7% paraformaldehyde. C: Merged image shows AURKB-eGFP in green and CREST in red. The experiments were repeated at least three times with approximately 20 oocytes analyzed at each stage.

Figure 4

Immunocytochemical detection of AURKC during meiotic maturation. A: GV-intact oocytes were collected from sexually mature mice and matured in vitro for 7 hr (Met I), or 16 hr (Met II) prior to fixation in cold methanol and staining with anti-AURKC and anti-β-tubulin antibodies. DNA was visualized with DAPI. Merged images show AURKC in red, β-tubulin in green, and DNA in blue. B: Staining with anti-AURKC antibody and CREST serum to mark centromeres. DNA was visualized with DAPI. Merged images show AURKC in red, CREST in green, and DNA in blue. C: GV-intact oocytes were microinjected with Aurkc-eGfp mRNA, held for 1 hr in maturation medium containing milrinone, and matured in vitro for 7 hr (Met I) prior to fixation in 3.7% paraformaldehyde. Experiments were repeated at least three times with approximately 20 oocytes per stage.

Figure 5

Effect of ZM447439 on meiotic progression and chromosome alignment. A: GV-intact oocytes were treated with concentrations of ZM447439 ranging from 0 to 10 μM for 1 hr in maturation medium containing milrinone, and matured in vitro in the same concentration of ZM447439 for 16 hr prior to fixation in cold methanol. The spindle was detected with an anti-β-tubulin antibody and DNA was visualized with DAPI. Confocal microscopy was used to determine the stage of meiosis. Data are shown as mean ± SEM from three independent experiments and were analyzed using two-way ANOVA. ***P < 0.001. B: As in (A) but confocal microscopy was used to determine chromosome alignment. Data are shown as mean ± SEM from three independent experiments and were analyzed using two-way ANOVA. **P < 0.01, ***P < 0.001. C: Representative images for scoring chromosome misalignment and spindle abnormalities. Merged images show β-tubulin in green and DNA in blue. D: GV-intact oocytes were treated with concentrations of ZM447439 ranging from 2 to 10 μM for 1 hr in maturation medium containing milrinone, and matured in vitro in the same concentration of ZM447439 for 8 hr. ZM447439 was washed out of the media (W) in some groups of oocytes and all groups were allowed to continue maturation in vitro for 10 hr prior to fixation in cold methanol. The spindle was detected with an anti-β-tubulin antibody and DNA was visualized with DAPI. Confocal microscopy was used to determine chromosome misalignment. Data are shown as mean ± SEM from three independent experiments and were analyzed using two-way ANOVA. **P < 0.01, ***P < 0.001. E: As in (D) but confocal microscopy was used to determine the stage of meiosis. Data are shown as mean ± SEM from three independent experiments and were analyzed using two-way ANOVA. *P < 0.05.

Figure 6

Effect of ZM447439 on chromosome alignment at Met I and Met II. A: GV-intact oocytes were treated with concentrations of ZM447439 ranging from 0 to 10 μM for 1 hr in maturation medium containing milrinone, and matured in vitro in the same concentration of ZM447439 for 8 hr prior to fixation in cold methanol. The spindle was detected with an anti-β-tubulin antibody and DNA was visualized with DAPI. Confocal microscopy was used to determine chromosome alignment at Met I. Data are shown as mean ± SEM from three independent experiments and were analyzed using two-way ANOVA. *P < 0.05. B: GV-intact oocytes were held for 1 hr in maturation medium containing milrinone and matured in vitro for 10 hr, a time at which most oocytes have passed Met I. Concentrations of ZM447439 ranging from 0 to 10 μM were added to the media and oocytes were allowed to continue maturation in vitro for 8 hr prior to fixation in cold methanol. The spindle was detected with an anti-β-tubulin antibody and DNA was visualized with DAPI. Confocal microscopy was used to determine chromosome alignment at Met II. Data are shown as mean ± SEM from three independent experiments and were analyzed using two-way ANOVA. ***P < 0.001.

Figure 7

AURKB rescue of chromosome alignment defect caused by ZM447439. GV-intact oocytes were microinjected with Gfp, Aurka-eGfp, Aurkb-eGfp, or Aurkc-eGfp mRNA and held for 14 hr in medium containing milrinone. These oocytes were treated with 1.5 μM ZM447439 for 1 hr in maturation medium containing milrinone and matured in vitro in the same concentration of ZM447439 for 8 hr prior to fixation in 3.7% paraformaldehyde. DNA was visualized with propidium iodide. Confocal microscopy was used to determine chromosome alignment. Data are shown as mean ± SEM from three independent experiments and were analyzed using Student’s t-test. *P < 0.05.