Mos–MAPK reduces aster growth to minimize polar body size. (A) Selected frames from a time-lapse recording of the microtubule spindle in an oocyte undergoing MI on a spinning disk confocal microscope. Chromosomes are labeled with H1-Alexa568 (red) and microtubule plus tips by EB3-mClover3 protein (cyan). Before inducing maturation, the oocyte was treated with either 20 µM U0126 or an equal amount of DMSO. (Left) overlay of the two fluorescent channels; (middle) the EB3-mClover3 channel is shown with adjusted gamma; (right) 3D rendering of the centrosome (cyan) and chromosomes (red). Maximum intensity projections are shown; scale bars are 20 µm. (B and C) 3D distances of the spindle poles and the maximal length of astral microtubules at different stages of MI are quantified on recordings similar to A. n refers to the number of individual oocytes analyzed. (D) Boxplot showing quantification of polar body sizes on transmitted light microscopy images. n refers to the number of individual oocytes analyzed. ***P < 0.0001, statistical comparison was done using a two-tailed t test. (E) Bar chart showing the success rate of polar body formation. Polar bodies were counted by live imaging in H1-Alexa568–injected oocytes after completion of meiosis. n refers to the number of individual oocytes analyzed. (F) Immunofluorescence images of oocytes stained for microtubules (anti-tubulin, cyan) and chromatin (Hoechst 33342, red). Oocytes were treated with 20 µM U0126 or an equal amount of DMSO and then fixed at the stages indicated. Imaging was done using spinning disk confocal microscopy. Maximum intensity projections are shown; scale bar is 20 µm. Lower panels show the tubulin channel separately and with adjusted gamma (0.5). (G) Quantification of the 3D distance between spindle poles on data similar to that shown in F. **P < 0.05, ***P < 0.0001 in two-tailed t tests. (H) Single optical sections crossing the centrosomes selected from the projected images shown in F. Images are shown after gamma adjustment (0.5); scale bar is 10 µm.
Figure 4.

Mos–MAPK reduces aster growth to minimize polar body size. (A) Selected frames from a time-lapse recording of the microtubule spindle in an oocyte undergoing MI on a spinning disk confocal microscope. Chromosomes are labeled with H1-Alexa568 (red) and microtubule plus tips by EB3-mClover3 protein (cyan). Before inducing maturation, the oocyte was treated with either 20 µM U0126 or an equal amount of DMSO. (Left) overlay of the two fluorescent channels; (middle) the EB3-mClover3 channel is shown with adjusted gamma; (right) 3D rendering of the centrosome (cyan) and chromosomes (red). Maximum intensity projections are shown; scale bars are 20 µm. (B and C) 3D distances of the spindle poles and the maximal length of astral microtubules at different stages of MI are quantified on recordings similar to A. n refers to the number of individual oocytes analyzed. (D) Boxplot showing quantification of polar body sizes on transmitted light microscopy images. n refers to the number of individual oocytes analyzed. ***P < 0.0001, statistical comparison was done using a two-tailed t test. (E) Bar chart showing the success rate of polar body formation. Polar bodies were counted by live imaging in H1-Alexa568–injected oocytes after completion of meiosis. n refers to the number of individual oocytes analyzed. (F) Immunofluorescence images of oocytes stained for microtubules (anti-tubulin, cyan) and chromatin (Hoechst 33342, red). Oocytes were treated with 20 µM U0126 or an equal amount of DMSO and then fixed at the stages indicated. Imaging was done using spinning disk confocal microscopy. Maximum intensity projections are shown; scale bar is 20 µm. Lower panels show the tubulin channel separately and with adjusted gamma (0.5). (G) Quantification of the 3D distance between spindle poles on data similar to that shown in F. **P < 0.05, ***P < 0.0001 in two-tailed t tests. (H) Single optical sections crossing the centrosomes selected from the projected images shown in F. Images are shown after gamma adjustment (0.5); scale bar is 10 µm.

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