1. Large Cytoplasm Is Linked to the Error-Prone Nature of Oocytes
Hirohisa Kyogoku, Tomoya S. Kitajima 
Developmental Cell 
Volume 41, Issue 3, Pages e4 (May 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 41, 287-298. e4DOI: (10. 1016/j. devcel. 2017
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3. Figure 1 Cytoplasmic Size Controls Spindle Size and Anaphase Timing
(A) Experimental scheme. Image shows the generated oocytes. Scale bar, 100 μm.
(B) Live imaging of meiosis I. Maximum z-projection images of EGFP-MAP4 (microtubules, green) and H2B-mCherry (chromosomes, red). Time after NEBD (hr:min). Scale bar, 5 μm.
(C) Spindle scaling. Spindle volume was determined from 3D-reconstructed images (n = 16, 15, 14, and 16 oocytes). Error bars denote SD. Scale bar, 7 μm.
(D) Cytoplasmic size controls anaphase timing. Line plot shows the cumulative percentage of oocytes that entered anaphase (n = 4 experiments). Error bars denote SD. Box plot shows the time of anaphase onset (n = 53, 35, 42, and 35 oocytes). Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t test was performed. ∗∗∗p < 0.001.
See also Figures S1 and S2; and Movies S1, S2, and S3.
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4. Figure 2 The Amount of Cytoplasmic Factors Controls the Spindle Size
(A) Experimental scheme. The oocytes were halved before or after NEBD. Spindle formation was synchronized through nocodazole washout. The concentration of nucleus-derived factors is doubled in oocytes halved before NEBD, whereas it is intact in oocytes halved after NEBD.
(B) The spindle is half-sized in oocytes halved after NEBD. Spindle volume was determined at late metaphase (n = 17, 12, and 16 oocytes).
(C) Experimental scheme. Oocytes were fused and then meiotic resumption was induced in the presence of 200 nM jasplakinolide to inhibit cell rounding (dumbbell-shaped doubled).
(D) The spindle is double-sized in the dumbbell-shaped doubled oocytes. Spindle volume was determined at late metaphase (n = 44, 23, and 22 oocytes). Control and round-shaped doubled oocytes were treated with 200 nM jasplakinolide at meiotic resumption as was performed in the dumbbell-shaped doubled oocytes.
Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t tests were performed. N.S., not significant. ∗∗∗p < Scale bars, 5 μm.
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5. Figure 3 Cytoplasmic Size-Dependent Deformation of Spindle Poles
(A) MTOC distribution in 3D. Images of mNeonGreen-Cep192 (MTOCs, green) and H2B-mCherry (chromosomes, red) were reconstructed in 3D and surface rendered. In each panel, a side view of the spindle (left) and its pole (right top) and a top view of the pole (right bottom) at late prometaphase (3 hr after NEBD) or late metaphase (6 hr after NEBD) are shown.
(B) Progressive anisotropic deformation of polar MTOCs. Top-view images of polar MTOCs were aligned, averaged, and color-coded (n = 23, 21, and 20 oocytes).
(C) Quantification of MTOC deformation. The width and aspect ratio of the MTOC distribution in top-view images are measured as shown in the diagram in (B) (n = 23, 20, 21, and 20 oocytes). Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t tests were performed. N.S., not significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
Scale bars, 5 μm. See also Figure S2 and Movie S4.
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6. Figure 4
Cytoplasmic Size Affects the Efficiency of Chromosome Alignment
(A) Images of mNeonGreen-Cep192 (MTOCs, green) and H2B-mCherry (chromosomes, red) were reconstructed in 3D and surface rendered. Views from the side of the spindle are shown. Scale bar, 5 μm.
(B) Cytoplasmic size affects chromosome alignment. Aligned chromosomes were defined as chromosomes with chromosome-equator distances shorter than 12.5% of the metaphase spindle length (Figure S2; halved 21.6 μm, control 26.8 μm, doubled 33.8 μm) in 3D. Error bars denote SD. Box plot shows the time at which 80% of the chromosomes were aligned (n = 22, 27, 21, and 34 oocytes). Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t test was performed. ∗∗p < 0.01, ∗∗∗p <
See also Figures S3 and S4.
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7. Figure 5
Cytoplasmic Size Affects the Efficiency of Chromosome Biorientation
(A) 3D kinetochore tracking. Kinetochore and chromosome positions were determined from images of oocytes expressing 2mNeonGreen-CENP-C and H2B-mCherry. Kinetochore positions are shown in the 3D plot as green spheres. Red bars connect the homologous kinetochores. The view along the spindle equator is shown. Black arrowheads indicate the non-bioriented chromosomes. The unit of the grid is 5 μm.
(B) A fraction of the chromosomes remain near the spindle poles. Dynamics of individual chromosomes along the spindle axis are shown (n = 100, 160, and 80 chromosomes). The positions of the poles of the metaphase spindle are shown as dashed red lines. Pink boxes indicate the positions within 4 μm of the spindle poles. Chromosome tracks that remained within 4 μm of the spindle poles for more than 30 min are indicated by red lines.
(C) Prolonged stay near the spindle poles. Chromosomes that remained within 4 μm of the spindle poles for more than 30 min (indicated by red lines in B) were counted (n = 100, 160, and 80 chromosomes). A chi-square test was performed. ∗p < 0.05, ∗∗p < 0.01.
(D) Intra-polar chromosome stretching. Maximum z-projection images of doubled oocytes expressing 2mNeonGreen-CENP-C (kinetochores, green) and H2B-mCherry (chromosomes, red). Kinetochore signals are peak enhanced and background subtracted. Chromosomes marked with squares are magnified at the bottom. Note that the chromosomes exhibited a transient stretching perpendicular to the spindle axis as shown in the diagram. Time after NEBD (hr:min). Scale bar, 5 μm.
See also Movies S5 and S6.
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8. Figure 6
A Large Cytoplasmic Volume Limits the Stringency of the Spindle Checkpoint
(A) Cytoplasmic size-dependent anaphase delay depends on Mps1. Meiotic resumption was induced in oocytes expressing H2B-mCherry in the presence of 1 μM reversine, and cells were monitored for anaphase onset. The cumulative percentage of oocytes that entered anaphase is plotted (n = 4 experiments). Box graph shows the time of anaphase onset (n = 37, 35, 37, and 35 oocytes). Error bars denote SD. Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t tests were performed. N.S., not significant. ∗∗∗p <
(B) Experimental scheme. H2B-mCherry-expressing oocytes were halved before or after NEBD and monitored for anaphase onset. Note that oocytes halved after NEBD carry a double concentration of kinetochores and an intact concentration of nucleus-derived factors.
(C) The nucleocytoplasmic ratio controls the timing of anaphase. The cumulative percentage of oocytes that entered anaphase is plotted (n = 4 experiments). Error bars denote SD. Box graph shows the time of anaphase onset (n = 22, 39, and 32 oocytes). Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t tests were performed. N.S., not significant. ∗p < 0.05, ∗∗∗p <
(D) Immunostaining of C-Mad2 before NEBD. Oocytes were fixed at the GV stage for the staining of C-Mad2 (green), nuclear membrane (Lamin A/C, red), and DNA (Hoechst 33342, blue). Total signal intensity of C-Mad2 at the nuclear rim was measured in 3D. Normalized values are shown (n = 29, 26, 27, and 32 oocytes). Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t test was performed. N.S., not significant. Scale bars, 5 μm.
(E) Immunostaining of kinetochore C-Mad2. Oocytes were fixed 2.5 hr after NEBD for the staining of C-Mad2 (green), kinetochores (ACA, red), and DNA (Hoechst 33342, blue). Image contrast is adjusted based on cytoplasmic signals. Normalized relative signal intensities on kinetochores (n = 28, 36, 30, and 32 oocytes) are shown. Boxes show the 25th–75th percentiles and whiskers the 10th–90th percentiles. Two-tailed, unpaired Student's t test was performed. ∗∗∗p < Scale bars, 5 μm.
(F) Schematic representation of the construct used in (G) and (H).
(G) Checkpoint response to on-kinetochore activation. Oocytes expressing Mad1-mNeonGreen-CENP-C and H2B-mCherry were monitored. Bar graph shows the percentages of metaphase-arrested oocytes 11 hr after NEBD (n = 64, 37 oocytes). Chi-square tests were performed. ∗∗∗p <
(H) Oocytes mildly expressing Mad1-mNeonGreen-CENP-C were monitored. Bar graph shows the percentages of metaphase-arrested oocytes 11 hr after NEBD (n = 42, 38, and 34 oocytes). Chi-square tests were performed. N.S., not significant. ∗p < 0.05, ∗∗∗p <
See also Figures S5 and S6.
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9. Figure 7
Cytoplasmic Size Affects the Number of Misaligned Chromosomes Necessary for Anaphase Block
(A) Sycp3 knockout (KO) oocytes with misaligned univalents. Maximum z-projection images of Sycp3 knockout oocytes expressing 2mNeonGreen-CENP-C (kinetochores, green) and H2B-mCherry (chromosomes, red). Kinetochore signals are peak enhanced and background subtracted. Arrowheads indicate misaligned univalents. Time after NEBD (hr:min). Scale bar, 5 μm.
(B) Efficient anaphase block with a few misaligned univalents in halved oocytes. The number of misaligned chromosomes was determined by tracking individual kinetochores in 3D throughout metaphase. The percentage of metaphase-arrested oocytes 14 hr after NEBD was determined (n = 26, 39, 18, 19, 17, and 18 oocytes). Chi-square tests were performed. ∗p < 0.05, ∗∗p < 0.01.
See also Movie S7.
Developmental Cell  , e4DOI: ( /j.devcel )
Copyright © 2017 Elsevier Inc. Terms and Conditions