Data supplements
Files in this Data Supplement:
- Supplemental Materials (PDF)
- Video 1
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Nonaligned bivalents do not prevent anaphase in B3 oocytes. B3 oocytes expressing H2B-mCherry (red) and CenpC-GFP (green) with time shown (h:min) relative to anaphase onset. Bar, 10 µm. Inset shows the bright-field image of the same oocyte at low magnification, with H2B-mCherry in red. - Video 2
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Nocodazole-induced nonaligned bivalents do not prevent anaphase in B3 oocytes. B3 oocytes expressing H2B-mCherry (red) and CenpC-GFP (green) with time shown (h:min) relative to anaphase onset. Bar, 10 µm. Nocodazole present throughout at 25 nM. Inset shows the bright-field image of the same oocyte at low magnification, with H2B-mCherry in red.
Article Figures & Data
Figures
- Figure 1.
Repeated oocyte bisection produces small oocytes that undergo MI. (A) Schematic of the triple bisection procedure showing the generation of B0, B1, B2, and B3 oocytes. The same colors are adopted for all figures throughout. (B) Oocyte volumes expressed in pL (left axis) or as a percentage of full size oocytes (right axis). Dashed horizontal lines show the expected sizes of half, quarter, and eighth oocytes. (C) Oocyte radii after bisection. (D) Representative images of oocytes expressing tubulin-GFP and H2B-mCherry, with cartoon depiction underneath. Bar, 50 µm. (E) Percentage of oocytes completing MI; n.s. not significant. Error bars indicate 95% confidence interval. In B, C, and E, numbers of oocytes used are indicated in parentheses. In B and C, groups without common letters indicate significant differences (analysis of variance with Tukey’s correction for multiple comparisons; P < 0.0001).
- Figure 2.
Smaller oocytes spend longer in prometaphase but then have a faster rate of securin loss. (A) Relative securin degradation traces from B3 cytoplasts lacking chromosomes after bisection. (B) Representative time lapse of B0 (top) and B3 (bottom) oocytes expressing securin-YFP and H2B-mCherry relative to time after NEB. Bar, 10 µm. (C) Timing of anaphase relative to NEB, determined from images as in B. There is no significant difference in the time of anaphase between B0 and B3 oocytes. (D) Timing of NEB (left) and anaphase (right) relative to activation of the APC. Plots for individual oocytes are displayed above, with means below. (E) Individual (pale) and mean (bold) securin traces from B0 and B3 oocytes, arranged relative to the time of APC activation. Horizontal bars show the corresponding timing of anaphase. (F) Maximal rate of securin destruction recorded from traces as in E. In C, D, and F, numbers of oocytes used are indicated in parentheses. In C, D, and F, statistical test was unpaired t test. Error bars indicate SD.
- Figure 3.
Non-bioriented bivalents do not prevent APC activation in smaller oocytes. (A) Representative time-lapse images of B0 and B3 oocytes expressing H2B-mCherry (green) and CenpC-GFP (red) with time relative to anaphase. Arrowheads indicate non-bioriented bivalents in both types of oocyte. Bar, 10 µm. (B) Cartoon depicting measurements of stretch, displacement and θ on a bivalent with sister kinetochore pairs (red). (C) Number of bivalents per oocyte classed as non-bioriented (>3 SDs from the control mean in any of the parameters of stretch, displacement, or θ) at times relative to anaphase; n.s. not significant. (D) 3D plot of bivalents in B3 oocytes 5 min before anaphase, indicating the spread of stretch, displacement, and θ. Colors indicate the number of SDs a bivalent is, according to its worst-performing measure, from mean values defined by metaphase oocytes treated without nocodazole (green <1, yellow ≤2, orange ≤3, and red >3). (E and F) Proportion of bivalents falling within the stated number of SDs at anaphase −5 min for B0 (E) and B3 (F) oocytes. Error bars indicate SD.
- Figure 4.
Several non-bioriented bivalents fail to prevent APC activation in small oocytes. (A) Percentage of B0 and B3 oocytes extruding polar bodies with or without addition of 25 nM nocodazole to the culture medium. Statistical test was Fisher’s exact. Error bars indicate 95% confidence interval. (B) Representative time-lapse images of oocytes expressing H2B-mCherry and CenpC-GFP with time relative to anaphase. Bar, 10 µm. (C) Number of bivalents per oocyte classed as non-bioriented (>3 SDs from the control mean in any of the parameters of stretch, displacement, or θ) at times relative to anaphase. Statistical test is Dunn’s multiple comparison test; n.s. not significant; *, P < 0.05; **, P < 0.01. (D) 3D scatterplot of bivalents 5 min before anaphase in B3 oocytes cultured in 25 nM nocodazole. Colors indicate the number of SDs a bivalent is, according to its worst-performing measure, from mean values defined by metaphase oocytes treated without nocodazole (green <1, yellow ≤2, orange ≤3, and red >3). (E) Proportion of bivalents for B0 oocytes falling within the stated number of SDs at anaphase −5 min.
- Figure 5.
Matched doses of nocodazole reveal differences in APC activity with reduced oocyte volume. (A) B0 oocytes were matured in the indicated concentrations of nocodazole and assessed for bivalent biorientation 6 h after NEB. Colors indicate the number of SDs a bivalent is, according to its worst-performing measure, from mean values defined by metaphase oocytes treated without nocodazole (green <1, yellow ≤2, orange ≤3, and red >3). (B and C) 3D scatterplots showing the extent of chromosome biorientation in B3 oocytes (25 nM nocodazole; B) and B0 oocytes (35 nM nocodazole; C) as in A. (D) Percentage of oocytes of the indicated size extruding polar bodies after 15-h culture in the indicated concentration of nocodazole. (E) Duration of MI, defined as the time from NEB to anaphase. (F) Individual (pale) and mean (bold) securin traces from B0 and B3 oocytes, arranged relative to the time of APC activation. Horizontal bars show the corresponding timing of anaphase. (G) Representative traces of two modes of securin degradation found in B3 oocytes matured in 25 nM nocodazole. Arrowheads indicate the timing of anaphase. Red color indicates periods of stalling. (H) Proportion of MI-arrested oocytes that exhibited stalling. (I) Proportion of oocytes that completed MI that exhibited stalling. (J) Results from experiments with and without nocodazole addition are presented together for comparison of the effects of oocyte volume reduction and addition of matched doses of nocodazole. Blue points, B3 oocytes; red, B0; round points, no stalling event was detected; square points, stalling. Back border indicates the presence of nocodazole (25 nM for B3 and 35 nM for B0). Parentheses indicate number of oocytes. Error bars are SDs. Gray dashed lines indicate points where the duration of MI (prometaphase + metaphase) is the same. Statistical comparisons between all points are indicated in Fig. S3. In A, D, E, and G–I, numbers of oocytes are indicated in parenthesis. Statistical tests used were Fisher’s exact test (D, H, and I) and unpaired t test (E).
Current Issue
December 2, 2019
Volume 218, No. 12