Figure 2.
Figure 2. Overexpression of Plk4 during oocyte growth induces aMTOC fragmentation. (A) Schematic of the two methods for achieving mCherry-Plk4 OE in mouse oocytes: by either a transgenic approach allowing Plk4 OE at the beginning of the growth phase (in blue) or mCherry-Plk4 cRNA injection allowing Plk4 OE after the growth phase (in pink). mCherry-Plk4 cRNA was injected into competent/fully grown control oocytes and expressed 3 h before live imaging. Plk4 OE oocytes were imaged directly upon isolation. Gray, chromosome; pink, kinetochores; green, MTs. MII, metaphase II. (B) Upper panels correspond to the merge of transmitted light images with the mCherry-Plk4 fluorescent image in cRNA-injected (left) versus transgenic Plk4 OE (right) oocytes observed in prophase I (mCherry-Plk4, magenta). The white dotted square highlights the nuclear area, and the mCherry-Plk4 signal from this region is displayed on the lower panels. The signal intensity lookup table is depicted on the right side. Scale bar is 10 µm (upper panels) or 2 µm (lower panels). (C) Total levels of mCherry-Plk4 expression measured as absolute signal intensity from oocytes in prophase I, either cRNA-injected (purple dots) or from Plk4 OE (blue squares) as observed in B; ****, P < 0.0001 (two tailed t test with Welch correction). (D) aMTOC-associated levels of mCherry-Plk4 overexpression measured as absolute signal intensity from oocytes in prophase I, either cRNA-injected (purple dots) or from Plk4 OE (blue squares); **, P = 0.0046. (E) 2D quantitative analysis of aMTOC number in oocytes injected with mCherry-Plk4 cRNA (purple dots) and from Plk4 OE (blue squares); P = 0.8752. (F) 2D quantitative analysis of aMTOC area in oocytes injected with mCherry-Plk4 cRNA (purple dots) and from Plk4 OE (blue squares); ****, P < 0.0001. (G) EB3-GFP (green) is presented to visualize MT organization in cRNA-injected (left) versus Plk4 OE (right) prophase I oocytes. The EB3-GFP signal (green) is merged with the mCherry-Plk4 signal (magenta). The white dotted boxes highlight the nuclear regions. Insets are higher magnifications of signals from nuclear regions. In insets, the EB3-GFP signal appears black while mCherry-Plk4 appears magenta. The black arrows point toward aMTOCs. Scale bars are 10 µm (upper panels) and 5 µm (lower insets). (H) Normalized signal intensity of the EB3-GFP signal around chromosomes in oocytes observed at NEBD from controls (black dots) or injected with mCherry-Plk4 cRNA (pink dots). P = 0.1358. In D–F and H, the statistical tests used were two-tailed Mann–Whitney. Error bars correspond to SD. In all graphs, n is the number of oocytes.

Overexpression of Plk4 during oocyte growth induces aMTOC fragmentation. (A) Schematic of the two methods for achieving mCherry-Plk4 OE in mouse oocytes: by either a transgenic approach allowing Plk4 OE at the beginning of the growth phase (in blue) or mCherry-Plk4 cRNA injection allowing Plk4 OE after the growth phase (in pink). mCherry-Plk4 cRNA was injected into competent/fully grown control oocytes and expressed 3 h before live imaging. Plk4 OE oocytes were imaged directly upon isolation. Gray, chromosome; pink, kinetochores; green, MTs. MII, metaphase II. (B) Upper panels correspond to the merge of transmitted light images with the mCherry-Plk4 fluorescent image in cRNA-injected (left) versus transgenic Plk4 OE (right) oocytes observed in prophase I (mCherry-Plk4, magenta). The white dotted square highlights the nuclear area, and the mCherry-Plk4 signal from this region is displayed on the lower panels. The signal intensity lookup table is depicted on the right side. Scale bar is 10 µm (upper panels) or 2 µm (lower panels). (C) Total levels of mCherry-Plk4 expression measured as absolute signal intensity from oocytes in prophase I, either cRNA-injected (purple dots) or from Plk4 OE (blue squares) as observed in B; ****, P < 0.0001 (two tailed t test with Welch correction). (D) aMTOC-associated levels of mCherry-Plk4 overexpression measured as absolute signal intensity from oocytes in prophase I, either cRNA-injected (purple dots) or from Plk4 OE (blue squares); **, P = 0.0046. (E) 2D quantitative analysis of aMTOC number in oocytes injected with mCherry-Plk4 cRNA (purple dots) and from Plk4 OE (blue squares); P = 0.8752. (F) 2D quantitative analysis of aMTOC area in oocytes injected with mCherry-Plk4 cRNA (purple dots) and from Plk4 OE (blue squares); ****, P < 0.0001. (G) EB3-GFP (green) is presented to visualize MT organization in cRNA-injected (left) versus Plk4 OE (right) prophase I oocytes. The EB3-GFP signal (green) is merged with the mCherry-Plk4 signal (magenta). The white dotted boxes highlight the nuclear regions. Insets are higher magnifications of signals from nuclear regions. In insets, the EB3-GFP signal appears black while mCherry-Plk4 appears magenta. The black arrows point toward aMTOCs. Scale bars are 10 µm (upper panels) and 5 µm (lower insets). (H) Normalized signal intensity of the EB3-GFP signal around chromosomes in oocytes observed at NEBD from controls (black dots) or injected with mCherry-Plk4 cRNA (pink dots). P = 0.1358. In D–F and H, the statistical tests used were two-tailed Mann–Whitney. Error bars correspond to SD. In all graphs, n is the number of oocytes.

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