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. 2023 Oct 24;9:105. doi: 10.1038/s41421-023-00599-z

Fig. 3. Ectopic expression of TUBB8 induced erroneous K–MT attachments by reducing tension of stretched bivalents in mouse oocytes.

Fig. 3

a Representative images of K–MT attachments, including amphitelic attachments, merotelic attachments, and lateral interactions in mouse oocytes expressing different human tubulin isotypes. Mouse GV oocytes were injected with vehicle, 5’FLAG-TUBB4A-cRNA (200 ng/μL), or 5’FLAG-TUBB8-cRNA (200 ng/μL), maintained for 1 h in 2.5 μM milrinone, and then washed and transferred into milrinone-free M2 medium to allow the resumption of meiosis. At 5 h after NEBD, oocytes were placed on ice for 10 min to induce depolymerization of unstable microtubules and then immediately fixed and immunostained for tubulin, ACA, and DNA (using Hoechst). Where indicated, oocytes injected with TUBB8 cRNA at 5 h after NEBD were treated either with ZM447439 (10 μM) or AZD1152 (500 nM) for 15 min or 30 min prior to the cold treatment. Scale bar, 5 μm. b Percentages of erroneous K–MT attachments in mouse oocytes expressing different human tubulin isotypes. Fisher’s exact test. ***P < 0.001, ****P < 0.0001. c Representative images show HEC1 pS55 (phosphorylation of HEC1 on Ser55) at late MI stage in mouse oocytes expressing different human tubulin isotypes. Oocytes injected with TUBB8 cRNA were treated either with ZM447439 (10 μM) or AZD1152 (500 nM) for 15 min or 30 min to inhibit AURKB/C and then fixed and immunostained for HEC1 pS55, ACA, and DNA (using Hoechst). Scale bar, 2 μm. d Relative intensities of HEC1 pS55 normalized to that of ACA. n, number of kinetochores. One-way ANOVA with multiple comparisons test. ****P < 0.0001. e Representative full overlays of bivalents and kinetochores in the same oocytes expressing different human tubulin isotypes. Magnifications of stretched chromosomes in the different groups were shown in the yellow insets. Interkinetochore distances were determined as indicated in the magnifications. Scale bar, 5 μm (top) and 2 μm (bottom). f Distribution of interkinetochore distances. n, number of bivalents. One-way ANOVA with multiple comparisons test. ****P < 0.0001. Data in b, d, and f were collected from at least three independent experiments.