Fig. 4. Reducing tubulin tyrosination in peripheral GV oocytes rescued the abnormal K-MT attachments.

(A) Representative images of oocytes with centrally and peripherally located spindles stained for tyrosinated α-tubulin and β-tubulin. (B) Quantification of tubulin intensities within the spindle. n.s., not significant. (C) Representative images of a control oocyte and an oocyte expressing CDC42 dominant-negative mutant (CDC42T17N) stained for tyrosinated α-tubulin. (D) Quantification of tyrosinated α-tubulin levels within the spindle for the oocyte populations is illustrated in (C). (E to G) Full-grown GV oocytes were microinjected with vasohibin 2 (VASH2)/small VASH binding protein (SVBP) cRNAs and cultured overnight in the presence of milrinone to prevent meiotic resumption before allowing the oocytes to mature (in the absence of milrinone) to metaphase I stage. Representative images of oocytes expressing VASH2 and VASH2/SVBP stained for tyrosinated α-tubulin (E) and β-tubulin (F). (G) Quantification of tubulin intensities within the spindle for the oocyte populations is illustrated in (E) and (F). (H) Representative images of K-MT attachments in a central GV oocyte (as a control), a peripheral GV oocyte, and a peripheral GV oocyte expressing VASH2 and VASH2/SVBP. Full-grown GV oocytes were collected and immediately microinjected with VASH2/SVBP cRNAs during IVM (in the absence of milrinone). Metaphase I oocytes were fixed and assessed for K-MT attachments. (I) Quantification of abnormal K-MT attachments for the oocyte populations is illustrated in (H). Scale bars, 10 μm. One-way ANOVA and Tukey’s post hoc test were performed to determine statistical significance. Data are displayed as means ± SEM. Values with asterisks vary significantly, *P < 0.05; **P < 0.01; ***P < 0.001. The total number of analyzed oocytes is specified above each graph.