Fig. 3

NET1 is essential for spindle assembly, K-MT attachments, and cytoskeletal organization in mouse oocytes. (A) Control and Net1-KD oocytes were stained with α-tubulin antibody to visualize the spindle (green) and counterstained with PI to observe chromosomes (red). (a) Control oocytes show the characteristic barrel-shaped spindles (arrows) and well-aligned chromosomes. (b-d) Three examples illustrating the disorganized spindles (arrows) and misaligned chromosomes (arrowheads) that were frequently observed in Net1-KD oocytes (scale bar, 20 μm). (B). Quantification of control and Net1-KD oocytes with spindle/chromosomal defects (9.6 ± 1.39% vs. 28.9± 2.58%). (C). Control and Net1-KD metaphase oocytes were labeled with CREST antibody for kinetochores (purple), anti-tubulin antibody for microtubules (green), and Hoechst 33,342 for chromosomes (blue). Representative confocal images are shown. (D). Quantitative analysis of K-MT attachments in control and Net1-KD oocytes (20.1 ± 2.70% vs. 58.7 ± 1.99%). Kinetochores in regions where fibers were not easily visualized were not included in the analysis. (E) Metaphase I oocytes were labeled with phalloidin to visualize actin (green) and counterstained with propidium iodide for chromosomes (red). (F) Quantification of control and Net1-KD oocytes with formation of a normal actin cap (79.2 ± 2.55% vs. 38.2 ± 2.45%). The Figure depicts the mean percentage ± SD of the results obtained from three independent experiments (*significantly different at P < 0.05, **significantly different at P < 0.01)