Treatment with SMIFH2 decreases the actin level in oocytes and impairs spindle formation.

<p><b>A</b>. Treatment with SMIFH2 decreases the cortical actin level in maturing mouse oocytes. Control (left) and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Chromatin was stained with Hoechst 33342 (blue). Bar = 10 μm. <b>B</b>. Quantification of the cortical actin levels in SMIFH2-treated (300 and 500 μM) and control oocytes. Actin in the cortex regions excluding the cortical actin cap and polar body regions was quantified using ImageJ[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123438#pone.0123438.ref042" target="_blank">42</a>]. The box represents the interquartile range; the whiskers show 1.5x the interquartile range; line inside the box represents the median (control: n = 14; 300 μM: n = 12; 500 μM: n = 12). N.S.: not significant (p > 0.05); ***: significantly different from control oocytes (p < 0.001). <b>C</b>. Phalloidin-stained cytoplasmic actin mesh in oocytes. Untreated (control) oocytes and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Bar = 3 μm. <b>D.</b> Quantification of the cytoplasmic actin stained with phalloidin in control (n = 27) and SMIFH2-treated (300 μM: n = 21; 500 μM: n = 7) cells. <b>E.</b> Abnormal spindle formation induced by SMIFH2 treatment. Spindles of control or SMIFH2-treated (300 and 500 μM) oocytes were visualized by immunostaining with anti-α-tubulin (green), and chromatin was stained with Hoechst 33342 (blue). No spindle structures are detected in oocytes treated with 500 μM of SMIFH2. Bar: 10 μm. <b>F.</b> Effect of various SMIFH2 concentrations on spindle formation and morphology. Oocytes were classified based on the spindle morphology. Normal: oocytes with normal spindle shape with proper chromatin alignment, similar to the spindle shape of the control cells in panel E; Abnormal: oocytes containing a spindle with improper chromatin alignment or irregular shape, similar to that of cells treated with 300 μM SMIFH2 in panel E; No spindle: oocytes with no detectable spindle structure, similar to that of cells treated with 500 μM SMIFH2. The ratio of each spindle type of oocytes to total oocytes was plotted. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter(a,b and c).