Fig. 3. Inhibition of Src-related kinases with SKI606 induced abnormal spindles and misalignment of chromosomes during meiosis.

Denuded oocytes (eggs) and intact COC matured 17h in 0, 1, 5 or 10 μM SKI606 (fig. 2) were fixed and co-labeled with monoclonal mouse anti-αβ tubulin (microtubules; green) and goat anti-mouse Alexa-488, phalloidin-Alexa-568 (f-actin; red) and Hoechst 33258 (DNA; white) and oocytes were confocal imaged and examined as described previously. Three-dimensional reconstructions were used to examine oocyte meiotic status and the associations of tubulin, actin and DNA. The majority of control oocytes (A = 0 μM SKI606) matured and arrested at metaphase of meiosis II with normal spindle morphology, chromatin aligned on the metaphase plate and an extruded polar body. Oocytes exposed to SKI606 arrested at various stages of meiosis, many with displaced chromosomes and spindle microtubules that branch abnormally in seeming attempts to enclose these aberrant chromosomes (B–H). Fewer abnormal oocytes occurred in the lower doses of SKI606 as compared to the higher doses, however the general types of abnormalities were the same across all concentrations of the inhibitor (malformed spindles and wayward chromosomes: B, C, G = 10 μM; D, E, F = 5 μM; H = 1 μM SKI606, respectively). Panels E (tubulin label turned-off) and F (with tubulin (green)) are the same oocyte. Both images are shown to demonstrate the stretched chromosomes that failed to separate (E) even though the spindle microtubules have attempted to elongate and undergo anaphase (F). Panel H demonstrates two oocytes cultured in low (1 μM) SKI606 which extruded polar bodies but have displaced chromosomes. One oocyte has a chromosome that failed to segregate and was trapped in the constriction site between the polar body and the oocyte (*) while the second oocyte has multiple chromosomes displaced along the spindle (**). [scale bar = 10 μm]