Aurora A activity is required for the correct size and number of MTOCs in oocytes. (A) Localization of Plk4 and Aurora A phosphorylated at threonine residue 288 (AurApT288) revealed by immunostaining of oocytes with anti–Plk4 (red), anti–α-tubulin (green), and anti–AurApT288 (red) antibodies. Separated channels in gray are also shown as indicated; DNA is shown in blue. Both kinases colocalize at MTOCs. (B) Localization of PlK1 at MTOCs revealed by immunostaining of meiosis I oocytes with anti-Plk1 (red) and anti–α-tubulin (green). Plk1 and Plk4 channels are also shown in monochrome and DNA in blue. (C) Meiosis I oocytes stained to visualize DNA (blue), γ-tubulin (red), and phospho-TACC3 (pTACC3, green; top) or α-tubulin (red, middle) and pTACC3 (monochrome, bottom). Depicted are controls compared with conditions in which Aurora A and Plk4 were inhibited by treatment with Aurora A inhibitor 1 or centrinone, respectively. Aurora A inhibition causes a significant reduction of pTACC3 at MTOCs (see bottom panel, pTACC3 in monochrome). (D) Oocytes stained with antibodies against α−tubulin (gray, bottom), DNA (blue, top), γ-tubulin (red or monochrome), and CEP192 (green or monochrome, bottom; A) or stained to visualize α−tubulin (gray, top), DNA (blue, top), γ-tubulin (red or monochrome), and NEDD1 (green or monochrome, bottom; E). Shown are representative images of controls and of oocytes treated with Aurora A inhibitor 1 and centrinone. Bars, 10 µm. (F) Measurement of pTACC3 pixel intensity at the MTOCs relative to the background (*, P < 0.05). Error bars represent SD of the mean. (G and H) Quantifications of the size (G) and number (H) of γ-tubulin–positive MTOCs. Aurora A inhibition, but not treatment of oocytes with the Plk4 inhibitor centrinone, causes a significant reduction in size and number of MTOCs (**, P < 0.01).