Fig. 6. DOC1R depletion induces formation of numerous microtubule asters at spindle poles as well as in the cytoplasm of MII oocytes. Immature oocytes were microinjected with antisense RNA (asDOC1R) or double-stranded RNA (dsDOC1R or dsFrz) directed against DOC1R or Xenopus Frizzled mRNA, or they were injected with RNA encoding Xenopus DOC1R with or without dsDOC1R, further cultured and collected at different stages of meiotic maturation. Oocytes were fixed with formaldehyde and analyzed by confocal microscopy. (A) Control non-injected oocyte (NI) collected in metaphase II. (B) Control oocyte injected with dsFrz collected in metaphase II. (C) Control oocyte injected with RNA encoding Xenopus DOC1R and collected in metaphase II. (D) Oocyte injected with asDOC1R collected in metaphase II. (E) Oocyte injected with dsDOC1R collected in metaphase II. (F) Oocyte injected with dsDOC1R and with RNA encoding Xenopus DOC1R, then collected in metaphase II. (G,H) Non-injected oocyte collected in metaphase I (MI, G) or metaphase II (MII, H). (I,J) Oocyte injected with the dsDOC1R collected in metaphase I (I) or II (J). For A-F, microtubules appear in green, for G,H, the endogenous DOC1R staining appears in green. For all images, chromosomes appear in red. Scale bars: 10 µm in A-F; 6 µm in G-J. (K) Statistics of the experiment described above. Percent MII: percentage of oocytes presenting a normal bipolar metaphase II spindle. Percent phenotypes: percentage of oocytes presenting spindle defects and numerous asters in the cytoplasm. The numbers in brackets correspond to the total number of oocytes analyzed. These experiments have been repeated between three to five times.

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