Figure 7.

The effect of Cdc20-4AVGR injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with the Cdc20-4AVGR mutant were treated as in Fig. 3 A. Red arrows indicate the extruded PB. The oocyte marked by the green box failed, despite several attempts, to extrude its PB. The pink arrow indicates the spot where the PB should have been extruded. The oocyte marked by the blue box, however, divided abnormally and extruded two PBs, indicated by the light blue arrow. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of uninjected, and Cdc20-4AVGR–injected oocytes. Oocytes were treated as in Fig. 4 B. (Left) The whole oocyte is shown in an overview. (Right) The area of the spindle is enlarged, and the DNA and tubulin staining are shown individually, as well as overlaid (merge). The uninjected oocyte extruded the PB, and the chromosomes are aligned on a metaphase plate with an intact spindle apparatus. Examples of the various phenotypes of Cdc20-4AVGR–injected oocytes are shown below. In b–d, the oocytes did not extrude their first PB. The abnormal spindles in c and d are marked with red arrows. In e, an oocyte with an interphase nucleus in the absence of any extruded PB is shown. In f, a typical example of an oocyte with one PB extruded is shown. In g, an oocyte is shown with two extruded PBs. Bars, 10 μm. (C) Activation of Cdc20-4AVGR–injected oocytes. The oocytes were treated as in Fig. 4 C. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. (D) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Cdc20-, Cdc20-4AV–, and Cdc20-4AVGR–injected oocytes. The autoradiograph is shown.