Bivalent-to-dyad transition in the absence of spindle microtubules. (A) An oocyte injected with mChe-H2B and EMTB-3GFP was imaged 1 h after GVBD (left). Nocodazole was added to the imaging well (3.3 µM in OR2), followed by time-lapse imaging for >2 h. Shown on the right are images 4 min after the addition of nocodazole. The schematic depicts the position of spindle/chromosomes in live oocyte before (left) and after (right) nocodazole treatment. See Video 1 for the entire series. The images are representative of three movies. (B) 1 h after GVBD, the oocytes were placed in OR2 plus 3.3 µM nocodazole. Oocytes were karyotyped in the presence of 3.3 µM nocodazole such that the mini-cells burst onto glass slides either <100 min after GVBD (metaphase I) or 3 h after GVBD (metaphase II, inset showing one dyad). The slides were stained with Sytox orange and anti–Aur-B. (C) Oocytes were treated as described in B, except that all oocytes were karyotyped between 110 and 130 min after GVBD. The graph summarizes three experiments (n = 25). (D) Control oocytes, or oocytes treated with nocodazole, were lysed individually at germinal vesicles (no progesterone) 2 h after the addition of progesterone but before GVBD (GV′), at GVBD (BD), or at the indicated time after GVBD. Extracts were immunoblotted with anti–cyclin B2. Each lane represents half the extract from one oocyte. The red box indicates time of metaphase I–to–anaphase I transition in frog oocytes. Data are representative of three experiments. (E) Control oocytes, nocodazole-treated oocytes (from GVDB, 1 h), and oocytes injected with xSecurin-DM mRNA were selected individually at 3 h after GVBD. The oocytes were either lysed immediately (metaphase II) or 10 min after being pricked. The resulting extracts were subjected to immunoblotting with anti–cyclin B2, followed by anti–α-tubulin of the same membrane. Note the residual cyclin B2 signal on the α-tubulin blot. Data shown are representative of five experiments.