Shao H , Li R , Ma C , Chen E , Liu XJ .

MOP89973 Canadian Institutes of Health Research

	Figure 1. Karyotyping Xenopus oocytes during meiosis. (A) Schematic illustration of karyotyping Xenopus oocytes: two mini-cells are shown, one without a polar body (left, with overhead light) and the other with a first polar body (pb) still attached (right, photographed with transmission light). (B) Representative images of oocytes at metaphase I, anaphase I, cytokinesis, and metaphase II. The graph summarizes karyotypes of 94 oocytes (>10 experiments) analyzed between 115 and 145 min after GVBD. Numbers are used to facilitate chromosome counting, not to imply chromosome identities. The inset shows two dyads. (C) A representative karyotype image of oocytes injected with xSecurin-DM mRNA at 3 h after GVBD. The inset depicts a single bivalent. (D) Metaphase I (left) and metaphase II (right) chromosome spreads double-stained with Sytox orange and anti–Aur-B. In metaphase I spread, each bivalent (inset) has two pairs of Aur-B foci representing two maternal and two paternal sister centromeres, respectively. In metaphase II spread, each dyad (inset) has two closely associated Aur-B foci representing the two sister centromeres. (E) Metaphase II oocytes before (left) and 15 min after (right) prick activation (n = 7). Note that metaphase II chromosome dyads in these oocytes have more elongated arms than those shortly after polar body emission (see B). Anaphase II spread consists of two sets of sister chromatids (arrow). Bars: (insets) 10 µm.
	Figure 2. 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.
	Figure 3. Colcemid-treated oocytes are arrested in metaphase II. (A) A representative colcemid-treated (50 µM) oocyte karyotyped 3 h after GVBD, stained with Sytox orange and anti–Aur-B (n = 9). The inset depicts a single dyad. (B) A colcemid-treated (50 µM) oocyte was imaged live, beginning at 3 h after GVBD (00:00). The oocyte was then exposed to UV excitation until the end of the imaging experiment. The oocyte was pricked immediately after the scan at 00:16. The schematic depicts position of the spindle/chromosomes in a live oocyte. Note that the egg chromosomes at 00:53 would be forming the female pronucleus and be beyond the depth of the confocal imaging system. See Video 2 for the entire series.
	Figure 4. Monopolar Aur-B122R oocytes underwent anaphase I and anaphase II. (A, top) Time series (side view) of a representative oocyte injected with wild-type Aur-B (as control), depicting metaphase I (00:04), anaphase I (00:08 and 00:12), and cytokinesis (00:14 and 00:18). See Video 3 for the entire series. The far right image (metaphase II) was from a different oocyte in the same experiment. (A, bottom) Time series (side view) of a representative Aur-B122R mRNA-injected oocyte exhibiting a monopolar spindle (00:14), monopolar anaphase (00:17 and 00:26), and metaphase II with another monopolar spindle (01:06). Cross-sectional views of selective time points are also shown to depict the transient membrane protrusion (00:26, arrows). The schematic depicts the position of the monopolar spindle/chromosomes in live oocyte. See Video 4 for the entire series (n = 9). (B, top) Time series (side view) of a control oocyte undergoing meiosis I, via imaging chromosomes (H2B) and the spindle poles (Alexa Fluor 488 anti–Aur-A). See Video 5 for the entire series (n = 9). (B, bottom) Time series (side view) of a representative monopolar Aur-B122R oocyte, depicting chromosome poleward movement (00:15 and 00:27) followed by the formation of another monopolar (meiosis II) spindle (00:48). See Video 6 for the entire series (n = 13). (C) Karyotype analyses of monopolar Aur-B122R oocytes at metaphase I (top), metaphase II (middle), and 15 min after prick activation (bottom). (D) Monopolar Aur-B122R oocytes were karyotyped between 110 and 145 min after GVBD. The graph summarizes >10 experiments (n = 72).
	Figure 5. Monopolar anaphase in STLC-treated oocytes. (A) Oocytes injected with mChe-H2B and EMTB-3GFP were incubated overnight with progesterone (control) or progesterone plus 2 µM STLC. Oocytes were imaged live. Shown are representative images (side view) of a control oocyte (left) and an STLC-treated oocyte (right). (B) Time series of an STLC (2 µM)-treated oocyte during metaphase I–to–anaphase I transition (00:00–00:17), and arrested at metaphase II (02:18; n = 5). See Video 8 for the entire series. (C) Typical karyotype image of STLC-treated oocytes 3 h after GVBD. The inset depicts a dyad.

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