![Figure 3. Cell cycle arrest in Ccnb3−/− oocytes is associated with incomplete APC/C activation. (A) Schematic of separase activity sensor. See text for details. (B) Failure to activate separase in the absence of cyclin B3. Separase activity sensor mRNA was injected into GV oocytes, which were released into meiosis I and visualized by spinning-disk confocal microscopy. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) from a representative video are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. White asterisks indicate PBs. n: number of oocytes from three independent experiments. (C) Western blot analysis of cyclin B1 and securin during oocyte maturation, at the time points, indicated as hours:minutes after GVBD. β-Actin served as the loading control. The number of oocytes used and the presence or absence of a PB are indicated. Two mice of each genotype were used per experiment. The data shown are representative of results from two independent experiments. (D) Total cyclin B–CDK1 activity during oocyte maturation, at the time points indicated as hours:minutes after GVBD. Histone H1 was used as a substrate. Five oocytes were used per kinase reaction; presence or absence of a PB is indicated. The graph shows quantification of phosphate incorporation from three independent experiments, and error bars indicate SD (means ± SD: lane 1: 100 [used for normalization]; lane 2: 31.27 ± 37.28; lane 3: 109.34 ± 17.09; lane 4: 104.05 ± 19.33; lane 5: 98.67 ± 50.56; lane 6: 65.40 ± 22.12). (E) CDK inhibition rescues meiosis I division. Oocytes were incubated with siR-DNA to visualize chromosomes. In metaphase I, 6 h 20 min after GVBD, oocytes were treated with 0.2 mM roscovitine (final concentration), where indicated, and the video was started. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) of DIC far-red channel from a representative movie of Ccnb3−/− oocytes with or without roscovitine treatment are shown. The asterisk indicates chromosome segregation in anaphase I. Time points after GVBD are indicated as hours:minutes. Scale bar: 20 µm. n: number of oocytes from three independent experiments. (F) Degradation of exogenous APC/C substrates. Securin-YFP (top) or cyclin B1–GFP (bottom) mRNA was injected into GV oocytes. Stills from representative videos are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. n: number of oocytes from two independent experiment); white asterisk indicates PB extrusion (PBE). Fluorescence intensities (mean ± SD) were quantified from the indicated number of oocytes imaged (securin-YFP: 4 Ccnb3−/−, 6 Ccnb3+/−; cyclin B1–GFP injections: 6 Ccnb3−/−, 8 Ccnb3+/−).](https://cdn.rupress.org/rup/content_public/journal/jcb/218/4/10.1083_jcb.201808091/9/jcb_201808091_fig3.jpeg?Expires=1768779905&Signature=chdAlEy9UzelDFh4~KEdXN084A0lvZzpwE9zeEdeUNcdkHVggkuAjU8yHAmUBkzH6yfwyWUAK4KgVEIEuMSNJ7tdDiHQhhdpfMufHhKrjzKWh39BWPCoQy4LVDtm0-cgUjcR4v~7o2gIEMOlEFz0sWLBv7IdnUt~JssI1EgmCqXtLHf9F68qdw4sJlc9l1fB9bFR-47oXmzK5TTi-labXdqzRl9W6Syk2yQzN2YSHWOqCZ-XyPXWnAd3bkPgb7pTkhOsqE2Z1uImRs5s0HK-KXJ4XNMb6BCl4ujnfk4OszpAoAlfMQhsipivr15eEKCEd7GDC~~Ckdnq4FwJ076x7g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Cell cycle arrest in Ccnb3−/− oocytes is associated with incomplete APC/C activation. (A) Schematic of separase activity sensor. See text for details. (B) Failure to activate separase in the absence of cyclin B3. Separase activity sensor mRNA was injected into GV oocytes, which were released into meiosis I and visualized by spinning-disk confocal microscopy. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) from a representative video are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. White asterisks indicate PBs. n: number of oocytes from three independent experiments. (C) Western blot analysis of cyclin B1 and securin during oocyte maturation, at the time points, indicated as hours:minutes after GVBD. β-Actin served as the loading control. The number of oocytes used and the presence or absence of a PB are indicated. Two mice of each genotype were used per experiment. The data shown are representative of results from two independent experiments. (D) Total cyclin B–CDK1 activity during oocyte maturation, at the time points indicated as hours:minutes after GVBD. Histone H1 was used as a substrate. Five oocytes were used per kinase reaction; presence or absence of a PB is indicated. The graph shows quantification of phosphate incorporation from three independent experiments, and error bars indicate SD (means ± SD: lane 1: 100 [used for normalization]; lane 2: 31.27 ± 37.28; lane 3: 109.34 ± 17.09; lane 4: 104.05 ± 19.33; lane 5: 98.67 ± 50.56; lane 6: 65.40 ± 22.12). (E) CDK inhibition rescues meiosis I division. Oocytes were incubated with siR-DNA to visualize chromosomes. In metaphase I, 6 h 20 min after GVBD, oocytes were treated with 0.2 mM roscovitine (final concentration), where indicated, and the video was started. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) of DIC far-red channel from a representative movie of Ccnb3−/− oocytes with or without roscovitine treatment are shown. The asterisk indicates chromosome segregation in anaphase I. Time points after GVBD are indicated as hours:minutes. Scale bar: 20 µm. n: number of oocytes from three independent experiments. (F) Degradation of exogenous APC/C substrates. Securin-YFP (top) or cyclin B1–GFP (bottom) mRNA was injected into GV oocytes. Stills from representative videos are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. n: number of oocytes from two independent experiment); white asterisk indicates PB extrusion (PBE). Fluorescence intensities (mean ± SD) were quantified from the indicated number of oocytes imaged (securin-YFP: 4 Ccnb3−/−, 6 Ccnb3+/−; cyclin B1–GFP injections: 6 Ccnb3−/−, 8 Ccnb3+/−).
