![Figure 6. Only cyclin B3 that can support in vitro kinase activity can rescue Ccnb3−/− oocytes. (A) Affinity purification of cyclin B3–CDK1 complexes. MBPHiscyclin B3 or MBPHiscyclin B3 MRL mutant were expressed in insect cells alone or coexpressed with either untagged or HA-tagged CDK1. Left: The eluates from purification on amylose resin were separated on SDS-PAGE and stained with Coomassie. Right: Representative autoradiograph (top) and quantification (bottom) from histone H1 kinase assays. In the graph, values in each experiment (n = 3) were normalized to the signal from the MBPHiscyclin B3 MRL sample (lane 4 in the autoradiograph); lines indicate means (lane 1: 1.733; lane 2: 4.351; lane 3: 3.878; lane 4: 0 [used for normalization]; lane 5: 0.121; lane 6: −0.202). (B) Rescue of Ccnb3−/− oocytes by expression of exogenous cyclin B3. Ccnb3−/− oocytes were sham injected or injected with the indicated cyclin mRNA, then released into meiosis I. Frames of representative videos are shown. Times after GVBD are indicated as hours:minutes, and percentages of oocytes of the shown phenotypes are indicated. Scale bar, 20 µm. White asterisks: PBs. n: number of oocytes from three independent experiments. (C) Total cyclin B–CDK1 activity during oocyte maturation, in control (lane 1–3), and Ccnb3−/− (lanes 4–9, labeled in red) oocytes expressing wild-type cyclin B3 (lanes 6 and 7) or ΔDbox cyclin B3 (lanes 8 and 9), at the time points indicated as hours:minutes after GVBD. Oocytes extruding PBs are indicated. Histone H1 was used as a substrate. A representative example from two independent experiments is shown above; quantification of both experiments is shown below (32P-H1 signal normalized to the signal in lane 2; points are values from each experiment; lines indicate means: lane 1: 6.61; lane 2: 100 [used for normalization]; lane 3: 14.38; lane 4: 191.58; lane 5: 138.53; lane 6: 109.88; lane 7: 9.59; lane 8: 37.36; lane 9: 1.5). (D) Representative chromosome spreads 16 h after GVBD. Chromosomes were stained with Hoechst (blue) and kinetochores with CREST (green). Insets show typical chromosome figures observed (chromosomes are shown in grayscale). Scale bar, 5 µm. n: number of oocytes from three independent experiments. Schematics of metaphase I bivalents or metaphase II univalent chromosomes are shown to aid interpretation.](https://cdn.rupress.org/rup/content_public/journal/jcb/218/4/10.1083_jcb.201808091/10/jcb_201808091_fig6.jpeg?Expires=1773569663&Signature=z6XI5JKpFHefWTnZepWsQIgsofGglXJupSRNezM4BN7c7JIr6EXYTnxyuLya9~adxvSu7uF9OU42VVJiuDipGATzxKT51vsa6GdY5SVMyO0OL9q3KHJQbwnpafUV1OaFibMi4g6sfSt3fTgpwkVxHppt0qnLIbjmclBcUhKLdHrfmch1A2cuonFxfulb09hmXw1kGmdp89pIO7PfnSER369zGxQHkQbjoQ~A3fTftR562Cp20oKniUFEllR~u4EtsBKuouAVIIT9WKo0Ppdum5djFBxW3kDUBGRPjpuzYmImvd2Tj0jQTLGuIimCp9ohSrtGwMdtT6hicUBN20ZFvA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Only cyclin B3 that can support in vitro kinase activity can rescue Ccnb3−/− oocytes. (A) Affinity purification of cyclin B3–CDK1 complexes. MBPHiscyclin B3 or MBPHiscyclin B3 MRL mutant were expressed in insect cells alone or coexpressed with either untagged or HA-tagged CDK1. Left: The eluates from purification on amylose resin were separated on SDS-PAGE and stained with Coomassie. Right: Representative autoradiograph (top) and quantification (bottom) from histone H1 kinase assays. In the graph, values in each experiment (n = 3) were normalized to the signal from the MBPHiscyclin B3 MRL sample (lane 4 in the autoradiograph); lines indicate means (lane 1: 1.733; lane 2: 4.351; lane 3: 3.878; lane 4: 0 [used for normalization]; lane 5: 0.121; lane 6: −0.202). (B) Rescue of Ccnb3−/− oocytes by expression of exogenous cyclin B3. Ccnb3−/− oocytes were sham injected or injected with the indicated cyclin mRNA, then released into meiosis I. Frames of representative videos are shown. Times after GVBD are indicated as hours:minutes, and percentages of oocytes of the shown phenotypes are indicated. Scale bar, 20 µm. White asterisks: PBs. n: number of oocytes from three independent experiments. (C) Total cyclin B–CDK1 activity during oocyte maturation, in control (lane 1–3), and Ccnb3−/− (lanes 4–9, labeled in red) oocytes expressing wild-type cyclin B3 (lanes 6 and 7) or ΔDbox cyclin B3 (lanes 8 and 9), at the time points indicated as hours:minutes after GVBD. Oocytes extruding PBs are indicated. Histone H1 was used as a substrate. A representative example from two independent experiments is shown above; quantification of both experiments is shown below (32P-H1 signal normalized to the signal in lane 2; points are values from each experiment; lines indicate means: lane 1: 6.61; lane 2: 100 [used for normalization]; lane 3: 14.38; lane 4: 191.58; lane 5: 138.53; lane 6: 109.88; lane 7: 9.59; lane 8: 37.36; lane 9: 1.5). (D) Representative chromosome spreads 16 h after GVBD. Chromosomes were stained with Hoechst (blue) and kinetochores with CREST (green). Insets show typical chromosome figures observed (chromosomes are shown in grayscale). Scale bar, 5 µm. n: number of oocytes from three independent experiments. Schematics of metaphase I bivalents or metaphase II univalent chromosomes are shown to aid interpretation.
