XB-ART-56958
Sci Rep
2020 May 04;101:7455. doi: 10.1038/s41598-020-64418-1.
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Cyclin A1 in Oocytes Prevents Chromosome Segregation And Anaphase Entry.
Radonova L
,
Pauerova T
,
Jansova D
,
Danadova J
,
Skultety M
,
Kubelka M
,
Anger M
.
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In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.
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Figure 1. Expression of cyclin A1 in mouse GV oocytes blocks polar body extrusion and conversion of bivalents into univalent. (A) Frames from live cell imaging experiment of oocytes microinjected with cRNAs encoding tubulin (green) and histone (red) fused to fluorescent proteins, oocyte in lower panel was also co-injected with cRNA encoding cyclin A1. Scale bar represents 10 μm. (B) Scoring of maturation in control – uninjected oocytes (n = 84), oocytes injected with cyclin A1 (n = 44) or cyclin A2 (n = 52) cRNAs. The chart shows percentage of oocytes in meiosis I and meiosis II (control 92% MII; cyclin A1 0% MII; cyclin A2 85% MII) after overnight maturation in each category. The data were obtained in three independent experiments. The difference between control oocytes and oocytes injected with cyclin A1 cRNA was statistically significant (α < 0.05; ***P < 0.0001). The difference between control oocytes and oocytes injected with cyclin A2 cRNA was not statistically significant (α < 0.05; P = 0.2021). (C) Confocal microscopy images showing DNA stained by DAPI (red) and kinetochores stained by CREST antibody (green) in control – uninjected oocytes (left panel), oocytes injected with cyclin A1 cRNA (two central panels) and oocytes injected with cyclin A2. Scale bar represents 2 μm. (D) Scoring of chromosomes status after overnight maturation of control – uninjected oocytes (n = 36), oocytes injected with cyclin A1 cRNA (n = 37) and oocytes injected with cyclin A2 cRNA (n = 43). The percentage of oocytes carrying univalents (control 100%, cyclin A1 0%, cyclin A2 100%), bivalents (control 0%, cyclin A1 76%, cyclin A2 0%) or both (control 0%, cyclin A1 24%, cyclin A2 0%) after overnight meiotic maturation is indicated. The data were obtained in three independent experiments. The difference between control oocytes and oocytes injected with cyclin A1 cRNA was statistically significant (α < 0.05; ***P < 0.0001). The difference between control oocytes and oocytes injected with cyclin A2 cRNA was not statistically significant (α < 0.05; P > 0.9999). | |
Figure 2. Oocytes are unable to efficiently target cyclin A1 for degradation. (A) Frames from live cell imaging experiment of oocytes co-injected with cRNAs encoding histone (red) and either cyclin A2 or cyclin A1 (green) fused to fluorescent proteins. Scale bar represents 10 μm. (B) Profiles of fluorescent signal of cyclin A1 (orange, n = 12) and cyclin A2 (blue, n = 7) during meiotic maturation. The curves represent average curve for each construct and the standard deviation error bars are shown. The signal in each cell was normalized to the frame closest to the disassembly of the nuclear membrane (GVBD). The data were obtained from two independent experiments. (C) Profiles of fluorescent signal of securin fused to fluorescent protein in oocytes injected by cyclin A1 (orange, n = 14) or cyclin A2 (blue, n = 10) cRNAs during meiotic maturation. The curves represent average expression curve for all cells in the group and the standard deviation error bars are shown. The signal in each cell was normalized to the frame closest to GVBD. The data were obtained from two independent experiments. | |
Figure 3. Fragment of cyclin A1 which contains D-box is not efficiently targeted for degradation and alters dynamic of degradation of other APC substrates. (A) Schematic comparison of cyclin A1 and cyclin A2 and the relative positions of sequence motives (D-box, KEN box, ABBA motive) related to the ubiquitination and degradation of the molecule on proteasome. (B) Scoring of PBE in control oocytes (n = 8) and oocytes injected with either 113cyclin A1 (n = 12) or D113cyclin A1 (n = 12) cRNAs. Grey bars represent oocytes with underwent PBE (control 100%, 113cyclin A1 100% and D113cyclin A1 0%), black bars represent oocytes arrested in meiosis I. The data were obtained from two independent experiments. The difference between control oocytes and oocytes injected with D113cyclin A1 cRNA was statistically significant (α < 0.05; ***P < 0.0001). The difference between control oocytes and oocytes injected with 113cyclin A1 cRNA was not statistically significant (α < 0.05; P > 0.9999). (C) Relative expression curves of 113cyclin A1 (blue, n = 12) and D113cyclin A1 (orange, n = 12) during oocyte meiosis. GV oocytes were injected with histone and cyclin A1 cRNAs fragments fused to fluorescent proteins. The fluorescence was measured overnight using confocal time lapse microscopy. Signal in each cell was normalized to the level at GVBD and the average curves are shown together with standard deviation values at each time point. The data were obtained from two independent experiments. (D) Relative expression curves of securin in control cells (blue, n = 11), of securin in cells co-injected with cRNA encoding 113cyclin A1 (orange, n = 17) and relative expression curve of 113cyclin A1 itself (green, n = 17). The fluorescence was measured overnight using confocal time lapse microscopy, the signal in each cell was normalized to the level at GVBD. The average curves are shown for each time point. The data were obtained from two independent experiments. (E) Relative expression curves of 90cyclin B1 in control cells (blue, n = 16), and in cells co-injected with cRNAs encoding 90cyclin B1 and 113cyclin A1 (orange, n = 14) and relative expression curve of 113cyclin A1 itself (green, n = 14). The fluorescence was measured overnight using confocal time lapse microscopy, the signal in each cell was normalized to the level at GVBD. The average curves are shown for each time point. The data were obtained from two independent experiments. (F) Relative expression curves of securin in control cells (blue, n = 14) and in cells co-injected with cRNAs encoding securin and D113cyclin A1 (orange, n = 25). The fluorescence was measured overnight using confocal time lapse microscopy, the signal in each cell was normalized to the level at GVBD. The average curves, together with standard deviations are shown for each time point. The data were obtained in three independent experiments. (G) Scoring of spindle division phenotypes in oocytes injected with histone and tubulin cRNAs fused to fluorescent proteins. The frequency of each phenotype is indicated for control cells (n = 18; PBE 78%, no division 22%) and for cells injected with D113cyclin A1 cRNA (n = 25; two spindles inside 32%; internal spindle and partial chr. division 36%; no division 32%). The data were obtained in three independent experiments. Scale bar represents 10 μm. (H) Frames from time lapse movie of oocyte co-injected with cRNAs encoding histone, tubulin and D113cyclin A1. Scale bar represents 10 μm. (I) Chart demonstrating a dependency of expression levels of D113cyclin A1 on chromosomes and spindle division. GV oocytes were injected with cRNAs encoding histone and tubulin fused to fluorescent proteins and with various amount of D113cyclin A1 cRNA diluted in water containing fluorescently labelled dextran, allowing to quantify the amount of D113cyclin A1 in each individual cell. Presented are data from one experiment. | |
Figure 4. Cyclin A1 directly inhibits activity of separase. (A) Frames from confocal live cell imaging experiment of CD1 strain oocyte co-injected with cRNAs encoding histone (red) and tubulin (green) and cyclin A1 (without florescent tag). Oocyte showed on the right was co-injected also with separase mutated to alanine on position 1121. Scale bar represents 10 μm. (B) CD1 oocytes were injected with histone and tubulin fused to fluorescent proteins and also with cyclin A1 (n = 16) or cyclin A1 together with separase1121 (n = 16). Polar body extrusion (PBE) and chromosome segregation were then assessed by time lapse confocal microscopy. The left chart shows frequency of PBE in each group (both groups 0% PBE). The right chart shows the frequency of chromosomes segregation in each group (cyclin A1 0%, cyclin A1 + Sep1121 50%). The data were obtained from two independent experiments. The difference between oocytes microinjected with cyclin A1 and oocytes co-injected also with separase1121 in frequency of PBE was not statistically significant (α < 0.05; P > 0.9999). The difference between oocytes microinjected with cyclin A1 and oocytes co-injected also with separase1121 in frequency of chromosome division was statistically significant (α < 0.05; **P = 0.0024). (C) Separase Δ oocytes were injected with histone fused to fluorescent protein and with separase 1121 (n = 23) or separase 1121 together with cyclin A1 (n = 30). PBE and chromosome segregation were assessed by time lapse confocal microscopy. The left chart shows frequency of PBE in each group (separase 1121 70% PBE, separase1121 + cyclin A1 0% PBE). The right chart shows the frequency of chromosomes segregation in each group (separase 1121 96%, separase 1121 + cyclin A1 60% of chromosomes segregated). The data were obtained from three independent experiments. The difference between separase Δ oocytes microinjected with separase 1121 and separase Δ oocytes co-injected also with cyclin A1 in frequency of PBE was statistically significant (α < 0.05; ***P < 0.0001). The difference between separase Δ oocytes microinjected with separase1121 and separase Δ oocytes co-injected also with cyclin A1 in frequency of chromosome division was statistically significant (α < 0.05; **P = 0.0033). (D) Separase Δ oocytes were injected with histone fused to fluorescent protein and with wild type separase (separase WT, n = 13) or wild type separase together with cyclin A1 (n = 14). The left chart shows frequency of PBE in each group (separase WT 92% PBE, Separase WT + cyclin A1 0% PBE) and the right chart the frequency of chromosome segregation in each group (separase WT 92%, separase WT + cyclin A1 7%). The data were obtained from two independent experiments. The difference between separase Δ oocytes microinjected with separase WT and separase Δ oocytes co-injected also with cyclin A1 in frequency of PBE was statistically significant (α < 0.05; ***P < 0.0001). The difference between separase Δ oocytes microinjected with separase WT and separase Δ oocytes co-injected also with cyclin A1 in frequency of chromosome division was statistically significant (α < 0.05; ***P < 0.0001). | |
Figure 5. Expression of cyclin A1 does not prevent anaphase in zygotes and 2-cell embryos. (A) Frames from the time lapse experiment showing comparison between oocyte (upper panel) and 2-cell embryo (lower panel). Both oocytes and embryos were microinjected with cRNAs encoding histone and cyclin A1 fused to fluorescent proteins. Cell division and chromosome segregation were then assessed by time lapse confocal microscopy. Scale bar represents 10 μm. (B) Scoring of cell division of oocytes (n = 22) and 2-cell blastomeres (n = 42) described in panel (A). Cells which divided (oocytes 9%, embryos 95%) are indicated by grey bars and cell with no division (oocytes 91%, embryos 5%) observed during the duration of experiment are as black bars. The data were obtained from three independent experiments. The difference between oocytes and 2-cell blastomeres was statistically significant (α < 0.05; ***P < 0.0001). (C) Relative fluorescence signal of cyclin A1 in indicated cells and time intervals. GV oocytes – the relative fluorescence signal was measured in the first frame after GVBD (Oo GVBD, 43.24 AU) and in the frame of maximum fluorescence (Oo MAX, 102.6). 2-cell blastomeres – the relative fluorescence signal was measured in the first frame after NEBD (BL NEBD, 63.97 AU) and in the first frame of anaphase (BL ANA, 44.76). The difference between Oo GVBD and Oo MAX was statistically significant (α < 0.05; ***P < 0.0001), the difference between Oo GVBD and BL NEBD was statistically significant (α < 0.05; ***P < 0.0007) and the difference between BL NEBD and BL ANA was statistically significant (α < 0.05; **P < 0.0011). (D) Zygotes were microinjected with cRNAs encoding histone and either cyclin A1 (n = 13) or cyclin A2 (n = 12) fused to fluorescent proteins. Cell division was then assessed by time lapse confocal microscopy. Cells which divided (cyclin A1 100%, cyclin A2 75%) are indicated by grey bars and cell with no division (cyclin A1 0%, cyclin A2 25%) observed during the duration of experiment are as black bars. The data were obtained from two independent experiments. The difference in division between zygotes was not statistically significant (α < 0.05; P = 0.0957). (E) The length of the interval between disassembly of the nuclear membrane (NEBD) and anaphase was measured in cells described in panel (C). Average time interval between NEBD and anaphase was 1.79 h for cells injected with cyclin A1 cRNA and 1.83 h for cells injected with cyclin A2 cRNA. The data were obtained from two independent experiments. The difference between the length of mitosis in cells injected with cyclin A1 and cyclin A2 were not significant (α < 0.05; P = 0.7254). (F) The levels of fluorescence signal of cyclin A1 (red, n = 16) and cyclin A2 (green, n = 16) during mitotic division of zygotes. The signal in each cell from NEBD to anaphase is shown as individual curve. The signal was normalized to the maximum level. The data were obtained in two independent experiments. (G) The levels of fluorescence signal of D113cyclin A1 (red, n = 16) and cyclin A1 (green, n = 14) during mitotic division of zygotes. Individual curves for each analyzed cell. The signal in each cell from NEBD to anaphase is shown as individual curve. The signal was normalized to the maximum level. The data were obtained from two independent experiments. |
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