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First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02586

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An increase in [Ca²⁺]_i is sufficient but not necessary for driving mitosis in early mouse embryos

Greg FitzHarris^*, Mark Larman, Chris Richards and John Carroll

Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (29K): [in a new window]   Fig. 1. Repetitive global mitotic Ca2+ oscillations are sperm specific. [Ca2+]i was monitored during the first mitotic division using Fura-2/dextran and epifluorescence microscopy. (A) Ca2+ oscillations were detected at NEBD (open arrow) in all fertilized embryos and further mitotic transients were detected in eight of nine embryos (mean number of transients per embryo was 3.9±0.54). No transients were detected following cytokinesis (closed arrow). (B) A typical mitotic Ca2+ transient is presented as a series of pseudocoloured images, warmer colours indicating an increase in [Ca2+]i. Note that the increase in Ca2+ occurs throughout the embryo. (C) No Ca2+ transients are detected at NEBD or during mitosis in parthenogenetically activated embryos (n=6). A large increase in Ca2+ was subsequently detected when mitotic parthenotes were challenged with ionomycin.

View larger version (20K): [in a new window]   Fig. 2. Photorelease of cIns(1,4,5)P3 triggers precocious mitosis entry. Fertilized one-cell embryos were microinjected with cIns(1,4,5)P3 or water (vehicle) 32-34 hours after hCG, at which time approximately half of the embryos had undergone NEBD. Remaining interphase embryos were placed side-by-side on the microscope stage (cohorts of five to seven of each group) and exposed to UV light for 1 second. (A) An example of one such experiment in which six cIns(1,4,5)P3-injected and six control (vehicle) embryos were simultaneously exposed to UV light. Notice the occurrence of Ca2+ transients in all cIns(1,4,5)P3-injected embryos but not in controls. (B) Embryos were subsequently examined at 15 minute intervals to determine the timing of NEBD. The rate of mitosis entry was accelerated in cIns(1,4,5)P3-injected embryos compared with controls (n=24 for both groups). Inset shows the 15 minute and 30 minute time points as bar charts. C, control; I, cIns(1,4,5)P3. Data shown are from one of two similar replicates.

View larger version (21K): [in a new window]   Fig. 3. Increased cytoplasmic Ca2+-buffering does not prevent the first mitotic division. (A) Fertilized and parthenogenetic embryos were microinjected with BAPTA, Br2BAPTA, EGTA (final concentrations 10 mM) or injection buffer, or were loaded with BAPTA-AM (10 µM) 1-2 hours before the predicted time of NEBD. Data are expressed as the percentage of embryos that had undergone NEBD within 20 hours of fertilization or parthenogenetic activation. Only BAPTA-AM treatment caused a significant inhibition of NEBD compared with controls (P<0.01, 2 test). A minimum of two replicates were performed for each treatment. (B) One-cell embryos were microinjected with cIns(1,4,5)P3 and BAPTA (final concentration 10 mM; n=18) or cIns(1,4,5)P3 only (n=13). Injected embryos were loaded with Fura-red and [Ca2+]i was monitored during 10 millisecond, 100 millisecond, 1000 millisecond and 3000 millisecond exposures of UV light at 2 minute intervals. The peak change in Fura-red emission ratio was significantly reduced by BAPTA in response to 100 millisecond (#, P<0.05), 1000 millisecond and 3000 millisecond exposures (*, P<0.01). (C) BAPTA-injected embryos fail to exhibit repetitive Ca2+ transients in mitosis (n=19). Notice, however, that small fluctuations in Fura-2 baseline were detected at NEBD (ii, inset) (open arrow). Control embryos imaged at the same time exhibited characteristic Ca2+ oscillations (6.3±1.1 transients per embryo, n=14). No transients were detected following cytokinesis (closed arrow).

View larger version (29K): [in a new window]   Fig. 4. Adenophostin-A treatment prevents mitotic Ca2+ oscillations in fertilized embryos. (A) cIns(1,4,5)P3 was used to test the responsiveness of Ca2+ release in one-cell embryos 4 hours after Ad-A injection (2.5 µM). Peak Fura-red ratio change was significantly reduced in Ad-A injected embryos (n=16) compared with controls (uninjected, n=15; vehicle, n=6) in response to 300 millisecond, 1200 millisecond and 3000 millisecond exposures of UV light (*, P<0.01), indicating that Ad-A treatment inhibits Ins(1,4,5)P3-mediated Ca2+ release. (B) [Ca2+]i was monitored in Ad-A-treated embryos during mitosis using Fura-2/dextran. Mitotic Ca2+ oscillations were detected in all controls (Fura-2/dextran only; n=9) but not following Ad-A treatment (n=12). Open arrow, NEBD; closed arrow, cytokinesis. (C) Ad-A treatment had no effect upon the timing of NEBD or cytokinesis as determined using bright-field optics (data from two similar replicates; buffer, n=30; Ad-A, n=34). (D) Subsequent Hoechst labelling revealed chromatin within both blastomeres, indicating that chromosome disjunction at anaphase was uninhibited (two replicates, n=12 for Ad-A; n=19 for control).

View larger version (40K): [in a new window]   Fig. 5. Mitotic transients are dependent on extracellular Ca2+. Pronucleate embryos were transferred to Ca2+-free medium containing 1 mM EGTA 28-29 hours after hCG administration, at which time 10% had undergone NEBD. (A) No change in the Fura ratio was detected at NEBD or during mitosis in Ca2+-free medium in any case (n=14). (B) Transfer to Ca2+-free medium had no effect on the timing of NEBD and cytokinesis (data from two similar replicates; n=46 for Ca2+-containing medium, n=51 for Ca2+-free medium). (C) Hoechst labelling revealed chromatin within the nucleus of each resulting blastomere, indicating that chromosome disjunction is not dependent on extracellular Ca2+ (Ca2+-free medium, n=13; control, n=6). Open arrow, NEBD; closed arrow, cytokinesis.

View larger version (47K): [in a new window]   Fig. 6. Two-photon microscopy detects mitotic Ca2+ transients in fertilized embryos and reveals Ca2+-independent increases in indicator fluorescence within the nucleus at mitosis entry in parthenogenetic embryos. Ca2+ transients are readily detectable in a fertilized mitotic embryo using two-photon microscopy (A). (top) A series of images taken during the first transient plotted underneath. The mitotic Ca2+ transients cause a threefold increase in fluorescence. Ca2+-Green/dextran (B) and rhodamine-dextran (C) were monitored during NEBD in parthenotes. Bright-field optics were used to determine the timing of NEBD (Bi, top). In addition, the exclusion of the indicators from the nucleolus allowed mitosis entry to be established from the fluorescence images as the time at which the nucleolus disappeared (Bi, arrowheads indicate the position of the nucleolus). Notice the striking increase in fluorescence that occurs within the nucleus at the time of NEBD in both Ca2+-Green/dextran- and rhodamine/dextran-injected embryos (Bi,Ci). (Bii,Cii) The same images as in Bi and Ci but illustrating the regions of interest used for data analysis (grey, peripheral cytoplasm; black, nucleus). Analysis of fluorescence intensities using these regions of interest confirms that the fluorescence increase is restricted to the nucleus in both cases (Biii,Ciii). Lower-case letters indicate which image corresponds to given points on the graph. Notice also that ionomycin addition resulted in a dramatic fluorescence increase in Ca2+-Green/dextran-injected, but not rhodamine/dextran-injected, embryos.

View larger version (70K): [in a new window]   Fig. 7. The nuclear increase in indicator fluorescence occurs concomitantly with nuclear-membrane permeabilization. Parthenogenetic embryos were co-injected with Ca2+-Green/dextran and a 70 kDa rhodamine-dextran. The 70 kDa rhodamine-dextran was excluded from the nucleus, allowing NEBD to be determined accurately as the time at which the rhodamine signal entered the nucleoplasm (Aa'-d'). The changes in fluorescence of the two indicators were examined using regions of interest placed in the cytoplasm and nucleus (Bi). Lower-case letters indicate which image corresponds to given points on the graph. Notice that the nuclear increase in Ca2+-Green fluorescence occurs at precisely the same time that the 70 kDa rhodamine-dextran enters the nucleoplasm (Bii).

View larger version (23K): [in a new window]   Fig. 8. Mitotic Ca2+ transients cannot be detected during the second embryonic division. Two-cell embryos were co-injected with Fura-2/dextran and FITC-NLS-BSA to monitor [Ca2+]i and the presence of nuclei, respectively, and transferred to the microscope stage shortly before the second mitotic division. Notice the disappearance of the nucleus at mitosis entry and the formation of two new nuclei following cytokinesis. No Ca2+ transients were seen during the second mitotic division (n=9). A significant increase in Fura-2 ratio occurred when blastomeres were subsequently challenged with ionomycin (5 µM). Images were acquired at 10-second intervals.