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First published online May 14, 2007
doi: 10.1242/10.1242/jcs.003012

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A novel mechanism controls the Ca²⁺ oscillations triggered by activation of ascidian eggs and has an absolute requirement for Cdk1 activity

¹ Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle, Newcastle upon Tyne, UK
² Human Genetics Division, University of Southampton, Southampton General Hospital, Southampton, UK
³ "Biologie du Développement", UMR 7009, CNRS/Universite Pierre et Marie Curie, (Paris VI) Observatoire Océanologique, 06230 Villefranche-sur-Mer, France

View larger version (47K): [in this window] [in a new window]   Fig. 1. ASE-triggered Ca2+ oscillations stop in the absence of pronuclei. (A) A series of images showing the procedure by which eggs were separated into small and large cytoplasts. See Materials and Methods for details. Bar, 50 µm. (B) Pseudocolour images showing that the Ca2+ oscillations progress as waves through the cytoplasm of both large and small cytoplasts. Low Ca2+ is indicated in magenta, higher levels in blue and highest by yellow, 10-second time lapse between images in upper panel, 5 seconds in the lower panel. (Ca) The ASE-induced Ca2+ oscillations stop in both cytoplasts simultaneously. Each cytoplast was injected with ASE within 30 seconds of each other. The traces from both cytoplasts show the oscillations stop in the smaller (blue line) at approximately the same time as the larger (red line) which extruded two polar bodies, evidence that this cytoplast contained the chromatin. (b) Mean time (± s.e.m.) at which the oscillations stop in cytoplasts with (red bar) and without (blue bar) chromatin (P=0.5, n=10, nine animals).

View larger version (66K): [in this window] [in a new window]   Fig. 2. ASE-triggered Ca2+ oscillations in enucleated eggs mimic those induced by sperm in eggs. (A) A second series of large enucleated cytoplasts was made. A large bore micropipette was advanced using a hydraulic manipulator to the edge of the egg where the chromatin was positioned (a). Suction was then applied by mouth aspirator such that a portion of the egg started to enter the pipette (b). Suction was stopped as soon as the chromatin had been removed (c), the pipette withdrawn from the egg (d,e), and the fragment expelled under gentle pressure to leave an enucleated egg and a small intact egg fragment containing the chromatin (f).Chromatin (in blue) was stained with Hoechst, and the images overlaid on a series of brightfield images. Bar, 50 µm. (Ba) ASE was injected into these large cytoplasts and the time at which the Ca2+ oscillations stopped [23.3±1.46 minutes (mean ± s.e.m.) n=9, three animals] was not significantly different from the mean of the times recorded for control, unmanipulated eggs (23.09±1.42 minutes, P=0.91). (b) Confirmation that these large cytoplasts could oscillate for an extended period of time. The cytoplast was first injected with 90 cyclin B1::GFP protein, and the Ca2+ oscillations persisted throughout the entire period they were observed (n=6, two animals).

View larger version (5K): [in this window] [in a new window]   Fig. 3. Inhibition of Cdk1 activity inhibits the second phase of Ca2+ oscillations. (A) Normal pattern of Ca2+ oscillations after injection with ASE. (B) When p21::GFP protein was injected to inhibitory levels, injection of ASE resulted in only a single Ca2+ rise. Data in A and B are from eggs imaged simultaneously. Arrows indicate time of ASE injection and data is representative of every experiment (n=26, 16 animals).

View larger version (9K): [in this window] [in a new window]   Fig. 4. Long lasting 90 cyclin B1 mediated oscillations stop rapidly when Cdk1 activity is inhibited. (A) Long lasting oscillations in an egg injected with 90 cyclin B1:: GFP protein and then ASE. (B) Rapid termination of oscillations when an egg previously injected with 90 cyclin B::GFP and ASE is then injected with p21::GFP protein. Black arrows indicate time of ASE injection, open arrow the time of p21::GFP injection. Data in A and B are from eggs imaged simultaneously. Time of termination was 8.33±0.58 minutes (mean ± s.e.m.) (n=8, four animals).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Inhibition of Cdk1 activity does not decrease responsiveness to Ins(1,4,5)P3. (A) Determination of the threshold concentration of the potent Ins(1,4,5)P3 analogue adenophostin A required to give Ca2+ oscillations. (a) Injection of adenophostin A to a final concentration of approximately 0.1 µM gave a large Ca2+ rise and one (n=1, one animal) or two (n=2, two animals) small subsequent increases. (b) Injection of adenophostin A to a final concentration of approximately 0.25 µM gave a large initial Ca2+ rise that was always followed by at least four (n=2, two animals) or five (n=3, two animals) Ca2+ transients. Injection to higher final concentrations gave the same pattern of Ca2+ rises. 0.25 µM was thus used as the threshold concentration required to elicit oscillations. (Ba) Eggs remain responsive to this dose of adenophostin A when Cdk1 activity is low and they do not give second phase oscillations. Eggs were injected with p21::GFP protein, activated with ASE (black arrow) and then when an adjacent control egg (no p21) injected with ASE at approximately the same time was giving second phase oscillations, adenophostin A was injected (open arrow) into the p21-containing egg to a final concentration of 0.25 µM producing a large Ca2+ rise followed by several oscillations (four oscillations n=1, 5 oscillations, n=2, three animals). (Bb) Eggs that had exited meiosis did not respond to this dose of adenophostin A. Following injection of ASE (black arrow) and after allowing a full series of Ca2+ oscillations to proceed, as well as extrusion of both polar bodies, the egg was then injected with the same dose of adenophostin used in Bi (open arrow). In all cases this resulted in a single large rise in Ca2+ with no subsequent Ca2+ oscillations (n=6, three animals).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Inhibition of Cdk1 activity does not decrease responsiveness to Ins(1,4,5)P3. Pairs of eggs, A and B, were co-injected with 2 mM Ca2+ Green and 3.5 mM caged gPtdIns(4,5)P2 [a slowly hydrolysable Ins(1,4,5)P3 analogue] to give an intracellular concentration of approximately 20 µM Ca2+ Green and 35 µM gPtdIns(4,5)P2,and Ca2+ increases were measured simultaneously. Flashes of UV light (purple stars) were administered in increasing duration until a significant rise in Ca2+ release was observed (flash number 1). This flash duration (750 milliseconds) was the duration used for all further flashes. (A) Control egg with no p21. (B) An egg injected with p21::HcRed protein (the red fluorescent variant was used to avoid interfering with the Ca2+ Green signal). Each egg was injected with sperm extract and once the control had started to give second phase oscillations, UV-light flashes (numbered 2-6) were delivered in between oscillations in the control (indicated by white 2 on black background symbols). The pale blue dashed lines emphasise synchronicity of each UV-light-flash-induced Ca2+ release. After the second phase oscillations had stopped in the control and a second polar body had been extruded, two further flashes (numbers 7 and 8) were given (n=7, six animals).

View larger version (20K): [in this window] [in a new window]   Fig. 7. The ascidian (Ciona) type I Ins(1,4,5)P3R (IP3R1) sequence has diverged at the Cdk1 phosphorylation sites, which are conserved in Drosophila, Xenopus and human. Sequences are aligned, with differences outside the consensus S/T P motif shown in blue and differences in the consensus itself shown in red. Human mouse and Xenopus alignment is from Malathi et al. (Malathi et al., 2003). GenBank accession number for Drosophila is A43360, and the Ciona sequence was obtained from Nori Satoh's Ciona genome database, accession number ci0100150586.

View larger version (16K): [in this window] [in a new window]   Fig. 8. The model we propose for control of the second phase Ca2+ oscillations by Cdk1 activity in the ascidan. (Top) Sperm-egg fusion and extrusion of first and second polar bodies are shown diagrammatically to indicate relative times of fertilisation, transition from meiosis I to meiosis II and meiotic exit into interphase. The sperm head, coloured in red, depicts the sperm arriving to deliver fully active sperm factor to the egg. (Bottom) A typical pattern of fertilisation-triggered Ca2+ oscillations is shown by the black line. Previously determined Cdk1 activity (McDougall and Levasseur, 1998) is indicated by the blue line, and Ins(1,4,5)P3 responsiveness (Levasseur and McDougall, 2003) by the red line. Hypothetical sperm factor activity is indicated by the pseudocoloured bar, and correlates with Cdk1 activity.