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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00846

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Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes

Michael Carroll^1,*, Mark Levasseur¹, Chris Wood¹, Michael Whitaker¹, Keith T. Jones¹ and Alex McDougall^1,2,

¹ Cell and Developmental Physiology Group, School of Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
² Biologie du Développement, UMR 7009 CNRS, Universite Pierre et Marie Curie (Paris VI), Observatoire Océanologique, 06230 Villefranche-sur-Mer, France

View larger version (62K): [in a new window]   Fig. 1. Sperm-triggered Ca2+ waves in A. aspersa. Dechorionated eggs were injected with Fura-2-dextran (10-20 µM final concentration) and fertilized with preactivated sperm. As noted in several other species and previously also in A. aspersa, two phases of Ca2+ oscillations are observed, separated by a brief gap. (A) The temporal pattern of all Ca2+ oscillations is displayed. (B) The spatial pattern of the first Ca2+ wave is displayed. Time in seconds. (C) This Ca2+ wave is followed within 30-40 seconds by a visible cortical contraction that produces a surface protrusion termed the contraction pole (CP; arrow). Bar, 50 µm. Time in seconds. (D) All of the second-phase Ca2+ waves originate from the CP (arrow). As noted previously, these waves are faster than the initial Ca2+ wave and propagate more cortically (McDougall and Sardet, 1995). Time in seconds. Temperature 19°C, n=34 replicates.

View larger version (17K): [in a new window]   Fig. 5. Preventing ER accumulation during fertilization. Unfertilized eggs were injected with Ca2+-Green-1-dextran and treated with 2 µg ml–1 cytochalasin B or 2 µM nocodazole or a combination of both, and fertilized. (Ai) The pattern of Ca2+ oscillations in an egg treated with nocadazole alone; n=8. (Aii) The pattern of Ca2+ oscillations in an egg treated with cytochalasin B alone; n=9. (Bi) The pattern of Ca2+ oscillations in an egg treated with a combination of cytochalasin B (2 µg ml–1) and nocodazole (2 µM). (Bii) All of the Ca2+ waves originated at the same site in the egg (boxed). This boxed area is shown enlarged and contains the round sperm with condensed DNA (circled area). The black asterisk shows the elongated form of non-incorporated sperm DNA, which remains attached to the outer surface of the egg. Temperature 19°C, n=18 replicates, 5 animals.

View larger version (23K): [in a new window]   Fig. 2. Ca2+ influx during the second phase of Ca2+ waves. Fura-2-dextran-injected eggs were fertilized in natural (normal) sea water (SW) (shaded box). Following the first Ca2+ wave, individual eggs were then removed from the wedge and transferred to a large excess of Ca2+-free sea water (CaFSW: stippled box; approx. 5 µl into 2 ml) supplemented with 2 mM EGTA. Once the eggs had sunk to the bottom of the CaFSW imaging was restarted. (A) The temporal pattern of Ca2+ oscillations is displayed for an egg transferred to CaFSW. The transfer to CaFSW took 1 minute. Following transfer to CaFSW, the second phase of Ca2+ oscillations were present (7 waves in total in this particular egg). (B) The origin of each of the 7 waves is displayed. The white arrowhead indicates the location of the CP and the black arrowhead the location of the pb1. Temperature 19°C, n=5 replicates, 3 animals.

View larger version (84K): [in a new window]   Fig. 3. PtdIns(4,5)P2 accumulation at the CP. Unfertilized dechorionated eggs were injected with mRNA encoding either PH::GFP or a mutated form PH::GFP** whose PH domain does not recognise PtdIns(4,5)P2. Eggs were given sufficient time to produce translated protein products (5 hours). (A) Brightfield and fluorescence images are displayed following fertilization showing the accumulation of microvilli at the CP and the enrichment of PH::GFP at the same region of the fertilized egg. Temperature 19°C, n=7 replicates, 3 animals. (B) PH::GFP** did not localize to the PM before or during fertilization and had a more cytoplasmic distribution. This is seen in the three images showing an unfertilized egg (I), an egg 3 minutes after fertilization (II) and 4 minutes after fertilization, when the CP has formed (III). Bar, 50 µm. Temperature 19°C, n=5 replicates, 3 animals. (C) Line-scan analysis of eggs injected with PH::GFP or the mutant PH::GFP**. PH::GFP is clearly accumulated at the CP following fertilization, while some PH::GFP also remains accumulated at the PM. PH::GFP**, however, is not accumulated at the plasma membrane and displays a cytoplasmic distribution.

View larger version (94K): [in a new window]   Fig. 4. Removal of the CP following fertilization. Unfertilized eggs were injected with PH::GFP protein and cultured for 20 minutes to permit translocation to the PM. Following this, the eggs were injected with Fura-2-dextran and fertilized. Soon after extrusion of the first polar body pb1 (I), and once the CP was clearly visible (II) and the second series of Ca2+ waves had begun to originate from the CP (III), the eggs were treated with 2 µg ml–1 cytochalasin B. Rounded-up eggs (IV) reflect the loss of the actin basket; microvilli are also lost from the CP as is the accumulation of PH::GFP (V). Even though the CP has been lost and PtdIns(4,5)P2 is now redistributed, the Ca2+ waves still originate from the site where the CP previously resided (VI). To confirm that the site where the Ca2+ waves originate is identical to the site where the CP previously resided, we attached Nile Blue beads to the surface of the egg before fertilization [see arrow and bead (x) in I]. Following fertilization, the bead is located on the border of the CP (I); after the pharmacological removal of the CP the bead borders the site where the repetitive Ca2+ waves originated (IV, x). Finally, we noted the position of the sperm aster following removal of the CP (VII, marked *). The sperm aster is not near the site of origin of the Ca2+ waves (VII, arrow). Temperature 19°C, n=5 replicates, 3 animals.

View larger version (56K): [in a new window]   Fig. 6. Ca2+-Green-1-dextran was injected into unfertilized eggs. Subsequently, eggs were treated with cytochalasin B and nocodazole for 30 minutes and then injected with aSE. Thereafter, all Ca2+ waves originated at the cortex of the egg (A,B,C). Arrowheads mark the origin of three separate Ca2+ waves within the same egg. Temperature 14°C, n=6 replicates, 3 animals.