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

Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes
Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes
In ascidians, as in mammals, sperm trigger repetitive Ca2+-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes. In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca2+-wave pacemaker, but the mechanism that underlies the generation of a Ca2+-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca2+-wave pacemaker: (1) localized Ca2+ influx; (2) accumulation of phosphatidylinositol (4,5)bisphosphate [PtdIns(4,5)P2]; (3) accumulation of cortical endoplasmic reticulum (cER); and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase C1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P2. We found that eggs in Ca2+-free sea water displayed Ca2+ waves that originated from the CP, showing that enhanced CP Ca2+ influx does not determine the origin of the pacemaker. Also, disruption of the PH::GFP-labelled CP once it had formed did not dislodge the Ca2+-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca2+ waves came from the site of sperm-egg fusion and the frequency of Ca2+ oscillations was unaltered. These data show that local Ca2+ influx, the accumulation of PtdIns(4,5)P2 and cER at the CP are not required for Ca2+-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca2+-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubule-disrupting drugs, all the Ca2+ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We suggest that the Ca2+-releasing sperm factor might be tethered near or on the PM and that following the cortical contraction, it is translocated to the vegetal CP, thus making that site act as a Ca2+-wave pacemaker.
egg, fertilization, calcium, pacemaker
0021-9533
4997-5004
Carroll, Michael
527c3628-58dc-4502-98e6-d03983beac9e
Levasseur, Mark
4d33ce62-0062-4cbd-b6d3-7fe53b158ff7
Wood, Chris
3004b47a-88ce-469f-b801-5ea213e818ed
Whitaker, Michael
571a7044-ecce-4018-8697-34782882e8c6
Jones, Keith T.
7688cb4b-2f73-4b66-ac9e-db2d079fd21f
McDougall, Alex
23f6b2c7-b5fe-42b7-bf07-82e5a707e13a
Carroll, Michael
527c3628-58dc-4502-98e6-d03983beac9e
Levasseur, Mark
4d33ce62-0062-4cbd-b6d3-7fe53b158ff7
Wood, Chris
3004b47a-88ce-469f-b801-5ea213e818ed
Whitaker, Michael
571a7044-ecce-4018-8697-34782882e8c6
Jones, Keith T.
7688cb4b-2f73-4b66-ac9e-db2d079fd21f
McDougall, Alex
23f6b2c7-b5fe-42b7-bf07-82e5a707e13a

Carroll, Michael, Levasseur, Mark, Wood, Chris, Whitaker, Michael, Jones, Keith T. and McDougall, Alex (2003) Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes. Journal of Cell Science, 116 (24), 4997-5004. (doi:10.1242/10.1242/jcs.00846).

Record type: Article

Abstract

In ascidians, as in mammals, sperm trigger repetitive Ca2+-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes. In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca2+-wave pacemaker, but the mechanism that underlies the generation of a Ca2+-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca2+-wave pacemaker: (1) localized Ca2+ influx; (2) accumulation of phosphatidylinositol (4,5)bisphosphate [PtdIns(4,5)P2]; (3) accumulation of cortical endoplasmic reticulum (cER); and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase C1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P2. We found that eggs in Ca2+-free sea water displayed Ca2+ waves that originated from the CP, showing that enhanced CP Ca2+ influx does not determine the origin of the pacemaker. Also, disruption of the PH::GFP-labelled CP once it had formed did not dislodge the Ca2+-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca2+ waves came from the site of sperm-egg fusion and the frequency of Ca2+ oscillations was unaltered. These data show that local Ca2+ influx, the accumulation of PtdIns(4,5)P2 and cER at the CP are not required for Ca2+-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca2+-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubule-disrupting drugs, all the Ca2+ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We suggest that the Ca2+-releasing sperm factor might be tethered near or on the PM and that following the cortical contraction, it is translocated to the vegetal CP, thus making that site act as a Ca2+-wave pacemaker.

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Published date: December 2003
Keywords: egg, fertilization, calcium, pacemaker

Identifiers

Local EPrints ID: 24645
URI: https://eprints.soton.ac.uk/id/eprint/24645
ISSN: 0021-9533
PURE UUID: 6e720145-7a55-4456-90de-a9b56f02cd94

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Date deposited: 04 Apr 2006
Last modified: 17 Jul 2017 16:13

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