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 Cδ1 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 H::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 microtubuledisrupting 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.