Cell cycle-dependent repetitive Ca²⁺ waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization

Sperm-triggered Ca²⁺ oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca²⁺ oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca²⁺ oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca²⁺ oscillations that are all waves in ascidians. The first of these Ca²⁺ waves begins at the point of sperm-egg fusion while a second phase of Ca²⁺ waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca²⁺ waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca²⁺-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca²⁺ waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca²⁺ waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca²⁺ oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for spermtriggered Ca²⁺ oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophasestage oocytes, lacking CDK activity, failed to induce any Ca²⁺ release even though they responded to microinjection of the Ca²⁺-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca²⁺-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca²⁺-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca²⁺-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca²⁺ waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.