Cell cycle-dependent repetitive Ca(2+ )waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization.
Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) 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 Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) 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 Ca(2+) 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 Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-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 Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.
McDougall
A
A
Department of Physiological Sciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. a.d.mcdougall@ncl.ac.uk
Levasseur
M
M
O'Sullivan
A J
AJ
Jones
K T
KT
D000818:Animals; D020013:Calcium Signaling; D002453:Cell Cycle; D002845:Chromatography; D056744:Cyclin B1; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005306:Fertilization; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D008297:Male; D008540:Meiosis; D008845:Microinjections; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010063:Ovum / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000737:chemistry*; D014561:Urochordata / Q000502:physiology*
D056744:Cyclin B1; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D018844:Cyclin-Dependent Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D016257:Fura-2