title: Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei.
abstract: In mammals, the sperm triggers a series of cytosolic Ca(2+) oscillations that continue for approximately 4 hours, stopping close to the time of pronucleus formation. Ca(2+) transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca(2+) signalling is not known. Previous studies suggest two possible mechanisms: first, regulation of Ca(2+) oscillations by M-phase kinases; and second, regulation by the presence or absence of an intact nucleus. We describe experiments in mouse oocytes that differentiate between these mechanisms. We find that Ca(2+) oscillations continue after Cdk1-cyclin B1 activity falls at the time of polar body extrusion and after MAP kinase has been inhibited with UO126. This suggests that M-phase kinases are not necessary for continued Ca(2+) oscillations. A role for pronucleus formation in regulating Ca(2+) signalling is demonstrated in experiments where pronucleus formation is inhibited by microinjection of a lectin, WGA, without affecting the normal inactivation of the M-phase kinases. In oocytes with no pronuclei but with low M-phase kinase activity, sperm-induced Ca(2+) oscillations persist for nearly 10 hours. Furthermore, a dominant negative importin beta that inhibits nuclear transport, also prevents pronucleus formation and causes Ca(2+) oscillations that continue for nearly 12 hours. During mitosis, fluorescent tracers that mark nuclear envelope breakdown and the subsequent reformation of nuclei in the newly formed two-cell embryo establish that Ca(2+) oscillations are generated only in the absence of a patent nuclear membrane. We conclude by suggesting a model where nuclear sequestration and release of a Ca(2+)-releasing activity contributes to the temporal organization of Ca(2+) transients in meiosis and mitosis in mice.
authors.affiliation: Department of Physiology, UCL, Gower Street, London WC1E 6BT, UK.
authors.forename: Petros
authors.initials: P
authors.lastname: Marangos
authors.forename: Greg
authors.initials: G
authors.lastname: FitzHarris
authors.forename: John
authors.initials: J
authors.lastname: Carroll
mesh_terms: D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002970:Cleavage Stage, Ovum / Q000378:metabolism; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D005306:Fertilization / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015053:Zygote / Q000502:physiology*; D028961:beta Karyopherins / Q000378:metabolism
chemical_list: D019926:Cyclin B; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins; D016203:CDC2 Protein Kinase; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium