Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei

Development. 2003 Apr;130(7):1461-72. doi: 10.1242/dev.00340.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CDC2 Protein Kinase / metabolism
  • Calcium / metabolism*
  • Cleavage Stage, Ovum / metabolism
  • Cyclin B / genetics
  • Cyclin B / metabolism
  • Fertilization / physiology*
  • Green Fluorescent Proteins
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • Mitogen-Activated Protein Kinases / metabolism
  • Protein Synthesis Inhibitors / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Zygote / physiology*
  • beta Karyopherins / metabolism

Substances

  • Cyclin B
  • Luminescent Proteins
  • Protein Synthesis Inhibitors
  • Recombinant Fusion Proteins
  • beta Karyopherins
  • Green Fluorescent Proteins
  • CDC2 Protein Kinase
  • Mitogen-Activated Protein Kinases
  • Calcium