Fertilization is a fundamentally important event in which signaling pathways are activated that stimulate the initiation of embryonic development. In mammals, fertilization-associated oscillations of egg intracellular calcium (Ca2+) are necessary and sufficient to cause egg activation, including the block to polyspermy, cell cycle resumption for the completion of meiosis, changes in cell cycle regulatory kinases, and alterations in protein expression. Fertilization failure, abnormal early development, and polyspermy represent significant problems in humans and animals. The mechanisms by which Ca2+ and its oscillations stimulate the events of egg activation are not well understood and represent important gaps in our knowledge of how fertilization activates early development. Supported by preliminary data and recent publications, the Aims will determine the role(s) and Ca2+-dependence of an important Ca2+ sensor, CaMKII, in the initiation of mouse development.
AIM 1 will determine if crucial events of egg activation are CaMKII-dependent in fertilized eggs, including the decrease in activity of the cell cycle kinase MAPK, pronuclear formation, and changes in protein expression.
AIM 2 will test the hypothesis that CaMKII activity oscillates as a function of Ca2+ oscillations. The experimental strategy will be to directly correlate CaMKII activity with the intracellular Ca2+ level in the same egg. This innovative approach will demonstrate how CaMKII activity responds to Ca2+ oscillations in a living cell and may be the first demonstration directly correlating Ca2+ levels and kinase activity in the same cell.
AIM 3 will determine how CaMKII activity and CaMKII-dependent events are regulated by individual parameters of Ca2+ oscillations. By experimentally controlling these Ca2+ parameters (amplitude, frequency, number, etc.), it will be determined how CaMKII activity varies as a function of these oscillation parameters in vivo. In addition, the dependency of downstream cell cycle kinases (e.g., MPF, MAPK) on Ca2+ and CaMKII will be determined. These studies will significantly increase our knowledge of the signaling pathways downstream of the elevation of Ca2+, provide a more comprehensive biochemical mechanism for the initiation of individual events during the onset of mammalian development, and have implications for evaluating existing clinical (but artificial) egg activation procedures and for animal cloning.
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