Tonic fluxes, resulting in intracellular ionic activity changes, play a central role in activation of the metabolic processes which constitute early development. In the eggs of most animals studied, including both invertebrates and vertebrates fertilization triggers an immediate change in the plasma membrane's ion permeability as well as large changes in intracellular [CA2+] and pH. These ionic events must be important steps in the activation of development because simple manipulations of the egg's Ionic environment will often activate development parthenogenetically. This research will study the role of both intracellular [CA2+] and pH changes as well as transcellular ion currents in activation in two vertebrate eggs, that of the frog, Xenopus laevis, and the fish, Oryzias latipes. Techniques which will be used include: a dual pH/Ca ion-specific microelectrode which can detect pHi and [CA2+]i at the same point in the cell; and the patch clamp technique which will identify the ionic channels in conjunction with the extracellular vibrating probe; and 31P NMR to monitor ATP levels at the same time as pHi. The main goals of this research are 1) to determine the mechanisms responsible for the initiation and propagation of the Ca2+ wave triggered by fertilization; 2) identify the mechanisms generating the pHi increase following fertilization; 3) to investigate the coupling between [Ca2+]i and pHi in both unfertilized and fertilized eggs; 4) determine the nature of the channels contributing to the wave of inward current which propagates over the egg at fertilization. This work should improve our understanding of the mechanisms of the ionic changes accompanying activation of the vertebrate egg, and it will clarify the interrelationship between pHi and [Ca2+]i in a system which naturally undergoes physiological pHi and [Ca2+]i changes. Such information should prove valuable for the understanding of many cellular mechanisms which utilize pHi and/or Ca2+ as regulatory agents.
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