Ionic fluxes, resulting in intracellular ionic activity changes, play a central role in the activation of the metabolic processes which constitutes early development. In the eggs of most animal 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 pHi. 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 mechanisms by which sperm-egg interaction generates these ionic changes in the egg of the frog, Xenopus laevis. This comes under two main headings: 1) studies of the inositol lipid cascade and its role in Ca2+ release; 2) studies of the pHi and (Ca2+)i changes and interactions following activation. The studies of the role of inositol lipids and sugars in activation will include: 1) determining if asymmetries in the distribution of these lipids in the plasma membrane of the egg are associated with cell polarity or the activatability of the egg; 2) determining the normal time course of appearance and spatial distribution of the isomers of the inositol phosphates in the frog egg during activation; and 3) describing the Ca2+- release characteristics of each of the isomers of the inositol phosphates. The studies of the pHi and (Ca2+}i changes include: 1)determining the mechanism of Ca2+ removal that generates the transient, wave-like nature of the (Ca2+)i increase; 2) identifying the mechanisms responsible for the pHi changes which follow activation; and 3) determining how (Ca2+)i and pHi depend on one another in both unfertilized and fertilized eggs.
These specific aims will be investigated using a number of techniques including double-barrelled, ion-specific microelectrodes that allow us to measure the exact values of (Ca2+)i and pHi in the cytoplasm while microinjecting various solutions of inositol phosphates, and HPLC that allows us to monitor the natural production of these messengers in the activating egg. This research will clarify the relationships between the inositol lipid cascade, 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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