FERTILIZATION STIMULATES AN INCREASE IN INOSITOL TRISPHOSPHATE AND INOSITOL LIPID-LEVELS IN XENOPUS EGGS

Previous experiments from our lab have suggested that the hydrolysis o f phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-i nduced egg activation in Xenopus laevis. Here we measure the endogenou s production of both Ins(1,4,5)P-3 and PIP2 during the sperm-induced a nd ionomycin-induced calcium wave in the egg and find that both increa se following fertilization. Ins(1,4,5)P-3 increases 3.2-fold from an u nfertilized egg level of 0.13 pmole per egg (0.29 mu M) to a peak of 0 .42 pmole per egg (0.93 mu M) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P-3 du ring the time that the Ca2+ wave is propagating across the egg suggest s the involvement of Zns(1,4,5)P-3 in wave propagation. This increase in Ins(1,4,5)P-3 is smaller in ionomycin-activated eggs than in sperm- activated eggs, suggesting that the sperm-induced production of Ins(1, 4,5)P-3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a re sult of hydrolysis, we find that PIP2 actually increases P-fold. The t otal Lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per eg g and this increases to 1.5 pmole as the calcium wave reaches the anti pode. The PIP2 concentration peaks 2 min after the completion of the c alcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eg gs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of Lip id. Thus, there was an equivalent amount of PIP2 normalized to lipid i n each hemisphere. Isolated animal and vegetal hemisphere cortices exh ibit similar: PIP2 concentrations, suggesting that the 2-fold higher t otal PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membran es also contain PIP2. (C) 1996 Academic Press, Inc.