A wave of IP3 production accompanies the fertilization Ca2+ wave in the egg of the frog, Xenopus laevis: theoretical and experimental support.

The fertilization Ca2+ wave in Xenopus laevis is a single, large wave of elevated free Ca2+ that is initiated at the point of sperm-egg fusion and traverses the entire width of the egg. This Ca2+ wave involves an increase in inositol-1,4,5-trisphosphate (IP3) resulting from the interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca2+ release machinery. The extraordinarily large size of this cell (1.2 mm diameter) together with the small surface region of sperm-receptor activation makes special demands on the IP3-dependent Ca2+ mobilizing machinery. We propose a detailed model of the fertilization Ca2+ wave in Xenopus eggs that requires an accompanying wave of IP3 production. While the Ca2+ wave is initiated by a localized increase of IP3 near the site of sperm-egg fusion, the Ca2+ wave propagates via IP3 production correlated with the Ca2+ wave-possibly via Ca(2+)-mediated PLC activation. Such a Ca(2+)-mediated IP(3) production wave has not been required previously to explain the fertilization Ca2+ wave in eggs; we argue this is necessary to explain the observed IP3 dynamics in Xenopus eggs. To test our hypothesis, we have measured the IP3 levels from 20 nl "sips" of the egg cortex during wave propagation. We were unable to detect the low IP3 levels in unfertilized eggs, but after fertilization, [IP3] ranged from 175 to 430 nM at the sperm entry point and from 120 to 700 nM 90 degrees away once the Ca2+ wave passed that region about 2 min after fertilization. Prior to the Ca2+ wave reaching that region the IP3 levels were undetectable. Since significant IP3 could not diffuse to this region from the sperm entry point within 2 min, this observation is consistent with a regenerative wave of IP3 production.

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets.

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 degrees C (18.7+/-1.8 kcal/mol) than below that critical temperature (7.5+/-1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1+/-1.6 kcal/mol above 15 degrees C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 degrees C, and two smaller transitions at 26 and 37 degrees C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 degrees C and smaller transitions at 26 and 35 degrees C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-beta-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 degrees C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.

Sperm initiate a Ca2+ wave in frog eggs that is more similar to Ca2+ waves initiated by IP3 than by Ca2+.

We have measured the initial propagation velocity of the sperm-induced Ca(2+) wave in the egg of Xenopus laevis and have compared it with the initial propagation velocities of the inositol triphosphate (IP(3))-induced and Ca(2+)-induced Ca(2+) waves. The initial mean propagation velocity of the sperm-induced wave (13 microm/s) is very similar to that of the IP(3)-induced waves (12.3 microm/s) and two times faster than the mean Ca(2+)-induced wave velocity (6.6 microm/s). We have generated realistic simulations of the fertilization wave in the frog egg using a computational technique based on the finite difference method. Modeling refinements presented here include equations for the production, degradation, and diffusion of IP(3), a description for Ca(2+) dynamics in the endoplasmic reticulum, and a highly concentrated endoplasmic reticulum in the egg cortex. We conclude that models incorporating sperm-induced IP(3) generation fit the data best and those involving the influx of either Ca(2+) or a diffusible sperm factor fit the data poorly. This independence from Ca(2+) influx is also supported by electrophysiological data indicating that Ca(2+) influx is not needed to maintain open Cl(-) channels that generate the fertilization potential.