No abstract

We have used confocal microscopy to examine the [Ca2+]i increase in the albino eggs of the frog Xenopus laevis after fertilization. Eggs were placed in agar wells with their animal poles downward so that fertilization occurred preferentially in the equatorial plane, and confocal microscopy was used to provide a two-dimensional optical section through the three-dimensional Ca2+ wave. These data indicate that the wave of increased [Ca2+]i traverses the entire egg and converges uniformly on the antipode. We show that ratioing two different fluorescent dyes to correct for variations in cell thickness is not a reliable technique for this very thick cell due to differential absorption with depth. Indo-1-dextran proves to be a more reliable Ca2+ indicator in this respect. Indo-1-dextran measurements indicate that the resting [Ca2+]i is not uniform throughout the egg but exhibits a 15% higher [Ca2+]i in the cortex than deep in the cytoplasm. This difference is accentuated during wave propagation and is not dependent on extracellular Ca2+. The average peak [Ca2+]i in the center of the egg as the wave propagates through it is 0.7 microM, approximately 60% of the peak cortical [Ca2+]i. The wave velocity through the center of the egg (5.7 micron/s) is slower than that in the cortex (8.9 micron/s), and both velocities vary slightly during transit. The cortical wave speed is particularly high at the beginning (15.7 micron/s) and end (17.2 micron/s) of the wave. Eggs injected with 30-80 microM of 3 kD heparin to compete with inositol-1,4,5,-trisphosphate for binding to its receptor exhibited multiple localized spots of elevated [Ca2+]i, and many of these did not initiate a wave. For those that did lead to a wave, it was usually slow moving and exhibited a reduced (60% reduction) amplitude compared with controls.

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.

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.