A Confocal Microscope Study of the Fertilization-Induced Calcium Wave in the Frog Egg
Monday, February 9, 1998 - 11:00am - 12:00pm
Richard Nuccitelli (University of California)
We have used confocal microscopy to examine the [Ca2+]i increase in the albino eggs of the frog, Xenopus laevis, following 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 are the first confocal images of the sperm-induced calcium wave in the frog egg. 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 in order 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 µM, about 60% of the peak cortical [Ca2+]i. The wave velocity through the center of the egg (5.7 µm/s) is slower than that in the cortex (8.9 µs) and both velocities vary slightly during transit. The cortical wave speed is particularly high at the beginning (15.7 µm/s) and end (17.2 µm/s) of the wave. Eggs injected with 30-80 µM of 3 kD heparin to compete with Ins(1,4,5)P3 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 to controls.