Characterization of the Sperm-Induced Calcium Wave in Xenopus Eggs Using Confocal Microscopy

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.

Spatial Control of Ca2+ Signaling by Nicotinic Acid Adenine Dinucleotide Phosphate Diffusion and Gradients

“…These data suggest that the cortex is the area of the egg most sensitive to Ca 2ϩ -releasing messengers. Fertilization-induced Ca 2ϩ waves also have been shown to propagate preferentially through the cortex at velocities greater than through the center of the egg (31,32). The origin of this differential regional sensitivity to NAADP is unknown but may relate to the differentially organized endoplasmic reticulum in the cortex (33) or the relative density of NAADPsensitive Ca 2ϩ release channels in the cortex, as demonstrated for the IP 3 and ryanodine receptors (34, 35).…”

Ca2+ signaling differentiation during oocyte maturation

“…Fertilization of the Xenopus egg induces a local Ca 2þ rise at the site of sperm entry, followed by a Ca 2þ wave that slowly (3-4 min) sweeps across the entire egg (Fontanilla and Nuccitelli, 1998). The Ca 2þ wave is followed by a sustained Ca 2þ plateau that lasts for several minutes (Busa and Nuccitelli, 1985;El Jouni et al, 2005).…”

“…Furthermore, the Ca 2þ wave propagates at a more rapid rate in oocytes ($20 mm/sec) compared to eggs ($9 mm/sec; Fontanilla and Nuccitelli, 1998;Machaca, 2004), and the mode of wave propagation is modulated during maturation. In oocytes wave propagation is proposed to be saltatory (Lechleiter and Clapham, 1992;Callamaras et al, 1998;Machaca, 2004), whereas it appears to be continuous in eggs (Nuccitelli et al, 1993;Fontanilla and Nuccitelli, 1998;Machaca, 2004). The building blocks of Ca 2þ waves are quantal elementary Ca 2þ release events due to the opening of a few IP 3 receptors that in Xenopus are referred to as Ca 2þ puffs (Parker and Yao, 1995).…”

Endoplasmic reticulum reorganizations and Ca2+ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF

“…In mouse oocytes, however, the cortical ER clusters of mature oocytes disassemble at ~1.5-3.5 hours post-insemination, and this disassembly is apparently caused by a drop in MPF levels (FitzHarris et al, 2003). Similarly, in Xenopus, which produces a single Ca 2+ wave at fertilization (Fontanilla and Nuccitelli, 1998), ER clusters that are about 3-5 µm in diameter develop transiently at metaphase I and then again in MII-arrested specimens (Terasaki et al, 2001). Such clusters subsequently dissipate within a few minutes after MII-arrested oocytes generate a Ca 2+ wave in response to prick activation (Terasaki et al, 2001).…”