Intracellular free calcium oscillations in normal and cleavage-blocked embryos and artificially activated eggs of <i>Xenopus laevis</i>

Abstract

ABSTRACT We have measured levels of intracellular free calcium ([Ca2+]i) in albino Xenopus laevis embryos using recombinant aequorin and a photon-counting system. We observed sinusoidal oscillations in [Ca2+]i that had the same frequency as cleavage, with cleavage occurring when [Ca2+]i was lowest. An increase in calcium was seen to precede first cleavage. The cyclic changes in calcium were superimposed on a secondary pattern that increased, peaked between third and fifth cleavages and then slowly declined to a level similar to that measured before first cleavage. The amplitude of the oscillations was small during the first few cleavages but became larger with each cycle, with the largest oscillations occurring when the secondary pattern peaked (between third and fifth cleavage). As the secondary pattern declined, the amplitude of the oscillations also became smaller. The oscillations are due to release of calcium from intracellular stores, since the signal was the same in calcium-free solution as in normal medium. When cleavage was blocked with the microtubule-disrupting drugs colchicine or nocodazole, the [Ca2+]i oscillations persisted. Calcium oscillations of a similar magnitude and frequency were also present in artificially activated eggs. The secondary pattern was different in cleavage-blocked embryos and artificially activated eggs, the baseline increasing until about the third cycle and then remaining elevated for the rest of the recording (&amp;gt;8 hours). By fixing embryos at various points in the calcium cycle, we determined that mitosis began shortly after calcium levels reached their peak and was complete before the calcium level dropped to its lowest point. The fact that the calcium oscillations persist when nuclear division is not occurring suggests that either they operate independently of any downstream events that they might control or they are related to other cyclic activities in the eggs and embryos such as cycling of pHi, MPF, or surface contraction waves. When aequorin was injected into individual blastomeres of 64-cell embryos, the shape of the signal was substantially different from that seen in recordings from whole embryos. Compared to the whole-embryo recordings, the signal from a subregion of the embryo rose more quickly and had a slower, biphasic decline. These differences indicate that [Ca2+]i increases are not occurring uniformly across the embryo but are spatially localized, perhaps progressing as waves.