Analysis of the calcium transient at NEB during the first cell cycle in dividing sea urchin eggs.

Abstract

Accumulating evidence from several systems suggests that nuclear envelope breakdown (NEB) is triggered by an endogenous transient of free calcium. Using h- and f-semisynthetic aequorins as cytosolic calcium indicators, we have clearly and regularly visualized a single large, global calcium transient just before first NEB in normally developing, monospermic Lytechinus eggs. Although similar transients were not observed at NEB in subsequent cell cycles, microinjection of the calcium buffer BAPTA into one blastomere of the two-celled embryo resulted in the inhibition of NEB. The NEB transient in the first cell cycle was some five-fold smaller than the one associated with egg activation. Our data suggest that this transient takes the form of a calcium wave that spreads inwards from the periphery of the egg toward the nucleus. We confirmed that these NEB transients did not require extracellular Ca2+. In polyspermic eggs, NEB-associated transients were four-fold larger than in monospermic eggs and were periodically repeated. Examination of the distribution of fluorescein-conjugated aequorins with a laser scanning confocal microscope indicated that aequorin both enters the nucleus and is evenly distributed within the cytosol of the egg. The use of h- and f-aequorins did not reveal any NEB transients during subsequent cell cycles, nor did we detect transients associated with other cell cycle events. However, a complex train of calcium transients in the form of both localized pulses and propagated waves was detected from embryos beginning at about the morula-to-blastula transition and continuing through to hatching.