The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation.

Abstract

Abstract. We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 #m/s. A similar wave is seen in eggs fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in spermactivated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30--40 s after the calcium wave starts, a slower (1.4 #m/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 ° away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.

“…3 A). This contraction wave lasts --2 rain in PhaUusia mammillata Speksnijder et al, 1990a). At the end of it, a brightly fluorescent layer *2-6/~m thick is found at the contraction pole (Fig.…”

“…A cortical contraction wave originating from the animal pole propagates toward the vegetal pole region, and ends in a constricted region close to but not necessarily coincident with the vegetal pole. We have named this region the vegetal-contraction pole area (Speksnijder et al, 1990a). During this contraction wave, which lasts ,~2-3 rain, the subcortical myoplasmic layer becomes concentrated in the vegetal-contraction pole region.…”

“…The eggs were rinsed in 0.2 M Na-cacodylate pH 7.2, postfixed in 1% OSO4, 0.8% IGFe(CN)6 for 1 h, rinsed in distilled water, dehydrated in a graded series of ethanol, and embedded in Sport's plastic. animal-vegetal polarity manifest by the position of the meiotic spindle at the animal pole and the localiration of a subcortical myoplasmic domain in the vegetal hemisphere Gualtieri and Sardet, 1989;Speksnijder et al, 1990a;Sardet et al, 1992). Observations with a CLSM on 10 unfertilized eggs (five different egg batches) with their ER labeled using DiICI~(3), show those two features clearly.…”

“…Selective labeling of the ER was achieved by microinjecting a saturated solution of DiICit(3) (Molecular Probes, Inc., Eugene, OR) in Wesson oil (see Terasaki and Jaffe, 1991) using Hiramoto's low pressure method as described by Kiehart (1982). Unfertilized eggs were injected with the dye at random positions relative to their animal-vegetal axis, and held in position by slightly compressing them between two coverslips, a procedure which does not affect normal development (e.g., see Speksnijder et al, 1990a). At the site where the injected oil droplet comes in contact with membranes of the ER, the dye moves from the oil into the ER membrane and spreads within this membrane.…”

Actin in the Drosophila embryo: Is there a relationship to developmental cue localization?

“…5 and references therein). Calcium waves that are stimulated by sperm-egg fusion coincide with this contraction both in time and space (6) . Similarly in zygotes of the brown algae, Fucus pelvetia, a steady inward calcium current develops at the future rhizoid, and appears to be associated with the fixation of the embryonic axis which occurs in response to light (7) .…”