Abstract. Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell . We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at -0.5 Am/s . The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first T HE uncertain relationship of free calcium patterns to cytokinesis has been critically reviewed by Mabuchi (1986), by Hepler (1989), as well as by Salmon (1989) and considered by several investigators at a recentsymposium volume (Conrad and Schroeder, 1990). In our view, the most cogent evidence that a natural rise in calcium may favor furrow elongation as well as furrow initiation and apposition are Arnold's (1975) observations of the effects of A23187 on cleaving squid eggs. He reported that application ofthis calcium ionophore to cleaving eggs induces an immediate increase in the speed of furrow elongation as well as an immediate widening of the furrow. A minute or two later he saw a "relaxation" of the cleaving egg and a decrease in visibility ofthe furrow which probably indicated apposition ofthe separating cells. Longer term effects included an extension of the furrows beyond theirnormal extent. Moreover, ifthe ionophore is applied a few minutes before cleavage is scheduled, then the furrows appear prematurely. Also cogent is the more recent report of Conrad et al. (1987) that optimal concentrations of caffeine-well known to speed calcium release from the ER-substantially speeds the initiation of polar lobe furrows as well as cleavage furrows in Ilyanassa eggs.We have pursued this question in the large (1,200-,um diam), hyaline medaka egg because calcium patterns within this system can be easily and reliably studied with aequorin (Gilkey et al . 1978) . Fig . 1 diagrams the medaka egg's second cleavage . Each early cleavage furrow begins over (and is presumably initiated by) a centrally placed mitotic apparatus (Rappaport, 1990) . It then extends laterally to the edge

Using time-lapse video microscopy, we found that ooplasmic inclusions in the fertilized medaka egg displayed two types of movement during ooplasmic segregation. The first manifested itself as the movement of many inclusions (diameter = 1.5-11 μm) toward the animal pole at about 2.2 μm min-1; this type of movement appeared to be streaming. The second type of movement was faster (about 44 μm min-1) and saltatory; inclusions displaying this type of movement were smaller (diameter ≤1.0 μm) and moved toward the vegetal pole. The movement of oil droplets toward the vegetal pole of the egg may represent a third type of motion. All these movements began only after a strong contraction of the ooplasm toward the animal pole, which at 25°C began 10-12 min after fertilization and <3 min after formation of the second polar body. In eggs treated with microtubule poisons--colchicine, colcemid, or nocodazole--oil droplets did not move toward the vegetal pole, saltatory motion toward the vegetal pole was absent, and the growth of the blastodisc was slowed. Eggs treated with β-lumicolchicine, an inactive derivative of colchicine, showed normal movements. Colchicine, while not inhibiting formation of the second polar body, did inhibit pronuclear migration. These results suggest that microtubules are involved in the movement of some ooplasmic inclusions, including oil droplets, toward the vegetal pole; the movement of ooplasmic inclusions toward the animal pole; and pronuclear migration.

No abstract

We used time-lapse video microscopy to monitor the effects of cytochalasin D (CCD) and demecolcine on cytoplasmic streaming toward the animal pole of the medaka egg, the formation of the blastodisc at the animal pole, the movement of oil droplets in the cytoplasm toward the vegetal pole, and the saltatory movement of small cytoplasmic parcels toward the animal pole and vegetal pole. Cytochalasin D inhibited both cytoplasmic streaming toward the animal pole and the formation of the blastodisc, suggesting a role for microfilaments in these processes. However, CCD had no apparent effect on saltatory movement or on the movement of oil droplets toward the vegetal pole. Thus, the segregation of oil droplets toward the vegetal pole is not the result of the bulk movement of ooplasm toward the animal pole. In eggs treated with demecolcine, oil droplets did not move toward the vegetal pole but instead floated to the uppermost portion of the egg, and saltatory movement was absent, suggesting that microtubules are required for these movements. The effects of demecolcine on oil droplet movement and saltatory movement could be reversed by irradiating the eggs with UV light (360 nm). Using indirect immunofluorescence, we showed that irradiation of demecolcine-treated eggs with UV light regenerated microtubules within the irradiated region. The specificity of the mechanism responsible for the vegetal poleward movement of oil droplets was assessed by microinjecting droplets of five other fluids--mineral oil, silicone oil, vegetable oil, and two fluorinated aliphatic compounds--into the ooplasm. None of these fluids segregated with the endogenous oil droplets. These results suggest that a specific mechanism, probably involving microtubules, is responsible for the segregation of oil droplets to the vegetal pole.

We have injected medaka fish zygotes with recombinant aequorin and visualized the resulting patterns of luminescence to reveal patterns of free calcium during early development. We have co-injected fluorescein-labeled aequorin to correct for nonuniformities in aequorin (as opposed to calcium) distributions by visualizing the resulting patterns of fluorescence as opposed to luminescence. We have also coinjected a calcium buffer to facilitate calcium diffusion, dissipate apparent calcium gradients, and thus confirm their reality. An exploratory study shows zones of elevated free calcium at the vegetal as well as the animal pole during the first day of development and thus up to the beginning of gastrulation. A closer study during the first 6 h, and thus through ooplasmic segregation and early cleavage, shows a steady zone of high calcium at the vegetal pole and a slowly oscillating one at the animal pole. The latter is particularly strong during ooplasmic segregation and cytokinesis. This report contains the first unambiguous evidence of relatively steady zones of high cytosolic calcium during the development of an animal egg.