The inhibition of pronuclear movements by dibromo-BAPTA is consistent with the explanation that the buffer disrupts the organization of microtubules near the animal pole. In a number of species, including the medaka, the movement of the pronuclei is inhibited by microtubule poisons (Hiramoto et al., 1984; Sawada and Schatten, 1989; Abraham et al., 1993a), suggesting that microtubules are necessary for these movements.
The suggestion that the medaka egg has two independent microtubule networks is consistent with the situation in X, laevis, in which two independent networks of microtubules are present during the first cell cycle: one is near the animal pole and is associated with the pronuclei, while the other is the parallel array of microtubules near the vegetal pole (Elinson and Rowning, 1988; Houliston and Elinson, 1991; Elinson and Palacek, 1993; Sardet et al., 1994).
Apparently normal oil droplet movement was restored when we irradiated the animal pole or equatorial region en face, with oil droplets near the animal pole or equator moving away from the animal pole and toward the vegetal pole more or less along meridian lines [ILLUSTRATION FOR FIGURE 2A-F OMITTED].
The movements of droplets within the irradiated regions were very similar to those in control (not treated with demecolcine) eggs; that, is roughly along meridian lines and away from the animal pole [ILLUSTRATION FOR FIGURE 3 OMITTED].
Droplets of mineral oil were injected into five eggs, which were oriented with their animal pole uppermost.
We injected vegetable oil into 11 eggs and oriented them with their animal pole uppermost.
In other words, endogenous droplets, moving toward the vegetal pole, passed by the droplets of injected fluid, which were either stationary or moving toward the animal pole.
The inhibition suggests that the formation of the blastodisc in the medaka egg is the result of the streaming of ooplasm to the animal pole and that microfilaments are involved in this streaming.
The solation-contraction coupling hypothesis (Janson and Taylor, 1993) provides a model for how this streaming, which is likely driven by the interaction of actin and myosin, could be organized and driven by the zone of elevated cytosolic [Ca.sup.2+] present at the animal pole throughout the period of segregation (Fluck et al., 1992).
The experiments with CCD directly test the hypothesis that the movement of oil droplets toward the vegetal pole is caused by the movement of ooplasm in the opposite direction - toward the animal pole. The results are not consistent with this hypothesis, because oil droplets moved toward the vegetal pole even when we did not observe any cytoplasm streaming to the animal pole.
Moreover, the failure of injected droplets of several other fluids - fluorinated aliphatic compounds, silicone fluid, mineral oil, and vegetable oil - consistently to move away from the animal pole and toward the vegetal pole during segregation is inconsistent with the hypothesis.
These results suggest that UV irradiation at the equator lowered the concentration of demecolcine in the animal pole region (where cell division occurs) enough to permit the polymerization of tubulin.