Microtubule-Based Movements During Ooplasmic Segregation in the Medaka Fish Egg (Oryzias latipes)

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.