MICROTUBULE ARRAYS DURING OOPLASMIC SEGREGATION IN THE MEDAKA FISH EGG (ORYZIAS-LATIPES)

We used indirect immunofluorescence to study microtubule arrays in the medaka egg between fertilization (normalized time, T-n, = 0) and the first cleavage (T-n = 1.0). Eggs were fixed at various times after fer tilization and examined with conventional fluorescence microscopy, las er scanning confocal microscopy. and three-dimensional fluorescence mi croscopy. Soon after the eggs were fertilized (T-n = 0.02), we saw mic rotubules oriented perpendicular to the plane of the plasma membrane b ut none parallel to the plasma membrane. Later (T-n = 0.08), we saw an array of microtubules oriented more or less parallel to the plasma me mbrane but having no apparent preferred orientation with respect to th e animal-vegetal axis of the egg. In the interpolar regions of the egg , this network increased in density by T-n = 0.24 and remained a const ant feature of the ooplasm until the first cleavage. From T-n = 0.30 t o 0.76 the polar regions of the egg contained dense arrays of organize d microtubules. At the animal pole, microtubules radiated from a site near the pronuclei; while at the vegetal pole, an array of parallel mi crotubules was present. Injection of the weak (KD = 1.5 mu M) calcium buffer 5,5'-dibromo-BAPTA disrupted the radial pattern of microtubules near the animal pole but had no apparent effect on the parallel array of microtubules near the vegetal pole. Because this buffer has previo usly been shown to suppress a zone of elevated cytosolic calcium at th e animal pole and to disrupt ooplasmic segregation in this egg, the re sults of the present study (1) are consistent with a model in which mi crotubules are required for ooplasmic segregation in the medaka egg, a nd (2) suggest that the normal function of a microtubule-organizing ce nter at the animal pole of the egg requires a zone of elevated calcium .