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, Tn, - 0) and the first cleavage (Tn = 1.0). Eggs were fixed at various times after fertilization and examined with conventional fluorescence microscopy, laser scanning confocal microscopy, and three-dimensional fluorescence microscopy. Soon after the eggs were fertilized (Tn = 0.02), we saw microtubules oriented perpendicular to the plane of the plasma membrane but none parallel to the plasma membrane. Later (Tn = 0.08), we saw an array of microtubules oriented more or less parallel to the plasma membrane but having no apparent preferred orientation with respect to the animal-vegetal axis of the egg. In the interpolar regions of the egg, this network increased in density by Tn = 0.24 and remained a constant feature of the ooplasm until the first cleavage. From Tn = 0.30 to 0.76 the polar regions of the egg contained dense arrays of organized microtubules. At the animal pole, microtubules radiated from a site near the pronuclei; while at the vegetal pole, an array of parallel microtubules 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 previously been shown to suppress a zone of elevated cytosolic calcium at the animal pole and to disrupt ooplasmic segregation in this egg, the results of the present study (1) are consistent with a model in which microtubules are required for ooplasmic segregation in the medaka egg, and (2) suggest that the normal function of a microtubule-organizing center at the animal pole of the egg requires a zone of elevated calcium.