STUDY ON CELL MOTILITY UPON FERTILIZATION AND CELL DIVISION BY MEANS OF MICROMANIPULATION

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1. Cortical granule breakdown of sea urchin eggs was analyzed with a differential interference microscope equipped with a high-speed video system at 200 fields per second. At first, the diameter of cortical granules increased and then they disappeared gradually. The time required for the breakdown process was 30-40 msec even upon fertilization, after injection of a Ca buffer solution at high Ca^<2+> concentration into the eggs, and after perfusion of a solution at high Ca^<2+> concentration to the isolated cortex.2. When fluorescently labeled calf thymus histone was injected into starfish oocytes, chromosomes could be visualized at maturation division and cleavage. When DNA synthesis was inhibited with aphidicolin, chromosomes moved to the equator but did not move to the poles during anaphase and through telophase at the first cleavage. This was confirmed by staining DNA and microtubules with DAPI and anti-tubulin antibody, respectively. After the second cleavage, the mitotic apparatus composed only of two asters without the spindle or the chromosomes formed in the blastomeres, which had divided repeatedly.3. Female pronuclear migration upon fertilization is microtubule-dependent motility. When the microtubules connected from the sperm aster to the female pronucleus were cut with a microneedle by micromanipulation, the pronucleus stopped for a while and soon began to move. When the central part of the sperm aster was sucked into a micropipet, the pronucleus did not stop.4. The mitotic apparatus was observed after staining with several monoclonal antitubulin antibodies by indirect immunofluorescence. The mitotic stage was determined by observing chromosomes with a differential interference microscope. There were four groups of monoclonal antibodies against tubulin, which stained all parts of the mitotic apparatus, only the spindle, and only the mitotic asters, and did not stain the mitotic apparatus at all.