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

1. 利用配备200视场/秒高速视频系统的差示干涉显微镜，对海胆卵的皮质颗粒分解进行了分析。皮层颗粒先是直径增大，然后逐渐消失。即使在受精后，将高Ca^<2+>浓度的Ca缓冲溶液注射到卵中，以及将高Ca^<2+>浓度的溶液灌注到离体皮质后，分解过程所需的时间也为30-40毫秒。将荧光标记的小牛胸腺组蛋白注射到海星卵母细胞中，可以看到成熟分裂和卵裂的染色体。当DNA合成被阿霉素抑制时，染色体在第一次分裂的后期和末期向赤道移动而不向两极移动。用DAPI和抗微管蛋白抗体分别对DNA和微管进行染色，证实了这一点。第二次分裂后，有丝分裂装置仅由两条没有纺锤体的母粒组成，或在卵裂球中形成染色体，卵裂球多次分裂。受精后雌性原核的迁移是微管依赖的运动。当用微针通过显微操作切断连接精子和雌性原核的微管时，原核停止了一会儿，很快开始移动。当精子的中心部分被吸进微移液管时，原核并没有停止。用几种单克隆抗微管蛋白抗体间接免疫荧光染色观察有丝分裂器。用差示干涉显微镜观察染色体，确定有丝分裂阶段。抗微管蛋白的单克隆抗体有四组，它们能染色有丝分裂器的所有部分，只染色纺锤体，只染色有丝分裂母细胞，而不染色有丝分裂器。

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.