Spindle Observation in Living Mammalian Oocytes with the Polarization Microscope and Its Practical Use

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Abstract
The meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis. Conventional methods to image spindles rely on fixation and transmission electron microscope or immunofluorescence staining and fluorescence microscope, so they provide limited value to studies of spindle dynamics and human clinical in vitro fertilization. A new orientation-independent polarized light microscope, the LC Polscope, was used to examine the bi-refringent spindles in living mammalian oocytes. It was found that spindles could be imaged with the Polscope in living oocytes in all mammals so far examined, including hamster, mouse, cattle, human, and rat. The first polar body did not accurately predict the spindle location in most metaphase II oocytes. Intracytoplasmic sperm injection (ICSI) could be performed by monitoring spindle position. Studies in humans indicated that, aftr ICSI, higher fertilization and embryonic developmental rates could be achieved in oocytes with than without bi-refringent spindles. Because spindles in most mammalian oocytes are extremely sensitive to slight changes in temperature, maintenance of temperature at 37°C is crucial for normal spindle function. As chromosomes are usually associated with microtubule fibers in the spindles, the position of chromosomes could be indirectly located by imaging spindles. Removing spindles under the Polscope can achieve an enucleation efficiency rate of 100% in mouse oocytes. The Polscope can also be used to examine the spindle dynamics, detect spindle morphology, predict chromosome misalignment, and perform spindle transfer.