Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development

Abstract

Abstract. Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which 'out-of focus blur' cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research. Key words: Embryo assessment, Full-term development, Live-cell imaging, Preimplantation embryo, Spinning-disk confocal microscopy (J. Reprod. Dev. 55: [343][344][345][346][347][348][349][350] 2009) ammalian preimplantation development is a process in which the union of two highly specialized gametes endows the resulting zygote with the potential to form undifferentiated cell types. This is followed by cell proliferation and the beginning of differentiation during the brief period leading up to implantation. In these processes, a number of cellular components and structures are regulated spatially and temporally, as seen in repeated cell division, cell cycle progression and genomic reprogramming via global epigenetic modifications [1]. Hence, many researchers have been attracted to these fundamental biological processes. However, the numbers of oocytes and embryos that can be collected are very limited, especially in mammals. Therefore, analysis of molecular mechanisms is hampered because of difficulties in conducting biochemical analyses on sufficient materials. Also, immunostaining methods ...

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