■通过卵母细胞去核制备受体细胞质体是动物克隆和人类辅助生殖技术的基本任务。飞秒激光是一种精确和低侵入性的卵母细胞摘除工具,它应该是传统的微针穿刺摘除术的适当替代方法。然而,直到最近,激光去核仅在应用荧光染料的情况下进行。
■这项工作旨在(1)在不应用荧光染料的情况下实现飞秒激光卵母细胞摘除和(2)研究激光破坏染色体对纺锤体结构和动力学的影响。
■我们应用偏振光显微镜进行纺锤体可视化,并用1033nm飞秒激光进行了无染色的小鼠和人类卵母细胞摘除。此外,我们通过共聚焦显微镜研究了中期板消除后纺锤体的转化。
■我们证明了在不施加荧光染料的情况下,通过1033nm飞秒激光辐射使小鼠和人卵母细胞中的中期板失活的基本可能性。主轴区域的辐照,通过偏振光显微镜观察,导致部分或完全的中期板破坏,但避免了微管损伤。中期平板消除后,重新组织了主轴,然而,这不是完全解聚。
■这种受体细胞质体制备的方法有望用于动物克隆和辅助生殖技术。
UNASSIGNED: Preparation of a recipient cytoplast by oocyte
enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte
enucleation, and it should be an appropriate alternative to traditional
enucleation by a microneedle aspiration. However, until recently, the laser
enucleation was performed only with applying a fluorescent dye.
UNASSIGNED: This work is aimed to (1) achieve femtosecond laser oocyte
enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle.
UNASSIGNED: We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy.
UNASSIGNED: We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization.
UNASSIGNED: This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.