Three-dimensional evaluation of murine ovarian follicles using a modified CUBIC tissue clearing method.
Three-dimensional evaluation of murine ovarian follicles using a modified CUBIC tissue clearing method.
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DOI：
10.1186/s12958-018-0381-7
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发表时间：
2018-08-02
期刊：
影响因子：
--
通讯作者：
Fujiwara H
中科院分区：
文献类型：
--
作者：
Kagami K;Shinmyo Y;Ono M;Kawasaki H;Fujiwara H
最近，我们证明了三维（3D）定位的小鼠滋养层巨细胞在妊娠子宫中使用一个修改后的清晰的无障碍脑成像鸡尾酒和计算分析（CUBIC）的组织清除方法和杂交构建的巨细胞病毒增强子融合到鸡β-肌动蛋白启动子（CAG）共轭增强绿色荧光蛋白（EGFP）转基因小鼠。在这项研究中，我们应用这种方法来获得一个透明的整体图像的卵巢和观察的三维定位个别卵母细胞在发育中的卵泡。从增强型绿色荧光蛋白转基因小鼠中获得卵巢样本，并用碘化丙啶（PI）和CUBIC处理进行核染色。分别通过光片显微镜和计算机程序检测双荧光信号（绿色和红色）并随后重建整个卵巢的3D图像。用CUBIC法观察卵巢透明，PI灌注法对卵泡组成细胞的每个细胞核进行均匀荧光染色。相反，卵母细胞中的EGFP信号很强，而周围颗粒细胞的信号很弱。卵母细胞、颗粒细胞和卵泡膜间质细胞之间EGFP表达的这些信号差异产生了生长卵泡的对比度良好的图像，提供了单个卵母细胞3D定位的清晰信息。结果表明，该方法是分析整个卵巢卵泡三维结构的有效方法之一。本文的在线版本（10.1186/s12958-018-0381-7）包含补充材料，可供授权用户使用。
Recently, we demonstrated the three-dimensional (3D) localization of murine trophoblast giant cells in the pregnant uterus using a modified Clear Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) tissue-clearing method and hybrid construct consisting of the cytomegalovirus enhancer fused to the chicken beta-actin promoter (CAG) conjugated enhanced green fluorescent protein (EGFP) transgenic mice. In this study, we applied this method to obtain a transparent whole-image of the ovary and observed the 3D localization of individual oocytes in the developing follicles. Ovarian samples were obtained from EGFP transgenic mice and subjected to nuclear staining with propidium iodide (PI) and CUBIC treatment. The detection of double fluorescence signals (green and red) and subsequent reconstruction of 3D images of the whole ovary were performed by light-sheet microscopy and computer programs, respectively. The ovary became transparent using the CUBIC method and each nucleus of the follicle component cells was uniformly fluoro-stained by PI perfusion. In contrast, EGFP signals were strong in oocytes, whereas those of surrounding granulosa cells were faint. These signal differences in EGFP expression among oocytes, granulosa cells, and theca-interstitial cells produce well-contrasted images of the growing follicles, providing clear information of the 3D localization of individual oocytes. These results indicate that this procedure is one of the effective approaches to analyze the 3D structure of follicles in the whole ovary. The online version of this article (10.1186/s12958-018-0381-7) contains supplementary material, which is available to authorized users.
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影响因子：
64.8
作者：
通讯作者：
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影响因子：
4.6
作者：
Kagami K;Shinmyo Y;Ono M;Kawasaki H;Fujiwara H
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