Deciphering mechanisms of oocyte development in the fish ovary still remain challenging, and a comprehensive overview of this process at the level of the organ is still needed. The recent development of optical tissue clearing methods has tremendously boosted the three-dimensional (3D) imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the nontoxic solvent-based ethyl cinnamate (ECi) method for efficient clearing and the Clear Unobstructed Brain Imaging Cocktails and Computational (CUBIC) method to enhance lipid removal and reduce nonspecific staining. The methyl green fluorescent dye was used to stain nuclei and delineate the follicular structures that include oocytes. Using this procedure (named CUBIC-ECi [C-ECi]), ovaries of both medaka and trout could be imaged in 3D and follicles analyzed. To our knowledge, this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-ECi method thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further developmental and morphological studies.

中文翻译：

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C-ECi：用于鱼类卵巢 3D 卵泡含量分析的立方-ECi 组合清除方法†。

鱼类卵巢中卵母细胞发育的破译机制仍然具有挑战性，仍然需要在器官水平上对该过程进行全面概述。光学组织清除方法的最新发展极大地促进了自然不透明的大尺寸生物样本的三维 (3D) 成像。然而，迄今为止，还没有报道试图清除在卵母细胞内积累了极高浓度脂质的鱼卵巢。为了应对这种卵巢特有的挑战，我们结合了两种现有的清除方法，用于有效清除的无毒溶剂型肉桂酸乙酯 (ECi) 方法和 Clear Unobstructed Brain Imaging鸡尾酒和计算 (CUBIC) 方法可增强脂质去除并减少非特异性染色。甲基绿荧光染料用于染色细胞核并描绘包括卵母细胞在内的卵泡结构。使用此程序（命名为 CUBIC-ECi [C-ECi]），青鳉和鳟鱼的卵巢可以在 3D 中成像并分析卵泡。据我们所知，这是第一个在 3D 中清除和成像鱼卵巢的过程。因此，C-ECi 方法提供了一种有趣的工具，可用于获取有关鱼类卵巢中卵泡含量的精确定量数据，并有望用于进一步的发育和形态学研究。