Methods for the analysis of early oogenesis in Zebrafish
Methods for the analysis of early oogenesis in Zebrafish
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DOI：
10.1016/j.ydbio.2016.12.014
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发表时间：
2017-10-15
影响因子：
2.7
通讯作者：
Mullins, Mary C.
中科院分区：
文献类型：
--
作者：
Elkouby, Yaniv M.;Mullins, Mary C.
卵母细胞分化是一个高度动态和复杂的发育过程，其机制的理解促进了女性生殖、生育和卵巢癌生物学的发展。尽管斑马鱼模型具有许多特性，但它尚未被充分利用于早期卵母细胞分化和卵巢发育的研究。这部分是因为成年斑马鱼卵巢的特性使得获取早期卵母细胞在技术上具有挑战性。因此，这些阶段的特征一直缺乏，分析工具也不足。为了克服这些技术障碍，我们利用了斑马鱼幼鱼的卵巢，在那里可以很容易地发现大量早期卵母细胞，并且以可预测的方式发展。我们描述了卵母细胞分化和卵巢发育的早期阶段，并定义了准确的分期标准。我们进一步开发了定量显微镜、实时延时成像、卵巢培养和分离特定阶段卵母细胞进行生化分析的方案。这些方法最近为我们提供了一个前所未有的早期卵子发生的视角，使我们能够研究Balbiani体的形成，Balbiani体是一种通用的卵母细胞颗粒，与小鼠的卵母细胞存活有关，也是鱼和青蛙的卵母细胞和卵极性所必需的。尽管它具有巨大的发育意义，但对Bb的研究很少，两个多世纪以来，它是如何形成的，在任何物种中都是未知的。我们能够追踪巴尔比亚尼体的形成和卵母细胞的对称性破坏到减数分裂的开始。通过这项研究，我们揭示了卵母细胞中新的细胞骨架结构和特化细胞组织对分化的贡献。总之，幼斑马鱼卵巢是细胞和发育生物学研究的一个令人兴奋的模型。我们在本期《发育生物学》的一篇随附手稿中回顾了这些和其他脊椎动物卵子发生的最新进展。在这里，我们描述了我们在斑马鱼中开发的卵巢研究方案，包括所有的实验步骤，这些步骤将很容易地允许其他人复制这样的分析。这个幼鱼卵巢工具箱也有助于建立斑马鱼作为幼虫后发育阶段的模型。
Oocyte differentiation is a highly dynamic and intricate developmental process whose mechanistic understanding advances female reproduction, fertility, and ovarian cancer biology. Despite the many attributes of the zebrafish model, it has yet to be fully exploited for the investigation of early oocyte differentiation and ovarian development. This is partly because the properties of the adult zebrafish ovary make it technically challenging to access early stage oocytes. As a result, characterization of these stages has been lacking and tools for their analysis have been insufficient. To overcome these technical hurdles, we took advantage of the juvenile zebrafish ovary, where early stage oocytes can readily be found in high numbers and progress in a predictable manner. We characterized the earliest stages of oocyte differentiation and ovarian development and defined accurate staging criteria. We further developed protocols for quantitative microscopy, live time-lapse imaging, ovarian culture, and isolation of stage-specific oocytes for biochemical analysis. These methods have recently provided us with an unprecedented view of early oogenesis, allowing us to study formation of the Balbiani body, a universal oocyte granule that is associated with oocyte survival in mice and required for oocyte and egg polarity in fish and frogs. Despite its tremendous developmental significance, the Bb has been little investigated and how it forms was unknown in any species for over two centuries. We were able to trace Balbiani body formation and oocyte symmetry breaking to the onset of meiosis. Through this investigation we revealed novel cytoskeletal structures in oocytes and the contribution of specialized cellular organization to differentiation. Overall, the juvenile zebrafish ovary arises as an exciting model for studies of cell and developmental biology. We review these and other recent advances in vertebrate oogenesis in an accompanying manuscript in this issue of Developmental Biology. Here, we describe the protocols for ovarian investigation that we developed in the zebrafish, including all experimental steps that will easily allow others to reproduce such analysis. This juvenile ovary toolbox also contributes to establishing the zebrafish as a model for post-larval developmental stages.