PDF(Pubmed)
Abstract：
Mammalian oocytes accumulate more than ten thousand mRNAs, of which three to four thousand mRNAs are translationally repressed. The timings and sites of translational activation of these dormant mRNAs are crucial for promoting oocyte maturation and embryonic development. How these mRNAs are accumulated and distributed in oocytes is therefore a fundamental issue to be explored. A method that enables visualization of mRNA molecules with high resolution in a simple manner would be valuable for understanding how oocytes accumulate and regulate the dormant mRNAs. We have developed a highly sensitive whole-mount in situ hybridization method using in vitro-synthesized RNA probes and the tyramide signal amplification (TSA) system optimized for mouse oocytes and embryos. By using this method, Pou5f1/Oct4, Emi2, and cyclin B1 mRNAs were detected in immature oocytes and 2-cell stage embryos. Confocal microscopy showed that these mRNAs formed granular structures in the oocyte cytoplasm. The structures of Pou5f1/Oct4 and cyclin B1 mRNAs persisted in 2-cell stage embryos. Pou5f1/Oct4 RNA granules exhibited a solid-like property in immature oocytes and became liquid-like droplets in 2-cell stage embryos. Double-staining of cyclin B1 mRNA with Emi2 or Pou5f1/Oct4 mRNA revealed that these mRNAs were distributed as different RNA granules without overlapping each other and that the size of cyclin B1 RNA granules tended to be larger than that of Emi2 RNA granules. The structures and distribution patterns of these mRNAs were further analyzed by N-SIM super-resolution microscopy. This analysis revealed that the large-sized RNA granules consist of many small-sized granules, suggesting the accumulation and regulation of dormant mRNAs as basal-sized RNA granules. The method established in this study can easily visualize the structure and distribution of mRNAs accumulated in mammalian oocytes and embryos with high sensitivity and super-resolution. This method is useful for investigating the cellular and molecular mechanisms of translational control of mRNAs by which maturation and early developmental processes are promoted.
摘要：
哺乳动物卵母细胞积累了超过一万个mRNA，其中三到四千个mRNA被翻译抑制。这些休眠mRNA的翻译激活的时间和位点对于促进卵母细胞成熟和胚胎发育至关重要。因此，这些mRNA如何在卵母细胞中积累和分布是一个有待探索的基本问题。能够以简单的方式高分辨率可视化mRNA分子的方法对于理解卵母细胞如何积累和调节休眠的mRNA将是有价值的。我们使用体外合成的RNA探针和针对小鼠卵母细胞和胚胎优化的酪胺信号放大（TSA）系统，开发了一种高度敏感的全装原位杂交方法。通过使用此方法，在未成熟卵母细胞和2细胞期胚胎中检测到Pou5f1/Oct4，Emi2和细胞周期蛋白B1mRNA。共聚焦显微镜显示这些mRNA在卵母细胞细胞质中形成颗粒状结构。Pou5f1/Oct4和细胞周期蛋白B1mRNA的结构在2细胞期胚胎中持续存在。Pou5f1/Oct4RNA颗粒在未成熟卵母细胞中表现出固体样特性，并在2细胞期胚胎中变成液体样液滴。用Emi2或Pou5f1/Oct4mRNA对细胞周期蛋白B1mRNA进行双重染色表明，这些mRNA以不同的RNA颗粒分布，彼此不重叠，并且细胞周期蛋白B1RNA颗粒的大小倾向于大于Emi2RNA颗粒。通过N-SIM超分辨率显微镜进一步分析了这些mRNA的结构和分布模式。这项分析表明，大尺寸的RNA颗粒由许多小尺寸的颗粒组成，提示休眠mRNA作为基础大小的RNA颗粒的积累和调节。本研究建立的方法可以很容易地可视化哺乳动物卵母细胞和胚胎中积累的mRNA的结构和分布，具有高灵敏度和超分辨率。该方法可用于研究促进成熟和早期发育过程的mRNA翻译控制的细胞和分子机制。
