BACKGROUND Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. RESULTS The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. CONCLUSIONS This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.

中文翻译：

---

脊椎动物卵巢和睾丸中编码和长链非编码 RNA 的高灵敏度和高分辨率原位杂交。

背景编码和非编码RNA的亚细胞定位已经成为各种细胞类型和许多生物体中基因表达的主要调节机制。然而，能够以高灵敏度和高分辨率检测这些 RNA 的亚细胞分布的技术仍然有限，特别是在脊椎动物成人组织和器官中。在这项研究中，我们通过将基于酪胺信号放大 (TSA) 的原位杂交与石蜡结合，检测了斑马鱼和小鼠卵巢中无精子症样 (Dazl) 中编码 Pou5f1/Oct4、Mos、Cyclin B1 和 Deleted 的 mRNA 的表达和定位。可以在亚细胞水平上保存组织和器官的细胞形态的切片。此外，用相同的方法检查了长链非编码 RNA (lncRNA) lncRNA-HSVIII 在小鼠睾丸中的分布。结果发现编码Mos、Cyclin B1和Dazl的mRNAs组装成不同的颗粒，分布在斑马鱼和小鼠卵母细胞的不同亚细胞区域，表明这些mRNAs的保守和特异性调控。lncRNA-HSVIII首先在减数分裂细胞周期前期的精母细胞核中检测到，然后在圆形精子细胞的细胞质中发现，揭示了生殖细胞发育过程中lncRNA的表达模式。总的来说，本研究展示的原位杂交方法实现了对成人组织和器官单细胞中编码和非编码RNA在亚细胞水平上的精确分布模式的检测和比较。