Meng Lan, Xu Caoling, Cao Yuzhu, Wu Limin, Zhu Yuzhang, Zou Jiaqi, Uddin Islam, Zafar Iqra, Muhammad Azhar, Xing Xuemei, Jin Ren-Tao, He Li, Liu Hongbin, Li Wenqing, Bao Jianqiang
Center for Reproduction and Genetics, Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230001, China.
Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Hefei National Laboratory for Physical Sciences at Microscale, Biomedical Sciences and Health Laboratory of Anhui Province, University of Science and Technology of China (USTC), Hefei, Anhui, China.
Cell Death Differ. 2025 Apr 23. doi: 10.1038/s41418-025-01495-7.
A large resource of epitope-tagged and Cre/CreERT2-expressing mouse models are available for studying germ granules and germline development. Germ granules are proteinaceous, membraneless organelles (MLO) involved in germ cell differentiation and maturation; however, their protein and RNA transcript constituents, as well as their functional mechanisms remain incompletely understood. Herein, we generated a versatile germline mouse model through combinatorially tagging DDX4 to enable simultaneous expression of three cistronic coding products (C-terminally tagged DDX4 - DDX4, EGFP, and CreERT2) under the control of the endogenous Ddx4 promoter. By leveraging the high-affinity HA tag, we optimized an efficient workflow to purify germ granules (Chromatoid body, CB) from spermatids, and characterized their protein and RNA transcript composition. Moreover, we explored and ascertained that DDX4-mediated, phase-separation dependent CB integrity is functionally important for recruiting distinctive long RNA transcripts and for the biogenesis of pachytene- and TE-derived piRNAs. Together, our study generated a versatile germline mouse model with a multiplicity of applications for germline study, and provided mechanistic insights into germline development as dictated by germ granules.
有大量带有表位标签并表达Cre/CreERT2的小鼠模型资源可用于研究生殖颗粒和生殖系发育。生殖颗粒是参与生殖细胞分化和成熟的蛋白质性无膜细胞器（MLO）；然而，它们的蛋白质和RNA转录本成分以及功能机制仍未完全了解。在此，我们通过组合标记DDX4生成了一种通用的生殖系小鼠模型，以在内源性Ddx4启动子的控制下同时表达三种顺反子编码产物（C端标记的DDX4 - DDX4、EGFP和CreERT2）。通过利用高亲和力HA标签，我们优化了一种从精子细胞中纯化生殖颗粒（拟染色体，CB）的高效工作流程，并对其蛋白质和RNA转录本组成进行了表征。此外，我们探索并确定DDX4介导的、相分离依赖的CB完整性对于募集独特的长RNA转录本以及粗线期和TE衍生的piRNA的生物发生在功能上很重要。总之，我们的研究生成了一种具有多种生殖系研究应用的通用生殖系小鼠模型，并为生殖颗粒所决定的生殖系发育提供了机制性见解。
