Germ cell mitotic arrest is conserved in many vertebrates, including birds, although the time of entry or exit into quiescence phase differs. Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch. However, mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage. We performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens. Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages (E12, E16, and hatch), we found that metabolic and signaling pathways were regulated, and that the epigenome was reprogrammed during mitotic arrest. In particular, we found that histone H3K9 and H3K14 acetylation (by HDAC2) and DNA demethylation (by DNMT3B and HELLS) led to a transcriptionally permissive chromatin state. Furthermore, we found that global DNA demethylation occurred gradually after the onset of mitotic arrest, indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome. DNA hypomethylation persisted after hatching, and methylation was slowly re-established 3 weeks later. We found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching. This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described. Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

中文翻译：

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用 DAZL::GFP 对有丝分裂停止的精原细胞进行单细胞 RNA 测序并揭示鸡独特的表观遗传重编程

生殖细胞有丝分裂停滞在包括鸟类在内的许多脊椎动物中是保守的，尽管进入或退出静止期的时间不同。有丝分裂停滞对于雄性生殖细胞正常分化为精原细胞至关重要，并且伴随着从胚胎发育到孵化后的表观遗传重编程和减数分裂抑制。然而，由于难以从相关发育阶段获得纯生殖细胞，因此在鸡中有丝分裂停滞没有得到很好的研究。我们进行了单细胞 RNA 测序，以研究 DAZL::GFP 鸡有丝分裂停滞期间雄性生殖细胞的转录动力学。使用差异表达基因分析和 K-means 聚类分析处于不同发育阶段（E12、E16 和孵化）的细胞，我们发现代谢和信号通路受到调节，并且表观基因组在有丝分裂停滞期间被重新编程。特别是，我们发现组蛋白 H3K9 和 H3K14 乙酰化（通过 HDAC2）和 DNA 去甲基化（通过 DNMT3B 和 HELLS）导致转录许可的染色质状态。此外，我们发现全球 DNA 去甲基化在有丝分裂停滞开始后逐渐发生，表明鸡基因组的表观遗传重编程时间表与哺乳动物基因组的不同。孵化后DNA低甲基化持续存在，3周后甲基化缓慢重建。我们发现了有丝分裂停滞的鸡精原细胞和孵化后延长的低甲基化的独特表观遗传重编程时间表。这将为理解几个物种的生殖细胞表观遗传调控过程提供基础，而这些物种的这一过程没有被清楚地描述。我们对与精原细胞性别特异性分化相关的生物学过程的研究结果有助于更详细地研究体外生殖系发育。