Choi Hyeon Jeong, Jung Kyung Min, Rengaraj Deivendran, Lee Kyung Youn, Yoo Eunhui, Kim Tae Hyun, Han Jae Yong
Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, South Korea.
Department of Animal Science, Pennsylvania State University, State College, PA, 16801, USA.
J Anim Sci Biotechnol. 2022 Jun 6;13(1):64. doi: 10.1186/s40104-022-00712-4.
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.
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都存在，尽管进入或退出静止期的时间有所不同。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，鸡的有丝分裂停滞尚未得到充分研究。
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态。通过差异表达基因分析和K均值聚类分析不同发育阶段（E12、E16和孵化时）的细胞，我们发现代谢和信号通路受到调控，并且在有丝分裂停滞期间表观基因组发生了重编程。特别是，我们发现组蛋白H3K9和H3K14乙酰化（由HDAC2介导）以及DNA去甲基化（由DNMT3B和HELLS介导）导致了转录允许的染色质状态。此外，我们发现有丝分裂停滞开始后，全基因组DNA去甲基化逐渐发生，这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不同。DNA低甲基化在孵化后持续存在，3周后甲基化缓慢重新建立。
我们发现了有丝分裂停滞的鸡原生殖细胞独特的表观遗传重编程时间表以及孵化后延长的低甲基化状态。这将为理解几种尚未明确描述此过程的物种的生殖细胞表观遗传调控过程提供基础。我们关于原生殖细胞性别特异性分化相关生物学过程的发现有助于更深入地研究体外生殖系发育。
