Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

Hyeon Jeong Choi; Kyung Min Jung; Deivendran Rengaraj; Kyung Youn Lee; Eunhui Yoo; Tae Hyun Kim; Jae Yong Han

doi:10.1186/s40104-022-00712-4

Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

J Anim Sci Biotechnol. 2022 Jun 6;13(1):64. doi: 10.1186/s40104-022-00712-4.

Authors

Hyeon Jeong Choi¹, Kyung Min Jung¹, Deivendran Rengaraj¹, Kyung Youn Lee¹, Eunhui Yoo¹, Tae Hyun Kim², Jae Yong Han³

Affiliations

¹ Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, South Korea.
² Department of Animal Science, Pennsylvania State University, State College, PA, 16801, USA.
³ Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, South Korea. jaehan@snu.ac.kr.

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: DAZL::GFP chickens; DNA methylation; Epigenetic reprogramming; Histone acetylation; Male germ cell; Mitotic arrest; Prospermatogonia; Single-cell RNA sequencing.

Grants and funding

2015R1A3A2033826/National Research Foundation of Korea