Proper development of fetal germ cells (FGCs) is vital for the precise transmission of genetic and epigenetic information through generations. The transcriptional landscapes of human FGC development have been revealed; however, the epigenetic reprogramming process of FGCs remains elusive. Here, we profiled the genome-wide DNA methylation and chromatin accessibility of human FGCs at different phases as well as gonadal niche cells at single-cell resolution. First, we found that DNA methylation levels of FGCs changed in a temporal manner, whereas FGCs at different phases in the same embryo exhibited comparable DNA methylation levels and patterns. Second, we revealed the phase-specific chromatin accessibility signatures at the promoter regions of a large set of critical transcription factors and signaling pathway genes. We also identified potential distal regulatory elements including enhancers in FGCs. Third, compared with other hominid-specific retrotransposons, SVA_D might have a broad spectrum of binding capacity for transcription factors, including SOX15 and SOX17. Finally, using an in vitro culture system of human FGCs, we showed that the BMP signaling pathway promoted the cell proliferation of FGCs, and regulated the WNT signaling pathway by orchestrating the chromatin accessibility of its ligand genes. Our single-cell epigenomic atlas and functional assays provide valuable insights for understanding the strongly heterogeneous, unsynchronized, yet highly robust nature of human germ cell development.

Round spermatid injection (ROSI) technique holds great promise for clinical treatment of a proportion of infertile men. However, the compromised developmental potential of ROSI embryos largely limits the clinical application, and the mechanisms are not fully understood. Here, we describe the transcriptome, chromatin accessibility, and DNA methylation landscapes of mouse ROSI embryos derived from early-stage round spermatids using a single-cell multiomics sequencing approach. By interrogating these data, we identify the reprogramming defects in ROSI embryos at the pronuclear stages, which are mainly associated with the misexpression of a cohort of minor zygotic genome activation genes. We screen a small compound, A366, that can significantly increase the developmental potential of ROSI embryos, in which A366 can partially overcome the reprogramming defects by amending the epigenetic and transcriptomic states. Collectively, our study uncovers the reprogramming defects in ROSI embryos for understanding the mechanisms underlying compromised developmental potential and offers an avenue for ROSI technique optimization.

Long non-coding RNAs (lncRNAs) are emerging as a critical regulator in controlling the expression level of genes involved in cell differentiation and development. Primordial follicle activation (PFA) is the first step for follicle maturation, and excessive PFA results in premature ovarian insufficiency (POI). However, the correlation between lncRNA and cell differentiation was largely unknown, especially during PFA. In this study, we observed the expression level of lncRNA was more specific than protein-coding genes in both follicles and granulosa cells, suggesting lncRNA might play a crucial role in follicle development. Hence, a systematical framework was needed to infer the functions of lncRNAs during PFA. Additionally, an increasing number of studies indicate that the subpathway is more precise in reflecting biological processes than the entire pathway. Given the complex expression patterns of lncRNA target genes, target genes were further clustered based on their expression similarity and classification performance to reveal the activated/inhibited gene modules, which intuitively illustrated the diversity of lncRNA regulation. Moreover, the knockdown of SBF2-AS1 in the A549 cell line and ZFAS1 in the SK-Hep1 cell line further validated the function of SBF2-AS1 in regulating the Hippo signaling subpathway and ZFAS1 in the cell cycle subpathway. Overall, our findings demonstrated the importance of subpathway analysis in uncovering the functions of lncRNAs during PFA, and paved new avenues for future lncRNA-associated research.