Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells’ identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development. Here, using a time-series single-cell RNA sequencing (scRNA-seq) strategy, we analyzed fetal germ cells (FGCs) and gonadal somatic cells in human embryos and fetuses. Clustering analysis of testes and ovaries revealed several novel cell subsets, including POU5F1+SPARC+ FGCs and KRT19+ somatic cells. Furthermore, our data indicated that the bone morphogenetic protein (BMP) signaling pathway plays cell type-specific and developmental stage-specific roles in testis development and promotes the gonocyte-to-spermatogonium transition (GST) in late-stage testicular mitotic arrest FGCs. Intriguingly, testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a stepwise manner. In our study, potential interactions between gonadal somatic cells were systematically explored and we identified cell type-specific developmental defects in both FGCs and gonadal somatic cells in a Turner syndrome embryo (45, XO). Our work provides a blueprint of the complex yet highly ordered development of and the interactions among human FGCs and gonadal somatic cells.

Gonadotropin releasing hormone (GnRH) plays an important role in the regulation of vertebrate reproduction. Studies have shown that immunization against GnRHa can induce sexually sterile tilapia. To explore the mechanism behind this, in this study, RNA-seq and data-independent acquisition (DIA) techniques were used to study the transcriptome and proteome of the gonad of tilapia immunized with GnRHa. 644 differentially expressed genes (80 upregulated and 564 downregulated) and 1150 differentially expressed proteins (351 upregulated and 799 downregulated) were identified. There were 209 genes with consistent differential expression patterns in the transcriptomic and proteomic analyses, of which 9 were upregulated and 200 downregulated, indicating that the gonad gene expression was inhibited by GnRHa immunization. The downregulated genes were particularly involved in the functions of single-organism process, binding, cellular process, metabolic process and catalytic activity, and associated with the pathways including ECM-receptor interaction, focal adhesion, cardiac muscle contraction and oxidative phosphorylation. The expression of six differentially expressed genes involved in the GnRH signaling pathway was all downregulated. In addition, several important functional genes related to gonadal development after GnRHa immunization were screened. This study confirmed the expression of corresponding genes was affected by GnRHa on the gonad development in tilapia at the molecular level, and laid a foundation for elucidating the mechanism of GnRHa immunization.