In sexual reproduction, germ cells carry the task of passing genetic information to the next generation, which also constitutes the basis for the multiplication of life. The development of embryonic and adolescent male germ cells is crucial to the establishment of male fertility, however the pivotal development stages and regulatory mechanisms remain to be elucidated: (1) What the gene expression dynamics of primordial germ cells before colonization to genital ridge? (2) What are the key regulatory processes in the transition from mitotic to mitotic arrest state of male germ cells after colonization? (3) What is the biological significance of male germ cells entering the quiescence, and which factors or signal pathways drive this process? Answering the above questions will help clarify the gene expression regulation mechanism of male germ cells throughout the development, and have important guiding significance for our understanding, diagnosis and treatment of male infertility-related diseases.
Here, our study systematically elucidated the fate transition process and regulatory mechanism of male germ cells for the first time. It was found that the transitional PGCs are an important subset during the transition period, and revealed that the Notch signaling pathway plays important roles in the process of mitotic arrest. Using Helq knockout mouse proved that the embryonic germ cell identity transition is essential for the maintenance of male fertility. Finally, the conserved regulatory factors of human-mouse germ cells were identified through integrated data.
Establish a single-cell transcriptome map of the full-time development of mouse male germ cells
First, we used double transgenic fluorescent reporter mice (Blimp1-mVenus and Stella-ECFP, BVSC) to establish a transcriptome dataset containing 11,896 single cells in 28 developmental stages, with an average of 9413 genes and 302772 mRNAs detected per cell. Through dimensionality reduction and cluster analysis, 18 types of germ cells were identified, including specification PGCs, migrating PGCs, mitotic PGCs, and mitotic arrest PGC, etc.
Identification of new cell subpopulations and exploration of potential regulatory signals in the process of male germ cell fate transition
We analyzed the proliferation state and transcriptome characteristics of male germ cells after colonization and found a transitional cell type in the transition from mitotic to mitosis arrest state. They clustered independently and exhibit unique gene expression characteristics so that were defined as Transitional PGC. Subsequently, we used simultaneous immunofluorescence staining and RNAscope analysis to identify the existence of the above three cell subgroups in situ in the genital ridge. Bioinformatics analysis and verification experiments show that the transition of primordial germ cells from mitotic to mitotic arrest state is accompanied by a shift in cell cycle, pluripotency, and cell metabolism. More importantly, we identified a series of potentially functional regulatory factors during this transition, including: Ascl2, Hesx1, Hes1, Tgif1, and Sp5. We then revealed the key role of the Notch signaling pathway in initiating the mitotic arrest process through small molecule inhibition experiments in vivo. In addition, we used the Helq knockout mouse model to further prove that the correct regulation of the cell cycle is very important for the development of male germ cells.
Interspecies comparison of germ cell development between human and mouse
In order to analyze the conservation and difference of male germ cell development among species, we combined mouse single-cell transcriptome data with human fetal germ cell single-cell transcriptome data produced on the same platform. we found that the developmental trajectories of human and mouse primordial germ cells, prospermatogonia and spermatogonia are highly similar. Based on this, we further inferred a series of relatively conservative regulatory factors for mammalian germ cell development. It is worth mentioning that among the dynamically changing genes in the transition of mitotic PGC to mitotic arrest PGC, we found 435 genes that are closely related to diseases such as male infertility, non-obstructive azoospermia, and testicular germ cell tumors, which provides valuable clues for prenatal testing and disease diagnosis, etc.
In summary, this study reported the most comprehensive map of mammalian male germ cell development so far. The established high-quality data set provides accurate coordinates for the analysis of the regulatory mechanism of mammalian germ cell fate determination, and also provides a great basis for exploration of inducing germ cell in vitro and a theoretical reference for treating male infertility.