Revealing cellular and molecular transitions in neonatal germ cell differentiation using single cell RNA sequencing

Neonatal germ cell development provides the foundation of spermatogenesis. However, a systematic understanding of this process is still limited. To resolve cellular and molecular heterogeneity in this process, we profiled single cell transcriptomes of undifferentiated germ cells from neonatal mouse testes and employed unbiased clustering and pseudotime ordering analysis to assign cells to distinct cell states in the developmental continuum. We defined the unique transcriptional programs underlying migratory capacity, resting cellular states and apoptosis regulation in transitional gonocytes. We also identified a subpopulation of primitive spermatogonia marked by CD87 (plasminogen activator, urokinase receptor), which exhibited a higher level of self-renewal gene expression and migration potential. We further revealed a differentiation-primed state within the undifferentiated compartment, in which elevated Oct4 expression correlates with lower expression of self-renewal pathway factors, higher Rarg expression, and enhanced retinoic acid responsiveness. Lastly, a knockdown experiment revealed the role of Oct4 in the regulation of gene expression related to the MAPK pathway and cell adhesion, which may contribute to stem cell differentiation. Our study thus provides novel insights into cellular and molecular regulation during early germ cell development.