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

Abstract

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.

Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis

“…In the postnatal mouse testis, prospermatogonia give rise to spermatogonia, exhausting the prospermatogonia population by postnatal day 5 (P5) ( Drumond et al., 2011 ; Kluin et al., 1982 ; Yoshida et al., 2006 ). By P6-8, distinct spermatogonial subtypes (SSC, progenitor, differentiating) can be distinguished on the basis of specific marker proteins ( Buaas et al., 2004 ; Chan et al., 2014 ; Valli et al., 2014 ), lineage tracing ( Hara et al., 2014 ; Sun et al., 2015 ), bulk ( Helsel et al., 2017 ), and single-cell (sc) ( Chen et al., 2018 ; Ernst et al., 2019 ; Green et al., 2018 ; Grive et al., 2019 ; Guo et al., 2018 ; Hermann et al., 2018 ; Jung et al., 2019 ; Law et al., 2019 ; Liao et al., 2019 ; Sohni et al., 2019 ; Velte et al., 2019 ) RNA-sequencing (RNA-seq), or transplantation analysis ( McLaren, 2003 ; Shinohara et al., 2000 ). SSCs retain regenerative capacity and divide to either self-renew or generate progenitors that lose regenerative capacity as they become primed to initiate spermatogenic differentiation giving rise to differentiating spermatogonia ( Drumond et al., 2011 ; Kluin et al., 1982 ; Yang et al., 2013 ).…”

Contribution of Single-Cell Transcriptomics to the Characterization of Human Spermatogonial Stem Cells: Toward an Application in Male Fertility Regenerative Medicine?

“…Many scRNAseq experiments have been conducted on murine testicular cells [8,9,10,11,12,13,14,15,16,17,18,19,97]. Green and Wang’s works, both of which were published in 2018, mapped the gene expression dynamics of adult germ cell maturation in mice and humans, respectively [9,19].…”

“…In addition to the many scRNAseq studies on spermatogenesis in neonatal, prepubertal, and adult mice [7,8,9,10,11,12,13,14,15,16,17,18], which provided unbiased classifications of spermatogonial stem cell (SSC) pools and differentiation states, an atlas of human spermatogenesis at three different developmental stages (neonatal, prepubertal, and adult) has also been described recently thanks to this method [12,19,20,21,22,23]. The atlas reveals the striking heterogeneity among testicular somatic and germinal cell populations and assesses the identity of human SSC (hSSC) and spermatogonial progenitors.…”

scATAC-Seq reveals epigenetic heterogeneity associated with an EMT-like process in male germline stem cells and its regulation by G9a

“…For instance, Cxcr4 and Foxc2 promote migration and invasiveness through EMT in different types of cancer [14][15][16][17][18] . CD87 (urokinase-type plasminogen activator (uPA) receptor/uPAR), a marker for neonatal undifferentiated spermatogonia, can be regulated by TGFβ and participates in tumor invasion and metastasis [19,20] . Recent whole transcriptome analysis also confirmed that THY1+ subpopulations of neonatal germ cells were mesenchymal-like due to higher levels of EMT genes ( Vim , Zeb2 ), while the ID4+ population (SSC-enriched) expressed lower levels of EMT genes [21] .…”

“…To address this knowledge gap, we searched for potential candidates from our previous single cell RNA-seq study on neonatal undifferentiated germ cells. We found Euchromatic histone-lysine N-methyltransferase 2 (EHMT2), also known as G9a, particularly interesting as it is one of the transcriptional regulators upregulated in differentiation-primed spermatogonia ( Gfra1 −/ Kit − cells) [19] . Moreover, neonatal differentiation-primed germ cells expressed higher levels of mesenchymal markers (e.g.…”