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Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate

ABSTRACT: Undifferentiated spermatogonia (uSPG), including spermatogonial stem cells, are essential for long-term spermatogenesis and hold therapeutic potential for treating male infertility. While rodent uSPG are well characterized, human uSPG remain poorly understood. Here, we screened antibodies against 21 proteins encoded by genes enriched in human uSPG subsets identified from single-cell RNA sequencing analysis. Among them, we identified FSD1 as a pan-uSPG cell-surface marker suitable for purifying the entire uSPG population. Transcriptomic profiling of FSD1+ uSPG uncovered candidate signaling pathways and transcriptional networks involved in uSPG development. PIWIL4 and EGR4 label primitive, quiescent uSPG, whereas PPP1R36 and NANOS3 mark proliferative uSPG subsets. PIWIL4+ and NANOS3+ cells do not overlap, highlighting distinct uSPG developmental states. We provide empirical evidence that RAS signaling regulates the transition between these states. Together, our study provides valuable tools for studying uSPG development and facilitating future translational advances.

ORGANISM(S): Homo sapiens

PROVIDER: GSE297876 | GEO | 2025/12/15

REPOSITORIES: GEO

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Project description:Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression (Ccnd1) to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network.Spermatogonial stem cells (SSCs) balance self-renewal and differentiation to ensure continuous sperm production in the testis. The transcription factor Zbtb16 (PLZF) supports undifferentiated SSC maintenance through partly unknown mechanisms. We combined genomics (RNA-seq and ChIP-seq) and genetic approaches to reveal multiple functions of Zbtb16 in juvenile mouse SSCs. Zbtb16-bound loci show a striking correlation with active promoters bearing H3K4me3 and the activator Sall4. Zbtb16 activates genes that support SSC self-renewal and cell cycle progression (e.g., Ccnd1) that help maintain undifferentiated SSC pools, including both self-renewing SSCs and transit-amplifying progenitors. Zbtb16 also attenuates certain genes, including meiotic genes and specific retrotransposons that confer genome instability. Notably, Zbtb16 genome localization and its impact on the transcriptome are dynamic, displaying mesenchymal gene targets in vivo, which are not maintained in cultured SSCs. Our data reveal dynamic roles for Zbtb16 in ensuring SSC identity, amplification, and maintenance in vivo.

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Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate

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