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

ABSTRACT: Spermatogonial stem cells (SSCs) are essential for long-term spermatogenesis and hold therapeutic potential for treating male infertility. While rodent SSCs are well characterized, human SSCs remain poorly understood. Here, we screen antibodies against proteins encoded by genes enriched in specific subsets of human undifferentiated spermatogonia (uSPG) identified by single-cell RNA sequencing. We characterize four markers labeling distinct uSPG subsets: PIWIL4 marks primitive, quiescent uSPG; EGR4 marks uSPG at a proliferative crossroads; and PPP1R36 and NANOS3 label distinct proliferative subsets poised for differentiation. The most and least advanced subsets-PIWIL4+ and NANOS3+ cells-do not overlap. Comparative transcriptomics uncover candidate pathways involved in uSPG fate transitions, including RAS signaling. Using FSD1, a pan-uSPG cell-surface marker identified here, we purify the entire uSPG population and demonstrate that RAS signaling maintains the primitive uSPG state. These findings provide a framework for human uSPG identity, with broad implications for reproductive biology and regenerative medicine.

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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