Origin and functional significance of the spermatogonial stem cell transcriptome barcode

项目总结 哺乳动物的雄性生育能力取决于睾丸中基本SSC池的形成。中的缺陷 SSCs的形成或维持被认为是仅支持细胞(SCO)综合征的主要原因， 结果导致非梗阻性无精子症(NOA)和男性不育。啮齿动物SSCs起源于成熟原细胞 然而，在生命的第一周，负责它们的指定的机制却躲避了雄性 几十年来一直在繁衍领域。在这个项目中，我们将询问两个替代假设：1-基本假设 SSCs是预先确定的，出现在表达独特转录组的前体亚群中 条形码(“预定”)，或2-SSCs是从成熟原细胞诱导而来的，因此是唯一的 转录本来自相同的前体(“选择”)。通过检验这些替代假设，我们将 解决我们在精子发生基础是如何形成的知识方面的一个关键差距。 初步研究结果表明，P6ID4-EGFP+精原细胞具有基因表达 定义离散亚种群的异质性，包括那些高度丰富和枯竭的亚种群 基础性SSC。我们定义了一个独特的SSC转录组条形码，它是根据 单细胞rna-seq分析揭示了近乎纯种人的独特基因表达特征。 SSCS。这种SSC条形码似乎也由分离的雄性生殖细胞亚群表达 来自胎儿和新生儿的男性生殖细胞。最后，我们发现，丰富的人类种群未分化 精原细胞表现出与小鼠精原细胞相似的异质性，这可能使未来的识别成为可能 难以捉摸的人类SSC。这些结果表明SSC条形码在雄性小鼠体内是保守的 生殖系发育可能被用于广泛区分哺乳动物的基础SSCs。 我们提出了三个具体的目标来检验交替的预先决定和选择假设，通过 考察了SSC条形码的时间起源及其对SSC池形成的功能意义。 目标1将区分表达SSC条形码的细胞在不同的小鼠生殖细胞中的出现 胎儿晚期至出生后早期睾丸发育及其与SSC的关系 通过移植来发挥作用。目标2将确定是否需要表达该SSC条形码的胎儿生殖细胞 利用谱系消融和示踪方法在小鼠体内形成基本的SSc池。目标3将 确定小鼠SSC条形码在高等灵长类动物(狒狒和人类)中是否保守，并询问是否 它在灵长类动物中的临时出现与在老鼠身上看到的相似。 在整个提案中，我们将使用单细胞分辨率检查基因表达，并利用SSC 移植和谱系追踪/消融，以提供SSC命运的关键功能读数。建议数 研究将使我们能够解决男性生殖中的一个关键问题--SSCs池是如何建立的？

PROJECT SUMMARY Male fertility in mammals depends on formation of a foundational SSC pool in the testis. Defects in formation or maintenance of SSCs are considered primary causes of Sertoli cell-only (SCO) syndrome, which results in non-obstructive azoospermia (NOA) and male infertility. Rodent SSCs arise from prospermatogonia in the first week of life, yet the mechanisms responsible for their specification have eluded the male reproduction field for decades. In this project, we will interrogate two alternate hypotheses: 1- that foundational SSCs are predetermined and emerge from a precursor subpopulation expressing a unique transcriptome barcode (“predetermination”), or 2- that SSCs are induced from prospermatogonia such that unique transcriptomes emerge from equipotent precursors (“selection”). By testing these alternate hypotheses, we will address a critical gap in our knowledge of how the foundation of spermatogenesis is formed. Results of preliminary studies demonstrated that P6 ID4-EGFP+ spermatogonia exhibit gene expression heterogeneity which defined discrete subpopulations, including those highly-enriched and depleted for foundational SSCs. We defined a distinct SSC transcriptome barcode which was identified based on the unique gene expression signature revealed by single-cell RNA-seq analyses of a population of nearly pure SSCs. This SSC barcode also appeared to also be expressed by a subpopulation of male germ cells isolated from fetal and neonatal male germ cells. Lastly, we found that enriched populations of human undifferentiated spermatogonia exhibited similar heterogeneity to mouse spermatogonia which could enable future discernment of the elusive human SSC. These results suggest that the SSC barcode is conserved through mouse male germline development could potentially be used to broadly distinguish mammalian foundational SSCs. We propose three Specific Aims to test the alternate predetermination and selection hypotheses by examining the temporal origin the SSC barcode and its functional significance to the formation of the SSC pool. Aim 1 will distinguish emergence of cells expressing the SSC barcode among individual mouse germ cells from late fetal through early postnatal testis development and correlate appearance of this phenotype with SSC function by transplant. Aim 2 will determine whether fetal germ cells expressing this SSC barcode are required for formation of the foundational SSC pool using lineage ablation and tracing methods in mice. Aim 3 will establish whether the mouse SSC barcode is conserved in higher primates (baboons and humans) and ask if its temporal emergence in the primate parallels that seen in the mouse. Throughout this proposal, we will examine gene expression with single-cell resolution and leverage SSC transplantation and lineage tracing/ablation to provide pivotal functional readouts of SSC fate. The proposed studies will allow us to address a key question in male reproduction– how is the pool of SSCs established?