Origin and functional significance of the spermatogonial stem cell transcriptome barcode

精原干细胞转录组条形码的起源和功能意义

• 批准号: 9215401
• 负责人: Brian Peter Hermann
• 金额: $ 30.3万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9215401
• 关键词: Ablation; Address; Adult; Affect; Apoptosis; Appearance; Biological Assay; Cancer Patient; Cell Lineage; Cell physiology; Cells; Child; Contraceptive methods; Defect; Development; Disease; Etiology; Exhibits; Failure; Fertility; Foundations; Future; Gene Expression; Gene Expression Profile; Genes; Genetic; Germ Cells; Heterogeneity; Human; Individual; Infertility; Investigation; Knowledge; Life; Maintenance; Male Infertility; Mammals; Measures; Messenger RNA; Methods; Modeling; Molecular; Mus; Neonatal; Papio; Phenotype; Population; Population Heterogeneity; Primates; Radiation; Reproduction; Resolution; Rodent; Sertoli cell only syndrome; Spermatogenesis; Spermatogonia; Stem cell transplant; Stem cells; Testing; Testis; Translating; Transplantation; Undifferentiated; United States; aging population; base; cancer therapy; chemotherapy; differential expression; fetal; human male; male; man; men; novel; postnatal; prevent; prospective; reproductive; sperm cell; stem cell fate; stem cell fate specification; stemness; transcriptome; transcriptome sequencing

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?

项目概要 哺乳动物的雄性生育能力取决于睾丸中基础 SSC 池的形成。 SSC 的形成或维持被认为是纯支持细胞 (SCO) 综合征的主要原因， 导致非梗阻性无精子症 (NOA) 和男性不育症。 在生命的第一周，然而负责其规范的机制却让雄性无法识别 在这个项目中，我们将询问两个替代假设：1-基本假设。 SSC 是预先确定的，来自表达独特转录组的前体亚群 条形码（“预定”），或 2- SSCs 由原精原细胞诱导，因此具有独特的 转录组来自等位前体（“选择”）。通过测试这些替代假设，我们将。 解决了我们关于精子发生基础如何形成的知识中的一个关键差距。 初步研究结果表明P6 ID4-EGFP+精原细胞表现出基因表达 异质性定义了离散的亚群，包括那些高度富集和耗尽的亚群 我们定义了一个独特的 SSC 转录组条形码，该条形码是根据 通过对近乎纯的群体的单细胞 RNA-seq 分析揭示了独特的基因表达特征 这种 SSC 条形码似乎也由分离的雄性生殖细胞亚群表达。 最后，我们发现丰富的人类未分化群体。 精原细胞表现出与小鼠精原细胞相似的异质性，这可能有助于未来的辨别 这些结果表明，SSC 条形码在雄性小鼠中是保守的。 种系发育有可能用于广泛区分哺乳动物基础 SSC。 我们提出了三个具体目标来测试替代的预定和选择假设 检查 SSC 条形码的时间起源及其对 SSC 库形成的功能意义。 目标 1 将区分单个小鼠生殖细胞中表达 SSC 条形码的细胞的出现情况 胎儿晚期到产后早期睾丸发育，并将这种表型的外观与 SSC 相关联 目标 2 将确定是否需要表达该 SSC 条形码的胎儿生殖细胞。 Aim 3 将使用谱系消融和追踪方法来形成基础 SSC 库。 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出现时间与在小鼠中看到的相似。 在整个提案中，我们将以单细胞分辨率检查基因表达并利用 SSC 移植和谱系追踪/消融提供 SSC 命运的关键功能读数。 研究将使我们能够解决男性生殖中的一个关键问题——SSC库是如何建立的？