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?

项目摘要 哺乳动物中的男性生育能力取决于睾丸中基础SSC库的形成。缺陷 SSC的形成或维持被认为是仅Sertoli细胞综合征（SCO）综合征的主要原因， 导致非刺激性无植物植物（NOA）和男性不育症。啮齿动物SSC来自Prospermatogonia 在生命的第一周中 繁殖场数十年。在这个项目中，我们将审问两个替代假设：1-该基础 SSC是预先确定的，并从表达独特转录组的前体亚群中出现 条形码（“预先确定”），或2- SSC是由Prospermatogonia诱导的，如此独特 从等值前体出现转录组（“选择”）。通过检验这些替代假设，我们将 在我们了解如何形成精子发生的基础上，解决了一个关键的差距。 初步研究的结果表明，P6 ID4-EGFP+精子表达基因表达 定义离散亚群的异质性，包括高度富集和耗尽的子群 基础SSC。我们定义了一个不同的SSC转录组条形码，该条形码是根据 单细胞RNA-seq分析揭示的独特基因表达签名对几乎纯的人群 SSC。该SSC条形码似乎也通过分离的男性生殖细胞的亚群来表达 来自胎儿和新生儿男性生殖细胞。最后，我们发现富集的人群未分化 精子症暴露于与鼠标精子的类似异质性，这可能使未来的识别 难以捉摸的人类SSC。这些结果表明SSC条形码是通过小鼠男性保守的 种系发育可能可用于广泛区分哺乳动物基础SSC。 我们提出了三个特定的目的，以测试替代性预先确定和选择假设 检查临时起源SSC条形码及其对SSC池形成的功能意义。 AIM 1将区分单个小鼠生殖细胞之间表达SSC条形码的细胞的出现与 胎儿晚期通过产后睾丸发育，并将这种表型的外观与SSC相关 通过移植功能。 AIM 2将确定是否需要表达此SSC条形码的胎儿生殖细胞 为了形成基础SSC池，使用小鼠的谱系消融和追踪方法。目标3意志 确定小鼠SSC条形码是否在较高的灵长类动物（狒狒和人类）中保存 它在小鼠中看到的主要相似之处中的暂时出现。 通过此提案，我们将通过单细胞分辨率检查基因表达并利用SSC 移植和谱系跟踪/消融，以提供SSC命运的关键功能读数。提议 研究将使我们能够在男性繁殖中解决一个关键问题 - 如何建立SSC的库？