High Resolution Transcriptome Analysis of the Primate Testis

灵长类动物睾丸的高分辨率转录组分析

• 批准号: 10261389
• 负责人: Sarah Munyoki
• 金额: $ 3.26万
• 依托单位: MAGEE-WOMEN'S RES INST AND FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-23 至 2022-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10261389
• 关键词: Adult; Affect; Apoptosis; Assisted Reproductive Technology; Biology; Biopsy; Cancer Etiology; Cell Culture Techniques; Cell surface; Cells; Chemotherapy and/or radiation; Clinic; Coupled; Couples; Cryopreservation; Data; Data Set; Databases; Development; Dimensions; Endocrine; Ensure; Equilibrium; Erinaceidae; FGF2 gene; FMR1; Fertility; Fibroblast Growth Factor; Flow Cytometry; Fluorescence-Activated Cell Sorting; Foundations; Freezing; Future; GPC3 gene; Gene Expression; Genes; Genetic Transcription; Germ Cells; Goals; Growth Factor; Homeostasis; Human; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Infertility; Knowledge; Life; Life Style; Macaca mulatta; Male Infertility; Mediating; Metabolism; Methods; Molecular; Molecular Abnormality; Monkeys; Mus; Natural regeneration; Neuroglia; Nuclear; Nude Mice; Ontology; Paracrine Communication; Patients; Pattern; Population; Primates; Production; Proto-Oncogene Proteins c-akt; Research; Resolution; Rodent; Secondary to; Signal Pathway; Signal Transduction; Somatic Cell; Spermatogenesis; Spermatogonia; Stains; Stem Cell Development; Stem cell transplant; Surface; System; TCF3 gene; Technology; Testicular Tissue; Testing; Testis; Tissues; Transplantation; Undifferentiated; United States; Xenograft procedure; aged; base; boys; cancer genetics; cancer therapy; candidate marker; career; cell type; chemotherapy; differential expression; improved; in vivo; infertility treatment; insight; male; men; neurotrophic factor; novel; novel marker; practical application; prepuberty; progenitor; protein biomarkers; receptor; reproductive; secondary infertility; self-renewal; single-cell RNA sequencing; sperm cell; sperm cryopreservation; stem; stem cell biology; stem cell growth; stem cell niche; stem cell proliferation; stem cell self renewal; stem cell therapy; stem cells; tool; transcriptome; transcriptomics

ABSTRACT Infertility is a prevalent condition that affects 15% of couples globally, with male factor infertility contributing to 50% of cases. Assisted reproductive technologies (ARTs), fertility treatments and changes in lifestyle are helping infertile men achieve their reproductive goals. However, these therapies are dependent on the infertile male producing at least a few mature sperm which is impossible in many cases such as infertility secondary to cancer treatment or genetic abnormalities. Testicular biopsies containing spermatogonial stem cells (SSCs), can be obtained from these men and cryopreserved for experimental stem cell-based therapies for infertility. SSCs balance self-renewal and differentiation to ensure continuous sperm production throughout an adult males reproductive life. But the molecular mechanisms governing this balance are still not well defined particularly, in higher primates. Identifying the protein markers of human and monkey SSCs as well as the signaling pathways regulating their self-renewal and proliferation, will facilitate the development of efficient, effective and safe SSC-based therapies for the treatment of male infertility. We performed high throughput, unbiased, single-cell RNA-sequencing of healthy adult primate (human and rhesus macaque) testicular tissue, generating ~33,800 single cell transcriptomes. Clustering coupled with the analysis of the expression patterns of validated marker genes, have identified most of the known cell types of the primate testis. Based on preliminary data, I hypothesize that primate single cell transcriptomic data will reveal spermatogonial markers/niche signaling pathways that can be exploited to isolate and enrich primate SSCs (pSSCs) and expand them in culture. We have identified novel genes CDK17, GPX1, MORC1, GPC4 and GPC3, DNAJB6, MAGEB2, FMR1, TCF3 as potential markers stem/progenitor spermatogonia that are conserved in human and monkey. These genes are implicated in signaling pathways (i.e. WNT, BMP, FGF) that have been associated with regulating rodent SSC self-renewal and proliferation. I will validate the expression of our candidate markers by immunohistochemistry and colorimetric staining, to correlate the expression of these genes with classic descriptions of nuclear staining intensity of Adark and Apale spermatogonia. Cell surface markers like GPC4, GPC3 and TSPAN33 may be used to enrich SSCs. We will investigate this possibility through immunohistochemistry, flow cytometry analysis and SSC transplantation to quantify transplantable stem cell activity in marker positive cell populations. I will also use the primate scRNAseq data to determine niche growth factor signals influencing pSSC self-renewal and proliferation. This will provide candidate growth factors to test in primate SSC culture to establish conditions that promote their long-term propagation in vitro. Our single cell data may contribute an improved understanding of primate SSCs biology and may enable stem cell applications in the male infertility clinic. Completion of this project will greatly expand my scientific and technical knowledge, giving me the strong foundation I need to have a successful research career.

抽象的 不孕不育是一种普遍存在的疾病，影响着全球 15% 的夫妇，其中男性因素不孕症导致 50%的情况。辅助生殖技术 (ART)、生育治疗和生活方式的改变 帮助不育男性实现生育目标。然而，这些疗法依赖于不孕症患者 男性至少产生一些成熟的精子，这在许多情况下是不可能的，例如继发性不孕症 癌症治疗或基因异常。含有精原干细胞（SSC）的睾丸活检， 可以从这些男性身上获得并冷冻保存，用于基于干细胞的不孕不育实验疗法。 SSC 平衡自我更新和分化，以确保在整个成年过程中持续产生精子 男性的生殖生活。但控制这种平衡的分子机制仍然没有明确定义 尤其是高等灵长类动物。鉴定人和猴 SSC 的蛋白质标记以及 调节其自我更新和增殖的信号通路将促进高效、 有效且安全的基于 SSC 的疗法用于治疗男性不育症。我们执行了高吞吐量， 对健康成年灵长类动物（人类和恒河猴）睾丸组织进行无偏倚的单细胞 RNA 测序， 生成约 33,800 个单细胞转录组。聚类与表达模式分析相结合 经过验证的标记基因，已经鉴定了灵长类动物睾丸的大多数已知细胞类型。基于 初步数据，我假设灵长类单细胞转录组数据将揭示精原细胞 可用于分离和富集灵长类 SSC (pSSC) 的标记/利基信号通路 在文化中扩展它们。我们已经鉴定出新基因 CDK17、GPX1、MORC1、GPC4 和 GPC3、DNAJB6、 MAGEB2、FMR1、TCF3 作为人类精原细胞中保守的干细胞/祖细胞潜在标记物 猴。这些基因涉及与相关的信号通路（即 WNT、BMP、FGF） 具有调节啮齿动物SSC自我更新和增殖的作用。我将验证我们候选人的表达 通过免疫组织化学和比色染色进行标记，将这些基因的表达与 Adark 和 Apale 精原细胞核染色强度的经典描述。细胞表面标记如 GPC4、GPC3 和 TSPAN33 可用于富集 SSC。我们将通过以下方式调查这种可能性 免疫组织化学、流式细胞术分析和 SSC 移植来量化可移植干细胞 标记阳性细胞群中的活性。我还将使用灵长类动物 scRNAseq 数据来确定利基 生长因子信号影响 pSSC 自我更新和增殖。这将为候选人提供成长因素 在灵长类 SSC 培养物中进行测试，以建立促进其体外长期繁殖的条件。我们的 单细胞数据可能有助于加深对灵长类 SSC 生物学的理解，并可能使干细胞成为可能 在男性不育症临床中的应用。这个项目的完成将极大地拓展我的科学和 技术知识为我提供了成功的研究生涯所需的坚实基础。

项目成果

Perspectives: Methods for Evaluating Primate Spermatogonial Stem Cells.

观点：灵长类精原干细胞的评估方法。

• DOI: 10.1007/978-1-0716-3139-3_18
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Munyoki,SarahK;Orwig,KyleE
• 通讯作者: Orwig,KyleE

Single-Cell RNA Sequencing of Human, Macaque, and Mouse Testes Uncovers Conserved and Divergent Features of Mammalian Spermatogenesis.

• DOI: 10.1016/j.devcel.2020.05.010
• 发表时间: 2020-08-24
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Shami AN;Zheng X;Munyoki SK;Ma Q;Manske GL;Green CD;Sukhwani M;Orwig KE;Li JZ;Hammoud SS
• 通讯作者: Hammoud SS

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

• DOI: 10.1007/s00439-021-02287-y
• 发表时间: 2021-08
• 期刊: Human genetics
• 影响因子: 5.3
• 作者: Hardy JJ;Wyrwoll MJ;Mcfadden W;Malcher A;Rotte N;Pollock NC;Munyoki S;Veroli MV;Houston BJ;Xavier MJ;Kasak L;Punab M;Laan M;Kliesch S;Schlegel P;Jaffe T;Hwang K;Vukina J;Brieño-Enríquez MA;Orwig K;Yanowitz J;Buszczak M;Veltman JA;Oud M;Nagirnaja L;Olszewska M;O'Bryan MK;Conrad DF;Kurpisz M;Tüttelmann F;Yatsenko AN;GEMINI Consortium
• 通讯作者: GEMINI Consortium