High Resolution Transcriptome Analysis of the Primate Testis

摘要 不孕症是一种流行的疾病，影响着全球15%的夫妇，其中男性因素不孕是导致 50%的病例。辅助生殖技术(ARTS)、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，这些治疗方法依赖于不孕症患者。 男性至少产生几个成熟精子，这在许多情况下是不可能的，如继发性不育症 癌症治疗或基因异常。含有精原干细胞(SSCs)的睾丸活检， 可以从这些人身上获得并冷冻保存，用于实验性的基于干细胞的不孕症治疗。 SSCS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。但支配这种平衡的分子机制仍然没有很好地确定。 尤其是在高等灵长类动物中。鉴定人和猴SSCs的蛋白质标志物以及 调节其自我更新和增殖的信号通路，将促进高效、 以SSc为基础的治疗男性不育的有效和安全的方法。我们实现了高吞吐量， 健康成年灵长类动物(人和猕猴)睾丸组织的无偏单细胞RNA测序， 产生约33,800个单细胞转录本。结合表达模式分析的聚类 已经确定了灵长类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数据将揭示精原细胞 可用于分离和丰富灵长类SSCs(PSSCs)和 在文化上扩展它们。我们已经发现了新基因CDK17、GPX1、MORC1、GPC4和GPC3、DNAJB6、 MAGEB2、FMR1、TCF3作为干细胞/祖精原细胞的潜在标记 猴子。这些基因与信号通路(即WNT、BMP、成纤维细胞生长因子)有关 具有调节啮齿动物SSC自我更新和增殖的作用。我会验证我们候选人的表达方式 标记物通过免疫组织化学和比色染色，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。细胞表面标志物，如 GPC4、GPC3和TSPAN33可用于富集SSCs。我们将通过以下途径调查这一可能性 免疫组织化学、流式细胞术和干细胞移植定量检测可移植干细胞 标记阳性细胞群体中的活性。我还将使用灵长类的scRNAseq数据来确定生态位 生长因子信号影响PSSC的自我更新和增殖。这将提供候选的增长因素 在灵长类SSC培养中进行测试，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改善对灵长类SSCs生物学的理解，并可能使干细胞 在男性不育临床中的应用。该项目的完成将极大地扩大我的科学和 技术知识，给了我成功的研究事业所需的坚实基础。

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.