STUDY QUESTION Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development? SUMMARY ANSWER Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism. WHAT IS KNOWN ALREADY Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous. STUDY DESIGN, SIZE, DURATION To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations. PARTICIPANTS/MATERIALS, SETTING, METHODS Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition. LARGE SCALE DATA New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819. LIMITATIONS, REASONS FOR CAUTION Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.

中文翻译：

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代谢转变定义精原干细胞的成熟


研究问题 精原细胞，包括精原干细胞 (SSC)，在青春期前发育过程中是否经历代谢变化？摘要答案在此，我们表明青春期前人类精原细胞的代谢表型与成年精原细胞不同，并且 SSC 发育的特点是从氧化磷酸化 (OXPHOS) 到无氧代谢的独特代谢转变。已知信息 小鼠和人类成年 SSC 的维持都依赖于糖酵解，而胚胎 SSC 前体、原始生殖细胞 (PGC) 对 OXPHOS 表现出较高的依赖性。据报道，新生猪 SSC 前体在发育 2 个月时开始向成年 SSC 代谢表型转变。然而，人类何时以及是否发生这种代谢转变尚不清楚。研究设计、规模、持续时间 为了解决我们的研究问题：(i) 我们对公开可用的和新生成的（当前研究）单细胞 RNA 测序 (scRNA-Seq) 数据集进行了荟萃分析，以建立 SSC 路线图人类从胚胎阶段（胚胎第6周）到成年（25岁）的代谢发育，共十组； (ii) 同时，我们分析了分离的幼犬 (n = 3) 和成年 (n = 2) 小鼠精原细胞的单细胞 RNA 测序数据集，以确定是否发生类似的代谢转换； (iii)我们通过使用两个不同年龄的青春期前猪精原细胞作为模型（每个模型有四个独立收集的细胞群）进行定量蛋白质组学分析，描述了SSC成熟期间调节这些代谢转变的机制。 参与者/材料、环境、方法 从 1 岁、2 岁和 7 岁人类男性中收集单个睾丸细胞，并在 6 至 8 天（n = 3）和 4 个月（n = 2）对老年小鼠进行scRNA-Seq。人类序列经过单独处理，然后与公开可用的数据集合并，使用 Seurat V4 软件包进行荟萃分析。然后，我们在年龄组之间进行了成对差异基因表达分析，然后使用基因集富集分析进行通路富集分析（错误发现率的截止值％3C 0.05）。来自小鼠的序列经历了与人类描述的类似的工作流程。通过免疫组织化学、蛋白质印迹、qRT-PCR、定量蛋白质组学和培养实验，分析了青春期前猪精原细胞的早期（1 周龄）和晚期（8 周龄），以揭示代谢转变的潜在细胞机制。主要结果和机会的作用 人类 PGC 和青春期前的人类精原细胞显示 OXPHOS 相关基因的富集，该基因在青春期开始时下调（P < 0.0001）。此外，我们证明在小鼠中可以检测到幼犬和成年精原细胞之间相似的代谢变化（P％3C 0.0001）。在人类中，青春期代谢转变之前也会出现 11 岁时 SSC 形状的剧烈变化 (P < 0.0001)。使用猪模型，我们揭示了这种代谢转变可以通过哺乳动物雷帕霉素靶标和蛋白酶体抑制，通过胰岛素生长因子-1依赖性信号传导途径进行调节。大规模数据 从本研究中获得的新单细胞 RNA 测序数据集可通过 NCBI GEO 免费获取，登录号为 GSE196819。 局限性、注意原因人类青春期前组织样本稀缺，这导致每个年龄的样本数量较少。基因富集分析仅给出有关细胞功能状态的指示。由于青春期前人类精原细胞数量有限，猪精原细胞被用于进一步的蛋白质组学和体外分析。研究结果的更广泛意义 我们发现，青春期前的人类精原细胞表现出高 OXHPOS，并且仅在 11 岁后才转变为类似成人的代谢。青春期前的癌症幸存者在成年后常常患有不孕症。 SSC移植可以为治疗不孕症提供有力的工具；然而，它需要大量的细胞。这项工作提供了对人类 SSC 在整个发育过程中的动态代谢需求的关键见解，这对于建立离体系统以支持 SSC 的临床应用扩展和持续功能至关重要。研究经费/竞争利益 这项工作由 NIH/NICHD R01 HD091068 和 NIH/ORIP R01 OD016575 资助，IDKEO 得到 R01 HD100197 的支持。 T32 HD087194 和 F31 HD101323 支持 SKM。作者声明不存在利益冲突。