Metabolic Transitions Define the Spermatogonial Stem Cell Maturation [human]

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis and therefore of male fertility. Transplantation of SSCs, isolated before treatment for cancer, and cultured in vitro, could be a potential treatment for infertility. Such clinical usage requires an understanding of the metabolic requirements during SSC development. Adult SSCs mainly use glycolysis for their maintenance in the mouse and human. However, SSCs embryonic precursors, primordial germ cells (PGCs), require a high mitochondrial metabolism in the mouse. Similarly, pig neonatal SSC precursors have been described to rely on oxidative phosphorylation (OXPHOS) for the first 2 months of development, when a transition to an adult SSC metabolic phenotype is initiated. When and if such a metabolic transition occurs in humans is ambiguous. We show here for the first time using single-cell RNA sequencing, that human PGCs and prepubertal human spermatogonia have an enrichment of oxidative phosphorylation associated genes, which is downregulated by 13 years of age. Furthermore, we show, that similar metabolic differences are detectable after birth also in mouse. The metabolic transition in humans with puberty was preceded by a drastic change of SSC shape. Using a pig model, we reveal that these metabolic changes could be regulated by IGF-1 dependent signaling via mTOR and proteasomic inhibition. Understanding the metabolic requirements of SSCs during development is crucial to establish a culture system and enable clinical use of SSCs. Overall design: Human samples (homo sapiens) were obtained from the Fertility Preservation Program in Pittsburgh and processed at the University of Pittsburgh. Testes were collected from 1-, 2- and 7-year-old males through the University of Pittsburgh Health Sciences Tissue Bank and Center for Organ recovery under the University of Pittsburgh CORID 686. Human samples were enzymatically digested and processed as previously described (Shami et al. Dev Cell . 2020 Aug 24;54(4):529-547.e12). Human samples were processed with approval from the University of Pittsburgh Institutional Review Board (Protocol #STUDY09020220). Isolated primary human testicular cells were processed using Chromium TM Single Cell 3' v3 Chemistry library prep kit according to the manufacturer's instructions (10X Genomics, San Francisco, CA, USA). Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. A shallow sequencing was performed first using Illumina MiSeq SR50 to estimate the number of recovered cells in each sample (a total of 5089, 8447 and 7851 cells were collected from 1-, 2- and 7-year-old males, respectively). The sequencing of the libraries was performed a second time with a targeted sequencing depth of ~ 50,000 reads/cell using an Illumina NovaSeq 6000 sequencer (100pb single end reads) (CHGI, University of Calgary, Alberta, Canada). The raw reads were subsequently processed with a 10x Genomics CellRanger pipeline (version 3.1.0) using default settings. The outputs from the three human samples were combined using the CellRanger aggr function to normalize for the sequencing depth and produce aggregated gene-cell UMI count matrix for the downstream analysis.