Metabolic Transitions Define the Spermatogonial Stem Cell Maturation [mouse]

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: Testicular cells were collected from 6-8 day (n=3) and 4-month-old mouse testes (n=2) by 2 step enzymatic digestion as previously described (Yeh et al., 2007). Spermatogonia from 6-8 old mice were then isolated by size exclusion and granularity using FACS light scatter properties. The primary contaminating cell type in the enriched spermatogonia population were somatic peritubular myoid and Sertoli cells. Spermatogonia from 4-month-old were isolated by fluorescence assisted cell sorting (FACS) (FACSAria II, BD Biosciences), using antibodies against GFR?1 ([1:100] Neuromics, goat polyclonal) and cKIT ([1:100] AbCam, rat polyclonal). The primary contaminating cell type in the enriched spermatogonia population after FACS was further differentiating germ cells (spermatocytes and round spermatids). Porcine cells were isolated from the testis by a two-step enzymatic digestion, as previously described (Sakib et al. 2019). Enrichment was performed by differential plating in alpha MEM advanced (ThermoFisher,#12492013), 5% FBS, 1x P/S with subsequent sort as previously described (Voigt et al. 2021). Isolated primary murine spermatogonia samples were resuspended in PBS supplemented with 1% BSA and 6000–10,000 cells per sample were processed using ChromiumTM Single Cell 3' v2 Chemistry library prep kit according to the manufacturer's instructions (10X Genomics, San Francisco, CA, USA). Samples were sequenced on an Illumina HiSeq4000 with paired-end 50 base pair reads by Genome Quebec (Montreal, QC, Canada). The raw counts were subsequently processed with a 10x Genomics CellRanger pipeline using default settings, after which the outputs from the individual samples were combined using the CellRanger aggr function to normalize the sequencing depth and produce aggregated gene-cell UMI count matrix. Sequencing data was analyzed using the cell ranger pipeline. A total of 19,166 cells were included in the analysis and normalized to a read depth of 104,230 reads per cell.