Genetic ablation of the adult Leydig cell lineage reveals stem Leydig cells and a peritubular regenerative niche in mice

Abstract Differentiated Leydig cells (LCs) produce testosterone and INSL3, two hormones essential for male sex differentiation and fertility. In mice, the developmental origin of adult Leydig cells (ALCs) and the presence of stem Leydig cells (SLCs) in adulthood remain unresolved. To investigate this, we used the Leydig cell-specific Insl3iCre line combined with Rosa26lox-STOP-lox-iDTR/lox-STOP-lox-TdTomato to genetically ablate the committed LC lineage in adult mice. These mice displayed a normal phenotype without diphtheria toxin (DT). Following DT administration, TdTomato-positive and CYP17A1-positive LCs were eliminated within 4-7 days, leading to marked reductions in testosterone levels and androgen-dependent organ weights. Activated Caspase 3 confirmed apoptosis of targeted Leydig cells during the acute ablation phase. This was accompanied by transient macrophage recruitment as shown by F4-80 immunofluorescence, consistent with their involvement in clearing apoptotic Leydig cells. Importantly, COUP-TFII-positive/αSMA-negative/CYP17A1-negative/TdTomato-negative cells persisted adjacent to peritubular and perivascular myoid cells, identifying a candidate Insl3-negative precursor population. Newly emerging TdTomato-positive cells subsequently appeared predominantly around seminiferous tubules and, to a lesser extent, blood vessels, marking the onset of Leydig cell regeneration. Testosterone levels progressively recovered as these cells acquired CYP17A1 expression and populated the interstitial compartment. By 65 days post-DT, the ALC population and endocrine function were fully restored. These findings provide strong functional evidence for adult SLCs capable of repopulating the LC lineage after complete ablation and identify the peritubular compartment as a major regenerative niche. This inducible LC-depletion mouse model provides a powerful tool to investigate SLC biology and the mechanisms governing LC differentiation and testicular endocrine homeostasis.