A single-cell atlas of the testicular interstitium defines Leydig progenitor networks sustaining Leydig cell homeostasis across the lifespan

Declining rates of male fertility pose a significant clinical challenge, while the mechanisms underlying testicular interstitial function remain incompletely understood. Here, we conducted a comprehensive analysis of the single-cell transcriptomic landscape of the murine testicular interstitium across the postnatal lifespan. The investigation unveiled a previously unrecognized population of Cd34⁺/Sox4⁺ mesenchymal cells nestled within the interstitium, hinting at their potential as Leydig cell (LC) progenitors. With the aging process of Cd34⁺/Sox4⁺ mesenchymal cells, we observed a decline in glutathione levels within the testicular interstitium. Remarkably, these Cd34⁺/Sox4⁺ mesenchymal cells exhibited clonogenic self-renewal capacity and a robust propensity to differentiate into LCs. Intriguingly, when transplanted into LC-disrupted or failure models, Cd34⁺/Sox4⁺ cells efficiently colonized the testicular interstitium, resulting in a notable increase in testosterone production. Exploring the epigenetic landscape, we identified critical transcription factors, most notably Sox4, governing the stem cell fate of Cd34⁺/Sox4⁺ mesenchymal cells. Overall, this comprehensive lifespan-resolved single-cell atlas of testicular interstitial cells provides fundamental insights into LC progenitor biology and regenerative capacity during aging.