Abstract
Background: Testicular macrophages (TMs) are key regulators of testicular immune privilege and endocrine function in the testis. However, their age-related heterogeneity and role in testicular degeneration remain poorly characterized. Methods: We performed spatial transcriptomics and FACS-enriched single-cell RNA sequencing (scRNA-seq) to characterize testicular macrophage heterogeneity across ageing. Key findings were validated through intratesticular injection of recombinant CCL8 protein, organotypic culture of seminiferous tubules, and immunofluorescence analysis. Results: Using spatial transcriptomics, we identified pronounced Leydig niche senescence in aged testes, mechanistically linked to TM-mediated inflammatory remodelling. Coupling FACS-enriched TM isolation with scRNA-seq resolved seven transcriptionally distinct subpopulations, including ageing-associated subsets (Ccl8hi and Cxcl13hi). These subsets exhibited inflammatory signalling rewiring (e.g., CCL8-CCR2 axis) and activation of senescence transcriptional regulators (ASCL2, SPI1, CEBPB, JUNB), with conserved mediators (CCL8, TREM2, IL1β, and CXCL2) across murine and human testes. Functional validation showed that intratesticular injection of recombinant CCL8 protein in 3-month-old mice recapitulated ageing phenotypes, such as germ cell apoptosis and steroidogenic decline. Conclusions: Our multi-omics atlas highlights TM heterogeneity as a driver of testicular inflammaging and identifies CCL8 as a conserved target for therapeutic interventions aimed at mitigating age-associated male reproductive decline. Key points: Spatiotemporal multi‑omics establish inflammaging as a defining feature of testicular ageing. A refined CD74‑based sorting strategy improves enrichment of testicular macrophages. scRNA-seq resolves seven TM subsets with age‑dependent shifts. TM‑secreted inflammatory mediators-especially CCL8-drive testicular inflammaging.