To investigate the critical role of hypoxia in impairing spermatogenesis via M1 macrophage polarization and subsequent induction of lysosome-dependent cell death (LDCD) in Leydig cells.

Ten eight-week-old male SD rats (weight 220~250 g) were randomly divided into normoxia (n=5) and hypoxia groups (n=5). Hypoxic models were established by housing rats in hypoxic chambers simulating 5 000 m altitude for 4 weeks. TM3 mouse Leydig cells underwent 1% O₂ exposure for 2 d. A transwell co-culture system integrated RAW 264.7 macrophages and TM3 cells. Testicular histopathology was evaluated via HE staining (spermatogenic cells/tubular area). Testosterone levels were quantified by ELISA (BCA-quantified). Immunofluorescence assessed Leydig cells (CYP11A1⁺), M1 macrophages (CD68⁺/CD86⁺ in tissue; iNOS⁺ in cells), and lysosomal integrity (LysoTracker/CTSB staining). Single-cell RNA-seq data (PRJNA926018) with GSEA identified programmed cell death pathways.

Hypoxia significantly reduced spermatogenic cell density (P<0.01), testosterone levels (P<0.01), and Leydig cell counts (P<0.01). scRNA-seq revealed decreased proportions of elongated spermatids, Leydig cells, and sertoli cells (P<0.01), but increased spermatocytes/spermatogonia (P<0.01), with significant LDCD enrichment in Leydig cells (P=0.002 7). Hypoxia induced testicular M1 macrophage infiltration (CD68⁺/CD86⁺) and elevated iNOS⁺ in RAW 264.7 cells (P<0.01). Transwell co-culture showed hypoxia-treated macrophages,but not hypoxia alone,disrupted lysosomal membrane stability in TM3 cells.

Hypoxia impairs spermatogenesis by driving M1 macrophage polarization, triggering LDCD in Leydig cells, and reducing testosterone synthesis.