Single-cell analysis of testicular bacterial microbiome changes during aging and effect on reproductive capacity

Recent studies have highlighted connections between bacterial microbiome (BM) of the gut and seminal fluid and male reproductive health. However, knowledge regarding the BM within the testis—the site of spermatogenesis—remains notably limited. Here, we employed INVADEseq, an improved single-cell RNA sequencing technology, to explore the testicular BM in terms of its composition, distribution, age-related changes, host-microbiome interactions and impact on reproductive capacity. Our analyses unveiled a widespread yet sparse distribution of BM across diverse testicular cell types. Notably, we identified significant spatial heterogeneity in BM load, with somatic cells and early-stage germ cells located outside the blood-testis barrier (BTB) exhibiting relatively higher BM abundance. Additionally, we observed an age-dependent increase in BM load, potentially attributable to compromised BTB integrity in aged testes. Functionally, the presence of BM induced distinct transcriptional responses across testicular cell types. Specifically, it enhanced testosterone production in Leydig cells, promoted endocytosis and autophagy functions in M2 macrophages, and reshaped the ligand-receptor interaction network. Collectively, our study presents the first single-cell resolution atlas of host-microbiome interactions in the testis. These findings not only deepen our understanding of testicular microbiome biology but also hold promise for the discovery of novel diagnostic biomarkers and therapeutic targets for BM-related and age-associated male subfertility. C57BL/6 male mice at 5 months (young) and 20 months (older) were used, with 3 biological replicates per group.