Gut microbiota induces dysspermatogenesis via microbial-derived phenylacetylglycine in Ggt1-deficient mice

Male infertility represents a global health concern, with emerging evidence linking gut microbiota dysbiosis to dysspermatogenesis and subfertility. However, the molecular mediators and regulatory mechanisms by which gut microbiota influences testicular functions remain poorly defined. This study demonstrates that male gamma-glutamyl transferase 1-deletion (Ggt1−/−) mice exhibits infertility phenotypes, including reduced germ and testicular Leydig cell numbers, increased rates of abnormal sperm, and altered reproductive hormone levels. Metabolomic analysis reveals elevated levels of the gut microbial-derived metabolite phenylacetylglycine (PAGly) in serum and testes of Ggt1−/− mice, with in vivo injection experiments indicating its role in impairing spermatogenesis. Moreover, blocking PAGly effectively restores the impaired spermatogenesis in Ggt1−/− mice. Fecal metagenomic and metabolomic analyses show that gut microbiota in Ggt1−/− mice induces elevation of phenylacetic acid, a precursor metabolite of PAGly. Strikingly, fecal microbiota transplantation from Ggt1−/− mice (Ggt1−/−-FMT) recapitulates the infertility phenotypes including reduced germ cells and increased rates of abnormal sperm. Mechanistically, integrated CUT&Tag and ATAC-Seq analyses reveal that transcription factor STAT5B occupies regulatory elements near Klk1b transcription start sites (TSS), confirming that transcription factor STAT5B directly regulates Klk1b gene transcription. Concretely, PAGly activates β2-adrenergic receptor (β2AR) on Leydig cells, triggering STAT3 phosphorylation, subsequent SOCS3 upregulation, and STAT5B phosphorylation suppression; p-STAT5B with transcriptional activation function is reduced, then Klk1b gene transcription is compromised, and therefore spermatogenesis is disrupted. Ggt1 deletion-induced gut microbiota dysbiosis disrupts spermatogenesis via β2AR-STAT3-SOCS3-STAT5B-Klk1bs signaling pathway. Specifically, PAGly-induced β2AR activation promotes STAT3 phosphorylation, which induces SOCS3 to suppress p-STAT5B dependent Klk1bs transcription. This mechanism underscores the critical role of gut-derived metabolites in regulating testicular function and identifies potential targets for microbiota-modulated male infertility.