Fig. 4

3-HPAA treatment promotes spermatogenesis of old mice through its metabolism pathway. A The experimental workflow of 3-HPAA treatment of old mice. Administration of normal saline was used as control. B–D Sperm concentration (B) and sperm motility including grade A + B sperm (C) and grade A + B + C sperm (D) of old mice with or without 3-HPAA treatment. E H&E staining on testis tissues of vehicle and 3-HPAA mice. Scale bar = 100 μm. F Representative images of immunofluorescence staining for DAZL (spermatogonia marker), SYCP3 (spermatocyte marker), TNP1 (spermatid marker), PGK2 (spermatozoa marker), and WT1 (Sertoli cell marker) in the testis tissues of old and 3-HPAA mice. Scale bar = 25 μm. G–I Unsupervised principal component analysis (PCA) (G) and the volcano plots (H) showed the differentiated metabolites as well as the abundance of 2-HPAA, 4-coumaric acid, and pyroglutamic acid (I) in plasma of the two groups. J–L PCA (J) and the volcano plots (K) showed the differentiated metabolites as well as the abundance of L-malic acid, 4-coumaric acid, and 3-D-hydroxybutyric acid (3-DHBA) (L) in testis of these two groups. Significantly regulated metabolites were determined by absolute FC (fold change) > 1, p value < 0.05 and representative metabolites were indicated (H and K). All data are presented as mean ± SEM. *P < 0.05, ***P < 0.001. Data are analyzed by two-tailed unpaired Student’s t test or Wilcoxon test. n = 7–9 mice per group