Fig. 6: Enhancing ketogenesis in LCs of aged mice alleviates testicular aging.

a Schematic of AAV-mediated Hmgcs2 overexpression. b Left: 2 weeks post-injection, representative testicular sections from AAV-Control and AAV-O.E. infected mice, stained with LCs marker HSD3β, HMGCS2, and DAPI. NC: negative control. Scale bar: 50μm. Right: quantitative analysis of HMGCS2⁺ HSD3β⁺ cells. n = 3 per group. c Testicular BHB concentration 2 weeks post-injection. n = 5 per group. d Left: 2 weeks post-injection, representative western blots for H3K9ac and Histone H3 in testes of different mice. Right: quantitative analysis of H3K9ac protein level, relative to Histone H3. n = 3 per group. e Left: 2 weeks post-injection, representative western blots for FOXO3a and GAPDH in testes of different mice. Right: quantitative analysis of FOXO3a protein level, relative to GAPDH. n = 3 per group. f Left: 2 months post-injection, representative testicular sections from different mice stained with LC marker CYP17A1, and SA-β-gal. Scale bar: 75μm. Right: quantitative analysis of CYP17A1⁺ SA-β-gal⁺ cells. n = 3 per group. g Left: 2 months post-injection, representative testicular sections from different mice stained with senescence marker p21, HSD3β, and DAPI. Scale bar: 50μm. Right: quantitative analysis of p21⁺ HSD3β⁺ cells. n = 3 per group. h, i 2 months post-injection, quantitative RT-PCR analysis of senescence markers (p21, Cxcl10) in LCs. n = 3 per group. j–m Serum testosterone (j), intratesticular testosterone (k), serum Insl3 (l), serum LH (m) level of the indicated groups. n = 12 per group in (j), n = 4 per group in (k), n = 12 per group in (l), n = 7 per group in (m). n–p 2 months post-injection, representative HE stained testicular sections. Scale bar: 500 μm (n). The percentages of seminiferous tubules with varying diameters (o) and epithelial thickness (p). n = 3 per group. q–t Representative light micrographs of sperm from the indicated groups. Scale bar: 100 μm (q). Sperm concentration (r), proportion of sperm with motility (s), proportion of sperm with progressive motility (t). n = 10 per group. Data were presented as mean ± SEM. Significance was determined by one-way ANOVA (b–i, k, l, o–t) or Kruskal-Wallis test (j, m). Illustrations were created with BioRender. Source data are provided as a Source Data file.