Testosterone is synthesized from cholesterol in the Leydig cells of the testes, and its deficiency can lead to primary or late-onset hypogonadism (LOH). APOE is a critical protein for maintaining cholesterol homeostasis, and its knockout has been found to reduce cholesterol in Leydig cells, and ultimately result in an LOH-like phenotype in Apoe-knockout mice. Therefore, this study aims to evaluate whether Apoe-supplement therapy can alleviate LOH-associated symptoms and provide a novel therapeutic approach for LOH in clinical settings.

Testis tissues underwent single-cell transcriptomic and lipidomic profiling. Primary Leydig cells were isolated via a two-step enzymatic digestion protocol and analyzed using transcriptomic and proteomic approaches. Recombinant adeno-associated virus (AAV) encoding ApoE (ApoE-AAV) was injected into the testicular interstitium of 20-month-old WT and ApoE-KO mice.

Lipidomic profiling revealed a marked reduction in testicular cholesterol content in aged mice. Single-cell transcriptome analysis and Proteomic analysis further highlighted alterations in lipid-associated proteins, including diminished APOE levels. Following ApoE-AAV delivery, APOE protein expression was selectively restored in Leydig cells, reaching levels comparable to young controls. Treated aged and ApoE-KO mice exhibited reduced senescent Leydig cells and increased lipid droplets.

Age-related declines in Leydig cell APOE expression and disrupted lipid homeostasis are key contributors to late-onset hypogonadism (LOH). Targeted intratesticular delivery of ApoE via AAV effectively restores APOE protein levels, mitigates cellular senescence, and rescues steroidogenic function in aged mice.