The addition of 5-aminolevulinic acid to HBSS protects testis grafts during hypothermic transportation: a novel preservation strategy

The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank's balanced salt solution (HBSS; n = 5), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group (P < 0.05) and the αMEM group (P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l-1, 2 mmol l-1, and 5 mmol l-1) to determine the most effective concentration of 5-ALA. The 2 mmol l-1 5-ALA group (n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l-1, and 5 mmol l-1 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control (n = 3) and 2 mmol l-1 5-ALA (n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes (n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l-1 5-ALA group compared with that in the control group (normalized enrichment score [NES] = -1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l-1 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.