Protective effects of seminal exosomes on cryopreserved sperm via inhibiting oxidative damage

This study aimed to explore the protective effect of seminal plasma exosomes (SPEs) on human sperm structure and function during cryopreservation and its potential mechanism. The samples were divided into two groups: the control group was treated solely with sperm cryoprotectant before freezing, while the exosome group was supplemented with SPEs. After cryopreservation and thawing, sperm progressive motility, normal morphological rate, and survival rate were evaluated. Furthermore, PKH67 labeling experiments were performed, and oxidative stress markers as well as energy metabolism indicators in sperm were detected. Subsequent mechanism exploration was conducted via proteomic analysis and protein validation assays. This work reveals that adding SPEs at a concentration of 1 or 2 mg/ml effectively improves sperm progressive motility after cryopreservation. After supplementing with SPEs, sperm glucose levels are reduced and mitochondrial membrane potential is enhanced. Simultaneously, SPEs alleviate oxidative stress by decreasing reactive oxygen species (ROS) and DNA fragment index (DFI) while increasing superoxide dismutase (SOD) activity. Functional annotation of proteomics reveals that 14 of the differentially expressed proteins (DEPs) are associated with sperm motility. Enriched metabolic pathways related to sperm motility and sperm protein validation experiments indicate that the expression of MAPK, p-MAPK, and p-JNK proteins in sperm is higher in the Exosome group than in the Control group. This study provides important theoretical support for the application of SPEs in mitigating cryopreservation damage to sperm by enhancing antioxidant capacity. The specific mechanism may be mediated by the MAPK/p-JNK pathway.