Proteomic Profiling of Cryopreservation‐Induced Alterations in Rhesus Macaque Sperm
Xiao‐Jing Fan, Xiao‐Lu Fan, Hai‐Tao Zhang, Jun‐Wen Zhang, Yun‐Hui Liu, Xu‐Dong Zhuang, Lu Chen, Xin‐Rui Wang, Ya‐Lan WeiABSTRACT
Background
Sperm cryopreservation is a fundamental technique in assisted reproductive technologies. While human sperm freezing is clinically routine, preserving nonhuman primate sperm remains challenging due to species‐specific characteristics and low post‐thaw viability.
Objectives
This study aims to investigate the proteomic changes in rhesus macaque sperm before and after cryopreservation, focusing on identifying the molecular mechanisms behind cryoinjury.
Materials and Methods
Semen was collected from six sexually mature male rhesus macaques (6–10 years) using rectal electroejaculation. Motility parameters were quantified pre‐ and postcryopreservation using computer‐assisted semen analysis (CASA) with vapor‐phase liquid nitrogen protocols. Data‐independent acquisition (DIA) quantitative proteomics, combined with LC–MS/MS, was used to profile global protein expression changes. Differential protein analysis was performed using multibioinformatics tools for functional annotation and network modeling. Papanicolaou and acridine orange (AO) staining were used to assess sperm morphology and DNA integrity, while reactive oxygen species (ROS) assay and immunofluorescence were employed to validate the proteomic analysis results.
Results
We identified 512 differentially expressed proteins (32 upregulated, 480 downregulated), which are predominantly enriched in pathways related to metabolic reprogramming, oxidative stress response, and protein homeostasis. Immunofluorescence staining further confirmed markedly reduced levels of SOD1, PGK2, PKC‑alpha, and phospho‑tyrosine in sperm after freeze–thaw cycling. These molecular hubs are associated with sperm motility and functional stability and may be essential for regulating energy metabolism, oxidative balance, and proteotoxicity during cryopreservation‐induced stress in rhesus macaque sperm.
Conclusion
Cryopreservation induces profound proteomic remodeling in rhesus macaque sperm, mitochondrial electron transport, and metabolic homeostasis. Our findings reveal conserved and species‐specific cryoinjury mechanisms, offering molecular targets for optimizing cryopreservation protocols in nonhuman primates to advance reproductive conservation strategies.