Related Experiment Video
Updated: Jul 2, 2026
Phosphopeptide Analysis of Rodent Epididymal Spermatozoa
Published on: December 30, 2014
Proteomic Profiling of Cryopreservation-Induced Alterations in Rhesus Macaque Sperm
Xiao-Jing Fan1,2,3, Xiao-Lu Fan2,3, Hai-Tao Zhang1,2,3
1Medical Research Center, Fujian Children's Hospital (Fujian Branch of Shanghai Children's Medical Center), College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University, Fuzhou, Fujian, China.
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.
