Stress preconditioning of sperm for cryopreservation: new approach based on proteomics characteristics

There is an association between dysfunctional spermatozoa and proteomic changes due to cryo-injury. Recently, the application of high-throughput techniques proteomics allows us to identify low-abundance protein changes in human sperm cells throughout cryopreservation. Stress preconditioning of sperm (NO:0.01 μM,60 min) improved function frozen–thawed sperm with increase expression stress-related proteins and some intracellular antioxidants. Stress-induced proteins reduce activation of the apoptotic cascade and maintain the quality of sperm after freezing. This approach can modulate proteins that may be important in fertility sperm, and this response may be regulated at translational or posttranslational levels. We selected 36 semen samples from normozoospermic men according to the WorldHealth Organization guidelines which did not have the oxidative stress (ROS-TAC score). After processing sperm with PureSperm gradient, each sample was divided into 3aliquots according to the following groups: fresh, freeze and group exposed to 0.01 μM Sodium Nitroprusside (NO donor) for 60 minute before freezing. Proteins extracted for three different experimental conditions and proteins from each group were pooled and peptide labeled with tandem mass tags (TMTs) coupled to LC-MS/MS. Bioinformatics analyses were performed using DAVID. Candidate proteins were further validated by western blot analysis. We detected 2,912 proteins in human sperm where 238 and 191 proteins were respectively up and down-regulated in cryopreserved sperm compared to fresh sperm. The main down-regulated proteins were involved in metabolic processes and sperm-egg recognition. Furthermore , we identified 85 proteins expressed differentially in two groups with and without NO treatment. The main over expressed proteins were associated with the cell stress response and cytoskeleton proteins such as thioredoxin and centrin-1. Mild oxidative stress induces biosynthesis kinetics of Chaperon-related proteins and enzymes in redox regulation. These proteins eliminate oxidative stress and protect sperm against cryo-injury. This strategy may improve sperm conservation techniques in assisted reproductive techniques

Source of Funding: Royan Institute

Conflict of Interest: None to disclose