Proteomics study reveals the molecular mechanisms underlying cryotolerance induced by mild sublethal stress in human sperm

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01 μM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p < 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated that the abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm.