Proteomic characteristics of human sperm after freezing–thawing treatment

Background: Despite protective capacity of the seminal fluid during cryopreservation, sperm preparation methods should use to select mature and functional spermatozoa with low rates of apoptosis before cryopreservation. On the other hand, there is an association between dysfunctional spermatozoa due to cryoinjury and protein changes. The application of high-throughput proteomics to study the human sperm cell allows us to identify proteomic changes in human sperm cells throughout the cryopreservation.Methods: we selected semen samples from normozoospermic men (n=36), after processing sperm with PureSperm gradient, each sample was divided into 2 aliquots: fresh and cryopreserved groups. Sperm quality parameters (motility ,apoptosis status, DNA fragmentation) evaluated after freezing-thawing, then proteins extracted for two different experimental conditions. Extracted proteins from each group were pooled and labeled with tandem mass tags (TMTs) coupled to LC-MS/MS. Bioinformatic analyses were performed using DAVID software. Candidate proteins were further validated by western blot analysis.Result: 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.Conclusion: Several proteins detected as deregulated could be candidates for diagnostic markers in pathogenic mechanisms involved in cryopreservation and given the unknown impact of some of these proteins on offspring health .This is the first study to compare protein levels in fresh and cryopreserved sperm without seminal plasma using the TMT technology coupled to LC-MS/MS.

Keywords:

Sperm , Cryopreservation , Proteomics , Seminal plasma