Abstract
During sperm cryopreservation, the most significant phenotype of cryodamage is the decrease in sperm motility. Several proteomics studies have already been performed to search for key regulators at the protein level. However, sperm functions are known to be highly regulated by phosphorylation signaling. Here, we constructed a quantitative phosphoproteome to investigate the expression change of phosphorylated sites during sperm cryopreservation. A total of 3107 phosphorylated sites are identified and 848 of them are found to be significantly differentially expressed (DE). Bioinformatics analysis showed that the corresponding genes of these regulated sites are highly associated with sperm motility, providing a connection between the molecular basis and the phenotype of cryodamage. We then performed kinase enrichment analysis and successfully identified glycogen synthase kinase-3α (GSK3A) as the key kinase that may play an important role in the regulation of sperm motility. We further constructed a GSK3A centric network that could help us better understand the molecular mechanism of cryodamage in sperm motility. Finally, we also verified that GSK3A was abnormally activated during this process. The presented phosphoproteome and functional associations provide abundant research resources for us to learn the regulation of sperm functions, as well as to optimize the cryoprotectant for sperm cryopreservation.
Highlights
Sperm cryopreservation via liquid nitrogen is a valuable technology for male fertility preservation
We checked the morphological features by light microscopy and found no obvious alterations during each step (Supplementary Figure S1). These results are consistent with previous study based on large dataset and verify that the impaired sperm motility is the main phenotype of cryodamage [4]
Among the genes associated with differentially expressed (DE) sites, we found that many diseases or phenotypes that directly associated with sperm functions are statistically enriched (Figure 4B; Supplementary Data S2)
Summary
Sperm cryopreservation via liquid nitrogen is a valuable technology for male fertility preservation. It was widely applied in clinical cases such as long-term storage for sperm donation, sperm preservation before cancer therapy or vasoligation, and sperm preparation for severe oligoathenospermia. It is important to study the corresponding molecular changes during the process of sperm cryopreservation and thawing, which may help us to understand the mechanism of cryodamage or to improve the technology of cryopreservation. It may provide novel hints for the diagnosis and treatment of asthenozoospermia
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