Glycolysis metabolic changes in sperm cryopreservation based on a targeted metabolomic strategy.

Our aim was to identify potential metabolomic pathway changes in the sperm cryopreservation process and to find new markers of human sperm freezability. Targeted metabolomic experiments were used to identify the quantitative metabolomic compound characterization of human sperm cryopreservation. A KEGG pathway analysis was used for these deregulated compounds. A total of 16 significantly deregulated compounds was identified between fresh and post-thawed sperm; of these, 7 were downregulated and 9 were upregulated in the frozen-thawed group. A bioinformatics analysis revealed that metabolic pathways play an important role in cryopreservation, including the citrate cycle (TCA cycle), glycolysis or gluconeogenesis, glyoxylate and dicarboxylate metabolism, pyruvate metabolism and galactose metabolism. We used immunoblotting and immunofluorescence to analyze the expression and localization of the three key enzymes in glycolysis. The glycolytic metabolic changes were noted in sperm cryopreservation. HK2 expression levels in fresh sperm were significantly higher than the levels in freeze-thawed sperm.