Expression changes of superoxide dismutase 2 and peroxiredoxin 6 in asthenospermia and their correlation with sperm motility

Abstract

Objective To explore the damaging effects of reactive oxygen species (ROS) on spermatozoa, and investigate the expression changes of superoxide dismutase 2 (SOD2) and peroxiredoxin 6 (PRDX6) in spermatozoa of asthenospermia patients, so as to analyze their correlation with sperm motility. Methods A total of 84 semen samples from asthenospermia patients [progressive motility (PR) < 32%, PR+ non-progressive (NP) < 40%, sperm density ≥15×106/mL] and 55 normal semen samples (PR ≥32%, PR+NP≥40%, sperm density ≥15×106/mL) were collected in our center from January to November 2020. The normal semen parameters were detected using a computer-aided sperm analysis (CASA) system. After the samples were treated with different concentrations of H2O2, and the altered parameters were measured by CASA. The changes in sperm survival rate were determined by eosin staining, and the expression changes of SOD2 and PRDX6 were evaluated by Western blotting. Finally, RT-qPCR and Western blotting were adopted to compare the levels of SOD2 and PRDX6 in spermatozoa between the asthenospermia patients and normal controls, and the correlation of the levels with semen parameters were further analyzed. Results CASA results showed that sperm motility was significantly restrained after 0.10, 0.35 and 1.00 mmol/L H2O2 treatment; and the motility parameters like curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and mean angular displacement (MAD) were decreased in a dose-dependent manner with the increase of H2O2 concentration, while the amplitude of lateral head displacement (ALH), linearity (LIN), wobble (WOB) and straightness (STR) were reduced at concentrations of 0.10 and 0.35 mmol/L. Eosin staining indicated that the sperm survival rate was decreased after 0.35 and 1.00 mmol/L H2O2 treatment, and Western blotting showed the expression of SOD2 and PRDX6 in spermatozoa was up-regulated at a dose-dependent manner with the increase of H2O2 concentration. In addition, both the mRNA levels of SOD2 (0.297±0.038 vs 0.895±0.140, P < 0.01) and PRDX6 (0.743±0.107 vs 1.416±0.163, P < 0.01) in spermatozoa were significantly lower in the asthenospermia patients than the normal controls, and so were their protein levels (SOD: 20.689±0.147 vs 1.466±0.212, P < 0.01; PRDX6: 0.726±0.084 vs 1.183±0.100, P < 0.01). Moreover, the mRNA expression level of SOD2 was positively correlated with the sperm progressive motility (r=0.267, P < 0.01) and total motility (r=0.329, P < 0.01), and that of PRDX6 was positively with sperm progressive motility (r=0.453, P < 0.01), total motility (r=0.476, P < 0.01), STR (r=0.270, P < 0.01), VCL (r=0.313, P < 0.01), VSL (r=0.349, P < 0.01) and VAP (r=0.410, P < 0.01). Conclusion ROS reduces sperm motility and survival rate, and up-regulates the expression of SOD2 and PRDX6. The down-regulated levels of SOD2 and PRDX6 are positively correlated with sperm motility in asthenospermia patients.

Keywords

• asthenospermia
• sperm motility
• reactive oxygen species
• superoxide dismutase 2
• peroxiredoxin