Inhibition of HSP70 during prolonged liquid storage at 17 °C increases mitochondrial membrane potential and calcium levels

Abstract

Serine phosphorylation of heat shock protein 70 (HSP70/HSPA1A) is known to be involved in sperm cryotolerance. This study aimed to determine whether HSP70 is implicated in the resilience of pig semen to prolonged storage at 17 °C. For this purpose, HSP70 was blocked with various concentrations of YM-1 (0, 0.05, 0.1 and 0.2 µM), an allosteric inhibitor of this chaperone, and samples were stored for 21 d. At various time points (0, 4, 10, 14, and 21 d), sperm motility and kinetic parameters by computer-assisted semen analysis, and viability (SYBR14/PI), mitochondrial membrane potential (JC1) and intracellular levels of calcium (FLUO3), total ROS (H2DCFDA) and superoxides (HE) through flow cytometry were assessed. No significant differences in sperm motility parameters were observed, except a reduction of VSL in the treatment containing 0.2 µM YM-1 at day 14 (P<0.05). Although no YM-1 concentration affected viability and intracellular concentrations of total ROS and superoxides, inhibiting HSP70 with YM-1, regardless of inhibitor concentration, increased intracellular calcium concentrations at day 10 compared to the control (P<0.05). Moreover, the treatment containing 0.05 µM YM-1 had higher JC1agg/JC1mon ratios than the control at day 10 (P<0.05). In conclusion, although inhibition of HSP70 with YM-1 did not affect pig sperm survival during long-term storage at 17 °C, it increased mitochondrial activity and intracellular calcium concentrations. This work was supported by the Ministry of Science and Innovation, Spain (PID2020-113320RB-I00) and the Regional Government of Catalonia, Spain (2017-SGR-1229).