Autophagic Degradation of GPX4 Mediates Ferroptosis During Sheep Sperm Cryopreservation.

Ferroptosis is implicated in cryodamage to sheep sperm, potentially due to glutathione peroxidase 4 (GPX4) degradation during freezing; however, the pathway underlying GPX4 degradation remains unclear. In this study, a comparison of cryoprotective effects between the autophagy inhibitor chloroquine (CQ) and the ubiquitination inhibitor MG132 revealed that 5 μM CQ treatment significantly enhanced the motility (p < 0.01) and sperm plasma membrane integrity rate (p < 0.01) of frozen-thawed sperm; no protective effects were observed in any MG132 treatment group. Mechanistic analysis indicated that CQ treatment substantially restored GPX4 protein expression (p < 0.01), and concurrently reduced lipid peroxidation (p < 0.01) and free iron ion accumulation (p < 0.01), in frozen-thawed sperm. These findings suggest that GPX4 degradation during cryopreservation occurs via the autophagy pathway. This study established a ferroptosis-GPX4-autophagy axis during sheep sperm cryopreservation and identified autophagy-mediated GPX4 loss as a potential target for enhancing sperm cryoprotection.