Regulation of autophagy–Ferroptosis crosstalk by the SLC7A11–GPX4 axis during sperm capacitation

Abstract

Sperm capacitation exposes porcine spermatozoa to an acute redox challenge while they remain transcriptionally silent and unable to synthesize new proteins. To elucidate how the SLC7A11–GPX4 antioxidant axis functions as a key regulatory node to maintain redox homeostasis under these constraints, we combined ubiquitin-focused proteomics with linkage-specific co-immunoprecipitation. This approach revealed a shift in GPX4 (glutathione peroxidase 4) ubiquitin chains from K48- to K63-linkage and an overall reduction in GPX4 ubiquitination during capacitation. Immunofluorescence showed that GPX4 relocalizes from a diffuse distribution across the sperm head to a marked enrichment in the posterior head region. Pharmacology combined with immunoblotting and GPX4 activity assays demonstrated that proteasome inhibition (MG132) preserves GPX4 abundance and activity, whereas USP8 inhibition (DUB-IN-2) increases GPX4 ubiquitination and reduces its level and activity; conversely, the GPX4 inhibitor RSL3 nearly abolishes activity. Autophagy blockade with chloroquine, assessed by LC3/p62 immunoblotting and SLC7A11 immunofluorescence, disrupted polarized SLC7A11 localization and, as shown by cystine-uptake and glutamate-efflux assays, impaired transporter function. Ferroptotic stress markers—ROS (BDP 581/591), labile Fe²⁺ (RhoNox-1), and malondialdehyde (TBARS assay)—were increased by erastin or RSL3, and In vitro fertilization assays further showed that pharmacological disruption of the autophagy–SLC7A11–GPX4 axis reduced sperm–oocyte binding and early embryo cleavage. Together, these data indicate that ubiquitin-linkage remodeling and deubiquitination cooperate with autophagy-dependent SLC7A11 trafficking to maintain a catalytically competent GPX4 pool during capacitation, thereby buffering ferroptotic stress and supporting fertilization competence. Pharmacological inhibition further nominates USP8 as a candidate GPX4 deubiquitinase, pending targeted biochemical and genetic validation.