Ubiquitin-mediated degradation of RPN13 promotes porcine sperm capacitation and sperm-zona pellucida binding.

Sperm capacitation involves biochemical and functional changes necessary for fertilization. The ubiquitin-proteasome system (UPS) regulates protein turnover, but the roles of proteasomal receptors during capacitation remain unclear. This study investigates the regulation and functional impact of proteasomal subunits RPN13 (ADRM1) and RPN2 (PSMD1) during in vitro capacitation of boar sperm. Semen samples (n = 6) were assigned to four groups: non-capacitating control (Non-Cap, 4 h in medium lacking bovine serum albumin (BSA)), capacitating control (Cap, 4 h with BSA) and groups treated with vehicle (CM + dimethyl sulfoxide (DMSO)) or the RPN13 inhibitor RA190. We measured RPN2/RPN13 levels, RPN13-associated ubiquitin signal and RPN13-RPN2 interactions (Western blot, Co-IP) and assessed sperm kinematics, intracellular Ca2+, protein tyrosine phosphorylation (pTyr), mitochondrial membrane potential (ΔΨm), ATP, ZP binding and early embryo cleavage after IVF. The Cap group showed successful capacitation, confirmed by increases in pTyr, sperm hyperactivation and Ca2+-associated Fluo-4 fluorescence. During capacitation, RPN2 and RPN13 abundance decreased, while RPN2-RPN13 association increased. RA190 treatment increased RPN13 levels, inhibited RPN13 turnover and decreased its interaction with RPN2 and the associated ubiquitin signal. Functionally, RA190 attenuated sperm hyperactivation, Ca2+-associated Fluo-4 fluorescence, protein tyrosine phosphorylation, mitochondrial membrane potential and ATP levels and was associated with reduced fertilization-related outcomes, including ZP binding and early embryo cleavage after IVF. These findings highlight the role of RPN13 in sperm capacitation and fertilization.