Significance: Maternal inheritance of mitochondria and mitochondrial genes is a major developmental paradigm in mammals. Propagation of paternal, sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals, due to postfertilization targeting and degradation of sperm mitochondria, referred to as “sperm mitophagy.” Our and others’ recent results suggest that postfertilization sperm mitophagy is mediated by the ubiquitin–proteasome system, the major protein-turnover pathway that degrades proteins and the autophagic pathway. Here we demonstrate that the co-inhibition of the ubiquitin-binding autophagy receptors, sequestosome 1 (SQSTM1) and gamma-aminobutyric acid receptor-associated protein (GABARAP), and the ubiquitinated protein dislocase valosin-containing protein (VCP)-dependent pathways delayed postfertilization sperm mitophagy. Our findings provide the mechanisms guiding sperm mitochondrion recognition and disposal during preimplantation embryo development, which prevents a potentially detrimental effect of heteroplasmy.

Sperm capacitation, the ultimate maturation event preparing mammalian spermatozoa for fertilization, was first described in 1951, yet its regulatory mechanisms remain poorly understood. The capacitation process encompasses an influx of bicarbonate and calcium ions, removal of decapacitating factors, changes of pH and sperm proteasomal activities, and the increased protein tyrosine phosphorylation. Here, we document a novel biological phenomenon of a unique zinc (Zn²⁺) ion redistribution associated with mammalian sperm in vitro capacitation (IVC). Using image-based flow cytometry (IBFC), we identified four distinct types of sperm zinc ion distribution patterns (further zinc signature) and their changes during IVC. The zinc signature was altered after sperm capacitation, reduced by proteasomal inhibitors, removed by zinc chelators, and maintained with addition of external ZnCl₂. These findings represent a fundamental shift in the understanding of mammalian fertilization, paving the way for improved semen analysis, in vitro fertilization (IVF), and artificial insemination (AI).

The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al.

The omnipresent ubiquitin–proteasome system (UPS) is an ATP-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. The current knowledge of UPS involvement in the process of sperm penetration through vitelline coat (VC) during human and animal fertilization is reviewed in this study, with attention also being given to sperm capacitation and acrosome reaction/exocytosis. In ascidians, spermatozoa release ubiquitin-activating and conjugating enzymes, proteasomes, and unconjugated ubiquitin to first ubiquitinate and then degrade the sperm receptor on the VC; in echinoderms and mammals, the VC (zona pellucida/ZP in mammals) is ubiquitinated during oogenesis and the sperm receptor degraded during fertilization. Various proteasomal subunits and associated enzymes have been detected in spermatozoa and localized to sperm acrosome and other sperm structures. By using specific fluorometric substrates, proteasome-specific proteolytic and deubiquitinating activities can be measured in live, intact spermatozoa and in sperm protein extracts. The requirement of proteasomal proteolysis during fertilization has been documented by the application of various proteasome-specific inhibitors and antibodies. A similar effect was achieved by depletion of sperm-surface ATP. Degradation of VC/ZP-associated sperm receptor proteins by sperm-borne proteasomes has been demonstrated in ascidians and sea urchins. On the applied side, polyspermy has been ameliorated by modulating sperm-associated deubiquitinating enzymes. Diagnostic and therapeutic applications could emerge in human reproductive medicine. Altogether, the studies on sperm proteasome indicate that animal fertilization is controlled in part by a unique, gamete associated, extracellular UPS.