Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

Department of Obstetrics Gynecology and Women's Health University of Missouri Columbia MO 65211 USA

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Czech Republic

Division of Animal Sciences University of Missouri Columbia MO 65211 USA

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences Vestec 25250 Czech Republic

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Bottom-up approach to deciphering the targets of the ubiquitin-proteasome system in porcine sperm capacitation

Modulatory effect of MG-132 proteasomal inhibition on boar sperm motility during in vitro capacitation

Evidence of endogenously produced hydrogen sulfide (H2S) and persulfidation in male reproduction

Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals

The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation