This study was undertaken to investigate the dynamics of protein ubiquitination in pig gametes and their micro-environments, as well as to explore the action of deubiquitinases (DUBs) in sperm-oocyte binding. Protein ubiquitination states were evaluated by in the ejaculated sperm, seminal plasma, epididymal sperm, oocytes, zona pellucida (ZP) and follicular fluid (FF) by western blotting. Different concentrations of PR-619, a non-selective inhibitor of DUBs, were used to treat oocytes during in vitro maturation (IVM), the maturation rate, amount of ubiquitinated ZP proteins, and ZP solubility were assessed. The PR-619 was also used to treat sperms during capacitation, then the ubiquitinated amounts of acrosin inhibitor (AI) proteins were evaluated. The number of sperm attached to the ZP of each oocyte was subsequently determined after gamete co-incubation. The study indicates the existence of ubiquitinated proteins (76kDa) in sperm, seminal plasma, oocytes, and follicular fluid (FF). The amount of ubiquitinated ZP proteins changed as growth of follicles progressed. Treatment with PR-619 at 10 and 15μM concentrations during IVM reduced the maturation rate of pig oocytes (P<0.05), while treatments with 10μM of PR-619 extended the ZP dissolution time (P<0.05). Treatment with PR-619 enhanced AI ubiquitination and improved amounts of 30-kDa ubiquitinated proteins (P<0.05). Treatment with PR-619 at the 10μM dose effectively reduced the number of sperm attached to per oocyte (P<0.05). Ubiquitinated proteins were present in gametes and their micro-environments. The DUBs were important in regulating pig gamete ubiquitination and sperm-oocyte binding.
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