Inhibition of PSMD4 alters ZP1 ubiquitination state and sperm-oocyte-binding ability in pigs

The aim of this study was to determine how the duration of culture affects the ubiquitination of zona pellucida (ZP) proteins (ZP1, ZP2 and ZP3) during porcine oocyte maturation in vitro. We analysed the changes in ZP protein ubiquitination under three conditions: (i) during oocyte maturation from stage GV to MII; (ii) in oocytes cultured for different periods of time; and (iii) in oocytes treated with an antibody against PSMD4. Our results show that ZP1 and ZP2 are ubiquitinated at the GV stage, while ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage, and band intensities for these proteins were significantly different between the GV and MII stages (p < .05). We also found that ubiquitination occurs in ZP1, ZP2 and ZP3 after cultured for 46, 52, 58 and 64 hr, and that the level of ubiquitinated ZP1 was significantly different in oocytes that were cultured for different time periods. Finally, treatment with an antibody against PSMD4 resulted in a significant decrease in ZP1 ubiquitination (p < .05), without affecting ZP2 or ZP3. The number of attached sperms per oocyte was also significantly different between control and anti-PSMD4-treated groups. Thus, we concluded that ZP1 and ZP2 are ubiquitinated at the GV stage, and ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage. As the duration of culture increases, the ubiquitination levels of ZP proteins decrease. We also found that PSMD4 improves ZP1 ubiquitination during in vitro culture of porcine oocytes and effectively inhibits sperm-oocyte binding.