Activation of proteolysis during oocyte in vitro maturation

In vitro maturation (IVM) is a form of assisted reproductive technology (ART) applied to obtain mature oocytes in culture. Decline in IVM success rates by age has led consideration of novel approaches based on cellular dynamics. Our aim was to achieve proteostasis in old bovine oocytes by removal of protein aggregates. Lysosomal activation was achieved through increasing concentrations of proton pump activators PIP2 (P1-P4), PMA (M1-M4) and DOG (D1-D4) at 6, 12, 18 and 24 hours of IVM in old bovine oocytes. Morphological analysis was performed and IVM rates were determined. DQ-Red BSA was applied to live oocytes to determine proteolytic activation while lysosome density was determined by Lysotracker probe. Protein carbonylation was detected through oxyblot analysis. PBE was observed in P1, P2-6h; P4-12h; M1-18h; P1, P2-24h groups. Oocyte diameter was the highest in D2-6h, M1-12h, P3-18h and P2-24h groups. Morphological scores of oocytes were higher in young and old control groups. Lower concentrations of activators resulted in similar oocyte scores to control groups at 6, 12 and 18h of culture. Proteolytic activation was achieved in all groups at 6h of culture. At all other time points PIP2 and PMA groups showed a better response to proteolytic activation. Lysosome density was increased in P4-6h; P1,P3-12h; P3, P4-18h as well as M1-6h; M2, M3-12h; M2-18h; D4-6h and D1-12h. Protein carbonylation was the lowest in P1 groups at 12h, 18h and 24h. PIP2, PMA and DOG affected oocyte quality and proteostasis through lysosome activation during IVM in a time and dose-dependent manner.