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 from 13 to 16-year-old bovine with a lower potential for fertilization. Lysosomal activation was achieved through increasing concentrations of proton pump activators PIP2 (0.1, 0.5, 1, and 5 μM), PMA (0.1, 1, 10, and 50 μM), and DOG (0.1, 1, 10, and 50 μM) at 6, 12, 18, and 24 h 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. Polar body extrusion (PBE), through which a haploid nonfunctional polar body is released in the perivitelline space after completion of the first meiotic division, was observed in PIP2-0.1 μM, -0.5μM-6h; PIP2-5μM-12h; PMA-0.1μM-18h; PIP2-0.1μM, -0.5μM-24h groups. Oocyte diameter was the highest in DOG-1μM-6h, PMA-0.1μM-12h, PIP2-1μM-18h, and PIP2-0.5μM-24h groups. Morphological scores of oocytes were higher in young and old control groups. PIP2, PMA, and DOG affected oocyte quality positively after 6 h of IVM yielding in oocyte scores similar to the control group oocytes. However, they had a negative impact on the oocyte scores in longer periods of IVM, except for lower doses PMA (0.1 and 1 μM) at 12 h and PIP2 (0.5 μM) and PMA (0.1 μM) at 18 h, which were able to maintain the scores relatively closer to the control oocytes. Proteolytic activation was achieved in all groups at 6 h of culture. At all other time points PIP2 and PMA groups showed a better response to proteolytic activation. Lysosome density was increased in PIP2-5μM-6h; PIP2-0.1μM, -1μM-12h; PIP2-1μM, -5μM-18h as well as PMA-0.1μM-6h; PMA-1μM, -10μM-12h; PMA-1μM-18h; DOG-50μM-6h and DOG-0.1μM-12h. Protein carbonylation was the lowest in PIP2-0.1 μM groups at 12, 18, and 24 h. This study suggests that proton pump activators PIP2 and PMA was found to have a positive impact on IVM in terms of both morphological scores and proteolytic activation in a time and dose dependant manner.