Proteasome activity assay

Proteasome activity assays were performed as previously reported with minor modifications [85]. To assess oocyte proteasome activity at 6 embryonic development stages of mouse biparental and uniparental embryos, 100 embryos per group were collected and washed 3 times in PBS/polyvinyl pyrrolidone (PVP). All cell suspensions were lysed in 20 µL protein extraction buffer composed of 150 mM sodium chloride, 50 mM Tris, and 0.5% Triton X-100 for 30 minutes under constant rotation at 4°C. After centrifugation at 16,300 g for 15 minutes, the supernatants were transferred to a clean tube and assessed for proteasome activity using a commercial proteasome assay kit, which uses an AMC-tagged peptide substrate, which releases free, highly fluorescent AMC in case of proteolytic activity (ab107921; Abcam). In brief, 10 µL of each sample was loaded into a 96-well plate in duplicate, alongside a Jurkat cell lysate-positive control (supplied) and AMC protein standards. A total of 50 µM of the proteasome inhibitor MG132 was added to 1 well of each sample to differentiate proteasome activity from other protease activity in the samples. Plates were incubated for 25 minutes and analyzed on a TECAN Sunrise™ plate reader (TECAN, Salzburg, Austria) at 350/440 nm excitation/emission. After a further 35-minute incubation at 37°C, plates were analyzed a second time to calculate the change in relative fluorescence units in each sample. Data were analyzed following the manufacturers’ instructions, and proteasome activity was calculated such that 1 unit of proteasome activity is equivalent to the amount of proteasome activity that generates 1.0 nmol of AMC per minute at 37°C. This experiment was repeated across 3 independent biological and technical replicates using 100 embryos per assay.