Figure 5.

PP1 inhibition enhances NEBD, reduces M-phase exit, and causes meiotic abnormalities in oocytes in culture conditions that allow partial maintenance of prophase I arrest. (a) Schematic representation of the experimental design. Prophase I oocytes were washed into medium containing 0.2 µM milrinone and increasing amounts of TMC (0.5–5 µM) or vehicle control (0.1% DMSO) for 16 h; under these culture conditions, ~65% of oocytes will undergo the G2/M transition, exiting from prophase I arrest. (b) Graphical representation of meiotic stage based on phase microscopy at 16 h into culture. IVM Ctrl oocytes were cultured in milrinone-free medium. Bar graph shows the percentages of oocytes at each meiotic stage. n = 87–182 oocytes over 4–7 replicates. * denotes a significant difference from Veh control oocytes (Fisher’s exact test). The number of oocytes at each meiotic stage is shown in Supplemental Table 2. (c) Table of the numbers and percentages of prophase I oocytes with SN or NSN morphology, with representative images of SN and NSN DNA morphology. Orange arrow points to the nucleolus and scale bar = 10 µM. n = 37–65 oocytes over 4–7 replicates. * denotes a significant difference from Veh control oocytes (Fisher’s exact test. (d) Graphical representation of the meiotic stage and DNA morphology of oocytes at 16 h into culture. Solid colors represent normal DNA morphology for the meiotic stage, and hatched colors represent abnormal DNA morphology for the meiotic stage. n = 38–107 oocytes over 4–7 replicates. For all bar graphs, * denotes a significant difference from Veh control oocytes (Fisher’s exact test).