Figure 3.

PP1 inhibition for the duration of oocyte meiotic maturation reduces meiosis I exit in oocytes and causes meiotic abnormalities. (a) Schematic representation of the experimental design. Prophase I oocytes were cultured in medium lacking meiotic arrest reagents and containing TMC (0.5–5 µM) or vehicle control (0.1% DMSO) for 16 h. (b) Graphical representation of meiotic stage based on phase microscopy at 16 h into culture. Bar graph shows the percentages of oocytes at each meiotic stage. n = 78–127 oocytes over 4–6 replicates. * denotes a significant difference from Veh control oocytes (Fisher’s exact test). The numbers of oocytes at each meiotic stage is shown in Supplemental Table 1. (c) Representative images of the oocyte phenotypes observed, apostrophe denotes zoomed-in image, scale bar = 10 µM. Orange dashed line denotes the PB boundary, and orange arrow points to single misaligned chromosomes. (i) Normal metaphase II oocyte. Oocytes that underwent NEBD but had no PB with a normal (ii) metaphase I spindle, with (iii and iii’) mild chromosome misalignment, with (iv and iv’) severe chromosome misalignment, or with (v) two spindles. (vi) Oocyte at cytokinesis. Metaphase II oocytes, with (vii and vii’) mild chromosome misalignment, or with (viii and viii’) severe chromosome misalignment. Oocytes with DNA ball, ± PB refers to very condensed chromatin in the oocyte, with no obvious meiotic spindle (not shown). (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 = 43–96 oocytes over 4–6 replicates. * denotes a significant difference from Veh control oocytes (Fisher’s exact test).