Full-grown GV oocytes were collected from CF-1 female mice aged 6 to 8 weeks. Oocyte collection and culture were carried out as previously described (23 (link), 34 (link)). Cumulus oocyte complexes were collected and cultured in bicarbonate-free minimal essential medium (MEM) supplemented with polyvinylpyrrolidone (PVP; 3 mg/ml) and 25 mM Hepes (pH 7.3) under mineral oil (MilliporeSigma, St. Louis, MO, USA, no. P2307, no. H3784, and no. M8410). Oocytes were manually denuded and sorted into three groups: central GV, intermediate GV, and peripheral GV oocytes. Careful rolling of the oocyte, as previously described, ensured correct classification (5 (link)). Denuded GV oocytes were microinjected with ∼5 pl of the indicated complementary RNA (cRNA) using Eppendorf FemtoJet 4i. The medium for oocyte microinjection was MEM supplemented with PVP (3 mg/ml) and 25 mM Hepes (pH 7.3) under mineral oil. Oocytes were then transferred to Chatot, Ziomek, and Bavister (CZB) maturation medium supplemented with 1 μM glutamine (MilliporeSigma, no. G8549) under mineral oil and matured at 37°C with humidified 5% CO2 in an incubator to prometaphase I, metaphase I, or metaphase II stages. The oocytes that do not undergo NEBD (within 2 hours after culture) were excluded from further experiments.
Nocodazole (Sigma-Aldrich, no. M1404), monastrol (Sigma-Aldrich, no. M8515), ML141 (Sigma-Aldrich, no. SML0407), ZM447439 (Tocris, no. 2458), and CK-666 (Sigma-Aldrich, no. 182515) were dissolved in dimethyl sulfoxide (DMSO) and added to CZB culture medium at a final concentration of 400 nM, 100 μM, 500 nM, 10 μM, and 50 μM, respectively. SiR-tubulin and SiR-DNA were added to the maturation medium (at a final concentration of 100 nM) during live imaging to label MTs and DNA, respectively.
Nocodazole (Sigma-Aldrich, no. M1404), monastrol (Sigma-Aldrich, no. M8515), ML141 (Sigma-Aldrich, no. SML0407), ZM447439 (Tocris, no. 2458), and CK-666 (Sigma-Aldrich, no. 182515) were dissolved in dimethyl sulfoxide (DMSO) and added to CZB culture medium at a final concentration of 400 nM, 100 μM, 500 nM, 10 μM, and 50 μM, respectively. SiR-tubulin and SiR-DNA were added to the maturation medium (at a final concentration of 100 nM) during live imaging to label MTs and DNA, respectively.
