Partial CDK1 inhibition slows stabilization of attachments. (A) Histone-H1 kinase activity was measured in oocytes at the indicated time points relative to GVBD. Each data point represents H1 phosphorylation averaged over three lysates, each with three oocytes. Arrows indicate time of GVBD, anaphase I (AI), and first polar body extrusion (PBE). (B) Oocytes were matured with low concentrations of CDK1 inhibitor RO-3306 (0.5 and 2 µM) or DMSO (control). Meiosis I progression from GV to AI was followed live by DIC microscopy. Times from milrinone washout to GVBD, GVBD to chromosome alignment, and alignment to anaphase I were averaged over multiple cells (n ≥ 18). (C–F) Oocytes were cultured with 0.5 µM RO-3306 or DMSO for 4.5, 6.5, or 11 h after GVBD, then analyzed for cold-stable MTs. Images in C are projections of a confocal z series or optical sections showing MTs (green) and kinetochores (CREST, red). Insets show individual kinetochores classified as attached (1), unattached (2), or lateral (3); bar, 0.5 µm. Percentages of each attachment state were averaged over multiple cells (n ≥ 23, 15 kinetochores per cell; *, P < 0.001) at each time point.