(a,b) Univalents in fixed aged oocytes. Oocytes from young (2 months old (a)) and aged (16 months old (b)) BDF1 mice were cultured and fixed at 6 h after NEBD. The oocytes were stained for KTs (ACA, green) and DNA (Hoechst 33342, red). KT signals are peak-enhanced and background-subtracted. All homologous KT positions were determined in 3D and the number of chromosomes was counted. Projection views from the top and side of the spindle are shown with signal interpolation in z. The outlines of chromosomes (bivalents, blue; univalents, red) and the positions of homologous KTs (circles) are shown. Representative bivalents and univalents are magnified. Scale bars, 2 μm. See also Supplementary Movie 6. (c) The rate of oocytes with univalents. In fixed oocytes, separated chromosome units that have one of the homologous KTs and an inter-homologous KT axis with a tilt angle of >25° with respect to the spindle axis were categorized as univalents (n=106, 118). The same criteria was used for live oocytes at 6 h after NEBD (n=180, 289). Note that the rates of univalents presented here is underestimated because this criteria excludes univalents that are indistinguishable from hyperstretched bivalents at a single time point. The rate of univalents determined by an analysis of multiple time points in live aged oocytes was 9.5% (Supplementary Table 1). Fisher’s exact test was performed. *P=0.015, ***P=0.0004. (d,e) Univalents are predisposed to predivision. The inter-sister KT distance (c) and the angle between inter-sister KT axis and the spindle axis (d) were measured (n=160, 152, 8). Boxes show the 25th–75th percentiles and whiskers show 1–99 percentiles. Two-tailed, unpaired Student’s t-test was performed. *P<0.05, ***P<0.001.