Comparing microtubule distribution in proximate bicentrosomal and distant monocentrosomal spindles in fixed and live bovine zygotes. (A and B) Intensity distribution of α-tubulin IF in 2D sections along the calculated spindle axis orthogonal to the metaphase chromosomes in both proximate bicentrosomal contralateral (A, n = 16) and distant monocentrosomal (B, n = 11 from six embryos) spindles (see also Fig. 2, A and B). Arrows indicate positions of centrosomes. (C–E) Respective 3D-rendered images of fluorescence from microtubule markers (EGFP-MAP4 and EB3-mEGFP2) in the pronuclear volumes of zygotes shown in Fig. 3, A–C to highlight dual spindles, and centrosome positions (arrowheads). Timings respective to synchronous pro-NEBD (NEBD) or NEBD of the first PN (PN1) in case of asynchrony. PN2, lagging PN. Projected scale bars, 10 µm. (F) 3D-rendered image of fluorescence from microtubule marker (EB3-mEGFP2, green) and chromatin marker (H2B-mCherry, magenta) of zygotic volume (same zygote as Fig. 3 C and Fig. S2, D and E) after background correction (median-based denoising). Arrows indicate multiple ingression sites at 140 min after NEBD as a consequence of distant dual spindles.