Stochastic MTOC assembly leads to formation of the multipolar spindle followed by progressive clustering into a barrel-shaped spindle. (A–I) Immunofluorescence staining of the mouse MII oocyte (A) and zygotes (B–I) fixed at consecutive stages of development: early interphase (18 h after hCG), in which the fertilization cone (dotted line) forms in response to sperm entry (B); mid (C)- and late (D) interphase (21 and 25 h after hCG, respectively); prophase (E); early (F), mid (G)-, and late (H) prometaphase; and metaphase (I; 28 h after hCG). Single-section images (C–H) or z-projected images of confocal sections (A, B, and I) show microtubules, pericentrin, and DNA. In B, arrowhead marks male chromatin delivered by the sperm; asterisk marks the second meiotic spindle. Note the absence of MTOC enhancement in the fertilization cone. Arrows in D and E mark MTOCs on the pronuclear surface. Upon NEBD, a multipolar spindle forms with no major axis (early prometaphase; F). Arrowheads in F and G mark the multipoles. A few major axes become visible in midprometaphase (G), consolidating into a single major axis with minor additional axes in late prometaphase (asterisks; H) and eventually forming a barrel-shaped spindle with pericentrin localized on two ring-shaped poles (metaphase; I). (J) Live imaging of mouse zygotes during the first division at prophase (left), prometaphase (middle), and metaphase (right). Z-projected images of confocal sections (3 µm thick) show microtubules (EGFP-MAP4; gray) and DNA (H2B-mRFP1). Circles and arrows mark MTOCs and the multipoles, respectively. Bars, 10 µm. Time is given in hours and minutes after NEBD.