Mechanism of the chromosome-induced polar body extrusion in mouse eggs

Abstract

Background: An oocyte undergoes two rounds of asymmetric division to generate a haploid gamete and two small polar bodies designed for apoptosis. Chromosomes play important roles in specifying the asymmetric meiotic divisions in the oocytes but the underlying mechanism is poorly understood.

Results: Chromosomes independently induce spindle formation and cortical actomyosin assembly into special cap and ring structures in the cortex of the oocyte. The spindle and the cortical cap/ring interact to generate mechanical forces, leading to polar body extrusion. Two distinct force-driven membrane changes were observed during 2nd polar body extrusion: a protrusion of the cortical cap and a membrane invagination induced by an anaphase spindle midzone. The cortical cap protrusion and invagination help rotate the spindle perpendicularly so that the spindle midzone can induce bilateral furrows at the shoulder of the protruding cap, leading to an abscission of the polar body. It is interesting to note that while the mitotic spindle midzone induces bilateral furrowing, leading to efficient symmetric division in the zygote, the meiotic spindle midzone induced cytokinetic furrowing only locally.

Conclusions: Distinct forces driving cortical cap protrusion and membrane invagination are involved in spindle rotation and polar body extrusion during meiosis II in mouse oocytes.

Figure 1

Differential induction of cortical polarity and spindle formation by chromatin. (A-C) DNA bead-induced an actin cap (A, red, arrowhead) and a myosin II ring (B, green, arrowhead) which are comparable to those induced by maternal chromosomes (indicated by MII, A-C, arrows). (D-F) DNA bead-induced cortical actin cap (shown in red) and cortical granule (CG) redistribution (shown in white). Note that the injected DNA beads induced formation of a CG free domain (E, arrowhead), which is overlapping with the actin cap (D and E, arrowhead). The arrows point to the MII chromosome region. DNA is shown in blue in all the figures unless otherwise stated. (G) A bipolar spindle induced by the injected DNA beads. In this image, the microtubules are shown in green. (H) A DIC image of G showing the DNA beads. (I) A comparison of the time required for the cortical actin cap and spindle formation induced by DNA beads. The scale bars shown in all of the figures represent 20 μm unless otherwise stated.

Figure 2

Sequential induction of cortical cap and myosin II ring by DNA beads. (A-C) DNA beads first induced a myosin II cap (A, green, arrowhead) and an overlapping actin cap (B, red, arrowhead) at 40 min after microinjection. Shown are representative images of 17 analyzed eggs. (D-F) DNA bead-induced myosin ring formation (D, green, arrowhead) surrounding an actin cap (E, red, arrowhead) observed at 90 min after injection (observation of over 50 eggs). (G-I) DNA bead-induced myosins II cap instead of ring formation (G, green, arrowhead) after disruption of actin by Lat-A. Note in H that actin (red) is not visible after Lat-A treatment.

Figure 3

Cortical protrusion and spindle midzone-induced membrane furrowing during polar body extrusion. (A-D) Different time points after SrCl₂ treatment showing the cortical cap protrusion (arrowheads) and the spindle midzone-induced membrane furrowing (arrows). Note the cortical protrusion overlying the chromosomes (arrowheads) and the spindle midzone-induced membrane furrows changing from the initial unilateral (B and C, arrowheads) to the eventual bilateral (D, arrowheads). (E) Cortical cap protrusion (arrowhead) after disruption of spindle microtubules by nocodazole. (F) Block of cortical protrusion by BAPTA/AM. (G, H) Block of cortical protrusion by blebbistatin and ML-7 during egg activation.

Figure 4

A distance-dependent membrane furrow induction by the spindle midzone. (A) DNA bead-induced spindle formation at the center of an MII egg. (B) After inducing anaphase by SrCl₂, the centrally positioned anaphase spindle was unable to induce membrane furrowing. Note that the cortically positioned maternal chromosomes and spindle (indicated by MII) induced the 2^nd polar body extrusion (PbII). (C) The DNA bead-induced spindles were able to induce membrane furrowing and polar body extrusion (indicated by Pb) if positioned close to the cortex.

Figure 5

A simplified model depicting the cortical cap protrusion and spindle midzone-induced membrane furrowing during polar body extrusion. (A) Chromosomes induce formation of a cortical actomyosin cap/ring prior to polar body extrusion. The squared region of the cortical cap/ring is shown on the top, an actin cap (red) surrounded by a myosin II ring (green). (B) Egg activation induces the cortical cap protrusion. (C) The anaphase spindle midzone induces unilateral furrowing. (D) Spindle rotation. (E) Spindle midzone induces bilateral furrowing and abscission of polar body.