Denuded mouse oocytes at the GV stage were collected and cultured in M16 medium for 90 min. Then, oocytes that had undergone GVBD were transferred into DMSO control medium or medium supplemented with different drugs and cultured for 16 hours. (A) Representative images of oocytes after a 16 hour incubation. Arrowheads indicate 2-cell oocytes. Scale bar: 100 µm. (B) Oocyte division results after 16 hours incubation. Data are the mean ±SEM from 3 independent experiments. ** P<0.01, 2×3 χ2-test, compared to concurrent control.

Representative images of oocytes before cytokinesis initiation (A) and after cytokinesis completion (B). Denuded oocytes were incubated in the presence of 0.3 mM dbcAMP or 0.2 mM IBMX for 24 hours, followed by incubation in M16 for 9 hours, or in control medium in the absence of dbcAMP or IBMX for 10 hours, and subsequently fixed and co-stained with Alexa594-phalloidin (red) and Hoechst 33342 (blue). Oocytes with (row 1 in panel A) or without (row 2 in panel A) an “F-actin cap” were observed in both the control group and treatment groups. Arrowheads indicate “F-actin cap” localization. Scale bar: 20 µm.

(A) Immunostaining of control (CTRL) oocytes cultured in control medium for 10 hours and oocytes cultured in M16 medium for 10 hours after a 20 hour incubation with 0.3 mM dbcAMP or 0.2 mM IBMX. Oocytes were stained with anti-phospho-MLC2 (Ser19) antibody (myosin II, green) and Hoechst 33342 (DNA, blue). (B) The myosin II activities of control or treated oocytes were assayed. Extracts from control, dbcAMP-treated and IBMX-treated oocytes were immunoblotted with antibodies against phospho-MLC2 or β actin. Each lane represents a protein sample derived from 300 oocytes.

Denuded mouse oocytes were cultured in DMSO control medium for 16 hours (control group), or in M16 medium containing 0.3 mM dbcAMP or 0.2 mM IBMX for 24 hours, followed by 16 hours of culture in M16 medium (dbcAMP and IBMX group). (A) Representative images of different oocyte phenotypes after incubation. GV designates oocytes remaining in the GV stage; 1Pb designates mature eggs that extruded a first polar body in the first meiotic division; 1-cell designates oocytes that displayed only one cell; 2-cell designates oocytes that produced two daughter cells of similar sizes in the first meiotic division. Arrowheads are used to indicate 2-cell oocytes. Scale Bar: 100 µm. (B) Comparison of cell division types between oocytes treated with or without IBMX or dbcAMP. Data are the mean ± SEM from 3 independent experiments. P values were calculated with a 2×4 χ2-test. N: number of cells analyzed.

Denuded mouse oocytes at the GV stage were collected and cultured in M16 medium for 90 min. Then, oocytes that had undergone GVBD were transferred into DMSO control medium or medium supplemented with 0.6 mM dbcAMP or 1.0 mM IBMX for subsequent live cell imaging. Representative live cell images were collected from oocytes undergoing normal cytokinesis with furrow abscission (A), symmetric cytokinesis with furrow abscission (B), asymmetric cytokinesis with furrow regression (C) and symmetric cytokinesis with furrow regression (D). White arrowheads: prometaphase lagging chromosome. Yellow arrowheads: anaphase/telophase lagging chromosome. The D_L and D_S in (A), (B), (C) and (D) represent the vertical distances from the far-end cortex to the cleavage furrow in the larger and smaller daughter cells, respectively. Time points (hours∶minutes) indicate the time elapsed from the beginning of imaging. Chromosomes were stained with Hoechst 33342, shown in red. Corresponding movies are provided in the supplemental materials. (E) Analysis of cell division in the control, dbcAMP and IBMX group, with each row representing a single oocyte division. Colour indicates whether the cell showed the presence (red) or absence (green) of abnormal anaphase chromosome localization (A.C.L., first column) or abnormal cleavage furrow localization (A.F.L., second column); occurrence of cleavage furrow regression (red) or not (green) (C.F.R., third column); presence (red) or absence (green) of lagging chromosomes at prometaphase (P, fourth column) or anaphase/telophase (A/T, fifth column). The proportion of red in each column is included below. Chromosome localization was deemed “abnormal” if the ratio of the shortest chromosome-cortex distance at anaphase initiation (D_A) over that at movie initiation (D_O) was greater than 0.68 (D_A/D₀ >0.68). Furrow localization was deemed “abnormal” if D_L/D_S was less than 1.70 (D_L/D_S <1.70).

Denuded GV stage oocytes were first cultured in M16 medium for 90 min; after GVBD, the oocytes were transferred into DMSO control medium or medium supplemented with 0.6 mM dbcAMP or 1.0 mM IBMX for live cell imaging. Chromosome movement was tracked during meiosis I. (A) Representative time-lapse images of chromosome movement from imaging initiation to anaphase onset. Time from imaging initiation is shown (hours∶minutes). Red: DNA. (B) Green dots indicate the position of the chromosome cluster. The shortest distance between the chromosomes and the cortex was measured at each time point (Dt) and divided by the distance to the cortex at the beginning of the experiment (D₀). Dt/D₀ was used as an indicator of chromosome movement and plotted against time. Panel (C) shows the relative position of the chromosome cluster at anaphase onset, as D_A/D₀. D_A is the shortest distance between the chromosomes and the cortex at anaphase initiation. ** P<0.01, Student's t-test, compared to concurrent control. N: number of cells analyzed.

(A) Representative images of oocytes taken 16 hours after microinjection with DAM-488 2^nd antibody (Control) and anti-phospho-MLC2 (Ser19) antibody (P-myosin). The arrowhead indicates a symmetrically dividing oocyte. Scale bar: 100 µm. (B) The frequency of 1 Pb, GV, 1-cell and 2-cell type divisions in the Control and P-myosin groups. The P value was calculated using a 2×4 χ2-test. (C) The shortest distance between chromosomes and the cortex was measured in 15 oocytes per group at GVBD (D_GV) and hourly time points after GVBD (Dt). Dt/D_GV plotted against time was used as an indicator of chromosome movement.