Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II

Abstract

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.

Figure 1.

Meiotic maturation of OoMastl^−/− oocytes. (A) Comparison of the kinetics of GVBD after release from dbcAMP. The total numbers of oocytes used (n) are indicated. (B) Representative images of immunostaining for DNA, CREST, and spindle showing normal progression to metaphase I in OoMastl^−/− oocytes. Oocytes were cultured for the indicated periods after GVBD and were fixed. 30 oocytes were analyzed for each time point. (C) Kinetics of PBE. Oocytes that had undergone GVBD within 2 h after release into dbcAMP-free M16 medium were selected (at time = 0) and cultured further. PBE was scored at 1-h intervals. The numbers of oocytes examined are indicated (n). (D) Comparable PBE rates of the ovulated oocytes collected at 16 h after hCG. The numbers of oocytes used (n) are shown. All of the experiments were repeated at least three times, and representative results are shown. (E) Western blots showing the timing of securin degradation and Cdc20 level during oocyte maturation. Lysate from 100 oocytes was loaded in each lane. The levels of β-actin were used as a control. The same β-actin panel is presented again in Fig. 3 B, which shows the expression of other proteins of interest in the same cell lysates.

Figure 2.

Failure of OoMastl^−/− oocytes to enter MII after PBE. (A) OoMastl^+/+ oocytes formed MetII spindles with condensed chromosomes (C) after PBE. (B) OoMastl^−/− oocytes extruded PB1s but formed nuclei with decondensed chromatin. In some oocytes (left inset), the nuclei had already formed, whereas the residual central spindle microtubules remained between the chromatin in the PB1 and the nucleus. 50 oocytes from each group were analyzed, and representative images are shown. (C) The percentages of oocytes with both a PB1 and a nucleus. For A–C, ovulated (16 h after hCG) oocytes were used. (D) Representative still images from Videos 1 and 2. Timestamps indicate hours and minutes after release from IBMX. H2B-mCherry (red fluorescence) shows the DNA, and Map7-EGFP (green fluorescence) labels the spindle microtubules. Arrows indicate the positions of PB1s.

Figure 3.

Cdk1 activity during oocyte maturation. (A) Comparable Cdk1 activities during prometaphase I (3 h) and metaphase I (6 h) but delayed reduction in Cdk1 activity in OoMastl^−/− oocytes (9 h). Cdk1 activity cannot be increased after PBE (14 h) in OoMastl^−/− oocytes (bottom right). (B) Western blots showing the dynamics of p-Cdk1 (Y15) during oocyte maturation. Lysate from 100 oocytes was loaded in each lane. The levels of β-actin and Cdk1 were used as controls. The same β-actin panel is presented in Fig. 1 E, which shows the expression of other proteins of interest in the same cell lysates. (C) Comparable PP1 phosphorylation (indicating inhibition) at GVBD in OoMastl^−/− and OoMastl^+/+ oocytes. (D) Expression of Mastl, cyclin B1, p-Cdk1 (Y15), Cdk1, phospho–lamin A/C (S22), and lamin A/C in ovulated oocytes. β-Actin was used as the loading control. Lysate from 100 oocytes was loaded in each lane.

Figure 4.

Rescue of MII entry in OoMastl^−/− oocytes upon Cdk1 activation and PP2A inhibition. (A) Ovulated OoMastl^−/− oocytes collected 16 h after hCG were treated with inhibitors that abolished the inhibitory phosphorylation on Cdk1 and enhanced Cdk1 activity in OoMastl^−/− oocytes. Lysate from 50 oocytes was loaded in each lane, and β-actin was used as the loading control. (B) Rescue of MII entry in OoMastl^−/− oocytes treated with PD166285 or OA. Error bars represent the SD. (C) Immunofluorescence of OoMastl^−/− oocytes treated with inhibitors showing formation of spindles and chromosome condensation. All experiments were repeated at least three times, and representative results are shown.

Figure 5.

Absence of Ensa protein expression in OoMastl^−/− oocytes. (A) Expression of Mastl, Ensa, and Arpp19 in ovulated OoMastl^+/+ and OoMastl^−/− oocytes. Lysate from 100 oocytes was loaded in each lane. (B) Relative mRNA levels of Ensa and Arpp19 in ovulated oocytes were determined by real-time PCR. The mRNA levels in the OoMastl^+/+ oocytes were set as 1.0, and the fold changes in OoMastl^−/− oocytes are shown. Error bars represent SDs.