CDC25A phosphatase controls meiosis I progression in mouse oocytes

Abstract

CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.

Figure 1

Expression of CDC25A protein and mRNA during meiotic maturation. a) At the indicated times oocytes were processed for and subjected to immunoblotting for CDC25A. GV-intact, aNEBD-(2 h), MI-(7h) and MII-(18h) stages. aNEBD; 2 h after transfer to IBMX-free medium, which corresponds to 1 h after NEBD. Each lane contained 400 oocytes/eggs. b) Quantification of immunoblots. The experiment was performed three times and the data are expressed as mean ± SEM. c) CDK1-dependent decrease in CDC25A protein. The samples were prepared as described in (a) but Roscovitine (0.025 mM, Rosc) was added after oocytes were transferred to IBMX-free medium. The experiment was done 3 times and quantification is included in b). Shown is a representative immunoblot; a loading control for Fig 1a and 1c) is included in Supplementary information d) Cdc25a mRNA expression in relative arbitrary units in GV-intact and MII-arrested eggs. Expression of both 5′ and 3′ ends of Cdc25a mRNA were normalized to exogenous Gfp mRNA, mRNA expression was measured in 4 independent experiments. The data are expressed as the mean ± SEM.

Figure 2

Exogenous CDC25A overcomes cAMP-mediated GV-stage block. a) Oocytes were scored for NEBD 18 h following injection of mRNAs encoding the indicated Gfp-containing constructs. About 100 oocytes were scored for each group. CDK1 (b) and MAPK (c) activities in oocytes resuming meiosis 2 h after microinjection of Gpf-Cdc25a mRNA in IBMX-arrested oocytes (CDC25A, NEBD), and control Gfp mRNA injected oocytes maintained in IBMX-containing medium (GFP, GV) and those spontaneously maturing after transfer to IBMX-free medium at 2 h (GFP, NEBD). The experiment was conducted 3 times and the data are expressed as the mean ± SEM. d) Immunocytochemical analysis of spindle formation and chromosome congression in defective metaphase I oocytes that were microinjected with Gpf-Cdc25a mRNA. Red, acetylated α-tubulin; blue, DAPI for DNA staining; CDK1

Figure 3

CDC25A and CDC25B exhibit different localization during resumption of meiosis. Localization of CDC25A and CDC25B in live oocytes after microinjection of Gfp-Cdc25a or Gfp-Cdc25b mRNA (150 ng/μl). GV-stage, shortly before (bNEBD) and shortly after (aNEBD) nuclear envelope break down. The experiment was performed 4 times and 120 oocytes were imaged. Shown are representative examples.

Figure 4

CDC25A protein degradation is essential for MI—MII transition. a) Concentration-dependent effect of microinjected Cdc25a mRNA on ability of oocytes to reach metaphase II by 18 h after transfer to IBMX-free medium. Gfp mRNA injected oocytes served as the control. Significant differences (p<0.05) in comparison to GFP controls are marked (*). About 50 oocytes in each group were scored; b) Immunocytochemical analysis of spindle formation and chromosome congression in control (Gfp mRNA injected) and Cdc25a mRNA-injected oocytes. The cells were processed for immunocytochemistry 7 h after microinjection; in both cases the injection solution was 250 ng/μl. Red, acetylated α-tubulin; blue, DAPI for DNA staining. About 75 of oocytes were injected and representative images are shown. CDK1 (c) and MAPK (d) activities at 7 h (MI in control GFP oocytes) and 18 h (MII in control GFP oocytes) after transfer to IBMX-free medium. In all cases the oocytes were injected with mRNAs at 250 ng/μl. Significant differences (p<0.05) in comparison to GFP controls at 7 h are marked (*). The experiment was performed three times and the data are expressed as mean ± SEM.

Figure 5

CDC25A is involved in resumption of meiosis and MI - MII transition. a) Effectiveness of RNAi-mediated knock-down of Cdc25a mRNA at 24 h in IBMX-supplemented medium after microinjection of long dsRNA Gfp RNAi (control) or long Cdc25a RNA. The experiment was performed 4 times and the data are expressed as the mean ± SEM. b) Meiotic maturation of control (GFP RNAi) and CDC25A RNAi oocytes scored 18 h after transfer to IBMX-free medium; ~100 oocytes for each group were scored. c) Immunocytochemical analysis of spindle formation and chromosome congression in GFP RNAi (control) and CDC25A RNAi oocytes at 7 h after transfer to IBMX-free medium. Red, acetylated α-tubulin; blue, DAPI for DNA staining. About 80 oocytes were injected and representative images are shown.