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Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption.

Oh JS, Han SJ, Conti M - J. Cell Biol. (2010)

Bottom Line: These movements are regulated by PKA inactivation and MPF activation, respectively.Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest.Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
After a long period of quiescence at dictyate prophase I, termed the germinal vesicle (GV) stage, mammalian oocytes reenter meiosis by activating the Cdc2-cyclin B complex (maturation-promoting factor [MPF]). The activity of MPF is regulated by Wee1/Myt1 kinases and Cdc25 phosphatases. In this study, we demonstrate that the sequestration of components that regulate MPF activity in distinct subcellular compartments is essential for their function during meiosis. Down-regulation of either Wee1B or Myt1 causes partial meiotic resumption, and oocytes reenter the cell cycle only when both proteins are down-regulated. Shortly before GV breakdown (GVBD), Cdc25B is translocated from the cytoplasm to the nucleus, whereas Wee1B is exported from the nucleus to the cytoplasm. These movements are regulated by PKA inactivation and MPF activation, respectively. Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest. Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

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Effect of down-regulation of Wee1 kinases on mouse oocyte meiotic arrest. (A and B) WT (A) or pde3a−/− (B) oocytes were injected with MO corresponding to Wee1A, Wee1B, or Myt1. Scrambled MO (Ctrl) was used as a negative control. Error bars indicate SEM. Numbers above the bars indicate the number of oocytes in the GVBD stage and number of total oocytes, respectively, as well as the number of experiments performed. *, P < 0.005; and **, P < 0.0001 compared with control. ***, P = 0.0192.
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fig1: Effect of down-regulation of Wee1 kinases on mouse oocyte meiotic arrest. (A and B) WT (A) or pde3a−/− (B) oocytes were injected with MO corresponding to Wee1A, Wee1B, or Myt1. Scrambled MO (Ctrl) was used as a negative control. Error bars indicate SEM. Numbers above the bars indicate the number of oocytes in the GVBD stage and number of total oocytes, respectively, as well as the number of experiments performed. *, P < 0.005; and **, P < 0.0001 compared with control. ***, P = 0.0192.

Mentions: We previously reported that down-regulation of the maternal Wee1B mRNA causes resumption of meiosis of mouse oocytes (Han et al., 2005), implicating this kinase in the maintenance of meiotic arrest. Because only a portion of the oocytes resumed meiosis, we hypothesized that this incomplete phenotype is caused by compensation of the loss of Wee1B activity by Myt1 and/or the somatic Wee1 (termed in this study Wee1A). To investigate the relative contribution of the three kinases in imposing the meiotic arrest, we designed morpholino oligonucleotides (MOs) corresponding to Wee1A, Myt1, and Wee1B by overlapping the initiator ATG of each mRNA. Less than 10% GVBD was induced with the injection of Wee1A MO, a value comparable with scrambled control MO (Fig. 1 A), indicating that this kinase is not involved in enforcing the meiotic arrest. This conclusion is consistent with the low mRNA levels for Wee1A detected in mature oocytes (Han et al., 2005). In contrast, 35% of the oocytes underwent GVBD after injection of Wee1B MO, which is a result consistent with our previous experiments (Han et al., 2005). Down-regulation of Myt1 also induced GVBD by ∼22% (Fig. 1 A). Only combined down-regulation of Wee1B and Myt1 produced GVBD in the majority of the oocytes (Fig. 1 A). Similar results were obtained in pde3a- oocytes, which are permanently arrested in GV stage because of high cAMP and PKA activity (Fig. 1 B). These results indicate that Wee1B and Myt1 but not Wee1A cooperate in maintaining the meiotic arrest in mouse oocytes. The presence of a fraction of oocytes that do not reenter meiosis after combined MO injection is likely caused by the incomplete knockdown of these proteins. Available antibodies do not detect the endogenous Myt1 and Wee1B in immunocytochemistry, and a large number of oocytes are required to obtain a significant signal in Western blot analysis, rendering technically difficult the detection of endogenous proteins in injected oocytes. Therefore, it has not been possible to directly monitor the efficiency of the MO down-regulation. Nevertheless, the specificity and efficacy of each MO was confirmed by down-regulation of recombinant protein expression in frog and mouse oocytes and by a rescue experiment (Fig. S1).


Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption.

Oh JS, Han SJ, Conti M - J. Cell Biol. (2010)

Effect of down-regulation of Wee1 kinases on mouse oocyte meiotic arrest. (A and B) WT (A) or pde3a−/− (B) oocytes were injected with MO corresponding to Wee1A, Wee1B, or Myt1. Scrambled MO (Ctrl) was used as a negative control. Error bars indicate SEM. Numbers above the bars indicate the number of oocytes in the GVBD stage and number of total oocytes, respectively, as well as the number of experiments performed. *, P < 0.005; and **, P < 0.0001 compared with control. ***, P = 0.0192.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812522&req=5

fig1: Effect of down-regulation of Wee1 kinases on mouse oocyte meiotic arrest. (A and B) WT (A) or pde3a−/− (B) oocytes were injected with MO corresponding to Wee1A, Wee1B, or Myt1. Scrambled MO (Ctrl) was used as a negative control. Error bars indicate SEM. Numbers above the bars indicate the number of oocytes in the GVBD stage and number of total oocytes, respectively, as well as the number of experiments performed. *, P < 0.005; and **, P < 0.0001 compared with control. ***, P = 0.0192.
Mentions: We previously reported that down-regulation of the maternal Wee1B mRNA causes resumption of meiosis of mouse oocytes (Han et al., 2005), implicating this kinase in the maintenance of meiotic arrest. Because only a portion of the oocytes resumed meiosis, we hypothesized that this incomplete phenotype is caused by compensation of the loss of Wee1B activity by Myt1 and/or the somatic Wee1 (termed in this study Wee1A). To investigate the relative contribution of the three kinases in imposing the meiotic arrest, we designed morpholino oligonucleotides (MOs) corresponding to Wee1A, Myt1, and Wee1B by overlapping the initiator ATG of each mRNA. Less than 10% GVBD was induced with the injection of Wee1A MO, a value comparable with scrambled control MO (Fig. 1 A), indicating that this kinase is not involved in enforcing the meiotic arrest. This conclusion is consistent with the low mRNA levels for Wee1A detected in mature oocytes (Han et al., 2005). In contrast, 35% of the oocytes underwent GVBD after injection of Wee1B MO, which is a result consistent with our previous experiments (Han et al., 2005). Down-regulation of Myt1 also induced GVBD by ∼22% (Fig. 1 A). Only combined down-regulation of Wee1B and Myt1 produced GVBD in the majority of the oocytes (Fig. 1 A). Similar results were obtained in pde3a- oocytes, which are permanently arrested in GV stage because of high cAMP and PKA activity (Fig. 1 B). These results indicate that Wee1B and Myt1 but not Wee1A cooperate in maintaining the meiotic arrest in mouse oocytes. The presence of a fraction of oocytes that do not reenter meiosis after combined MO injection is likely caused by the incomplete knockdown of these proteins. Available antibodies do not detect the endogenous Myt1 and Wee1B in immunocytochemistry, and a large number of oocytes are required to obtain a significant signal in Western blot analysis, rendering technically difficult the detection of endogenous proteins in injected oocytes. Therefore, it has not been possible to directly monitor the efficiency of the MO down-regulation. Nevertheless, the specificity and efficacy of each MO was confirmed by down-regulation of recombinant protein expression in frog and mouse oocytes and by a rescue experiment (Fig. S1).

Bottom Line: These movements are regulated by PKA inactivation and MPF activation, respectively.Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest.Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
After a long period of quiescence at dictyate prophase I, termed the germinal vesicle (GV) stage, mammalian oocytes reenter meiosis by activating the Cdc2-cyclin B complex (maturation-promoting factor [MPF]). The activity of MPF is regulated by Wee1/Myt1 kinases and Cdc25 phosphatases. In this study, we demonstrate that the sequestration of components that regulate MPF activity in distinct subcellular compartments is essential for their function during meiosis. Down-regulation of either Wee1B or Myt1 causes partial meiotic resumption, and oocytes reenter the cell cycle only when both proteins are down-regulated. Shortly before GV breakdown (GVBD), Cdc25B is translocated from the cytoplasm to the nucleus, whereas Wee1B is exported from the nucleus to the cytoplasm. These movements are regulated by PKA inactivation and MPF activation, respectively. Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest. Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

Show MeSH
Related in: MedlinePlus