We report here, for the first time, that CDC14A plays a role in meiotic maturation in mouse oocytes. During fetal development oocytes arrest in the dictyate stage prior to MI and then begin a prolonged growth phase. During this growth phase the oocyte acquires meiotic and developmental (the ability to support early embryonic development) and meiotic competence.2,20,21
Meiotic competence is fully acquired once an oocyte reaches ~80% of its full size, transcription is silenced and CDK1 protein accumulates.1-4,22-24
Furthermore, meiotically competent oocytes contain condensed chromatin that surrounds the nucleolus. Nuclear and cytoplasmic swap studies recently revealed that meiotic competence depends upon cytoplasmic factors whereas molecules required for developmental competence reside in the GV.25
We found that the nuclear localization of CDC14A highly correlated with chromatin configuration (). The configuration of chromatin in meiotically incompetent oocytes is not condensed and CDC14A is concentrated in the GV. In contrast, in meiotically competent oocytes where chromatin is condensed, CDC14A is dispersed between the GV and cytoplasm. Meiotic competence is also associated with an increase in CDK1 protein that is localized in the GV.22-24
Therefore, there is an inverse correlation between the nuclear localization of CDK1 and CDC14A at the time of meiotic competence acquisition. In fission yeast Cdk1 phosphorylates and inactivates Cdc14 (Clp1/Flp1).26
It will be interesting to see if this negative feedback loop is conserved in mouse oocytes, and will likely be important to understanding how meiotic competence acquisition is regulated.
In budding yeast, Cdc14 mutants are unable to form viable spores due to defects in proper MI exit.16
We demonstrate that overexpression of CDC14A prevents completion of meiotic maturation (), and that microinjection of a polyclonal antibody against CDC14A delays the transition between MI and MII and generates aneuploid eggs (). Although CDC14A was successfully depleted using siRNAs in human cells14
we find that in oocytes mCDC14A is surprisingly stable because 72 h after injection of long dsRNA CDC14A protein is not sufficiently reduced. Although there has not been a report demonstrating that the polyclonal antibody used in our studies neutralizes CDC14A activity, the delay in the timing of MI exit in our anti-CDC14A injected oocytes is consistent with what has been reported in lower eukaryotes. Moreover, CDC14A is dispersed at metaphase of MI and then localizes on the central meiotic spindle during the MI-to-MII transition, further suggesting that it plays a role in MI exit (Figs. and ).
In the mitotic cell cycle, DNA is replicated one time prior to separation of sister chromatids. DNA re-replication is prevented and DNA segregation is activated by CDKs.27,28
In yeast, Cdc14 antagonizes CDK action in late mitosis to promote DNA replication factor loading onto origins of replication and to promote mitotic exit.29
To generate gametes that contain half the number of chromosomes from diploid precursors in meiosis, two rounds of chromosome segregation must occur without an intervening round of DNA synthesis. This unique cell cycle situation demands that the mechanisms that regulate mitosis be altered so that correct chromosome segregation can occur during meiosis.
In budding yeast, one way this alteration is achieved is through uncoupling the spindle and chromosome cycles between MI and MII. During this transition, the spindle reduplicates while DNA replication is inhibited. Cdc14 is required for spindle duplication because Cdc14 mutants contain persistent MI spindles, but Cdc14 is not required to prevent DNA re-replication because Cdc14 mutants do not re-replicate their DNA.16
This meiosisspecific change in Cdc14 function occurs because yeast contain a CDK-related kinase called Ime2 that is only expressed during meiosis and whose consensus phosphorylation sites differs from that of CDKs and are not recognized by Cdc14.30
Although CDK and Ime2 phosphorylate different residues, their phosphorylation affects their substrates in similar ways. Therefore, during the MI-to-MII transition Ime2 phosphorylates DNA replication factors and Cdc14 does not recognize these phosphorylated residues. This mechanism prevents re-replication in a cell with low CDK activity. Mouse has several Ime2-like homologs (MAK, ICK and RAGE) and transcripts for all of these kinases are present in oocytes.31
It will be interesting to determine whether this regulatory mechanism is conserved in mammals.
In mammalian oocytes, CDK1 activity is partially reduced between MI and MII32
likely because cyclin B1 protein is only partially destroyed.33,34
To promote MI exit in mouse oocytes, separase binds to CDK1 and inhibits its activity.35
This mechanism contrasts to how MI exit in budding yeast is regulated because in yeast separase activates the release of Cdc14 from the nucleolus so that it can act upon its substrates. We found that injection of oocytes with a polyclonal antibody against CDC14A delays the MI-to-MII transition suggesting that it functions to promote MI exit in mammalian oocyte meiosis. As mentioned above, Cdk1 phosphorylates and inactivates Cdc14 (Clp1/Flp1) in fission yeast.26
During mitotic exit when Cdk1 activity decreases, Clp1/Flp1 is activated by auto-dephosphorylation in trans. Therefore, it is tempting to speculate that CDC14A is inactivated by CDK1 prior to Met I, and, because CDK1 activity declines during MI exit, CDC14A reactivates itself to promote Ana I by dephosphorylating CDK1 substrates. To address this model, a robust method that assesses changes in CDC14A activity during oocyte maturation must be developed.
As the spindle elongates and chromosomes segregate, a region called the central spindle, which is composed of anti-parallel, interdigitating microtubules, forms. In the center of this structure is an electron dense region called the spindle midzone that often appears as a dark band in immunofluorescent images because it is refractory to antibody staining. The central spindle and spindle midzone control cytokinesis and therefore the integrity of the central spindle is essential for cell division.36
CDC14A and CDC14B co-localize during this transition on the central meiotic spindle () suggesting that they are functionally redundant during this phase of meiosis. A subset of proteins required to coordinate central spindle formation are phosphorylated and inhibited by CDK1 during mitotic metaphase.37-39
CDC14 promotes mitotic anaphase, in part, by reversing the CDK1 phosphorylation on these substrates and thereby promote their localization to the central spindle and spindle midzone.8,13
We find that although oocytes with depleted CDC14B extrude polar bodies, they contain abnormal spindles and have cytokinesis defects.18
Collectively, these data indicate that CDC14A and CDC14B control proper MI exit through reversing CDK1 action to control spindle elongation. The generation of a Cdc14a
double mutant mouse strain will be a more robust way to address the requirement of these phosphatases during this meiosis-specific transition.