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RESEARCH |
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, 76100, Israel
Correspondence should be addressed to N Dekel; Email: nava.dekel{at}weizmann.ac.il
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The key regulator of meiosis is the maturation-promoting factor (MPF) (Masui & Markert 1971), which consists the catalytic p34cdc2 serine/threonine kinase and the regulatory cyclin B (Gautier et al. 1988, 1990). Activation of MPF is initially dependent on the availability of cyclin B that allows the formation of the p34cd2/cyclin B complex, known as pre-MPF. This pre-MPF stays inactive under the influence of the wee-1 and myt-1 kinases that phosphorylate the threonine 14 and tyrosine 15 residues of p34cdc2 (Russell & Nurse 1986, Gould & Nurse 1989, Mueller et al. 1995, Booher et al. 1997). The dual specificity phosphatase cdc25, which removes these inhibitory phosphates, converts the inactive pre-MPF into an active MPF (Gautier et al. 1991, Kumagai & Dunphy 1991, Millar et al. 1991, Strausfeld et al. 1991, Sebastian et al. 1993).
Homologs of cdc25 have been identified in different organisms including yeasts (Russell & Nurse 1986, Russell et al. 1989), Caenorhabditis elegans (Ashcroft et al. 1998), Drosophila (Edgar & O’Farrell 1989, Alphey et al. 1992, Courtot et al. 1992), and Xenopus (Izumi et al. 1992, Kumagai & Dunphy 1992). Mitosis in fission yeast is regulated by the rate of accumulation of cdc25 during G2 (Russell & Nurse 1986). On the other hand, the amount of cdc25 in Xenopus oocytes is constant and its activity is regulated posttranslationally by multiple phosphorylations. Phosphorylation of cdc25 is catalyzed by members of the polo-like family of kinases (Kumagai & Dunphy 1996, Qian et al. 1998) as well as by p34cdc2 kinase. The reciprocal phosphorylation of cdc25 and MPF creates an autocatalytic loop that sustains the activities of these two enzymes to drive the cell cycle into M-phase (Izumi et al. 1992, Kumagai & Dunphy 1992, Hoffmann et al. 1993). Conversely, phosphatase 2A is proposed to be involved in a negative regulation of cdc25 activity (Kumagai & Dunphy 1992, Clarke et al. 1993, Hoffmann et al. 1993).
Another mechanism that participates in regulation of cdc25 action involves alteration of its cellular distribution. Localization of cdc25 is determined by the 14-3-3 protein (Conklin et al. 1995, Furnari et al. 1997, Peng et al. 1997, Dalal et al. 1999, Kumagai & Dunphy 1999) that binds the phosphatase and prevents its transport into the nucleus (Zeng & Piwnica-Worms 1999, Davezac et al. 2000).
In mammalian cells, cdc25 represents a multigene family consisting of A, B, and C isoforms (Galaktionov & Beach 1991, Nagata et al. 1991, Jinno et al. 1994). Each member of the mammalian cdc25 family has a role at a particular phase of the cell cycle that is determined by its substrate specificity (Sebastian et al. 1993). Accordingly, cdc25A regulates the G1- to S-phase transition, by dephosphorylating the Cdk2-cyclin E kinase (Hoffmann et al. 1994, Jinno et al. 1994), whereas the entry into mitosis is regulated by cdc25C (Sadhu et al. 1990, Millar et al. 1991), which elicits its action towards the p34cdc2/ cyclin B complex (Gautier et al. 1991). Evidence regarding the role of cdc25B is still controversial. One report proposed that this form of cdc25 is required for progression into S-phase (Garner-Hamrick & Fisher 1998), while other studies have shown that cdc25B has a role in the G2-phase as a regulator of centrosomal microtubule nucleation (Millar et al. 1991, Gabrielli et al. 1996) and a starter of mitosis (Nishijima et al. 1997). This controversy can be possibly explained by the discovery of at least three splice variants of cdc25B that display differences in their biochemical properties (Baldin et al. 1997b).
A large body of information on the role of cdc25 in meiosis has been generated in Xenopus oocytes with hardly any available data on this key phosphatase in mammals. Furthermore, the specific member of the mammalian cdc25 family that functions in meiosis of mammalian oocytes is largely unknown. Early studies detected mRNA of cdc25B in murine ovaries particularly in oocytes, while mRNAs of cdc25A and cdc25C were observed in spermatids (Wickramasinghe et al. 1995, Wu & Wolgemuth 1995). Conflicting results were reported by a later study that identified cdc25C mRNA in mouse oocytes further demonstrating an increase in its protein product during oocyte maturation (Mitra & Schultz 1996). Along this line, it has been shown that cdc25C is essential for progression of porcine oocytes through late diakinesis and that de novo synthesis of this isoform is required for the transition from meiosis in oocytes to mitosis in early embryos (Dai et al. 2000). However, normal fertility exhibited by cdc25C deficient female mice (Chen et al. 2001) suggests that this phosphatase is either irrelevant or that another protein compensates for its function in the control of meiosis. A more recent report indeed demonstrated that oocytes of mice lacking cdc25B fail to activate MPF and remain meiotically arrested (Lincoln et al. 2002). Taking this information into account, we examined the pattern of cdc25B expression and investigated the mode of its regulation in rat oocytes resuming meiosis.
Supporting the above-mentioned observations, we show that microinjection of neutralizing antibodies against cdc25B, but not cdc25C impaired the ability of oocytes to resume meiosis. Along this line, we demonstrate changes in cdc25B localization that are associated with the progress of meiosis. Our findings also reveal that cdc25B exhibits periodic accumulation throughout meiosis that is translationally regulated and corresponds to the oscillatory pattern of MPF activation. We show herein that cdc25B translation is mediated by polyadenylation of its mRNA.
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Materials and Methods |
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The investigation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy of Science; Bethesda, MD, USA).
Antibodies and reagents
Cyclohexamid (CHX), isobutylmethylxanthine (IBMX), leupeptin, cordycepin and dibutyryl-cAMP (dbcAMP) were purchased from Sigma. MG132 (Z-leu-leu-CHO) was purchased from Calbiochem (Schwalbach, Germany). Syto13 was purchased from Molecular Probes (Paisley, Strathclyde UK). Goat anti-mouse-HRP, goat anti-rabbit-HRP and goat anti-mouse-Cy3 were purchased from Jackson Laboratories (West Grove, PA, USA). Monoclonal mouse anti-cdc25B antibodies were purchased from Transduction Laboratories (San Jose CA, USA). The cdc25B peptide was purchased from Transduction Laboratories; this peptide includes the amino acids in the 109–122 position of cdc25B protein sequence. Polyclonal rabbit anti-p34cdc2 antibodies were purchased from Santa Cruz (Santa Cruz CA, USA). Monoclonal mouse anti-tubulin antibody was purchased from Sigma.
Oocytes recovery and culture
Rat oocytes that resume meiosis spontaneously upon their removal from the ovarian follicles were employed for our study. The ability to resume meiosis is acquired progressively during oocyte growth. Accordingly, oocytes that are incompetent and competent to resume meiosis are referred to as growing and fully grown, respectively. A previous study in our laboratory (Goren & Dekel 1994) demonstrated that ovaries of 18-day-old Wistar rats contain growing oocytes, while fully grown oocytes can be recovered from ovaries of 25-day-old rats, primed (48 h earlier) with 10 IU of pregnant mare’s serum gonadotropin (PMSG, Chronogest Intervest). These two groups of animals served, therefore, as the source for the oocytes used for this study. The rats (Harlan, Rehovot, Israel) were sacrificed and their ovaries isolated and transferred into Leibovitz L-15 tissue culture medium (Gibco-BRL) containing 5% fetal bovine serum (Sigma). Follicles were punctured and the cumulus–oocyte complexes removed and placed into acidic L-15 medium (pH 6.0) to obtain cumulus-free oocytes. The oocytes were analyzed either immediatelyafter their isolation or after the indicated times of incubation in a 37 °C humidified incubator. Growing oocytes did not resume meiosis in vitro, whereas fully grown oocytes were maintained meiotically arrested by their incubation with dbcAMP (2 mM) and the cAMP phosphodiesterase inhibitor IBMX (0.02 mM). Meiotic arrest was identified by the presence of GV. Resumption of meiosis, which was indicated by GVB, occurred spontaneously in oocytes incubated in inhibitor-free medium for 4 h. These oocytes reached MI at 8 h after isolation, with PBI being emitted at 10 h of incubation. Oocytes examined after an overnight incubation were arrested at MII.
Western blot analysis
At the end of the indicated incubation time, the oocytes were lysed in Laemmli buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% ß-mercaptoethanol) and the samples were boiled for 5 min. The proteins were electrophoretically separated on a 10% acrylamide gel, followed by their transfer to a nitrocellulose membrane. After blocking with 5% skimmed milk, the membranes were incubated with the relevant primary antibodies overnight at 4 °C followed by the secondary antibodies for 1 h in room temperature. To confirm the specificity of the signal, the first antibody was preincubated with its relevant peptide (1:100) for 2 h at room temperature, prior to immunoblotting. The immunoreactive bands were detected by ECL (Amersham). The intensity of the signal was quantitated by computerized densitometry (quantity one).
Immunocytochemistry
Oocytes were fixed by 3% paraformaldehyde for 20 min, followed by their permeabilization with 1% triton (Sigma) for 4 min. These oocytes were incubated with monoclonal anti-cdc25B (1:200 dilution) at 4 °C, followed by further incubation with the secondary Cy3-conjugated anti-mouse antibody (1:300 dilution) for 1 h at room temperature. For fluorescent staining of the DNA, syto-13 was added along with the secondary antibody (1:1000). To confirm their specificity, the anti-cdc25B antibodies were preincubated with the relevant peptide (1:100) for 2 h at room temperature. All washes were done in GB-PBS (PBS containing 10 mg/ml of BSA and 10 mM glycine, pH 7.4). The oocytes were visualized by a laser scanning confocal system (BioRad) attached to an inverted microscope ECLIPSE TE-300 (Nikon; Melville, NY, USA).
Polyadenylation
The polyadenylation test (PAT) was preformed as described previously (Salles et al. 1999). Briefly, total RNA from 20 oocytes was extracted and dissolved in 5 µl double distilled water (DDW). Upon the addition of 1 µl of oligo(dt)-anchor (200 ng/µl, 5 '-GCGAGCTCCGCGGCCGCGT12–3'), the test tubes were warmed for 5 min at 65 °C, and immediately transferred to 42 °C for 2 min. This was followed by the addition of 14 µl of the reverse transcriptase (RT) cocktail [4 µl of 5 x superscript RNase H– RT buffer Gibco BRL); 2 µl of 0.1 M DTT; 1 µl of 10 mM dNTPs; 1 µl of RNasin (Promega), 4 µl of H2O, 2 µl (200 U/µl) superscript RNase H– RT (Gibco-BRL)]. The samples were incubated at 42°C for 1 h and kept at 65 °C for 15 min for RT inactivation. For the PCR, 1.2 µl of cDNAs was added to a 50 µl of standard PCR cocktail containing 0.5 µl [
-P32] dATP and 25 pmol of each cdc25B mRNA specific primer (5'-CTTTTCCTTTAAGACCCCCAG-3') and the oligo(dt)-anchor primer (amplification conditions: 93°C for 5 min; 30 cycles at 93°C for 30 s; 57°C for 1 min; 72°C for 1 min; with a final extension of 7 min at 72°C). After amplification, PCR products were ethanol-precipitated with 2.5 M ammonium acetate to remove unincorporated radioactivity. To confirm the specificity of the amplification, samples were digested by the restriction enzyme, BtsI (Promega). The radioactive products were electrophoretically separated on 5% non-denaturing polyacrylamide gel in 0.5X TBE buffer. Gels were dried and radioactivity was determined by their exposure to X-ray film over night.
Microinjection
For microinjection, the oocytes were placed in a 50 µl drop of L-15 tissue culture medium containing dbcAMP (2 mM) and IBMX (0.02 mM), under Paraffin oil. Oocyte were microinjected by 5–7 pl of anti-cdc25B or anti-cdc25C antibodies (83 µg/ml in 75% PBS). After microinjection, the oocytes were transferred into inhibitor-free medium, further incubated for 8 h at 37 °C and monitored for GVB by differential interference contrast (DIC) microscopy (Zeiss, Germany). Oocytes microinjected with anti-IgG (83 µg/ml in 75% PBS) served as control.
Statistical analysis
The data presented represent an average of the indicated number of individual experiments and are expressed as means ± S.E.M. Values were compared by F-test and considered statistically different when P < 0.02.
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Results |
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). On the other hand, microinjection of cdc25B antibodies did inhibit GVB that is the first morphological marker of reinitiation of meiosis. As expected, a large fraction (77.1 ± 8.55%) of control, non-injected oocytes resumed meiosis spontaneously. A slightly but not significantly (P < 0.05) lower fraction of anti-IgG-injected oocytes lost their GV throughout 8 h of incubation (67.95 ± 11.24%). On the other hand, microinjection of anti-cdc25B antibodies significantly (P < 0.02) affected the ability of the oocytes to reinitiate meiosis (GVB = 44.96 ± 8.65%).
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). On the other hand, in fully grown oocytes, that reinitiate meiosis a unique oscillatory pattern of cdc25B expression is demonstrated as follows. The amount of cdc25B that is elevated at GVB, decreased substantially at MI, followed by a later increase of the enzyme at MII (Fig. 2a–b). At all the developmental stages examined, the oocytes expressed one single protein that was recognized by the anti-cdc25B antibodies. Incubation of the antibodies with the relevant peptide eliminated immunoreactivity towards this protein indicating specificity towards cdc25B (Fig. 2c). Taken together, these results suggest that cdc25B action throughout meiosis is regulated by modulation of its availability rather than phosphorylation of this enzyme.
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). These concentrations of the inhibitors have been shown previously to elicit maximal effect in our oocyte system (Josefsberg et al. 2000). These findings indicate that cdc25B is degraded at MI by the proteasome, but not by the lysosome. Using GAP in the Analysis package Wisconsin sequence GCG software we indeed identified the presence of KEN, a domain that is required for proteasome catalyzed proteolysis (Pfleger & Kirschner 2000), in the molecule of cdc25B.
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, CHX blocked the reappearance of cdc25B at MII. This finding raised the possibility that the accumulation of cdc25B at MII can represent its translational regulation.
Fully grown oocytes that resume meiosis are transcriptionally dormant. These oocytes contain a pool of mRNA, some of which will be eventually translated. The translation of a selective preexiting mRNA is controlled by elongation of its polyA tail (Colgan & Manley 1997). For further confirmation of translational regulation, oocytes at different stages of meiosis were tested for their cdc25B mRNA polyadenylation. This experiment indeed revealed changes in the length of the cdc25B mRNA polyA tail that coincided with the modifications in the abundance of its protein product demonstrated in this study. Specifically, the GV oocytes possessed a short polyA tail that underwent elongation at GVB. The polyA tail of cdc25B mRNA was short again at MI, with another round of its elongation demonstrated at MII (Fig. 4a). No polyadenylation product was detected in the control samples that were not subjected to RT (data not shown). In order to prove specificity, the polyA samples were subjected to digestion by the restriction enzyme BtsI. The decrease in the size of the reaction product after digestion by 60 bp, confirmed its identity as cdc25B mRNA (Fig. 4b).
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, oocytes incubated in the presence of cordycepin failed to exhibit the elevated expression of cdc25B demonstrated in oocytes incubated in the absence of this inhibitor. This experiment demonstrates the absolute dependency of cdc25B accumulation on cytoplasmic polyadenylation further confirming our suggestion that the expression of this protein is regulated at the level of translation.
Translocation of cdc25B in oocytes resuming meiosis
Availability of cdc25B can serve as one mechanism for the control of its activity during meiosis. An additional mode of regulation of the catalytic activity of this enzyme could be possibly provided by its subcellular translocation. To examine this possibility changes in cdc25B localization during resumption of meiosis were monitored by immunostaining. We found that in meiotically arrested oocytes (Fig. 5a A, B) cdc25B is present in the cytoplasm and is absent in the nucleus (Fig. 5a C). After GVB (Fig. 5a E, F) cdc25B migrates towards the condensed chromosomes (Fig. 5a G). Oocytes arrested at MII (Fig. 5a I, J) exhibit low cytoplasmic abundance of cdc25B and colocalization of this protein with the MII spindle apparatus (Fig. 5a K). Preincubation of the antibody with the relevant peptide, abolished the staining, indicating its specificity towards cdc25B (Fig. 5b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The first clue linking cdc25 to the control of cell cycle was provided by observations that a G2-arrest in fission yeast is caused by cdc25– mutations (Fantes 1981). Later studies in fission yeast showed that the level of cdc25 mRNA oscillates through the cell cycle, reaching maximum at mitosis (Moreno et al. 1989, Ducommun et al. 1990). These studies raised the idea that periodic accumulation of the cdc25 protein is one of the rate-limiting processes regulating the transition into M-phase. Other studies demonstrated that inactivation of cdc25 results in a late G2-arrest that is accompanied by p34cdc2 phosphorylation (Gould & Nurse 1989). These and later demonstrations that cdc25 function is not required in strains expressing non-phosphorylatable p34cdc2 (Gould et al. 1990) were consistent with the idea that cdc25 functions to dephosphorylate this kinase.
The information generated in yeast on the role of cdc25 in regulating mitosis has been confirmed for the control of cell division in higher organisms (Sadhu et al. 1990, Gautier et al. 1991). Furthermore, studies in Xenopus oocytes disclosed a similar function for this enzyme in the control of meiosis (Izumi et al. 1992, Jessus & Beach 1992, Rime et al. 1994). On the other hand, hardly any information has been generated regarding cdc25 in mammalian oocytes. Since mammals express multiple cdc25 genes (Sadhu et al. 1990, Galaktionov & Beach 1991, Millar et al. 1991, Nagata et al. 1991) the identity of the specific isoform responsible for p34cdc2 dephosphorylation in mammalian oocytes resuming meiosis raised an additional point of interest.
As mentioned previously, it was commonly accepted that in mammals it is the cdc25C isoform that regulates the exit from G2 and entry into M-phase of the cell cycle (Gautier et al. 1991, Millar et al. 1991). Since reinitiation of meiosis represents a G2- to M-transition, the logical assumption was that resumption of meiosis in mammalian oocytes is subjected to regulation by cdc25C. However, the observation that knockout mice, lacking cdc25C, were fertile (Chen et al. 2001) denied a role for this isoform in regulation of meiosis.
The candidacy of cdc25B in regulating reinitiation of meiosis was initially postulated by demonstrations that microinjection of this isoform into Xenopus oocytes induced GVB (Powers et al. 2000). A more recent study showing that oocytes of female mice lacking cdc25B fail to activate MPF and remain meiotically arrested, confirmed this idea (Lincoln et al. 2002). The inhibitory effect of anti-cdc25B and not anti-cdc25C antibodies on reinitiation of meiosis demonstrated herein agrees with the notion that it is the B isoform of the cdc25 phosphatase family that plays a role in reinitiation of meiosis in rat oocytes. These results further suggest that cdc25B and cdc25C do not have redundant roles in regulating meiosis resumption. In fact, some supporting evidence of the role of cdc25B in entry into M-phase has also been generated in mitotically dividing cells. These studies demonstrated that injection of specific antibodies into human fibroblasts or overexpression of cdc25B mutants in HeLa cells inhibit entry into mitosis (Gabrielli et al. 1996, Lammer et al. 1998). Another study showed that overexpression of cdc25B in HeLa cells causes premature transition into M-phase (Karlsson et al. 1999).
A complementary evidence for the role of cdc25B in regulation of oocyte meiosis is provided by the oscillatory pattern of its expression. Cell cycle associated modulation of the expression of cdc25B has been previously reported in mitosis (Russell & Nurse 1986, Gabrielli et al. 1996, 1997a, Lammer et al. 1998). However, regulation of cdc25 by modulating its availability in the meiotic cell cycle is demonstrated herein. We show that the abundance of this enzyme substantially increases at GVB, transiently decreases at MI and is elevated again at MII. This periodic accumulation of cdc25B nicely corresponds to the kinetics of MPF activity throughout meiosis (Doree et al. 1983, Hashimoto & Kishimoto 1986, Choi et al. 1991, Fulka et al. 1992, Josefsberg et al. 2003). It is therefore plausible that the activity of MPF in rat oocytes is controlled by the availability of cdc25B. Accumulation of cdc25B shown herein represents apparently de novo synthesis of the enzyme mediated by polyadenylation.
Fully grown oocytes that resume meiosis are transcriptionally dormant. Protein synthesis in these oocytes is enhanced by recruitment of pooled mRNA and its selective cytoplasmic polyadenylation (Colgan & Manley 1997). So far, mRNAs for Mos, tPA, cyclin A1 and B1 as well as wee1 (Huarte et al. 1987, Gebauer et al. 1994, Sheets et al. 1994, Stebbins-Boaz et al. 1996, Charlesworth et al. 2000) have been shown to undergo polyadenylation throughout meiosis. We demonstrate herein for the first time that similar to the above-mentioned proteins, cdc25B translation in oocytes resuming meiosis is also regulated by its mRNA polyadenylation. Identification of both CPE and hexanucleotide sequence in the cdc25B mRNA agrees with our finding. Unlike the above-mentioned mRNAs, that exhibit an elongated polyA tail soon after reinitiation of meiosis that sustains throughout meiosis (Sheets et al. 1994, de Moor & Richter 1997, Lazar et al. 2002), polyadenylation of cdc25B mRNA and the subsequent accumulation of its protein product exhibits an oscillatory pattern.
It has been shown in Xenopus oocytes that poly-adenylation of cyclin B and cyclin A mRNAs is Mosdependent and that this effect of Mos is mediated by MPF (de Moor & Richter 1997). A later study in the rat revealed that Mos mRNA polyadenylation is subjected to negative regulation of cAMP and that this inhibitory effect is mediated by PKA (Lazar et al. 2002). A more recent report further demonstrated that Mos mRNA polyadenylation in rat oocytes will not take place in absence of an active MPF (Lazar et al. 2004). The mechanisms that regulate cdc25B expression during oocyte maturation remain to be elucidated.
The cdc25B mRNA polyadenylation followed by its translation in GVB oocytes, nicely corresponds with MPF activation at reinitiation of meiosis. However, the reduction in the amount of cdc25B at MI is somewhat unexpected, since inactivation of MPF upon completion of the first meiotic division that is subsequent to cyclin degradation, is not associated with p34cdc2 rephosphorylation (Choi et al. 1991, Goren & Dekel 1994). Furthermore, since reactivation of MPF upon the transition into MII is supposed to be solely dependent on cyclin synthesis, the second round of cdc25B mRNA polyadenylation was not anticipated. A clue for the possible role for cdc25B accumulation after completion of the first meiotic division is provided by a recent demonstration that co-expression of cdc25B and cyclin B in G1 cells drives the cells to enter mitosis without DNA replication (Karlsson et al. 1999). The concomitant elevation of cdc25B and cyclin B upon transition into MII may provide the mechanism that uncouples between cell division and DNA replication causing two sequential M-phases in meiosis. In addition, a previous study (Nakajo et al. 2000) claimed that wee1 decreases after reinitiation of meiosis and remains low until fertilization. Indeed, in the absence of wee1, there is no requirement for cdc25 to balance its effect on p34cdc2 phosphorylation. However, another report demonstrating that wee1 is present in oocytes resuming meiosis (Iwabuchi et al. 2000) disagrees with this idea further suggesting that accumulation of cdc25B may have a role in MPF reactivation at the entry into the second meiotic division.
The periodic pattern of cdc25B accumulation demonstrated herein in rat oocytes throughout meiosis is regulated by protein synthesis and apparently balanced by proteasomal degradation. Proteolysis of cdc25B that is brought about by the proteasome has been previously demonstrated (Baldin et al. 1997a, Cans et al. 1999). It has also been reported that at least in yeast cdc25 degradation is preceded by its ubiquitination (Nefsky & Beach 1996). This study further identified pub1, a major ubiquitin ligase of cdc25, the disruption of which dramatically reduces ubiquitination of cdc25 further stimulating premature entry into mitosis. Along this line, we report herein that the KEN motif, a domain on the molecule required for ubiquitin-mediated proteolysis (Pfleger & Kirschner 2000) can be identified in the cdc25B molecule.
Regulation of cdc25B by synthesis and degradation could be modulated by subcellular localization restricting its function to a precise cellular location. In support of this idea, a cell cycle-dependent transport of cdc25B in and out the nucleus, has been demonstrated in somatic (Girard et al. 1992, Garner-Hamrick & Fisher 1998, Dalal et al. 1999, Davezac et al. 2000). Our immunocytochemical experiments revealed a restricted cytoplasmatic localization of cdc25B in G2-arrested oocytes. We further demonstrate herein that cdc25B colocalizes with the spindle apparatus, which is of most interest. It has been shown that cyclin B and cdc25B translocate to the nucleus in concert (Gabrielli et al. 1996, 1997b) and that the cytoplasmatic localization of cdc25B depends upon cyclin B (Karlsson et al. 1999). Since cyclin B has also been demonstrated to be associated with the meiotic spindle (our unpublished observations), these findings may represent compartmentalization of the enzyme and its substrate that increases the efficiency of its catalytic activity. Finally, another support to this idea is provided by a recent study demonstrating that cdc25B has a role in activating the cyclin B–Cdk1 complex on the centrosomes at the G2/M transition in Hela cells undergoing mitosis (Lindqvist et al. 2005). In summary, our findings not only confirm the role of cdc25B in rodent oocytes, but also present a unique mode of regulation of the activity of this phosphatase by temporal and spatial modulation of its availability.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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