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Regular Article |
a Center for Research on Reproduction and Women's Health and
b Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
ABSTRACT
M-Phase promoting factor (MPF) is a complex of p34cdc2 and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34cdc2, rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34cdc2 is in excess to that of cyclin B, i.e., the accumulation of p34cdc2 is not a limiting factor. We report measurements of the absolute mass of p34cdc2 and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34cdc2, cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 x 106, 11.3 x 106, 24.6 x 106, 15.6 x 106, respectively, and in meiotically competent oocytes the numbers are 14.3 x 106, 95.5 x 106, 80.0 x 106, 40.1 x 106, respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34cdc2, and this is consistent with the hypothesis that the accumulation of p34cdc2 plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34cdc2 in both meiotically incompetent and competent oocytes.
gametogenesis, oocyte development
INTRODUCTION
Oocytes grow while arrested in the first meiotic prophase. During this period of growth, which is accompanied by the synthesis and accumulation of macromolecules and organelles (e.g., ribosomes, mitochondria) that will constitute the maternal contribution to early development following fertilization, oocytes acquire the ability to resume meiosis, i.e., oocyte maturation [1, 2]. In the mouse, as in other mammals, when fully-grown oocytes (
80 µm in diameter) obtained from preovulatory antral follicles are placed in a suitable culture medium, virtually all of the oocytes undergo chromosome condensation and germinal vesicle breakdown (GVBD), and progress to and arrest at the second meiotic metaphase (metaphase II) [3]; fertilization triggers the completion of the second meiotic reduction. In contrast, mouse oocytes <60 µm in diameter fail to undergo GVBD, i.e., they fail to resume oocyte maturation [2]. This acquired ability to resume meiosis is termed the acquisition of meiotic competence and is acquired in two broadly defined phases that are associated with increasing oocyte diameter, i.e., >60 µm in diameter. In the first phase an increasing fraction of the oocytes undergo GVBD [2] and arrest at metaphase I with the chromosomes aligned on a metaphase plate [4]. With further growth, an increasing fraction of the oocytes is able to progress to and arrest at metaphase II [2, 4].
The acquisition of meiotic competence during oocyte growth is associated with both cytoplasmic and nuclear changes reminiscent of somatic cells entering into M phase. For example, the chromosomes become more condensed [5]. In fact, meiotically competent oocytes are characterized by a rim of condensed chromatin surrounding the nucleolus [4, 6–8]. In addition, meiotically incompetent oocytes have nonphosphorylated centrosomes and long microtubule arrays characteristic of interphase cells, whereas meiotically competent oocytes have phosphorylated centrosomes that are associated with short microtubules [9].
Activation of M-phase promoting factor (MPF), which is a heterodimer composed of p34cdc2 and cyclin B [10, 11], plays a critical regulatory role in oocyte maturation. For example, MPF activation precedes GVBD in the mouse and inhibiting its activation prevents GVBD [12, 13]. Because MPF can phosphorylate centrosomes and convert an interphase microtubule array into an M-phase configuration in Xenopus egg extracts [14], changes in MPF concentration/activity during oocyte growth may underlie the observed changes in centrosome phosphorylation and microtubule length that occur during oocyte growth and the accompanying acquisition of meiotic competence. In fact, results of several studies employing either immunocytochemical or immunoblotting approaches demonstrate that while the concentration of cyclin B remains fairly constant during the acquisition of meiotic competence, the concentration of p34cdc2 increases by severalfold [15–17]. Such results have been interpreted to suggest that the accumulation of p34cdc2 may be a critical determining factor in the acquisition of meiotic competence. Although this is an attractive hypothesis, it should be noted that because different antibodies were used in these studies to measure the relative changes of cyclin B and p34cdc2 in meiotically incompetent and competent oocytes, only relative changes in mass of these two proteins, and not changes in their absolute amounts, were determined. Hence, it is formally possible that because functional MPF is a heterodimer, the molar amount of p34cdc2 could already be in excess of the molar amount of cyclin B in both incompetent and competent oocytes. Moreover, changes in the absolute amounts of the two proximal regulators of MPF, namely cdc25C and wee1, could also participate in the acquisition of meiotic competence. Cdc25C is a dual-specificity phosphatase that activates MPF, whereas wee1, which is a protein kinase, inactivates MPF [18, 19]. Therefore, measurements of the absolute molar amounts of p34cdc2, cyclin B, cdc25C, and wee1 in incompetent and competent oocytes would, in principle, provide better insight into the molecular basis for the acquisition of meiotic competence.
We report here the molar amounts of these aforementioned proteins. We find that the concentration of cyclin B1 is about seven times greater than that of p34cdc2 in both meiotically incompetent and fully grown, competent oocytes; the concentration of each increases during this period of oocyte growth. These results suggest that the accumulation of p34cdc2 is rate-limiting during the acquisition of meiotic competence. In addition, in both meiotically incompetent and competent oocytes, the concentration of both cdc25C and wee1 are greater than that of p34cdc2.
MATERIALS AND METHODS
Oocyte Collection
Fully-grown, meiotically competent cumulus-free oocytes were collected from 6-wk-old, eCG-primed female mice (CF-1, Harlan) as previously described [20]. The collection medium was bicarbonate-free minimum essential medium with Earle salts and supplemented with 100 µg/ml pyruvate, 10 µg/ml gentamycin, 3 mg/ml polyvinylpyrrolidone, and 25 mM Hepes, pH. 7.2. Meiotically incompetent oocytes were obtained from 12-day-old prepubertal mice by incubating pieces of ovarian tissue in Ca2+-, Mg2+-free Chatot, Ziomek, and Biggers (CZB) medium [21] containing 1 mg/ml collagenase and 0.2 mg/ml DNase at 37°C for up to 60 min. During this time, the oocytes present in the cohort of growing follicles were freed by repeated pipetting with a mouth-operated micropipet. In both cases, the oocytes were collected and transferred to CZB medium containing 0.2 mM 3-isobutyl-1 methylxanthine that inhibits GVBD of the meiotically competent oocytes [20].
Synthesis and Purification of Recombinant p34cdc2-, Cyclin B1-, cdc25C-, and wee1-Glutathione-S Transferase Fusion Proteins
In order to clone the full-length mouse p34cdc2 (residues 1–298) and cyclin B1 (residues 1–431) into pGEX-2T vector, the BamHI-EcoRI fragments of mouse cdc2 and cyclin B1 were produced by polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene, La Jolla, CA). pBluescript SK-cdc2 [22] (GeneBank M38724) and pBSSK-cyclin B1 [23] served as the template. The primers used for PCR amplification of cdc2 were 5'-GTTGAGGATCCATGGAAGACTAT-3' and 5'-GGGCTGAATTCTCTTAATCTGAT-3' and those for cyclin B1 were 5'-TGGGAGGATCCATGGCGCTCAGG-3' and 5'-TGGAGAATTCCCTTTGTCACGGC-3'. The PCR products were gel-purified, and the purified products were digested with BamHI-EcoRI and then cloned into pGEX-2T according to standard procedures. This vector is suitable for constructing glutathione-S transferase (GST)-fusion proteins in which GST is at the amino-terminal portion of the protein and is followed by a thrombin cleavage site.
To clone the full-length mouse cdc25C (residues 1–438) into pGEX-4T3 vector, the BamHI-XhoI fragment of mouse cdc25C was produced by PCR using pBluescript SK-cdc25C [24] (GenBank U15562) as the template. The primers for PCR amplification of cdc25C were 5'-GACTCCTGGATCCATGTCTACA-3' and 5'-TCATAGTCTCGAGTGTGGGCT-3'. The PCR product was then gel-purified, digested with BamHI-XhoI, and then cloned into pGEX-4T3.
To clone the full-length mouse wee1 (residues 1–647) into pGEX-2T, pBSSK-wee1 was digested with BamHI-EcoRI and the 668-base pair (bp) fragment was cloned into pGEX-2T. The pBSSK-wee1 clone was digested with EcoRI alone and the 1300-bp fragment was cloned into pGEX-2T that already contained the 668-bp fragment.
Escherichia coli BL21 (Novagen) were transformed with these constructs. The BL21 cells transformed with the pGEX vector containing the appropriate insert were cultured in 200 ml of 2x TY containing 100 µg/ml ampicillin to an optical density at 600 nm of 0.6–0.8, and fusion protein expression was induced by adding isopropyl-ß-D-thiogalactopyranoside (final concentration of 0.1–1.0 mM) followed by an incubation at 30°C for an additional 3 h. Following centrifugation, the bacterial pellet was suspended in 20 ml 1x PBS containing 0.1% NP-40, 1 mM PMSF, and 1 µg/ml each of leupeptin and pepstatin. The cells were then lysed by sonication. Insoluble material was removed by centrifugation, the supernatant transferred to a fresh tube, and then mixed with 1 ml of a 50% slurry of glutathione-conjugated Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ). The suspension was then agitated gently at 4°C for 1–2 h. The Sepharose 4B beads were collected by a brief centrifugation, and the beads were then washed three times with ice-cold PBS. Elution of recombinant cdc2, cyclin B1, and wee1 was achieved by incubating the beads for 10 min at room temperature in 600 µl of a buffer containing 50 mM Tris-HCl, pH 8.0, and 10 mM reduced glutathione. Elution of cdc25C was achieved under similar conditions using a buffer containing 100 mM Tris-HCl, pH 8.0, 120 mM NaCl, and 20 mM reduced glutathione. After the incubation, the beads containing the sample were centrifuged and the supernatant was then transferred to a fresh tube and incubated with 20 µl of thrombin (1 U/µl, Amersham Pharmacia Biotech) at 37°C for 2 h. After digestion, the proteins were aliquoted and stored at -80°C until use.
To measure the concentration of recombinant protein, the recombinant protein was added to Laemmli sample buffer [25] containing 10% 2-mercaptoethanol, boiled for 3 min, and then subjected to SDS-PAGE in 10% polyacrylamide gels along with known amounts of BSA. After silver staining, the gel was scanned on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). A standard curve was constructed using the values of the BSA samples, and the amount of recombinant protein was determined by interpolation of the value obtained for the recombinant protein against the standard curve. The density of the bands was analyzed with IP Lab Gel software (Molecular Dynamics).
Preparation of Protein Extracts, SDS-PAGE, and Western Blotting
Oocyte extracts were prepared by adding oocytes (the number indicated in the figures) in a minimum volume of collection medium (<5 µl) to 20–30 µl of protein extraction buffer (20 mM Tris-HCl, pH, 7.5, containing, 100 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM PMSF, and 1 µg/ml each of leupeptin and pepstatin). The extracts were rapidly frozen on dry ice and stored at -80°C and used within 3 h. Laemmli sample buffer containing 10% 2-mercaptoethanol was added to the protein extracts and the different recombinant proteins whose protein concentration was known (relative to BSA, see above). The samples were then boiled for 3 min, and then known amounts of the different recombinant proteins, as well as known amounts of oocyte protein extracts (using 25 ng protein/fully-grown oocyte [26]) were subjected to SDS-PAGE in 10% polyacrylamide gels. The fractionated proteins were transferred to Immobilon P (Millipore, Bedford, MA), and the membranes were then incubated for 1 h in PBS containing 0.27% Tween 20 and 10% fish gelatin (PBST). The blocked membranes were then incubated for 1 h with 2 µg/ml of the appropriate primary antibody. The blots were washed three times in PBST, incubated for 1 h with a biotinylated secondary antibody (Jackson Immunoresearch, West Grove, PA), and then washed three times with PBST. The blots were then incubated with the Vectastain ABC reagent (Vector Laboratories, Santa Clara, CA) for 30 min and then washed four times with PBST. Immunoreactive proteins were detected using the enhanced chemiluminescence detection system according to the manufacturer's instructions (Amersham Pharmacia Biotech). The x-ray film was scanned on a Personal Densitometer SI and the signal intensity was quantified using IP Lab Gel software (Molecular Dynamics). To determine the amount of the protein under investigation, a standard curve was constructed using the respective recombinant protein, and the values obtained for that protein in the oocyte extracts was determined by interpolation, using the linear portion of the standard curve (see Results and Discussion). The experiment was performed three times for each protein using both meiotically incompetent and competent oocytes.
Antibodies
Anti-p34cdc2 was a mouse monoclonal antibody (IgG2a) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal anti-human cyclin B1 was obtained from Upstate Biotechnology, Inc.; this antibody was raised against a bacterially produced GST-fusion protein (IgG2b) corresponding to residues 130–433 of human cyclin B1. A rabbit polyclonal antipeptide antibody to cdc25C (catalog no. SC-327) and its respective blocking peptide were from Santa Cruz Biotechnology. Anti-wee1 was a rabbit polyclonal antibody that was raised against a synthetic peptide based on the amino acid sequence (residues 505–532) of the human wee1 protein kinase (Calbiochem, LaJolla, CA).
Antibody specificity was established using both recombinant and oocyte extracts (Fig. 1). Using both oocyte extracts and recombinant p34cdc2, the anti-p34cdc2 antibody recognized a protein of Mr34 000, which is the expected relative mass of p34cdc2 (Fig. 1A). It should be noted that under the conditions of SDS-PAGE the phosphorylated forms of p34cdc2 that migrate with a slower electrophoretic mobility [16, 27] were intentionally not resolved in order to facilitate quantifying the mass amount of p34cdc2. When the primary antibody was replaced with mouse IgG this band was no longer detected.
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The anti-cyclin B1 antibody recognized a protein of Mr 60 000 when the recombinant protein was used and recognized proteins of Mr 60 000 and 62 000 in oocyte extracts; the protein of slower electrophoretic mobility likely represented a phosphorylated form of cyclin B ([28] and see Results and Discussion) (Fig. 1B); both forms were used to calculate the mass amounts of cyclin B1 in meiotically incompetent and competent oocytes. When the primary antibody was replaced with mouse IgG, this band was no longer detected.
The anti-cdc25C antibody recognized a single protein of Mr 82 000 when using recombinant cdc25C-GST fusion protein (Fig. 1C). This immunoreactive band was not observed when antibody incubated with the peptide to which it was generated was used. When the cdc25C-GST was cleaved with thrombin, two immunoreactive bands of Mr 57 000 and 55 000 were detected, and both of these bands were not observed when peptide-absorbed antibody was used (data not shown). We do not know why two bands were generated under these conditions. In order to simplify the quantification of the mass amount of cdc25C, the standard curves were constructed using the fusion protein. It should be noted, however, that when standard curves were constructed using these two cleavage products, similar results were obtained as those reported in Table 1. When oocyte extracts were used, a band of the anticipated Mr 53 000 was observed, and this immunoreactivity was specific, i.e., it was not detected when peptide-absorbed antibody was used.
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When recombinant wee1 was used, the anti-wee1 antibody recognized a protein of Mr 72 000, which is the anticipated size based on the amino acid sequence of the full-length cDNA (Fig. 1D). In contrast, the antibody recognized a protein of Mr 98 000 when oocyte extracts were probed. This anomalous electrophoretic behavior of wee1 detected in cellular extracts has been previously reported [29, 30]. Omission of the primary antibody resulted in the loss of the Mr 98 000 and Mr 72 000 bands in both the oocyte extracts and recombinant protein preparations, respectively. This result strongly supports the interpretation that the protein of Mr 98 000 in oocyte extracts is, in fact, wee1. For the data presented below, the mass measurements for wee1 were based on the predicted molecular weight of Mr 72 000.
For p34cdc2, cyclin B1, and wee1 it was not possible to determine antibody specificity by absorbing the antibody with the recombinant protein because only microgram quantities of recombinant protein could be isolated and purified.
RESULTS AND DISCUSSION
Figure 2 illustrates the experimental data used to calculate the mass amounts of p34cdc2, cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes. It should be noted that all of the measurements were made using the linear portion of the standard curve constructed with the appropriate recombinant proteins. The results of these experiments are summarized in Table 1. The concentrations of both p34cdc2 and cyclin B1 increased about threefold between meiotically incompetent oocytes (55 µm in diameter) and fully grown meiotically competent oocytes (78 µm in diameter). The concentration of cyclin B1, however, is about sevenfold higher than that of p34cdc2 at both stages, i.e., there is a molar excess of cyclin B1. This molar excess is in direct contrast to the situation observed in Xenopus laevis. In fully grown stage VI Xenopus oocytes, there are 1011 molecules of p34cdc2 (50 nM) and 2.4 x 109 molecules of cyclin B (1 nM) [31]. Thus, the majority of p34cdc2 is not complexed with cyclin B. This pool of uncomplexed p34cdc2 likely accounts for the observation that Xenopus oocytes injected with cyclin B mRNA resume meiosis in the absence of progesterone stimulation [32, 33].
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Our results, in which mass amounts were measured, are consistent with the previous proposal that p34cdc2 could be limiting in the meiotically incompetent oocyte and its accumulation a critical factor in the acquisition of meiotic competence [15–17]; it should be noted that this previous conclusion was based on measurements of relative changes of these cell cycle components. Nevertheless, changes in the ability of p34cdc2 and cyclin B1 to dimerize during the acquisition of meiotic competence are also likely to be a major locus of control. For example, although the injection of p34cdc2 mRNA into meiotically incompetent mouse oocytes results in protein levels of p34cdc2 similar to those found in meiotically competent oocytes, the injected oocytes nevertheless fail to resume meiosis [27]. The reason for this failure is the apparent inability of the newly synthesized p34cdc2 to become phosphorylated and thus be able to associate with the endogenous pool of cyclin; phosphorylation of p34cdc2 is required for it to associate with cyclin B and only the nonphosphorylated p34cdc2 was observed [27]. The failure of the p34cdc2 synthesized in the meiotically incompetent oocytes from the injected p34cdc2 mRNA to become phosphorylated is not a consequence of the meiotically incompetent oocytes lacking the enzymes that phosphorylate p34cdc2. Meiotically incompetent oocytes contain wee1 [17], which catalyzes an inhibitory phosphorylation of Tyr 15 of p34cdc2. Moreover, coinjecting meiotically incompetent oocytes with mRNAs encoding p34cdc2 and cyclin B1 results in the phosphorylation of the newly synthesized p34cdc2 (i.e., it associates with the newly synthesized cyclin B1) and resumption of meiosis [27]. The detection of active MPF also implies that meiotically incompetent oocytes possess an active cdk7 that is complexed with cyclin H. This cyclin-dependent protein kinase phosphorylates Thr 161 of p34cdc2 that is necessary for enzyme activity. In toto, these results suggest that although cyclin B1 is in excess in meiotically incompetent oocytes, it does not complex readily with p34cdc2 and the accumulation of p34cdc2 is not sufficient to establish meiotic competence.
Acquisition of meiotic competence also may entail changes in the ability of the p34cdc2-cyclin B complex to localize to the nucleus. Both p34cdc2 and cyclin B1 are almost exclusively localized to the cytoplasm in the meiotically incompetent oocyte, whereas their nuclear concentration is dramatically increased in meiotically competent oocytes [17]. This change in localization is consistent with meiotically competent oocytes possessing the M-phase characteristics that were described in the Introduction. In fact, MPF translocates to the nucleus in somatic cells concomitant with entry into M phase [34]. Phosphorylation of cyclin B may be a critical modulator of MPF localization. Cyclin B contains a nuclear export sequence, and phosphorylation of cyclin B inhibits its ability to interact with the nuclear export factor CRM1 [35]. This results in cyclin B accumulation in the nucleus. Although not an objective of this study, the results of our immunoblotting experiments (Fig. 2B) suggest that the acquisition of meiotic competence is associated with an increase in the ratio of the phosphorylated to nonphosphorylated forms of cyclin B1. Such a shift could provide a molecular basis for the observed accumulation of p34cdc2-cyclin B in the nucleus of meiotically competent oocytes.
Superimposed on these aforementioned changes in the concentration of p34cdc2 and cyclin B, changes in their association into a functional complex, and changes in their nuclear localization are two proximal regulators of MPF activity, namely cdc25C and wee1. Cdc25C activates MPF by catalyzing the dephosphorylation of Thr 14 and Tyr 15 of p34cdc2, whereas wee1 inhibits MPF by catalyzing the phosphorylation of Tyr 15 of p34cdc2 [18, 19]. Both proteins are present in meiotically incompetent and competent oocytes and results of a previous study in which relative amounts of cdc25C and wee1 were measured indicated that the concentration of cdc25C increased while that of wee1 decreased during the acquisition of meiotic competence [17]. As previously discussed, this reciprocal change in concentration could contribute to the acquisition of meiotic competence by shifting the relative balance of these to regulators toward MPF activation. Results of our study confirm the reciprocal changes in concentration of cdc25C and wee1 during the acquisition of meiotic competence but extend these observations by demonstrating that the concentration of both of these regulators is always greater than the concentration of functional p34cdc2/cyclin B complex. A similar reciprocal change in the concentration of cdc25C and wee1 also occurs during the acquisition of meiotic competence in Xenopus oocytes [36] and may be a general phenomenon that occurs during oocyte growth. The expression in mouse oocytes of the membrane-associated myt1 kinase [37] that catalyzes the phosphorylation of p34cdc2 on both Thr 14 and Tyr 15 (wee1 only catalyzes the phosphorylation of Tyr 15) [38, 39] has not been reported. In Xenopus oocytes, myt1 plays a direct role in prophase I arrest in fully grown oocytes but not in maturation, e.g., inhibiting myt1 results in dephosphorylation of Tyr 15 of p34cdc2 and GVBD in the absence of progesterone, but ectopic overexpression of myt1 has little, if any, effect on oocyte maturation [36]. The role of myt1 in the acquisition of meiotic competence in mouse oocytes warrants further investigation.
ACKNOWLEDGMENTS
The full-length cDNAs for cdc25C, p34cdc2, and cyclin B1 that were used as templates were the generous gift of Dr. Deborah Wolgemuth, Columbia University, and the cDNA for wee1 was the generous gift of Drs. Reiko Honda and Hideyo Yasuda, Tokyo University of Pharmacy and Life Science.
FOOTNOTES
1 This research was supported by a grant from the NIH to G.S.K. and R.M.S. (HD 22732). 
2 Correspondence: Gregory S. Kopf, Rm. 1315, Biomedical Research Building II, Center for Research on Reproduction and Women's Health, University of Pennsylvania, 421 Curie Blvd., Philadelphia, PA 19104-6142. FAX: 215 573 4337; kopf{at}mail.med.upenn.edu
3 Current address: Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Accepted: July 12, 2000.
Received: May 16, 2000.
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