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BOR - Papers in Press, published online ahead of print November 30, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046375
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BIOLOGY OF REPRODUCTION 74, 593–600 (2006)
DOI: 10.1095/biolreprod.105.046375
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Study of Germinal Vesicle Requirement for the Normal Kinetics of Maturation/M-Phase-Promoting Factor Activity During Porcine Oocyte Maturation1

Koji Sugiura 3 , Kunihiko Naito 2 , Tsutomu Endo , and Hideaki Tojo 

Department of Animal Resource Sciences, Graduate School of Agricultural Sciences, University of Tokyo, Tokyo 113-8657, Japan

ABSTRACT

Mammalian immature oocytes contain large nuclei referred to as germinal vesicles (GVs). The translocation of maturation/M-phase promoting factor (MPF) into GVs just before the activation of MPF has been reported in several species. To examine whether the GV is required for MPF activation in mammalian oocytes, porcine immature oocytes were enucleated and their MPF activity and CCNB (also known as cyclin B) levels were investigated. The activation of MPF at the start of maturation was detected at normal levels in enucleated oocytes, whereas reactivation to induce the second meiosis was not observed. Although protein synthesis was found to be normal both qualitatively and quantitatively, even in the absence of the nucleus, CCNB1 did not sufficiently accumulate in the enucleated oocytes. The defects in the enucleated oocytes were reversed by the injection of GV material into the enucleated oocytes. Furthermore, the inhibition of CCNB1 degradation revealed drastic accumulation of CCNB1, indicating active synthesis of CCNB1 in enucleated oocytes. The mitogen-activated protein kinase cascade remained unaffected by enucleation. These results indicate that GV is not required for the activation of MPF during the first meiosis, but that it is required for the second meiosis because of its promotion of CCNB1 accumulation.

gamete biology, gametogenesis, kinases, meiosis, oocyte development

INTRODUCTION

Oocytes arrested at the first meiotic prophase possess a distinctly large nucleus, which is referred to as the germinal vesicle (GV). Meiotic resumption of immature mammalian oocytes, defined by the breakdown of the GV membrane (GVBD), is induced by maturation/M-phase promoting factor (MPF), a key G2/M regulating kinase in eukaryotic cells that consists of a catalytic subunit, CDC2, and a regulatory subunit, CCNB (also known as cyclin B), as has also been reported in the case of lower animals [1, 2]. The fluctuation pattern of MPF activity during meiotic maturation of mammalian oocytes has been reported in many species [3–9]. The activation of MPF in mammalian oocytes correlates well with GVBD, and induces the first meiotic metaphase. Thereafter, the MPF activity transiently decreases when the oocyte emits the first polar body, and then MPF is consecutively reactivated to induce the second meiosis without DNA replication. These two consecutive M-phases without DNA replication are a principal characteristic of meiosis, which is accomplished via the consecutive reactivation of MPF after interkinesis; however, the mechanism of this meiosis-specific MPF regulation is not yet well understood.

Germinal vesicles are the enormous nuclei characteristic of immature oocytes, and the GV contents undergo mixture with the cytoplasm at the initiation of oocyte maturation by GVBD. Translocation of MPF into the GV just before MPF activation has been reported in studies of several species, including porcine oocytes [10–12]. Such findings have generated the expectation that the translocation of MPF into the GV, as well as the interaction of MPF with GV material, may be required for the activation and the maintenance of MPF activity. In starfish oocytes, the stimulus of maturation was unable to induce sufficient MPF activation in enucleated immature oocytes [13, 14], and meiotic resumption could not be induced by the injection of a small amount of active MPF, but was induced by injection of MPF with GV material [15, 16]. Based on the results of these reports, the requirement of GV material for MPF activation, especially for the activation of amplification machinery for MPF activity, has been accepted in the case of starfish oocytes. In contrast, the translocation and interaction of MPF with GV material has long been believed to be unnecessary for MPF activation in frog oocytes, because of the activation of MPF following the maturation stimulus in enucleated oocytes [17–19]. However, Iwashita et al. [20] revealed that the level of activation of MPF in enucleated Xenopus oocytes was lower than that of intact oocytes, and the time course of activation was also slower than that of intact oocytes. Moreover, they also demonstrated that the MPF in enucleated Xenopus oocytes was activated only once, followed by ongoing inactivation; the addition of nuclear material was then found to restore MPF activity after a transient decrease [20], thus suggesting the requirement of nuclear material for the reactivation of MPF after interkinesis.

In mammalian oocytes, Balakier and Masui [21] have demonstrated the activation of MPF in enucleated mouse oocyte fragments, suggesting the dispensability of the GV for MPF activation. In addition, we previously reported the MPF activation at the start of oocyte maturation in enucleated porcine oocytes [22]. Such findings have indicated that neither the translocation of MPF into the GV nor the GV material itself was required for the activation of MPF, at least not during the first meiosis. Recently, Polanski et al. [23] replaced the GV in immature mouse oocytes with various cell nuclei, and they observed the MPF activity and chromosome status in these oocytes after maturation culture. Their report revealed that both enucleated mouse oocytes and enucleated oocytes injected with cumulus nuclei exhibited low MPF activity as well as decondensed chromosomes after maturation culture, whereas enucleated and GV-injected oocytes showed high MPF activity and condensed chromosomes; such findings are suggestive of a GV requirement for achieving normal MPF kinetics [23]. However, at present, the details regarding maturation-phase GV requirements are unknown, and the reasons for such low levels of MPF activity in enucleated oocytes have not yet been examined in mammalian oocytes.

The present study was therefore conducted to elucidate whether or not the GV is required to produce the normal kinetics of MPF during porcine oocyte maturation, especially during the second meiosis. To this end, porcine immature oocytes were enucleated, and the kinetics of MPF and the levels of CCNB were evaluated. Moreover, kinetics of mitogen-activated protein kinase (MAPK)-ribosomal S6 kinase cascade in enucleated oocytes was also examined.

MATERIALS AND METHODS

Collection and Culture of Porcine Oocytes

Ovaries of prepubertal gilts were collected at a commercial slaughterhouse (Shibaura-zoki). Porcine cumulus cell-oocyte complexes were aspirated from the antral follicles (2–5 mm in diameter) in the ovaries as described in previous reports [22, 24]. The oocytes were denuded from the surrounding cumulus cells by gentle pipetting with a fine-bore pipette, and some of the oocytes were enucleated as described below. Denuded but not enucleated oocytes were used as control oocytes. Groups of 20–25 oocytes were cultured for up to 50 h in the culture medium, which consisted of modified Krebs-Ringer bicarbonate solution [25] containing 20% porcine follicular fluid, 1.0 IU/ml eCG (Pramex; Sankyo), and 3.2 mg/ml BSA (fraction V; WAKO Pure Chemical Ind.), at 37°C in 100% humidity and 5% CO2 in air.

Micromanipulation of Porcine Oocytes

Enucleation of porcine oocytes was performed as described previously [24, 26], soon after collection from the follicles. Briefly, denuded oocytes were centrifuged to translocate the lipid granules to one side of the oocyte and to render the GV visible. Then, the oocytes were treated with cytochalasin B (Sigma). Each oocyte was held with a holding pipette, and its zona close to the GV was cut with an injection pipette without damaging the oolemma. The oocyte was then aspirated by the holding pipette, so that GV with a small amount of cytoplasm was pushed out of the zona through the slit (see Supplemental Fig. 1, available online at http://www.biolreprod.org). The karyoplast was separated from the enucleated oocyte by gentle pipetting. The enucleation process was completed within 2 h. The time at the end of the manipulation was defined as 0 h of culture for both enucleated and control oocytes.


Figure 1
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  		FIG. 1. MPF activity and CCNB accumulation in enucleated porcine oocytes. A) MPF activity in enucleated (black circles) and control (white circles) oocytes was assayed as histone H1 kinase activity and expressed as value relative to that of the intact immature oocytes. The values indicate the mean ± SEM of more than three independent experiments. The values of the enucleated oocytes with asterisks were significantly different from those of the control oocytes at the same time point (P < 0.05). The values of enucleated oocytes indicated by different letters are significantly different (P < 0.05). The rate of GVBD and that of the first polar body extrusion in the control oocytes are shown in the insert. B) CDC2 kinase acivity in 50-h-cultured oocytes was assayed after immunoprecipitation with anti-CDC2 C-terminus antibody. As a negative control, normal rabbit serum was used for IP instead of the antibody. C) An enucleated oocyte at 30 h of culture was injected with a cumulus cell nucleus, and the nuclear status was examined after 50 h of culture. Arrowheads indicate the nuclear membrane. Bar = 30 µm. D) The accumulation of CCNB1/B2 during maturation culture of enucleated and control oocytes was detected by Western blotting analyses

Injection of GV material or cumulus cell nucleus into enucleated oocytes was performed using a Piezo-micromanipulating (PMM) system (Prime Tech Ltd.). The entire GV of the freshly isolated oocyte was sucked up into the pipette, and the nuclear membrane was broken by applying a PMM pulse. Cumulus cells were gently aspirated in and out of the injection pipette until their nuclei were largely devoid of visible cytoplasmic material. Then, the GV material or the cumulus cell nucleus was injected into a 30 h-cultured enucleated oocyte through the slit that had been made during the enucleation process (see Fig. 3A).


Figure 3
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  		FIG. 3. Effects of GV material replacement on MPF activity and CCNB accumulation in enucleated oocytes. A) A scheme of the experimental design. Enucleated oocytes (a) were cultured for 30 h, and the GV material from freshly isolated oocytes (b, c) was injected into the enucleated oocytes (d, e). An MPF activity assay and Western blotting were performed after an additional 20 h of culture (total, 50 h). Bars = 30 µm. B) The MPF activity in the indicated oocytes was assayed as histone H1 kinase activity, and the data are shown as activity relative to that in noncultured control oocytes. The values indicate the mean ± SEM of more than three independent experiments. The values indicated by different letters are significantly different (P < 0.05). C) CCNB1 accumulation in the indicated oocytes at 50 h of culture was detected by Western blot analyses. D) The nuclear status of a 50 h-cultured enucleated oocytes injected with GV material. The condensed chromosome is indicated by the arrowhead. Bar = 30 µm. E) CCNB1/B2 mRNAs in control and enucleated oocytes, and isolated GVs at 0 h of culture, were detected by RT-PCR

Kinase Assays

Assays for histone H1 kinase and myelin basic protein (MBP) kinase were performed as described in previous reports [22, 24]. To detect the transient decrease in histone H1 kinase activity at the first polar body emission, control oocytes at 35 h of culture were stained with Hoechst 33342, and those at the first anaphase or telophase (AT1) stage were collected as reported previously [4]. S6 kinase assays were performed as described in a previous report [27] using a S6 kinase assay kit (Upstate Biotechnology Inc.).

Antibodies, Western Blotting, Immunocytochemistry, and Immunoprecipitation

The antibodies against MAP2K (also known as MEK), MAPK3/MAPK1 (also known as ERK1/ERK2), RPS6KA (also known as RSK), and CCNB2 (also known as cyclin B2), were obtained from Santa Cruz Biotechnologies (sc-6250, sc-94, sc-231 and sc-5235, respectively). The antibody against CCNB1 (also known as cyclin B1) was obtained from Upstate Biotechnology (05–158). The rabbit polyclonal antiserum against mouse CDC2 C-terminus was a kind gift from Dr. F. Aoki (University of Tokyo, Tokyo, Japan).

Western blot analyses of MAP2K, MAPK3/MAPK1, and RPS6KA were performed as described in previous reports [24, 27], using a blotting detection kit in which the streptavidin-alkaline phosphatase conjugate was used as the signal-generating system (Amersham Pharmacia Biotech). For the detection of CCNB1 or CCNB2, the Western blots were probed with primary antibody and the appropriate secondary antibody horseradish peroxidase conjugate (Jackson Immunoresearch) and the results were revealed by ECL (Amersham Pharmacia Biotech) [12, 28]. Immunoprecipitation using the anti-CDC2 C-terminus was performed according to a method described in a previous report [29].

Analysis of Protein Synthesis

[35S]-methionine labeling was performed as reported previously [29]. Either control or enucleated oocytes were labeled in culture medium containing [35S]-methionine (1000 Ci/mmol; Amersham Pharmacia Biotech) at a radioactive concentration of 500 µCi/ml for 4 h. In some groups, 10 µg/ml cycloheximide (CHX) were added to the culture medium. After being washed thoroughly, the labeled oocytes were placed in 8 µl of saline supplemented with 0.1% polyvinylpyrrolidone (Sigma), to which 2 µl of 5 x Laemmli buffer [30] was added, and the proteins were denatured at 100°C for 5 min. Then, 1 ml of scintillation fluid (Amersham Pharmacia Biotech) was added to the solution, and the radioactivity was measured using a liquid scintillation counter (Aloka Co. Ltd.). For the detection of radiolabeled protein with SDS-PAGE, the oocytes were labeled for 3 h, and then 10 oocytes were subjected to SDS-PAGE. The labeled proteins were detected by autoradiography as described in a previous report [22].

RT-PCR Assays

Total RNA was isolated from 10 oocytes using Trizol reagent (Gibco BRL) according to the manufacturer's instructions, except for the addition of 20 µg of glycogen and 20 pg of mRNA encoding enhanced green fluorescent protein (EGFP) as the carrier and internal control, respectively. The RNA samples were reverse-transcribed into cDNA using SuperScript II (Gibco BRL) and random hexamers (Takara Shuzo Co., Ltd.) in a final volume of a 20-µl reaction mixture. The primers used for the amplification of CCNB1, CCNB2, and EGFP sequences were described previously [28, 31]. The reaction was performed with 32, 38, or 24 cycles for CCNB1, CCNB2, or EGFP, respectively. The PCR products were separated by electrophoresis in 2.0% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light.

Preparation and Injection of Destruction-Box mRNA

To obtain mRNA encoding destruction-box of CCNB1 (referred to as D-box mRNA), CCNB1/pGEM-3Z vector (see Fig. 4A) [28] containing a coding sequence of porcine CCNB1 was linearized by cutting with NcoI (Takara Shuzo Co., Ltd.); the linearized cDNA then was transcribed in vitro as reported previously [28]. EGFP mRNA was transcribed in vitro using EGFP/pGEM-3Z vector (see Fig. 4A) [32] as a template as reported previously [28]. The RNA transcripts were precipitated with absolute ethanol, washed, dried, and resuspended in RNase-free water. The RNA solutions were stored at –80°C until use as reported previously [28, 32].


Figure 4
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  		FIG. 4. CCNB1 accumulation in enucleated oocytes injected with mRNA of D-box in CCNB1 (A). Constructions of vectors used for in vitro synthesis of D-box mRNA and coinjected EGFP mRNA are shown schematically on the left, and the synthesized mRNAs are shown on the right. Full-length CCNB1 mRNA is also shown just for reference. B) An enucleated oocyte expressed EGFP illumination after 20 h of mRNA injection. A slit made for enucleation is indicated by the arrowhead. Bar = 30 µm. C) CCNB1 accumulation in indicated oocytes at 50 h of culture was detected by Western blotting analyses. Messenger RNAs were injected at 30 h of culture

Injection of mRNA into oocytes was performed after 30 h of culture as reported previously [28, 32]. After injection, the oocytes were cultured as described above and expression of EGFP was examined under fluorescent stereomicroscope (MZ FLIII; Leica). Only the oocytes expressing EGFP illumination were used for the Western blotting analysis.

Statistical Analysis

For paired comparisons, the Student t-test was used. To compare three or more groups, the Tukey-Kramer HSD test was used. A probability of P < 0.05 was considered to be statistically significant.

RESULTS

Effects of GV Removal on MPF Activity and the Components of MPF During Porcine Oocyte Maturation Culture

Because the MPF activity of denuded oocytes was comparable with that of sham-enucleated oocytes, which were not enucleated but removed the equivalent volume of cytoplasm (Supplemental Figs. 1 and 2, available online at http://www.biolreprod.org), we used denuded cumulus cell-free oocytes as the control throughout the present studies. In control oocytes cultured in our in vitro maturation system, GVBD took place mainly at 24 h of culture, and most of the oocytes emitted the first polar body at 35 h of culture (Fig. 1A, insert).


Figure 2
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  		FIG. 2. Protein synthesis in enucleated porcine oocytes. A) Control (white bars) and enucleated oocytes (black bars) cultured for 46 h were incubated for 4 h in a medium supplemented with [35S]-methionine with or without CHX, as indicated, and the amount of radioactivity incorporated into the oocytes was measured. *P < 0.05. B) Control and enucleated oocytes were radiolabeled with [35S]-methionine for 3 h as indicated under each lane, and the proteins synthesized soon after labeling were visualized by autoradiography. The major protein increases and decreases during culture are indicated by black and white arrowheads, respectively

MPF activity was low at the initiation of the maturation culture, and the activity in enucleated oocytes just after enucleation was the same as that of the control oocytes, indicating that the enucleation process had no deteriorative effect on MPF activity (Fig. 1A). Increases in MPF activity in the control oocytes were observed between 20 and 30 h of culture, which corresponded well with GVBD. In the enucleated oocytes, MPF activity was also increased at 20 h of culture, which was earlier than for control oocytes, and the level of activation was comparable to the peak level in the control oocytes at 30 h of culture (Fig. 1A). The time required for enucleation should not cause this earlier MPF activation in enucleated oocytes, because the same period was applied also for the control oocytes. This result confirms the findings of our previous report [22], indicating the dispensability of the GV for the initial activation of MPF and for the resumption of porcine oocyte maturation, as has also been reported in the case of frog and mouse oocytes [17, 21]. Thereafter, MPF activity decreased transiently at 35 h of culture in the control oocytes, i.e., during the first polar body emission, and then MPF activity was reactivated at 50 h of culture for the induction of the second meiosis. On the other hand, MPF activity in the enucleated oocytes decreased significantly at 30 h of culture, and did not increase again until 50 h of culture, which suggested an escape from the M-phase after the first meiosis (Fig. 1A).

To confirm the decrease in MPF activity in enucleated oocytes at 50 h of culture, a CDC2 kinase assay was conducted after immunoprecipitation of the samples with anti-CDC2 C-terminus antibody. As shown in Figure 1B, only a faint phosphorylated histone H1 band was observed in the enucleated oocytes (lane 3), whereas a strong band was detected in the control oocytes (lane 2). No band was detected among the negative controls, in which serum was used instead of the antibody (lane 1). To cytologically assess the MPF activity in enucleated oocytes at 50 h of culture, enucleated oocytes were injected with cumulus cell nuclei at 30 h of culture, and the nuclear state was examined at 50 h of culture. The oocytes were found to have a large expanded nucleus (Fig. 1C), which was characteristic of oocytes with low MPF activity. These results further confirmed the reduction in MPF activity after the first meiosis in enucleated oocytes.

During porcine oocyte maturation, the synthesis of CCNB is known to begin around the time of GVBD [28, 29], and then CCNB2 and CCNB1 peak at the first metaphase and the second metaphase, respectively [12, 28]. To assess the amount of CCNB protein in the enucleated oocytes, Western blotting analyses were performed (Fig. 1D). In the control oocytes, the levels of both types of CCNB during the maturation culture agreed well with those of our previous studies of intact porcine oocytes [12, 28]. In enucleated oocytes, the CCNB2 level, which peaked at 20 h and then decreased until 50 h of culture (Fig. 1D, lower right panel), was the same as that of control oocytes, indicating that GV material is not required for maintaining normal CCNB2 kinetics. In contrast, whereas CCNB1 levels in the control oocytes gradually increased and reached a peak after 50 h of culture (Fig. 1D, upper left panel), the levels of CCNB1 in the enucleated oocytes decreased drastically at 30 h, and became almost undetectable at 40 h and thereafter (Fig. 1D, upper right panel); thus, the present results are in good agreement with the decrease in MPF activity during the maturation periods. These results further suggest that GV material is required for maintaining normal CCNB1 protein kinetics, especially with respect to the accumulation of CCNB1 starting at 30 h of culture, which is required to induce the second meiosis.

Total Protein Synthesis of Enucleated Porcine Oocytes

We then investigated whether or not the above results could be attributed to a general decrease in metabolic activity, or the "death" of the enucleated oocytes, because of damage incurred during the enucleation process. To examine this possibility, the synthesis of new proteins was examined as an estimate of metabolic ability of both control and enucleated oocytes at 50 h of culture. As shown in Figure 2A, the radioactivity incorporated into the enucleated oocytes was comparable to that of the intact oocytes, and this incorporation was significantly reduced by treatment with a protein synthesis inhibitor, CHX (P < 0.05). The differences between the values obtained with and without CHX, which indicate the total amount of protein synthesis, were not different between the control and enucleated oocytes. This result demonstrated that the total protein synthesis activity of the enucleated oocytes had not deteriorated quantitatively.

Next, we applied SDS-PAGE and autoradiography to analyze the synthesized proteins to qualitatively examine protein synthesis activity (Fig. 2B). The protein synthesis patterns differed between before (lanes 1 and 2) and after GVBD (lanes 3 and 4) in the intact oocytes. In the enucleated oocytes, the patterns changed in the same manner as those of the intact oocytes between the periods before 20 h (lanes 5 and 6) and after 30 h (lanes 7 and 8) of culture, as was previously reported in studies of Rana pipiens [33], sheep [26], and cattle oocytes [34]. These results indicate that the enucleated porcine oocytes did not degenerate, and the proteins exhibited both quantitatively and qualitatively normal synthesis, even in the absence of a nucleus, until 50 h in culture.

Effects of Replacement of GV Material on the Accumulation of CCNB1 Protein and Reactivation of MPF

The above results strongly suggest that GV material is required for the reactivation of MPF in porcine oocytes, as has also been reported in the case of Xenopus oocytes [20]. To confirm this conclusion, enucleated oocytes were injected with the GV material of freshly isolated oocytes at 30 h of culture, and MPF activity and CCNB levels were examined at 50 h of culture (the experimental design is shown in Fig. 3A). In the enucleated oocytes injected with GV material, MPF activity was significantly increased, and approached that of the intact oocytes at 50 h of culture, whereas the MPF activity of the enucleated oocytes injected with the same volume of cytoplasm remained low (Fig. 3B).

To investigate whether or not GV material is capable of inducing CCNB1 accumulation in enucleated oocytes, Western blot analysis of CCNB1 was performed, and the results are shown in Figure 3C. The levels of CCNB1 at 50 h of culture increased substantially in the enucleated oocytes injected with GV material at 30 h of culture, whereas the CCNB1 levels in the enucleated oocytes injected with fresh cytoplasm remained unchanged from those of noninjected oocytes. Furthermore, the CCNB1 levels in GV material-injected enucleated oocytes were higher than those of the control oocytes (Fig. 3C). When the GV material-injected oocytes were examined in terms of their nuclear state at 50 h of culture, they exhibited condensed chromosomes, which were clearly observed in the oocytes with high MPF activity (Fig. 3D), thus suggesting that GV material has the ability to induce a reactivation of MPF in enucleated porcine oocytes.

Because the accumulation of CCNB1 into enucleated oocytes caused by the injection of GV material led us to presume the presence of CCNB1 mRNA in the nucleus of immature oocytes, RT-PCR analyses were conducted to address the issue of whether or not CCNB mRNA is indeed present in GVs (Fig. 3E). As a positive control of reverse transcription, mRNA encoding EGFP was included in each reaction mixture (see Materials and Methods). Comparable levels of mRNA encoding both CCNB1 and CCNB2 were detected in enucleated and intact oocytes, and there were no detectable CCNB mRNAs in the GVs. These results indicated that enucleation did not change the levels of CCNB1 and CCNB2 mRNAs in the enucleated oocytes. Taken together, the present results clearly demonstrate that certain GV factor(s), other than CCNB mRNAs, was/were essential for MPF reactivation and entry into second meiosis in porcine oocytes, as has also been demonstrated in the case of Xenopus oocytes [20].

Synthesis of CCNB1 During Maturation Culture in Enucleated Porcine Oocytes

The present results shown in Figure 2B indicate that most of the proteins in the enucleated oocytes were synthesized normally, in spite of the absence of a nucleus, thus suggesting that CCNB1 protein is synthesized normally and CCNB1 degradation is elevated in enucleated oocytes during the second meiosis period, when degradation of CCNB is normally inhibited [35]. If this is the case, then the inhibition of CCNB degradation in the enucleated oocytes should induce CCNB accumulation at levels comparable to those of control oocytes. To investigate this possibility, we attempted to express a competitor of CCNB destruction, CCNB1 D-box, which is known to be essential for the physiological ubiquitination and degradation of CCNB [36, 37]. In a previous report [32, 38], we found that collecting the oocytes with EGFP illumination after coinjecting EGFP mRNA with other mRNAs was a powerful method of selecting the viable oocytes and synthesizing the target proteins. Therefore, we employed this method for the present experiment. The constructions of the competitor mRNA (referred to as D-box mRNA) and EGFP mRNA were shown in Figure 4A, with full-length CCNB1 mRNA as a reference, and the translation of injected mRNA in enucleated oocytes was assessed by the expression of EGFP illumination (Fig. 4B). When D-box mRNA was injected into the enucleated oocytes at 30 h of culture, CCNB1 was shown to have accumulated at 50 h of culture to levels higher than those of control oocytes, whereas the CCNB1 levels in the oocytes injected with EGFP mRNA alone were low and remained unchanged from those in noninjected enucleated oocytes (Fig. 4C). These results strongly suggest that CCNB1 was synthesized at high levels, even in enucleated oocytes, during the maturation culture period; moreover, the failure of CCNB1 accumulation in the enucleated oocytes might be attributable to an abnormal elevation in CCNB1 degradation.

Activation of MAPK Cascade in Enucleated Oocytes

Because the MAPK cascade has been suggested to be related to the promotion of CCNB accumulation during the second meiosis in mouse and Xenopus oocytes [39–41], it was assumed that GV removal and replacement would exert some influence on the activity of this cascade. To investigate this possibility, we examined the activity and phosphorylation status of the MAPK cascade in enucleated oocytes. As shown in Figure 5A, MAPK activity, assessed as MBP kinase activity, was comparable to that of control oocytes during the entire maturation culture period. To confirm the activation of the MAPK cascade in enucleated oocytes, Western blotting analyses of MAPK3/MAPK1 (major MAPK in maturating mammalian oocytes), MAP2K (a dual specific activator of MAPK3/MAPK1), and ribosomal protein S6 kinase (RPS6KA; a major substrate of MAPK3/MAPK1) were performed (Fig. 5B). The phosphorylated bands of MAP2K and MAPK3/MAPK1, showing their activated forms, could be detected from 20 h to the end of the culture period in both control and enucleated oocytes. These results confirmed those of our previous reports [24], indicating that GV is not required for MAPK activation. The lowest-mobility band of RPS6KA, which represents an activated form of RPS6KA during porcine oocyte maturation [27], was detected throughout the maturation culture period in the present study; however, the intensity of the band markedly increased from 20 h to 50 h in both control and enucleated oocytes (Fig. 5B, lower panel). Consistent with the results of the Western blot analysis, the results showing S6 kinase activity revealed low activity at the initiation of oocyte maturation and elevation after 20 h of culture of these oocytes (Fig. 5C). These results indicate that the GV is not required for either the phosphorylation or the activation of the MAPK-RPS6KA cascade during porcine oocyte maturation.


Figure 5
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  		FIG. 5. MAPK-RPS6KA cascade in enucleated porcine oocytes. A) The MAPK activity of enucleated (black circles) and control (white circles) oocytes was assayed as MBP kinase activity and was expressed as a value relative to that of intact immature oocytes. Values indicate the mean ± SEM of more than three independent experiments. Values indicated by different letters are significantly different (P < 0.05). There were no statistically significant differences between control and enucleated oocytes at the same time point (P > 0.05). B) The phosphorylation status of MAP2K, MAPK3/MAPK1, and RPS6KA was examined by Western blot analysis. The phosphorylated and activated bands are indicated by asterisks. C) The S6 kinase activity of control (white bars) and enucleated (black bars) oocytes was assayed and was expressed as the molecular number (fmol) of phosphate incorporated into the substrate peptides. The values indicated by different letters were significantly different (P < 0.05). D) Effects of the injection of GV material, as described in Figure 3A, on MAPK activity in enucleated oocytes at 50 h of culture. The data are shown as activity relative to that in noncultured control oocytes. The values indicate the mean ± SEM of more than three independent experiments. There were no statistically significant differences between treatment groups (P > 0.05)

We next examined MAPK activity in enucleated oocytes that were injected with GV material after 30 h of culture; here, the same experimental design was used as that illustrated in Figure 3A. As shown in Figure 5D, MAPK activity in the enucleated oocytes injected with either nuclear material or cytoplasm was comparable to that of enucleated oocytes and intact oocytes, indicating that the replacement of the GV material exerted no influence on MAPK activity.

DISCUSSION

The objective of the present study was to investigate the possible requirement of the GV to attain normal MPF activity kinetics during porcine oocyte maturation. The present results indicated that the GV is not required for the initial activation of MPF, i.e., to initiate the oocyte maturation, but that the GV is indispensable for the reactivation of MPF for the induction of the second meiosis. This result is in support of conclusions of previous reports, showing elevations in MPF activity in enucleated mouse and porcine oocytes [21, 22], and showing low MPF activity in enucleated mouse oocytes after maturation culture [23]. In addition, the present results are in agreement with those of a recent study of Xenopus oocytes [20]. However, it should be noted that Iwashita et al. [20] reported observing a slower activation of MPF in enucleated Xenopus oocytes; in their study, they suggested the possibility that certain factors are required for the rapid activation of MPF in the GV of Xenopus oocytes. In contrast, the present results demonstrated an earlier activation of MPF in enucleated oocytes, indicating the complete dispensability of the GV for initial MPF activation in porcine oocytes. Moreover, in this regard, the present results reveal a clear difference from those obtained with Xenopus oocytes. The reasons for this accelerated MPF activation in enucleated oocytes remains unclear, but one possibility would be that the MPF in enucleated oocytes is amplified soon after the initial activation by a cytoplasmic feedback mechanism [42, 43]; on the other hand, the MPF of intact oocytes would have to translocate into the GV to induce GVBD before being amplified by this cytoplasmic mechanism.

In the present study, we injected either cumulus nuclei or GV material into enucleated porcine oocytes, and we revealed that although the former cells were unable to recover MPF activity, the latter could induce high MPF activity, i.e., that comparable with the activity observed in control oocytes, after a maturation culture period. In support of our results, Polanski et al. [23] examined the replacement of the mouse GV by the nuclei of various types of cellz, and they observed the maintenance of high MPF activity by the GVs from other oocytes or pronuclei from fertilized eggs, but not in the presence of cumulus nuclei. These results, taken together, strongly suggest that a factor or factors necessary for the meiosis-specific MPF reactivation is/are present in GVs. The candidates for the factors may include some proteins required for CDK2 activation, because importance of CDK2 activity for the induction and maintenance of the second meiosis have been reported in porcine oocytes [44] as well as in Xenopus oocytes [45, 46], although further studies are required to clarify this point.

We observed here that the levels of CCNB1 protein in enucleated oocytes were very low during the second meiotic period, whereas the levels of CCNB2 remained normal, and suggested that the reason for the failure of MPF reactivation in the enucleated oocytes could have been the low levels of CCNB1 accumulation during the second meiotic period. In a previous report, we examined the roles played by CCNB1 and CCNB2 by inhibiting the expression of each protein by specific antisense RNA injection, and demonstrated that the inhibition of CCNB2 did not induce any outstanding abnormalities in porcine oocyte maturation, but that the inhibition of CCNB1 did induce nuclear formation after the first meiotic metaphase instead of inducing the cell's entrance into the second meiosis [12, 28]. The importance of CCNB1, but not that of CCNB2, for the induction of the second meiosis has previously been reported in studies of mouse and Xenopus oocytes [47, 48]. The present results not only indicated the requirement of the GV for the accumulation of CCNB1 following interkinesis, but also confirmed the importance of CCNB1. In Xenopus oocytes, normal CCNB accumulation was observed in enucleated oocytes, and the defects in the enucleated oocytes were attributed to the abnormal phosphorylation of CDC2, which led MPF to inactivate pre-MPF [20]. The most reliable explanation for this discrepancy is thought to be the species difference. Additionally, because the antibody used for Western blotting analysis in the Xenopus study was anti-CCNB2 antibody, it remains possible that CCNB1 protein is not normally accumulated in enucleated Xenopus oocytes as it is in enucleated porcine oocytes, because the kinetics of CCNB2 were also normal in the enucleated porcine oocytes.

In general, it is conceivable that the failure of protein accumulation could be attributed either to a decrease of synthesis or to an increase of degradation, or both. In the present study, overall protein synthesis in the enucleated porcine oocytes was found to be almost normal, from both a qualitative and a quantitative perspective. It is well known that the types of protein that are synthesized change around GVBD [49, 50]. This change in the protein synthesis pattern was observed as occurring along a normal time course in the enucleated porcine oocytes examined here, thus clearly demonstrating the dispensability of the GV for protein synthesis during porcine oocyte maturation, as well as the absence of any deteriorative effects on oocyte metabolism in the present enucleation process. These results suggest normal synthesis of CCNB1 protein in enucleated porcine oocytes, although the 62-kDa CCNB1 band was not visible in the present [35S]-methionine labeling method as described previously [29]. The dispensability of the GV for protein synthesis during oocyte maturation has also been reported in previous studies of Rana pipiens [33], sheep [26], and cattle [34]. Furthermore, inhibition of CCNB1 degradation by the injection of competitor (CCNB1 D-box mRNA) resulted in a drastic increase of CCNB1 accumulation in the enucleated porcine oocytes. These results imply that the failure of these enucleated porcine oocytes to accumulate CCNB1 was caused by an increase in the degradation of CCNB1, rather than a decrease in CCNB1 synthesis.

It was previously reported in vertebrate oocytes that CCNB was incompletely degraded after the first meiosis, and that the presence of some remaining MPF activity is necessary for the cell's entrance into the second meiosis [51, 52]. The MAPK cascade has been well accepted as the principal factor regulating for the entrance into the second meiosis by inhibiting the degradation of CCNB after interkinesis [35]. The abnormal degradation of CCNB and the subsequent escape from meiosis soon after the first meiosis have been reported in mouse and Xenopus oocytes, in which MAPK activity was inhibited at interkinesis [40, 41, 53–55]. We have also previously reported on porcine oocytes injected with the antisense RNA of c-mos, the activator of the MAPK cascade; in that study, we observed that some of the MAPK-inhibited oocytes escaped from meiosis after the first meiosis [32]. In the present study, we confirmed the results of our previous report suggesting the dispensability of the GV for MAPK cascade activation in porcine oocytes [24], including a major MAPK substrate, RPS6KA, which was activated during porcine oocyte maturation [27]. Additionally, no further elevation of MAPK activity was observed after the injection of GV material into the enucleated oocytes. These results suggest that the MAPK cascade is not involved in GV function with respect to restoration of MPF reactivation in enucleated porcine oocytes.

FOOTNOTES

2 Correspondence: Kunihiko Naito, Department of Animal Resource Sciences, Graduate School of Agricultural Sciences, University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 5841–8191; aknaito{at}mail.ecc.u-tokyo.ac.jp Back

3 Current address: The Jackson Laboratory, Bar Harbor, Maine 04609. Back

1 Supported by grants-in-aid for scientific research 17380173 to K.N. and 16380197 to H.T. from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Back

Received: 5 August 2005.

First decision: 15 August 2005.

Accepted: 23 November 2005.

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