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Contribution of high p34^cdc2 kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes

¹ National Institute for Physiological Sciences, Okazaki 444-8787, Japan, ² The Graduate University of Advanced Studies, Okazaki 444-8787, Japan, ³ CREST of Japan Science and Technology Agency, Kawaguchi 332-0012, Japan, ⁴ Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan and ⁵ Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan

Correspondence should be addressed to S Hochi; Email: shochi{at}shinshu-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The present study was undertaken to clarify the relationship between the p34^cdc2 kinase activity of in vitro-aged or enucleated rat oocytes and the premature chromosome condensation (PCC) of microinjected cumulus cell nuclei. Wistar rat oocytes were placed in vitro up to 120 min after the animal was killed. The p34^cdc2 kinase activity of the oocytes decreased in a time-dependent manner. The incidence of PCC was higher when nuclear injection into intact oocytes was completed in 15–45 min rather than 46–120 min. When rat oocytes were enucleated for subsequent nuclear injection, the p34^cdc2 kinase activity transiently increased soon after enucleation but drastically decreased after 30 min. Removal of the cytoplasm instead of the meta-phase-plate did not affect the p34^cdc2 kinase activity even after 60 min. PCC occurred in intact and cytoplasm-removed oocytes but not in enucleated oocytes. In contrast, oocytes from BDF1 mice exhibited a p34^cdc2 kinase level twice that of rat oocytes and supported PCC despite enucleation. The p34^cdc2 kinase level of intact rat oocytes was reduced to the equivalent level of aged (120 min) or enucleated (+60 min) oocytes by a 45 min treatment with roscovitine, an inhibitor of p34^cdc2 kinase. None of the roscovitine-treated oocytes supported PCC while half of the control oocytes did. When rat oocytes were treated with MG132, a proteasome inhibitor, delayed inactivation of the p34^cdc2 kinase was observed in the MG132-treated oocytes. A significantly higher proportion of the MG132-treated oocytes supported PCC when compared with the control oocytes. Moreover, a higher proportion of MG132-treated and enucleated oocytes carried two pseudo-pronuclei after cumulus cell injection and developed to the two-cell stage when compared with the enucleated oocytes at the telophase-II stage. These results suggest that the decreased level of p34^cdc2 kinase activity in aged or enucleated rat oocytes is responsible for their inability to support PCC of microinjected donor cell nuclei and that inhibition of p34^cdc2 kinase inactivation by chemicals such as MG132 is in part effective for rat oocytes to promote PCC and further development.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Successful animal cloning by somatic cell nuclear transfer (NT) has been reported in large domestic species (Wilmut et al. 1997, Kato et al. 1998, Baguisi et al. 1999, Onishi et al. 2000, Galli et al. 2003). As for small rodents, Wakayama et al.(1998) were the first to produce cloned mice using G0/G1-phase cumulus cells, and very recently Zhou et al.(2003) reported the production of fertile cloned rats using M-phase fetal fibroblast cells. However, the success rates of producing cloned rodents by NT are still low.

In the mouse, nuclei injected into enucleated oocyte cytoplasm are directly exposed to reprogramming factors, resulting in premature chromosome condensation (PCC), with subsequent multiple pseudo-pronuclei formation following activation (Wakayama et al. 1998, 1999, Ogura et al. 2000). It is considered in rodents, at least, that PCC is a critical aspect contributing to successful cloning (Wakayama et al. 1999, Ogura et al. 2000). However, PCC is not essential for somatic cell cloning in all species, since telophase-II (TII) oocytes have promoted embryo development in goats (Baguisi et al. 1999) and cattle (Kurosaka et al. 2002).

There is a major difference between mouse and rat, in the behavior of somatic nuclei upon exposure to oocyte cytoplasm. We have reported that the PCC of both mouse and rat cumulus cell nuclei occurred in enucleated mouse oocytes but not at all in enucleated rat oocytes (Hirabayashi et al. 2003a). Even if cumulus nuclei were injected into the rat oocytes without enucleation, the rate of PCC formation was less than half (Hirabayashi et al. 2003b).

We also reported that the ability of rat oocytes to promote PCC of the injected nuclei was dependent on the characteristics of the oocytes, such as age (4- to 5-week-old, superior to > 10-week-old rats) or strain of the donor rats (Wistar and LEW, superior to the Donryu and F344 strains) and the timing of oocyte recovery (14 h post-human chorionic gonadotropin (hCG), superior to 17 h post hCG) (Hirabayashi et al. 2003b). In the same study, the time management of NT completion (less than 45 min after donor animal killing) and addition to the oocytehandling media of N-acetylleucylleucylnorleucinal (ALLN), a neutral cysteine protease inhibitor, were also found important for intact rat oocytes to promote PCC. The successful production of cloned rats (Zhou et al. 2003) may be attributed to injecting the metaphase-arrested fetal fibroblasts into oocytes treated with MG132, a proteasome inhibitor, followed by quick removal of the recipient metaphase-plate. Therefore, it is a possibility that recipient oocytes arrested at the metaphase-II (MII) stage by the inhibition of protein degradation have essential roles in nuclear reprogramming including PCC formation.

Ovulated rat oocytes are known to activate spontaneously but abortively during in vitro culture (Zernicka-Goetz 1991). We also confirmed that more than half of the oocytes from Wistar rats progressed to anaphase-II (AII)/TII stages at 70 min after killing of the donor rats (Kato et al. 2001). In vertebrate oocytes, it has been considered that high activity of maturation promoting factor (MPF) affects the chromosome condensation and the maintenance of the meiotic spindle, both of which are involved in the meiotic arrest at the MII stage (Jones 2004). MPF is a heterodimer composed of p34^cdc2 kinase and cyclin B1, controlling the cell cycle. When the activity was inactivated by the degradation of cyclin B (Nurse 1990), oocytes were released from the arrest at the MII stage. The p34^cdc2 kinase in oocytes was maintained at a high level until ovulation in many species (Kubiak et al. 1993, Naito et al. 1995, Abrieu et al. 2001, Ito & Shimada 2005), suggesting that p34^cdc2 kinase activity in rat ovulated oocytes was rapidly decreased in vitro. Moreover, destruction of the nuclear envelope in mitotic somatic cells occurred during the G2/M-phase transition by p34^cdc2 kinase-related phosphorylation of lamin, which is one of the nuclear membrane proteins (Peter et al. 1990). It has been reported that ALLN, which was used in our previous study (Hirabayashi et al. 2003b), has an effect on the inhibition of cyclin B destruction (Sherwood et al. 1993). Therefore, it is possible that the low rate of PCC of injected somatic cell nuclei resulted from rapidly decreasing p34^cdc2 kinase activity in rat ovulated oocytes, while direct evidence that p34^cdc2 kinase activity is involved in the PCC incidence has not yet been shown. Clarifying the relationship between p34^cdc2 kinase activity and PCC incidence would contribute to understanding the nuclear reprogramming mechanism in mammalian oocytes.

In the present study, two experiments were conducted to examine the effects of in vitro aging and enucleation of rat oocytes on the kinetics of p34^cdc2 kinase and the incidence of PCC of microinjected cumulus cell nuclei, with some comparisons with mouse oocytes. In addition, two more experiments were conducted to explain a possible role of p34^cdc2 kinase for promoting PCC, by treatment of the rat oocytes with roscovitine, an inhibitor of p34^cdc2 kinase (Lazar et al. 2002), and MG132, a proteasome inhibitor which has an effect on the inhibition of cyclin B1 degradation (Josefsberg et al. 2000).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Oocyte preparation
Specific-pathogen-free Crj:Wistar female rats (3–5 weeks old) or Crj:BDF1 female mice (7 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). They were housed under controlled lighting (lights on at 0600–1800 h), temperature (23 ± 2 °C) and humidity (55 ± 5%), with free access to laboratory diet (MF; Oriental Yeast Co., Tokyo, Japan) and water. Rats were superovulated by i.p. injections of 30 IU equine chorionic gonadotropin (eCG) (Nippon Zenyaku, Co., Fukushima, Japan) and 30 IU hCG (Sankyo Yell Yakuhin, Co., Tokyo, Japan) at 48 h intervals (Hirabayashi et al. 2001). Mice were superovulated as well with 5 IU eCG and 5 IU hCG.

Fourteen hours after the hCG injection, cumulus–oocyte complexes (COCs) were collected from the oviductal ampullae of donor rats or mice with modified Krebs–Ringer bicarbonate solution (mKRB) in Experiments 1 and 3 (Toyoda & Chang 1974) and modified rat one-cell embryo culture medium (mR1ECM) in Experiments 2, 4 and 5 (Oh et al. 1998) supplemented with 0.1% hyaluronidase (Sigma-Aldrich Corp., St Louis, MO, USA) respectively. Two to five minutes later, the denuded oocytes were washed three times with fresh mKRB or R1ECM and kept in the same medium at 37 °C until being subjected to the treatments. The time of killing of the donor animals was defined as 0 min, and it usually took 10–15 min from the animal killing to the preparation of denuded oocytes.

Measurement of p34^cdc2 kinase activity
The p34^cdc2 kinase assay was performed according to the method as described by Ito et al.(2001) with some modifications. In brief, the oocytes were washed several times with PBS and placed into cell lysis buffer composed of 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na₃VO₄, 1 mg/ml leupeptin (all chemicals from Cell Signaling Technology, Beverly, MA, USA) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). The oocyte lysates were stored at –80 °C until the ELISA assay.

The lysate (ten oocytes/5 µl cell lysis buffer) was mixed with 45 µl of kinase assay buffer-A composed of 25 mM Hepes buffer (pH 7.5) (MBL, Nagoya, Japan), 10 mM MgCl₂ (MBL), 10% (v/v) mouse vimentin peptide solution (SLYSSPGGAYC) (MBL) and 0.1 mM ATP (Sigma-Aldrich), and the mixture was incubated for 30 min at 30 °C. The reaction was terminated by the addition of 200 µl PBS containing 50 mM EGTA (MBL). The phosphorylation of mouse vimentin peptides was detected using an ELISA analysis (MESACUP cdc2 kinase assay kit (code no. 5234); MBL). Data were expressed in terms of the strength of p34^cdc2 kinase activity in denuded rat oocytes 15 min (Experiments 1, 3 and 4) or 45 min (Experiment 2) after animal killing.

Assessment of nuclear configuration
Rat oocytes immediately after recovery and 30, 60, 90 and 120 min after culture in mKRB were stained with 5 µg/ml Hoechst 33342 (Sigma-Aldrich). Their nuclear configuration was then assessed under UV light at 330–380 nm, according to the classifications reported previously (Kato et al. 2001): MII, characterized by a meta-phase spindle with the first polar body; AII, characterized by elongated spindle without the second polar body; and TII, characterized by elongated spindle with extrusion of the second polar body.

Nuclear injection and PCC evaluation
Cumulus cells dispersed from COCs by the hyaluronidase treatment were placed in mKRB (Experiments 1 and 3) or R1ECM (Experiments 2, 4 and 5) containing 22 mM Hepes and 5 mM NaHCO₃ (abbreviated hereafter as Hepes-mKRB and Hepes-R1ECM respectively). An aliquot (2 µl) of the cumulus cell suspension was transferred to 10 µl of Hepes-mKRB or Hepes-R1ECM supplemented with 12% w/v 360 kDa polyvinylpyrrolidone (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA). Nuclei were removed from cumulus cells and gently aspirated in and out of the injection pipette (7–10 µm) until their nuclei were largely devoid of visible cytoplasmic materials. Each nucleus was injected into a separate enucleated oocyte within 5 min of its isolation, as described by Kimura & Yanagimachi (1995). One hour after the nuclear injection, the oocytes were stained with 5 µg/ml Hoechst 33342 and evaluated for incidence of PCC under UV light at 330–380 nm, as described previously (Hirabayashi et al. 2003a,b). The injected somatic cell nucleus was distinguished from the oocyte nucleus by the position of the opening in the zona pellucida at the nuclear microinjection site.

Experimental design
Experiment 1: effect of in vitro aging
The effect of in vitro aging on the p34^cdc2 kinase activity and the PCC incidence was investigated using rat oocytes. Ten oocytes for each test were sampled for in vitro p34^cdc2 kinase assay immediately after recovery (15 min group), and after culture for 30 min (45 min group), 60 min (75 min group) or 105 min (120 min group) in mKRB at 37 °C in 5% CO₂ in air. Nuclear configuration of the oocytes was also examined immediately after recovery and every subsequent 30 min up to 120 min. The other oocytes were placed in Hepes-mKRB, and the cumulus cell nuclei were then injected into the oocytes up to 120 min. The injected oocytes were divided into two groups based on the time from the animal killing to NT completion (15–45 min group and 46–120 min group). The incidence of PCC was evaluated 1 h after the last injection among each group.

Experiment 2: effect of enucleation
The effect of oocyte enucleation on the p34^cdc2 kinase activity and the PCC incidence was investigated using both rat and mouse oocytes. For the in vitro p34^cdc2 kinase assay, oocytes derived from the same donor rats and mice were allocated to the non-enucleation group and the enucleation group, and the non-enucleated and enucleated oocytes were sampled 45 min after the animal killing. Enucleation was performed by aspiration of the metaphase-plate with a part of the volume ( 10–20%) of surrounding cytoplasm in Hepes-R1ECM containing 5 µg/ml cytochalasin B (Sigma-Aldrich). In addition to the non-enucleation and enucleation groups, denuded rat oocytes were allocated to a cytoplasm-removal group (a part of the cytoplasm was mechanically aspirated as a sham control.). All the treatments were completed within 45 min. The oocyte samples were harvested at 45, 75, 90 and 105 min (0, 30, 45 and 60 min after enucleation respectively). For PCC evaluation, nuclear injection into non-enucleated and enucleated mouse and rat oocytes, and into cytoplasm-removed rat oocytes, was completed within 45 min after animal killing.

Experiment 3: effect of roscovitine treatment
We investigated whether the decreased level of p34^cdc2 kinase activity in non-enucleated rat oocytes negatively affected the PCC incidence of injected cell nuclei. Roscovitine (Sigma-Aldrich) dissolved at 10 mM in DMSO and stored at –80 °C, a specific inhibitor of p34^cdc2 kinase, was added to all media throughout the process of oocyte recovery and the in vitro culture at the final concentration of 150 µM, as reported by Lazar et al.(2002). Oocytes were sampled for in vitro p34^cdc2 kinase assay at 15 (immediately after recovery), 45, 75 and 120 min as in Experiment 1. Because the 45 min treatment of rat oocytes with 150 µM roscovitine decreased their p34^cdc2 kinase activity to an equivalent level of oocytes 120 min after in vitro aging or 60 min after enucleation, nuclear injection into the roscovitine-treated oocytes was performed within 45 min, and the incidence of PCC was then evaluated after a further 1 h.

Experiment 4: effect of MG132 treatment
We investigated whether the treatment of rat oocytes with a proteasome inhibitor, by which the p34^cdc2 kinase activity would be maintained, could support the PCC incidence of the injected donor cell nuclei. MG132 (Sigma-Aldrich), an inhibitor which can block cyclin B1 degradation, was used at the final concentration of 5 µM, as reported by Josefsberg et al.(2000). Non-enucleated oocytes were exposed to MG132, throughout the process of oocyte recovery to in vitro culture or nuclear injection. Oocyte sampling for in vitro p34^cdc2 kinase assay was performed at 15 min (immediately after recovery), 45, 75 and 120 min. Nuclear injection into the MG132-treated oocytes and PCC evaluation were performed as in Experiment 3.

Experiment 5: effect of PCC on NT embryo development
In the first group, oocytes exposed to MG132 from oocyte recovery to just before enucleation were used as recipients. From the result of Experiment 4, MG132 treatment is expected to support PCC even in enucleated oocytes, because the p34^cdc2 kinase activity was maintained at a high level during the treatment. The procedure of enucleation was as described above. The cytoplasts were then injected with cumulus cell nuclei. One hour later, a high incidence of PCC was expected. In the second group, oocytes with the extrusion of a second polar body were selected from 2 h cultures of non-treated oocytes, and then enucleated and nuclear injected as well. The reduced incidence of PCC under a lowered MPF was expected. For both groups, the oocytes 1 h after nuclear injection were activated by two direct current pulses (100 V/mm, 99 µs) and 4 h treatment with 2 mM 6-dimethylaminopurine (6-DMAP) (Sigma-Aldrich) in mR1ECM medium. The reconstructed oocytes were cultured in 100 µl microdrops of mR1ECM at 37 °C in 5% CO₂ in air, and number of oocytes forming one or two pseudo-pronuclei and cleaving to the two-cell stage was recorded at 6 and 24 h of culture respectively.

Statistical analysis
Each experiment had at least three replicates. Data on p34^cdc2 kinase activity were compared by ANOVA and Fisher’s protected least significant difference test using the StatView program (Abacus Concepts, Inc., Berkeley, CA, USA). Data on PCC incidence and NT embryo development were compared by Fisher’s exact probability test. A value of P < 0.05 was chosen as an indication of statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Experiment 1: effect of in vitro aging
The time-dependent change in p34^cdc2 kinase activity in rat oocytes is shown in Fig. 1. The p34^cdc2 kinase activity of rat oocytes decreased significantly at 45 min (after 30 min cultivation) and more significantly at 75 min (after 60 min cultivation). The decreased activity was maintained until 120 min (after 105 min cultivation). Probably due to the decrease of p34^cdc2 kinase activity, the release from meiotic arrest of ovulated oocytes occurred in a time-dependent manner, as shown in Fig. 2. Within 75 min after animal killing, more than half of the oocytes progressed beyond the MII stage. At 135 min, most of the oocytes were at either the AII or the TII stage. The proportion of in vitro-aged rat oocytes promoting PCC of the injected cumulus cell nuclei is shown in Table 1. When nuclear injection was completed within 45 min from animal killing, a positive sign of PCC was observed in 32.5% of the NT oocytes. On the other hand, PCC occurred in only 2.6% of the oocytes into which cumulus cell nuclei were injected between 46 and 120 min.

View larger version (12K): [in this window] [in a new window]   Figure 1 Kinetics of p34cdc2 kinase activity in rat oocytes during in vitro culture. 1Data are expressed as relative percentage of the level of p34cdc2 kinase activity in rat oocytes immediately after recovery (15 min after the animal killing; defined as 100%). a– cDifferent superscripts on S.E.M. bars denote significant differences among the culture periods (P < 0.05).

View larger version (50K): [in this window] [in a new window]   Figure 2 Time-dependent changes of nuclear configuration during in vitro culture of ovulated rat oocytes. MII, metaphase-II; AII, anaphase-II; TII, telophase-II. 1Data are expressed as relative percentage of the level of p34cdc2 kinase activity in non-enucleated rat oocytes harvested at 45 min from the animal killing (defined as 100%).

View larger version (13K): [in this window] [in a new window]   Figure 3 Changes in p34cdc2 kinase activity by enucleation in rat and mouse oocytes. 1Data are expressed as relative percentage of the level of p34cdc2 kinase activity in non-enucleated rat oocytes harvested at 45 min from the animal killing (defined as 100%). *denotes significant difference from the corresponding value in the rat (P < 0.05).

View larger version (30K): [in this window] [in a new window]   Figure 4 Effect of enucleation and cytoplasm removal on the kinetics of p34cdc2 kinase activity in rat oocytes. 1Data are expressed as relative percentage of the level of p34cdc2 kinase activity in non-enucleated rat oocytes harvested at 45 min from the animal killing (defined as 100%). a, b; c, d; e, f; g, hDifferent superscripts on S.E.M. bars denote significant differences between the experimental groups in each culture period (P < 0.05). *An asterisk shows a significant difference compared with the activity in enucleated oocytes at 0 min (P < 0.05). NE, not examined.

View larger version (18K): [in this window] [in a new window]   Figure 5 Effect of roscovitine treatment on the kinetics of p34cdc2 kinase activity in rat oocytes. 1Data are expressed as relative percentage of the level of p34cdc2 kinase activity in non-treated rat oocytes immediately after recovery (15 min after the animal killing; defined as 100%). a –c; d –fDifferent superscripts on S.E.M. bars denote significant differences among different culture periods within each treatment group (P < 0.05). *Asterisk denotes a significant difference between the treatment groups (P < 0.05).

View larger version (19K): [in this window] [in a new window]   Figure 6 Effect of MG132 treatment on the kinetics of p34cdc2 kinase activity in rat oocytes. 1Data are expressed as relative percentage of the level of p34cdc2 kinase activity in non-treated rat oocytes immediately after recovery (15 min after the animal killing; defined as 100%). a, b; c –eDifferent superscripts on S.E.M. bars denote significant differences among culture periods within each treatment group (P < 0.05). *Asterisk denotes a significant difference between the treatment groups (P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Rat oocytes have poor potential for promoting PCC of injected somatic cell nuclei, and the rate was markedly decreased in aged or enucleated oocytes (Hirabayashi et al. 2003a,b). In the present study, rat oocytes into which cumulus cell nuclei were injected within 45 min promoted PCC (frequency 28–47% in Tables 1–4), but the incidence of PCC decreased with oocyte aging with a significant decrease in p34^cdc2 kinase activity (Table 1, Fig. 1). In mouse oocytes, PCC was initiated at least 30 min after nuclear injection and lasted until 2 h (Chen et al. 2004), therefore the timing of PCC observation in the present study (60 min after nuclear injection) might be optimal. When oocytes are treated with roscovitine, which decreases the level of p34^cdc2 kinase activity, PCC formation was never observed even though nuclear injection is completed within 45 min. Roscovitine is widely used as cyclin-dependent kinase inhibitor to suppress p34^cdc2 kinase in rodent oocytes (Phillips et al. 2002). Therefore, the rapid decrease of p34^cdc2 kinase activity in the roscovitine-treated oocytes found between 45 and 75 min (Fig. 5), when compared with the slow decrease in the non-treated oocytes during the same period, may influence the outcome of PCC. The high concentration of roscovitine affects the other cyclin-dependent protein kinases such as cdk2 (De Azevado et al. 1997). The unknown pathway and/or chemical toxicity of roscovitine cannot be ruled out for complete suppression of PCC induction. Moreover, the level of activity in rat oocytes was less than half compared with that in mouse oocytes, which had a high potential for promoting PCC. Judging from these results, we estimated that the high activity of p34^cdc2 kinase is associated with promoting PCC of injected somatic cell nuclei, and the low level of p34^cdc2 kinase activity is one of the reasons for the poor potential to promote PCC in rat oocytes compared with that of mouse oocytes. The reason why the potential level of p34^cdc2 kinase activity is different between mouse and rat oocytes remains unclear. Our previous study indicated, at least, that the PCC-inducing potential is determined by the origin of the recipient cytoplasm, not by the origin of the donor nucleus, in the interspecies NT experiment between mouse and rat (Hirabayashi et al. 2003a). In somatic cell division, dissolution of the nuclear envelope occurs during G2/M transition in mitosis (Nurse 1990) and is induced by phosphorylation of lamin, one of the major nuclear membrane proteins (Peter et al. 1990). In oocytes, p34^cdc2 kinase had the potential to phosphorylate lamins, inducing meiotic breakdown of the nuclear envelope and the meiotic resumption (Dessev & Goldman 1988). Therefore, it is possible that p34^cdc2 kinase plays an important role in the destruction of the nuclear membrane of the injected somatic cell through phosphorylation of lamin.

One possible pathway known for inactivation of p34^cdc2 kinase is the calmodulin-dependent pathway via Ca²⁺. We have reported that the Ca²⁺-dependent pathway triggers the degradation of cyclin B and results in inactivation of p34^cdc2 kinase in porcine oocytes (Ito et al. 2003, 2004a). In Xenopus oocytes (Lorca et al. 1994, Morin et al. 1994), activation of calmodulin-dependent protein kinase II induces the degradation of cyclin B via the ubiquitin proteasome pathway. Based on these literature sources and preliminary data, we have investigated the effect of a proteasome inhibitor MG132 on p34^cdc2 kinase activity and PCC incidence. The PCC incidence in rat oocytes was significantly improved by MG132 treatment (Table 4). This result matched well with the report by Josefsberg et al.(2000), who demonstrated that MG132-treated rat oocytes contained a high amount of cyclin B1. In the present study, MG132 also provided a suppressive effect on inactivation of p34^cdc2 kinase (Fig. 6). Use of ALLN, a neutral cysteine protease inhibitor, was also slightly effective for improving the PCC incidence in rat oocytes (Hirabayashi et al. 2003b). Thus, the Ca²⁺-dependent degradation pathway, which may induce a decrease in the cyclin B1 level, is activated in aged rat oocytes, which results in a low level of p34^cdc2 kinase activity, and a low potential to promote PCC. Our unpublished data (M Hirabayashi and J Ito, unpublished data) showing that the decreased concentration of extracellular free calcium in the handling medium partially inhibits spontaneous activation of rat oocytes, support this hypothesis.

The other pathway for inactivation by phosphorylation of ¹⁴Thr and ¹⁵Tyr of p34^cdc2 kinase has been reported in aged porcine oocytes (Kikuchi et al. 2000). In the same study, it has been reported that caffeine suppressed the shift from MPF to pre-MPF (¹⁴Thr/¹⁵Tyr-phosphorylated p34^cdc2 kinase + cyclin B1) in the aged porcine oocytes. Okadaic acid is also known as a protein phosphatase inhibitor which prevents phosphorylation of ¹⁴Thr and ¹⁵Tyr of p34^cdc2 kinase (Rime et al. 1995). In our preliminary study, PCC incidence of injected cumulus cell nuclei was slightly improved in rat oocytes treated with okadaic acid (M Hirabayashi and J Ito, unpublished data). Therefore, the phosphorylation of p34^cdc2 kinase as well as the cyclin degradation may be involved in spontaneous activation of rat oocytes during in vitro culture.

The level of p34^cdc2 kinase activity in non-treated rat oocytes was similar to that of cytoplasm-removed or enucleated oocytes in the case where the oocytes were sampled immediately after the treatment (Fig. 4). The mechanical puncture of the oocyte membrane and the aspiration of the cytoplasm or karyoplast did not influence the p34^cdc2 kinase activity immediately after the enucleation procedure beyond the level expected from the relative volume of aspirated cytoplasm ( 10–20%). These results strongly suggest that active p34^cdc2 kinase is not accumulated near the metaphase spindles in rat oocytes. There was significant difference in the p34^cdc2 kinase activity between 45 and 75 min in both non-treated and enucleated groups (Fig. 4). A dramatic decrease in the activity of p34^cdc2 kinase was observed in the rat oocytes enucleated and cultured for 30 min compared with the non-treated group (Fig. 4), suggesting that the oocyte metaphases per se are involved in the maintenance of p34^cdc2 kinase activity. We have previously reported in porcine oocytes that activation of p34^cdc2 kinase was dependent on the synthesis of cyclin B and that the synthesis was induced by the activation of mitogen-activated protein (MAP) kinase (Ito et al. 2004b). The MAP kinase suppressed Myt1 activity that phosphorylated p34^cdc2 kinase at ¹⁴Thr and ¹⁵Tyr (Mueller et al. 1995, Palmer et al. 1998). Because activated MAP kinase is localized on the meiotic spindle in porcine oocytes (Lee et al. 2000), it is possible that enucleation results in the loss of MAP kinase from the recipient oocytes.

Nuclear injection into the MG132-treated, MII-enucleated rat oocytes resulted in a higher development to the two-cell stage when compared with non-treated, TII-enucleated rat oocytes after 24 h cultivation (Table 5). The favorable circumstance of recipient cytoplasm with a high MPF activity must induce PCC and promote subsequent development of NT embryos. Wakayama & Yanagimachi (2001) reported in mice that the injected cumulus cell nuclei undergoing PCC transformation to the two pseudo-pronuclei and such NT embryos are able to develop to full-term. In contrast, a different situation has been reported in the cloning of large domestic animals. The somatic cell NT in the livestock species is successfully reproducible when enucleated and activated oocytes with a low MPF activity are used as recipient cytoplasm (Baguisi et al. 1999, Kurosaka et al. 2002, Shin et al. 2002). Such a difference between rodents and large domestic animals remains unclear, but our present data suggest that induction of PCC in reconstructed embryos is related to the high activity of p34^cdc2 kinase, which seems to play a key role in the successful rat cloning.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The authors thank Mrs A Ishikawa and Miss A Takata for their technical assistance. This work was supported in part by Grant-in-Aids for the Scientific Research (No. 15082211, No. 16300139; to M H and S H), and the 21st Century COE Program (to S H) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Research Fellowship for Young Scientists (No. 8254; to J I.) from the Japan Society for the Promotion of Science.

	Footnotes

 
Received 20 July 2004
First decision 16 August 2004
Revised manuscript received 8 September 2004
Accepted 12 October 2004

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