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research-article |
Department of Animal Resource Sciences, Graduate School of Agricultural Sciences, University of Tokyo, Tokyo 113-8657, Japan
ABSTRACT
RINGO, a protein with no homology to cyclin B, has been reported to be involved in activation of CDC2 and regulation of meiotic maturation in Xenopus oocytes. Although the presence of homologues of RINGO families, which are known as SPDY families, has been reported in mammals, their roles in meiotic maturation of mammalian oocytes have never been examined. In the present study, the effects of SPDY on meiotic maturation of porcine oocytes were examined. At first, Xenopus RINGO (xRINGO) mRNA was injected into immature porcine oocytes and found to significantly accelerate CDC2 activation and meiotic resumption. The CCNB (also known as cyclin B) synthesis was prematurely started at 12 h of culture, whereas it started at 18 h in normal oocytes. We next cloned RINGO A2 homologue in pig (pigSPDYA2) from total RNA of immature porcine oocytes by RT-PCR and obtained full-length cDNA that was more than 85% and 40% homologous with mammalian SPDYA2 and xRINGO, respectively. Acceleration effects similar to those by xRINGO were observed in CDC2 activation, meiotic resumption, and the start of CCNB synthesis in pigSPDYA2 mRNA-injected porcine oocytes. In clear contrast with the effects of xRINGO, which was accumulated abnormally in porcine oocytes and arrested them in the first meiotic metaphase (M1), pigSPDYA2 accelerated the meiotic progression, with about half of pigSPDYA2 mRNA-injected oocytes completing meiotic maturation within 30 h. These results suggest that pigSPDYA2 has important roles on meiotic maturation of porcine oocytes and that the rapid degradation of SPDY was necessary for the normal maturation of oocytes.
gamete biology, gametogenesis, kinases, meiosis, oocyte development
Vertebrate oocytes in the ovarian follicles are arrested at the first meiotic prophase, which is equivalent to the G2 phase of the somatic cell cycle, and have a large nucleus referred to as the germinal vesicle (GV). Meiotic resumption of these immature oocytes, defined by the breakdown of the GV membrane (GVBD), is directly induced by the intracellular activity of cyclin-dependent kinase 1 (CDK1), also known as CDC2. The catalytic activity of CDC2 is stimulated by binding with a regulatory subunit of CDC2 kinase, cyclin B, and the active CDC2/cyclin B heterodimer is called the maturation/M-phase promoting factor (MPF), and is also known to be a key G2/M regulating kinase in eukaryotic cells [1]. Although the intracellular regulatory mechanisms of CDC2 activity at the meiotic resumption of vertebrate oocytes have been fairly well investigated, especially in Xenopus [2–4], many uncertainties remain. For example, some protein syntheses are required for the meiotic resumption of oocytes in Xenopus and mammals other than rodents, but it has not been determined what types of proteins are synthesized.
In recent years, RINGO (rapid inducer of G2/M progression in oocytes) and Speedy have been identified as factors involved in the meiotic resumption of Xenopus oocytes [5, 6]. The amino acid sequences of RINGO and Speedy are 88% homologous, and the overexpression of either factor can induce meiotic resumption in Xenopus immature oocytes without disposing of maturation-inducing hormones. RINGO can bind and activate both CDC2 and CDK2, whereas Speedy can bind and activate primarily CDK2, which works mainly at the G1/S transition. Therefore, RINGO should be involved mainly in the G2/M regulation, including meiotic resumption of oocytes [5–7]. Indeed, the suppression of RINGO expression by injection of antisense RNA into Xenopus immature oocytes dramatically inhibited their GVBD, suggesting the physiological requirement of RINGO synthesis for their meiotic resumption [3, 5].
Although RINGO can bind and activate CDC2, it has no apparent homology with any known proteins, such as cyclins that can also activate CDC2. The kinase activity of CDC2/RINGO was lower than that of CDC2/cyclin B, when histone H1 was used for the substrate [7]. The phosphorylation of a conserved Thr-161 located in the T-loop of CDC2 is required for the activation of CDC2/cyclin B complexes, whereas it is dispensable for the full activation of CDC2 by RINGO. Moreover, the CDC2/RINGO complex was less sensitive than the CDC2/cyclin B complex to the negative regulation of Thr-14/Tyr-15 kinase Myt1 [7]. These observations might indicate that CDC2/RINGO has original roles different from CDC2/cyclin B on meiotic resumption of oocytes.
The presence of RINGO and Speedy homologues has been reported in mammals [8, 9], and they are termed as SPDY. Mammalian SPDY has been found to form families that possess a conserved sequence, RINGO box, which is necessary for CDC2 binding [10–12]. Mouse Spdy and human SPDY have been shown to induce meiotic resumption in Xenopus immature oocytes [8, 9, 11]. In contrast to the accumulated genetic data on mammalian SPDY families and their effects on Xenopus oocytes, their physiological roles on mammalian oocytes have never been studied. In regard to mammalian oocytes, only the presence of mouse Spdy mRNA in immature mouse oocytes has been reported [8]. The acceleration of meiotic resumption has also been reported in mouse oocytes, but Xenopus RINGO (xRINGO) was used in this experiment instead of mouse Spdy. Furthermore, these oocytes were prevented from spontaneous maturation by an inhibitory factor, dibutyryl cAMP [8]. At present, it is unclear whether the meiotic resumption of mammalian oocytes is accelerated by mammalian SPDY and/or Xenopus RINGO in a physiological state.
The aim of the present study was to clarify whether SPDY has an effect on the meiotic maturation of mammalian oocytes. Using porcine oocytes, we first investigated whether xRINGO possesses stimulatory effects on meiotic resumption without using inhibitory factors and presumed the presence and the roles of porcine SPDY (pigSPDY). Following this experiment, pigSPDY was cloned by RT-PCR and sequenced for comparison with other species. Finally, pigSPDY was overexpressed in porcine immature oocytes by injection of pigSPDY mRNA, and its effects on porcine oocyte maturation were studied.
Collection and Maturation of Porcine Oocytes
Ovaries of prepubertal gilts were collected at a commercial slaughterhouse and transported to our laboratory at about 37°C in saline. Cumulus oocyte complexes (COCs) were aspirated from follicles (about 2–5 mm in diameter) and washed four times in a culture medium consisting of a modified Krebs-Ringer bicarbonate solution [13] containing 20% porcine follicular fluid, 1.0 IU/ml eCG (Pramex; Sankyo, Tokyo, Japan), and 3.2 mg/ml BSA (fraction V; WAKO Pure Chemical Industries, Osaka, Japan). The washed COCs were subjected to microinjection as described below. Groups of 10–20 COCs were cultured up to 48 h in 100 µl of the culture medium, covered by liquid paraffin (Nakalai Tesque Inc., Kyoto, Japan) at 37°C, under an atmosphere of 5% CO2 in air and saturated humidity. After culturing, surrounding cumulus cells were removed by treatment with hyaluronidase (type IV; Sigma, St. Louis, MO) and gentle pipetting in saline supplemented with 0.1% polyvinyl-pyrrolidone (PVP, average molecular weight 10 000; Sigma). The denuded oocytes were subjected to immunoblotting and MPF activity assay. Some oocytes were examined for nuclear status after being mounted on a glass slide, fixed with acetic acid-ethanol (1:3), and stained with 0.75% acetoorcein solution.
Preparation of xRINGO mRNA and pigSPDY mRNA
Full-length xRINGO cDNA was kindly provided by Dr. Angel R. Nebreda, European Molecular Biology Laboratory, Heidelberg, Germany. Full-length pigSPDY cDNA was obtained by RT-PCR of total RNA extracted from porcine noncultured oocytes using a commercial RNA extraction solution (Trizol Reagent; Gibco BRL, Karlsruhe, Germany). The primer pairs were designed according to the two sequences (accession number AW312924 and CO941231) registered in the NCBI porcine EST database: forward primer, 5'-CCACCATGAGACATAATCAGCTGTGTTGCG-3'; and reverse primer, 5'-TCATTCTTCACTTCCTGTAAACCA-3'. The PCR product was cloned into a pcDNA3.1/V5-HisTOPO vector (Invitrogen Japan K.K., Tokyo, Japan). For sequencing, a commercial sequencing kit (Applied Biosystems, Foster City, CA) and a DNA sequencer (Applied Biosystems) were used according to the manufacturer's instructions.
For the in vitro synthesis of mRNA, xRINGO/FTX5 and pigSPDY/pGEM-TEasy were linearized by Xba I (TaKaRa Shuzo Co., Ltd., Tokyo, Japan) and Pme I (New England Biolabs Ipswich, Australia), respectively. The linearized cDNA was transcribed in vitro with T7-RNA-polymerase using the Cap-Scribe system (Nippon Roche Co., Ltd., Kamakura, Japan) according to the manufacturer's instructions. The reaction was performed in the presence of M7G(5')ppp(5')G to synthesize capped RNA transcripts. Enhanced green fluorescent protein (EGFP) mRNA was prepared as described previously [14]. 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.
In a previous report [14], we found that coinjecting EGFP mRNA with other mRNAs and then collecting the oocytes with EGFP illumination was a powerful method for selecting the oocytes expressing the objective proteins. In the present study, therefore, we employed this method for the injection of xRINGO mRNA or pigSPDY mRNA. The concentration of each RNA in the solution was adjusted to 0.5 µg/µl. The microinjection was performed in 150 µl of the culture medium using microinjectors (IM-5A/B; Narisige, Tokyo, Japan) equipped with manipulators (MO-202U; Narisige) mounted on an inverted microscope (Diaphot 200; Nikon, Kawasaki, Japan). Approximately 50 pl of RNA solution was injected into each ooplasm by continuous pneumatic pressure. After injection, all COCs were cultured as described previously and the expression of EGFP was examined under a fluorescent stereomicroscope (MZ FL III; Leica, Wetzlar, Germany). About 30% of oocytes were EGFP-positive (Fig. 1A). Only the oocytes expressing EGFP illumination were used for analysis in the present study.
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35S-methionine Labeling and In Vitro Translation
The oocytes were denuded soon after collection and injected with mRNAs as previously described. Soon after injection (about 3 h after the oocyte collection), the oocytes were added to the culture medium containing 35S-methionine (400 MBq/ml; Muromati, Tokyo, Japan) at a radioactive concentration of 500 µCi/ml, and labeled for 3 h at 37°C, under an atmosphere of 5% CO2 in air and saturated humidity. The noninjected control oocytes were also labeled in the same way (3–6 h of culturing). After the labeling culture, groups of 10 oocytes were put into 8 µl of saline supplemented with 0.1% (w/v) PVP (PVP-saline), to which 2 µl of 5 x Laemmli buffer [15] was added, and denatured at 100°C for 5 min. Proteins were separated on a 10% polyacrylamide gel by SDS-PAGE. Newly synthesized proteins were visualized by autoradiography, after drying the gels on filter paper, and exposed to BioMax Film (Eastman Kodak Company, Rochester, NY).
For in vitro translation of pigSPDY mRNA, 2.0 µg mRNA was translated in 50 µl translation mixture using an in vitro translation system (Rabbit Reticulocyte Lysate Systems, Nuclease Treated, Promega, Madison, WI) according to the manufacturer's instructions.
The denuded oocytes were lysed in 2 µl of assay buffer [16] and stored at –80°C until use. The activities of MPF were evaluated on the basis of the histone H1 kinase activity as described in previous reports [17]. The lysates (2 µl) were added to 15 µl of assay buffer containing 2.5 µl of 2.5 µM cAMP-dependent protein kinase inhibitor (Sigma), 5 µl of histone H1 (5 mg/ml; Sigma), and 5 µl of 0.1 mM [
-32p] ATP (0.4 mCi/ml; Americam-Pharmacia Biotech, Buckinghamshire, UK). The reaction took place at 37°C for 1 h and was stopped by adding 5 µl of 5 x Laemmli buffer into each assay tube; the mixture was then denatured at 100°C for 5 min and subjected to SDS-PAGE. The bands of phosphorylated histone H1 were visualized by autoradiography.
The micro-Western blotting [18], a small-size immunoblotting system, was used for the immunoblotting of the oocytes with several modifications. Ten oocytes were placed in 2 µl of PVP-saline, added with 0.5 µl of 5 x Laemmli buffer, denatured, and subjected to SDS-PAGE using a 10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride membrane (AE-6660; Atto Co., Tokyo, Japan). After blocking the membrane with 3% (w/v) skimmed milk for 20 min, the membrane was treated with anti-CCNB1 (also known as cyclin B1) monoclonal antibody (CB169; Upstate Biochemistry Inc., Waltham, MA), anti-CCNB2 (also known as cyclin B2) polyclonal antibody (N-20; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-CDC2 monoclonal antibody (sc-54; Santa Cruz Biotechnology). For the immunoblotting of the in vitro translated proteins, each 10 µl of diluted reaction mixture (1:12.5) was subjected to normal-size SDS-PAGE using a 10% polyacrylamide gel. After protein transfer and blocking as described, the membrane was treated with anti-His (C-terminal) monoclonal antibody (R930–25; Invitrogen Japan K.K., Tokyo, Japan) or anti-pigSPDYA polyclonal antibody generated from rats in our laboratory according to a reported method [19]. To visualize the protein-bound antibodies, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used for CCNB1, CDC2 and His, HRP-conjugated anti-rabbit IgG (AP132P; Chemicon International Inc., Temecula, CA) was used for CCNB2, and HRP-conjugated anti-rat IgG (81–9520, Zymed Laboratories, San Francisco, CA) was used for pigSPDYA as a second layer, followed by a detection procedure using an ECL detection kit (Amersham-Pharmacia) according to the manufacturer's instructions.
The results were evaluated with the chi-square test. Values of P < 0.05 were considered to indicate statistical significance.
Effects of xRINGO on Meiotic Maturation of Porcine Oocytes
The oocytes injected with xRINGO mRNA plus EGFP mRNA and exhibiting EGFP illumination after culturing, as shown in Figure 1A, were collected for the analyses. The injected xRINGO mRNA was detectable by RT-PCR at 12 h after the injection, indicating that it was sufficiently stable in the porcine ooplasm (Fig. 1B). Newly synthesized proteins were labeled with 35S-methionine during 3–6 h of culture, and expression of the xRINGO protein (myc-tagged) was detected at the expected molecular weight (approximately 42 kDa) by autoradiography (Fig. 1C). The GVBD rate of these xRINGO-expressing porcine oocytes is shown in Figure 1D along with the rates of noninjected oocytes and oocytes injected with only EGFP mRNA. About 80% of xRINGO mRNA-injected oocytes underwent GVBD at 12 h of culture, and their GVBD rates at 12 h and 18 h were significantly higher than those in noninjected and EGFP mRNA-injected oocytes (P < 0.001), in which GVBD took place at around 24 h of culture. Most of the xRINGO mRNA-injected oocytes were at the pro-metaphase of the first meiosis at 12 h of culture, as shown in the typical morphological appearance of the oocyte in Figure 1E, whereas all noninjected oocytes were at the GV stage.
The effects of xRINGO on the meiotic maturation of porcine oocytes were further examined until 48 h, a sufficient period for the completion of porcine oocyte maturation. The percentages of oocytes at the first meiotic metaphase (M1) was not significantly different among groups until 30 h of culture, but thereafter, the M1 oocytes were gradually increased in xRINGO-expressing oocytes, whereas they were decreased in the noninjected and only EGFP mRNA-injected groups and almost disappeared at 48 h of culture (Fig. 2A). At 48 h of culture, more than 90% of xRINGO-expressing oocytes were arrested at M1 and no oocytes had reached the second meiotic metaphase (M2), although more than 70% of oocytes had reached the M2 phase in the other two groups (Fig. 2B). The decrease of the injected-xRINGO mRNA dosage, one third or one tenth of original concentration, did not recover the abnormal M1 arrest (data not shown). Examples of typical appearances of xRINGO mRNA-injected and noninjected oocytes at 48 h of culture are shown in Figure 2C.
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Because the GVBD was dramatically accelerated and M1 arrest was induced by the xRINGO expression, we next examined the states of MPF in the oocytes. In the noninjected oocytes, the histone H1 kinase activity at 12 h of culture was as low as that at 0 h of culture, whereas high activity was detected at 48 h (Fig. 3A), as reported previously [16]. In contrast, the activity of xRINGO-expressing oocytes at 12 h of culture was already elevated to a level near that in normal oocytes cultured for 48 h, supporting the notion that xRINGO accelerates the effects of GVBD. The activity was further increased in xRINGO-expressing oocytes, and the level at 48 h of culture exceeded that in normal oocytes (Fig. 3A). The contents of the MPF subunits CCNB1, CCNB2, and CDC2 in the oocytes were investigated by immunoblotting, and the results are shown in Figure 3B. As reported previously, both CCNB1 and CCNB2 were increased from 18 h of culture and peaked at 30 h and 48 h in CCNB2 and CCNB1, respectively, whereas the CDC2 level was unchanged during maturation in normal oocytes [20]. The expression of xRINGO significantly accelerated the increase of both CCNB1 and CCNB2 to 12 h of culture and the peak levels were observed at 18 h of culture. CCNB1 and CCNB2 were decreased transiently at 24 h and 30 h, respectively, and then both were increased again at 48 h. These changes of CCNB levels were completely different from the physiological fluctuation patterns previously reported [20].
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Because xRINGO had significant effects on porcine oocyte maturation, we expected that the RINGO protein would also be present in porcine oocytes and would play physiological roles in porcine oocyte maturation. In order to clone a cDNA of porcine RINGO homologue, pigSPDY, by RT-PCR, we found sequences including probable 5'-end and 3'-end of the pigSPDY open reading frame (ORF) at the positions shown in Figure 4A by a database analysis. From these two sequences, forward and reverse primers were designed and an RT-PCR product of the expected length was obtained from total RNA of immature porcine oocytes. Sequencing of the product revealed that it contained 936 bp and 311 amino acids (Fig. 4B).
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We compared the amino acid alignment of the product with several RINGO or SPDY families available from databases, and found that the product was most closely related to human SPDYA2, and therefore was predicted to be pigSPDYA2 (Fig. 5A). The comparisons of pigSPDYA2 with human SPDYA2, mouse Spdya2, and xRINGO are shown in Figure 5B. The amino acid homologies with pigSPDYA2 were 89.5%, 86.5%, and 40.8%, respectively. The reported RINGO box [11] is indicated by shading, and the homologies with pigSPDYA2 within the RINGO box were 97.0%, 98.5%, and 59.4%, respectively (Fig. 5B). PEST sequences that have been reported to act as destruction signals in Xenopus, indicated by a box, but the homologies of these sequences are very low between xRINGO and other mammalian SPDYA2 (Fig. 5B). Using RT-PCR, we examined the presence of pigSPDYA2 mRNA during maturation. As shown in Figure 5C, pigSPDYA2 mRNA was detected throughout the maturation period, suggesting that it plays some roles in the meiotic maturation of porcine oocytes.
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Effects of pigSPDYA2 Overexpression on Meiotic Maturation of Porcine Oocytes
The GVBD rates of porcine oocytes injected with or without pigSPDYA2 mRNA are shown in Figure 6A. About 50% of pigSPDYA2 mRNA-injected oocytes underwent GVBD at 12 h of culture and most oocytes had begun the first meiotic metaphase (M1) by 18 h of culture, whereas all oocytes in the noninjected group were still arrested at GV stage. Typical examples of the morphological appearance of oocytes injected with pigSPDYA2 mRNA and cultured for 12 h (left panel) and 18 h (right panel) are shown in Figure 6B. When the pigSPDYA2 mRNA-injected oocytes were incubated with 35S-methionine during 3–6 h of culture, pigSPDYA2 protein (V5 and His tagged), which was expected to be about 46 kDa, was not accumulated and could not be detected by autoradiography, in spite of the clear biological effects of pigSPDYA2 expression (Fig. 6C). This result is in marked contrast to that of xRINGO, which was accumulated during 3–6 h of culture and was clearly detected by autoradiography (Fig. 1C). When pigSPDYA2 mRNA was translated in vitro using a rabbit reticulocyte lysate system, clear bands were detected by anti-His antibody and anti-pigSPDYA2 antibody at the expected position as shown in Figure 6D, indicating the normality of pigSPDYA2 mRNA.
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After GVBD, about 70% of pigSPDYA2 mRNA-injected oocytes reached the M1 stage by 24 h of culture, about 6 h earlier than normal oocytes (Fig. 7A). Thereafter, the rate of M1 oocytes was decreased in the pigSPDYA2 mRNA-injected group, and about 50% of oocytes in this group had reached the M2 stage at 30 h of culture (Fig. 7B), when most of the normal oocytes were at the M1 stage (Fig. 7A). At 48 h of culture, about 80% of oocytes were at the M2 stage, as shown in Figure 7C, and this rate was comparable to that in normal oocytes (Fig. 7B). This result was markedly different from the result in oocytes injected with xRINGO mRNA, which were mostly in M1 arrest.
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We next examined the effect of pigSPDYA2 on the MPF states of porcine oocytes. The histone H1 kinase activity in pigSPDYA2 mRNA-injected oocytes was activated at 12 h of culture at a level higher than that in 48 h–cultured normal oocytes, then declined thereafter to a level comparable to that of normal oocytes at 48 h of culture (Fig. 8A). The contents of CCNB1 and CCNB2 were increased from 18 h and 12 h of culture, respectively, then peaked at 30 h and 24 h of culture, respectively (Fig. 8B). These fluctuation patterns were 6–12 h earlier and the peak levels were higher than those in normal oocytes, but the fluctuation profiles themselves were roughly the same as the physiological patterns in the normal oocytes.
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In the present study, we cloned porcine RINGO A2 homologue, pigSPYDA2, for the purpose of understanding the effects of the SPDY protein on the meiotic maturation of porcine oocytes and found that SPDY accelerated the meiotic resumption of porcine oocytes. Although SPDY families have frequently been reported in mammals [8–12], no studies have examined their effects on mammalian oocytes. In the only report using mammalian oocytes to study the effects of SPDY, Xenopus RINGO (xRINGO) accelerated the meiotic resumption of mouse oocytes, but the oocytes were prevented from spontaneously maturing by an inhibitory factor, dibutyryl cAMP [8]. The present study is the first report to demonstrate effects of the xRINGO protein on mammalian oocytes without using inhibitory factors. More importantly, we showed for the first time that homogeneous mammalian SPDY, not heterogeneous xRINGO, stimulated meiotic resumption of mammalian oocytes. In addition, we showed the presence of pigSPDYA2 mRNA in porcine oocytes throughout the maturation period, suggesting that SPDY has the physiological significance for the meiotic maturation of mammalian oocytes.
The stimulation of CDC2 activity by RINGO has been reported in Xenopus oocytes [5]. The CDC2 activity was assayed as the histone H1 kinase activity in the present study and its premature activation at 12 h of culture was also observed in porcine oocytes, confirming its acceleration effect on meiotic resumption of porcine oocytes. At present, SPDY is the only protein proven to activate CDC2 through its expression in mammalian immature oocytes, with the exception of CCNB, which is known as a regulatory subunit of CDC2 kinase [21]. It has been reported that xRINGO binds with CDC2 and activates it directly without cyclin B mediation [7, 11, 12]. In the present study, because the CCNB synthesis in the SPDY mRNA-injected porcine oocytes was barely started at 12 h of culture, the high CDC2 activity in those oocytes at 12 h might have consisted of not only the direct activation by SPDY but also, at least in part, the activation by synthesized CCNB. However, most of the activity at 12 h should be attributed to the direct activation by SPDY, because the levels of synthesized CCNB at 12 h were much lower than those at 48 h, whereas the CDC2 activity at 48 h was almost comparable with that at 12 h.
In the present study, none of the xRINGO-expressing oocytes were able to complete the meiotic maturation; all were arrested at the first meiotic metaphase (M1). It is well known that, in normal oocytes, an inactivation of CDC2 after M1 and its subsequent reactivation are required for the escape from M1 and entry into the second meiosis, respectively, and that insufficient CDC2 inactivation after M1 induces M1 arrest [1, 21]. Because immunoblotting analysis revealed degradation of the CCNB1 and CCNB2 in the xRINGO-expressing oocytes, the failure of escape from M1 might be attributed not to the failure of CCNB degradation but to the continuous CDC2 activation by xRINGO, which might be accumulated in porcine oocytes throughout the maturation period. In contrast, the oocytes injected with pigSPDY mRNA reached the second meiotic metaphase at the normal rate, although the meiotic progression was greatly accelerated. This difference between xRINGO mRNA injection and pigSPDY mRNA injection was not attributed purely to the dose effect, because the abnormal M1 arrest was not recovered by the decrease of the injected xRINGO mRNA concentration to one third or one tenth of pigSPDY mRNA concentration. This indicates that pigSPDY was not accumulated during normal maturation in porcine oocytes.
It has been reported that Xenopus Speedy, a homologous protein of RINGO, has PEST sequences required for rapid degradation and has a very short half-life [6]. The presence of similar sequences in xRINGO suggests the rapid degradation and the short half-life of xRINGO. Because the PEST sequences are not present in mammalian SPDYA2, mammalian oocytes might not recognize this heterogeneous sequence and, therefore, might not degrade xRINGO efficiently, resulting in its accumulation. This idea was supported by our 35S-methionine labeling experiments, where xRINGO was clearly detected by autoradiography in the expected position, but pigSPDY was not observed at all in spite of the appearance of normal size SPDYA2 from the used mRNA by an in vitro translation system. This result agreed well with the idea that SPDY is degraded rapidly in nature but porcine oocytes cannot degrade xRINGO. Further experiments will be needed to clarify this concept, such as identification of the mammalian sequences required for rapid degradation and detail research into the synthesis and degradation of the endogenous SPDY protein.
In addition to the meiotic resumption, SPDY clearly accelerated the start of CCNB synthesis, which occurred normally at around 20 h of culture in porcine oocytes [20, 22]. Recently, RINGO has been reported to be involved in the protein translation in Xenopus oocytes through the activation of CPEB, which regulates poly (A) length of mos and cyclin B mRNAs [23]. Our results indicate that this might be the case also in porcine oocytes. In the present study, CCNB1 and CCNB2 were abnormally elevated at both 18 h and 48 h in xRINGO-expressing oocytes, while they showed only a single peak in normal oocytes and in pigSPDY mRNA-injected oocytes. The cause of this abnormality should be the abnormal accumulation of xRINGO, which persisted in porcine oocytes and stimulated the CCNB synthesis during second meiosis, when endogenous SPDY had already disappeared. These results support the reports that SPDY has an effect on the translational regulation of CCNB. We propose that the ability of SPDY to accelerate the meiotic maturation of porcine oocytes is attributable to the direct activation of CDC2 and the stimulation of CCNB synthesis.
It has long been known that some protein syntheses are required for the meiotic resumption of oocytes in Xenopus and mammals other than rodents [2, 24]. We have previously reported that the syntheses of an activator of the mitogen-activated-protein kinase cascade, Mos, and a CDC2 activator, CCNB, are not always required for the start of meiotic maturation in porcine oocytes [20, 25]. In Xenopus oocytes, RINGO has been shown to be one of proteins whose synthesis is required for meiotic resumption by injecting antisense oligonucleotides and inhibiting their expression [5]. It is probable that SPDY synthesis is also required for the start of maturation in the oocytes of mammals other than rodents. Although the inhibition of SPDY expression is prerequisite for addressing this question, mammalian SPDY has been found to form families from A to E [11, 12, 26], and we found the expression of several SPDY families in porcine oocytes (our preliminary data). Because it has been postulated that the various mammalian SPDY families have different expression patterns and intracellular localizations, it would be interesting to examine how these SPDY families interact to regulate the meiotic maturation in mammalian oocytes. To this end, we have begun to clone the other families of porcine SPDY and to inhibit their expression in combination during the meiotic maturation of porcine oocytes.
ACKNOWLEDGMENTS
We are grateful to Dr. Angel R. Nebreda, European Molecular Biology Laboratory, Heidelberg, Germany, for the generous gift of full-length xRINGO cDNA. We also thank Dr. M. Okabe, Osaka University, Japan, for the gift of EGFP cDNA.
FOOTNOTES
1Supported by a grant-in-aid for scientific research (no. 17380173 to K. N.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 
Correspondence: 2Kunihiko 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, e-mail: aknaito{at}mail.ecc.u-tokyo.ac.jp
Received: 21 September 2006.
First decision: 16 October 2006.
Accepted: 1 December 2006.
REFERENCES
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