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Analyses of Mitogen-Activated Protein Kinase Function in the Maturation of Porcine Oocytes¹

^a Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

	ABSTRACT

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The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase.

kinases, meiosis, oocyte development, ovum, signal transduction

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mitogen-activated protein kinase (MAPK) is a serine/threonine kinase belonging to an important intracellular signal transduction pathway. In all species investigated, MAPK is activated at germinal vesicle breakdown (GVBD) during oocyte maturation, and it maintains a high level of activity thereafter until the oocytes are matured [1–8]. The high level of MAPK activity in the second metaphase (M2) works as a cytostatic factor (CSF) in vertebrate oocytes and causes the arrest of oocytes at M2 until the oocytes are fertilized [9–11].

In Xenopus oocytes, progesterone triggers meiotic maturation by inducing the synthesis of c-mos protein, an upstream kinase of MAPK in oocytes, and subsequent MAPK activation [12, 13]. In the Xenopus species, the activation of MAPK mediates the activation of maturation/M phase-promoting factor (MPF), a key regulator of the M phase, and results in the induction of GVBD [10, 11]. Conversely, the inhibition of MAPK activity inhibits MPF activation and GVBD induction by progesterone [14, 15]. At present, it is well accepted that MAPK activation is both necessary and sufficient for the induction of GVBD in Xenopus oocytes [10, 11].

In contrast, the MAPK activation induced by c-mos mRNA injection was unable to induce MPF activation or GVBD in the oocytes of another frog, Rana japonica [16], or in goldfish oocytes [17]. Furthermore, injection of c-mos antisense oligonucleotide did not inhibit GVBD in these species, although their levels of MAPK activity remained low and those oocytes could not arrest at M2 [16, 17]. The authors of those previous studies indicated that MAPK has ubiquitous CSF activity in amphibian and fish oocytes but that MAPK activity was not required for the resumption of meiosis in these species except for Xenopus [18]. The dispensability of MAPK for the initiation of oocyte maturation has also been shown in c-mos knockout mouse studies, in which MAPK activity was absent but MPF activation and GVBD occurred normally [19–21].

In mammalian oocytes other than those of mouse, the requirement of MAPK activity for meiotic resumption is still controversial. The synthesis of c-mos protein before GVBD and the translocation of active MAPK into the nucleus just before GVBD have been reported in bovine and porcine oocytes, respectively [22, 23]. The increase in MAPK activity by an injection of c-mos mRNA and active MAPK protein accelerated GVBD in these species [6, 23]. These reports indicate the involvement of MAPK in the meiotic resumption in bovine and porcine oocytes. Recently, however, it has been reported that the inhibition of MAPK activity by expression of a MAPK-specific phosphatase, MKP-1, could not prevent GVBD in bovine oocytes [24]. In porcine oocytes, we previously reported a decrease in the rate of GVBD caused by the partial inhibition of MAPK activity by an upstream inhibitor, U0126 [25]. Nevertheless, further studies regarding more specific methods are required for understanding the MAPK function on porcine oocyte maturation.

In the present study, MAPK activity was stimulated or inhibited by an injection of porcine c-mos mRNA or antisense RNA, respectively, into porcine immature oocytes, and the effects were examined, especially at the initiation of meiotic maturation and the M2 arrest.

	MATERIALS AND METHODS

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Collection and Maturation of Porcine Oocytes In Vitro

Ovaries of prepubertal gilts were collected at a local abattoir and transported to the laboratory at approximately 37°C in saline. Cumulus-oocyte complexes (COCs) were aspirated from follicles (diameter, 2–5 mm) and washed three times in a modified Krebs-Ringer bicarbonate solution [26] containing 20% (w/v) porcine follicular fluid, 1.0 IU/ml of eCG (Pramex; Sankyo, Tokyo, Japan), and 3.2 mg/ml of BSA (fraction V; Wako Pure Chemical Ind., Osaka, Japan). The washed COCs were subjected to microinjection as described below. Groups of 10–20 COCs were cultured for up to 48 h in the same medium described above at 37°C with 5% CO₂ and saturated humidity in air. After culturing, oocytes were treated with 150 IU/ml of hyaluronidase (type IV; Sigma, St. Louis, MO) for a few minutes at room temperature, and the surrounding cumulus cells were removed by pipetting gently with a fine-bore pipette in saline supplemented with 0.1% polyvinylpyrrolidone (PVP; average molecular weight, 10 000; Sigma). The denuded oocytes were subjected to the immunoblotting and kinase assays. Some oocytes were examined for nuclear status by phase-contrast microscopy after fixation with acetic acid:ethanol (1:3 [v/v]) and staining with 0.75% aceto-orcein solution.

Preparation of Porcine c-mos mRNA and Antisense RNA

A full-length porcine c-mos cDNA, a gift from Dr. B. Newman, Babraham Institute, Cambridge, U.K. [27], was cloned into a pGEM-3Z vector (Promega, Tokyo, Japan) at the EcoRI/BamHI site. The cloned vector was linearized by cutting it with EcoRI (Takara Shuzo Co., Ltd., Tokyo, Japan) or BamHI (Takara Shuzo Co.) for mRNA or antisense RNA preparations, respectively, then the linearized cDNA was transcribed in vitro with SP6- or T7-mRNA-polymerase, respectively, 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. The RNA transcripts were precipitated with absolute ethanol, washed, dried, and resuspended in RNase-free water at a concentration of 1 µg/µl. The RNA solutions were stored at -80°C until use.

Microinjection

In a previous report, we found that coinjecting enhanced green fluorescent protein (EGFP) mRNA with objective mRNA and then collecting the oocytes with EGFP illumination was a powerful method for selecting not only the oocytes that were synthesizing the objective protein but also the viable oocytes [28]. Therefore, we employed this method not only for c-mos mRNA injection but also for c-mos antisense RNA injection as a marker of protein synthesis and oocyte viability, respectively. Approximately 30% of oocytes were EGFP positive. The c-mos mRNA solution (1 µg/µl) and the c-mos antisense RNA solution (1 µg/µl) were added with equal volume of EGFP mRNA solution (1 µg/µl) prepared as described previously [28]. Therefore, the concentrations of c-mos mRNA and c-mos antisense RNA in the RNA solutions were, finally, 0.5 µg/µl. Groups of 10 COCs were placed in 60-µl drops of culture medium covered by mineral oil. The microinjection was performed using microinjectors (IM-5A/B; Narisige, Tokyo, Japan) equipped with manipulators (Motorsteuerung; Zeiss, Oberkochen, Germany) mounted on an inverted microscope (Zeiss). Approximately 40 pl of RNA solution were injected into each ooplasm by continuous pneumatic pressure using a holding pipette (outer diameter, 150 µm; inner diameter, 50 µm) and an injection pipette (diameter, <0.1 µm) treated with heating (200°C, 2 h). After injection, all COCs were cultured as described above, and the expression of EGFP was examined under a fluorescent stereomicroscope (MZ FLIII; Leica, Wetzlar, Germany) at the oocyte collection. Only the oocytes expressing EGFP illumination were used for all analyses in the present study.

Assay of MAPK and MPF Activities

Ten denuded oocytes were lysed in 2.5 µl of assay buffer [29] and stored at -80°C until use. The activities of MPF and MAPK were evaluated in terms of the histone H1 kinase and myelin basic protein (MBP) kinase activities, respectively, as described in previous reports [4, 29]. The lysates (2.5 µl) were added to 2.5 µl of 2.5 µM cAMP-dependent protein kinase inhibitor (Sigma), 5 µl of a 2 mg/ml concentration of histone H1 (Sigma), 2.5 µl of a 10 mg/ml concentration of MBP (Sigma), and 5 µl of 0.1 mM [-³²P]ATP (0.4 mCi/ml; Amersham Pharmacia Biotech, Buckinghamshire, U.K.), and the reaction was performed at 37°C for 1 h. Next, 5 µl of 5x Laemmli buffer [30] were added to each lysate, which was then denatured at 100°C for 5 min and subjected to SDS-PAGE. The bands of phosphorylated histone H1 and MBP were visualized after autoradiography.

Immunoblotting

Micro-Western blotting method [31] was used with several modifications. Thirteen oocytes were put in 2 µl of saline supplemented with 0.1% PVP, added to 0.5 µl of 5x Laemmli buffer, and denatured at 100°C for 5 min. Proteins were separated on a modified 10% polyacrylamide gel [4] by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (AE-6660; Atto Co., Tokyo, Japan). After blocking the membrane with 5% milk for 1 h, the membrane was treated with anti-MAPK polyclonal antibody (K-23; Santa Cruz Biotechnology, Santa Cruz, CA). Signals were detected by a blotting detection kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Statistical Analysis

The chi-square test was used for evaluation of the results. A probability of P < 0.05 was considered to be statistically significant.

	RESULTS

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Effects of c-mos mRNA Injection on Meiotic Resumption of Porcine Oocytes

Porcine oocytes were injected (+) or not injected (-) with porcine c-mos mRNA and subjected to MAPK immunoblot (Fig. 1A, upper panel). In the noninjected oocytes, ERK1 and ERK2, major MAPKs in mammalian oocytes, were dephosphorylated in noncultured immature oocytes (lane 1) and phosphorylated in 48 h-cultured mature oocytes (lane 6) as reported previously [4, 25]. Although no phosphorylated bands were observed until 18 h of culture in the noninjected oocytes (lanes 2 and 4), the phosphorylated bands were detected slightly at 6 h of culture and increased at 18 h in c-mos mRNA injected oocytes (lanes 3 and 5). A typical result of MAPK activity assay is shown in Figure 1A (middle panel). It revealed that the activity was low and unchanged until 18 h of culture in noninjected oocytes (lanes 1, 2, and 4) and that the activity level was high in mature oocytes (lane 6). In contrast, the activity level in c-mos mRNA-injected oocytes was increased slightly and clearly at 6 and 18 h of culture, respectively (lanes 3 and 5).

View larger version (38K): [in this window] [in a new window]   FIG. 1. Effects of c-mos mRNA injection on meiotic resumption of porcine oocytes. A) Porcine oocytes injected (+) or not injected (-) with c-mos mRNA were cultured for the periods indicated and subjected to MAPK immunoblot (upper panel), MAPK activity assay (middle panel), and MPF activity assay (lower panel). In the upper panel, the dephosphorylated inactive forms of ERK1 and ERK2 (44 and 42 kDa, respectively), which are major MAPKs in mammalian oocytes, and their phosphorylated active forms are indicated on the right by arrowheads and asterisks, respectively. B) GVBD rate of porcine oocytes injected (mos+) or not injected (control) with c-mos mRNA were examined at the indicated culture periods. An asterisk shows a significant difference between mos+ and control values evaluated according to the chi-square test (P < 0.05)

We examined next the MPF activity and the meiotic progression in these MAPK-activated oocytes. Although MAPK activation had no detectable effect on MPF activity until 18 h of culture (Fig. 1A, lower panel), the GVBD rate at 24 h was significantly higher in c-mos mRNA-injected oocytes (78%) than in the noninjected oocytes (55%), as shown in Figure 1B. The final maturation rates at 48 h of culture were 90% in both the c-mos mRNA-injected and noninjected groups (Table 1), and the oocytes in both groups were morphologically normal (data not shown), indicating that the premature MAPK activation did not have deteriorative effects on the meiotic maturation of porcine oocytes.

Effects of c-mos Antisense RNA Injection on Meiotic Resumption of Porcine Oocytes

The phosphorylation states of ERK1 and ERK2 in the oocytes injected (+) or not injected (-) with c-mos antisense RNA are shown in Figure 2A. The phosphorylated forms were first detected at 30 h in the noninjected oocytes (lane 4), and the amounts increased until 48 h (lanes 6 and 8). In contrast, the ERK phosphorylation was completely inhibited by the injection of c-mos antisense RNA throughout the culture period (lanes 3, 5, 7, and 9). The changes in MAPK activity in those oocytes are shown in the upper panel of Figure 2B. The MAPK activation observed in the noninjected oocytes (lanes 2, 4, and 6) was also completely inhibited by the injection of c-mos antisense RNA (lanes 3, 5, and 7), confirming the absence of active MAPK in the oocytes injected with c-mos antisense RNA.

View larger version (39K): [in this window] [in a new window]   FIG. 2. Effects of c-mos antisense RNA injection on meiotic resumption of porcine oocytes. A) MAPK immunoblot of porcine oocytes injected (+) or not injected (-) with c-mos antisense RNA and cultured for the periods indicated. For both ERK1 and ERK2, the dephosphorylated inactive forms and the phosphorylated active forms are indicated by arrowheads and asterisks, respectively, on the right. B) MAPK (upper panel) and MPF (lower panel) activity assays of porcine oocytes injected (+) or not injected (-) with c-mos antisense RNA and cultured for the indicated hours. C) GVBD rate of porcine oocytes injected (mos-) or not injected (control) with c-mos antisense RNA were examined at the indicated culture periods. Chi-square tests were performed between mos- and control values, and no significant differences were detected in all culture periods

The MPF activity and the meiotic progression were next examined in those oocytes free from active MAPK. Interestingly, the absence of active MAPK clearly decreased the MPF activity, although the MPF activation was detected from 30 h even in the oocytes without MAPK activity (Fig. 2B, lower panel, lanes 5 and 7). In spite of the decrease in MPF activity, the GVBD rates in c-mos antisense-RNA-injected oocytes were not significantly different from those in noninjected oocytes (Fig. 2C). Furthermore, the rates of first metaphase (M1) and the progression into first anaphase were also unchanged between these two groups (Table 2). On the other hand, most of the oocytes injected with c-mos antisense RNA were abnormal in morphology and had decondensed chromosomes at 48 h of culture, whereas all noninjected oocytes were arrested at M2 and had condensed chromosomes (Table 3). We estimate that 27% of the oocytes having one pronucleus with one or no polar body were activated after the M1 and that 70% of oocytes having at least three pronuclei and polar bodies each were activated after M2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we investigated the role of the MAPK pathway during porcine oocyte maturation by injecting oocytes with either porcine c-mos mRNA or antisense RNA. When c-mos mRNA was injected into the porcine immature oocytes, the phosphorylation and activation of MAPK were observed at 6 h and increased at 18 h after the injection, whereas MAPK activation was detected from 24 h of culture in the intact oocytes, as reported previously [4, 23, 25]. This time course of MAPK phosphorylation in c-mos mRNA-injected oocytes agreed with our previous report, which found that the protein synthesis in porcine immature oocytes started within 6 h and continued up to 18 h after mRNA injection [28]. In the present study, the acceleration of GVBD was observed in the porcine oocytes injected with porcine c-mos mRNA. This acceleration was not the result of the microinjection process itself, because the injection of EGFP mRNA had no effect on the GVBD rate during our preliminary experiments (data not shown). The acceleration effect of high levels of MAPK activity on GVBD of mammalian oocytes has been reported in porcine oocytes injected with starfish active MAPK [23] and in bovine oocytes injected with Xenopus c-mos mRNA [6]. Furthermore, MAPK has been shown to promote GVBD in mouse oocytes, in which spontaneous GVBD was inhibited by a phosphodiesterase inhibitor, isobutylmethylxanthine [32]. The acceleration of GVBD by stimulation of MAPK activity has also been reported in frog and fish oocytes, although MAPK activation alone cannot induce GVBD in these species except for Xenopus [16]. These reports, combined with our present results, indicate that the role of MAPK in promoting GVBD induction might be common in all vertebrates.

In contrast, the absence of active MAPK with the injection of c-mos antisense RNA had no effect on the start of meiotic maturation in the porcine oocytes. This occurred also with c-mos knockout mouse oocytes, in which MAPK activation was completely inhibited yet GVBD occurred normally [21, 32, 33]. The dispensability of MAPK activity for GVBD of mammalian oocytes was also shown in bovine oocytes by the expression of a MAPK-specific phosphatase, MKP-1 [24]. In our previous report, in which MAPK activation of porcine COCs was inhibited by the use of U0126, an upstream inhibitor of MAPK, the meiotic resumption was inhibited in approximately half the examined oocytes. At that time, we suggested the involvement of MAPK activation in the meiotic resumption in porcine oocytes [25]. The inhibitory effect of U0126 was, however, observed only in the presence of cumulus cells, and U0126 had no effect on the GVBD rate or on the MAPK activity in denuded oocytes, probably because of U0126's nonpermeability into the denuded oocytes [25]. Recently, Su et al. [34] showed that gonadotropin-induced GVBD required the participation of MAPK activity in cumulus cells in mouse COCs. Because the presence of MAPK in porcine granulosa cells and its activation by gonadotropins have been reported [35], it can be considered that the MAPK in cumulus cells might be activated by premeiotic gonadotropin release and involved in the meiotic resumption in porcine oocytes as well. Our present result clearly shows that MAPK activation in oocyte cytoplasm is not required for GVBD in porcine oocytes, which is the same result as for other reported vertebrates except for Xenopus [16–21, 24, 32].

In spite of the normality of GVBD, the degree of MPF activation was very low in the present c-mos antisense RNA-injected porcine oocytes. It has been suggested that the polyadenylation of cyclin B1 mRNA, the regulatory subunit of MPF, requires MAPK activity in Xenopus oocytes [36–38]. The decrease of cyclin B accumulation was also reported in mouse oocytes injected with c-mos antisense RNA [39]. In addition, c-mos and MAPK have been reported to phosphorylate and inactivate Myt1 kinase, which converts active MPF into inactive pre-MPF, and subsequently to increase active MPF [40, 41]. Therefore, the high level of MAPK activity might contribute the cyclin B translation and/or pre-MPF to MPF conversion and, therefore, enhance MPF activity in porcine oocytes, although MAPK is not strictly needed to trigger MPF activation. The present result indicates that the low level of MPF activity that we observed is enough to escape from G₂ arrest and to induce GVBD in porcine oocytes.

Another noteworthy point was the abnormality we observed after M1 in the oocytes injected with c-mos antisense RNA. Some oocytes were transferred interphase from M1 directly without arriving at M2, and most of the other oocytes, even those that were transferred into second meiosis, did not arrest at M2 but activated spontaneously. It has been well accepted that the high level of MAPK activity in M2 oocytes works as CSF and causes the M2 arrest in vertebrate oocytes, including mammals [19–21, 24, 42, 43]. The spontaneous activation of M2 oocytes in the present study agrees well with this consensus. In Xenopus, CSF function is mediated by RSK, a downstream kinase of MAPK [44, 45]. Recently, we reported that RSK was present in the downstream of MAPK in porcine oocytes [46], suggesting the RSK mediation of CSF function in porcine oocytes as well. The direct transition of M1 oocytes into interphase was also observed in c-mos knockout mouse oocytes [21] and MAPK-inhibited Xenopus oocytes [47]. Recently, Mos was found in starfish, in which the oocytes were arrested not in M2 but in the pronuclear stage. The proposed function of Mos in that case was to prevent the meiosis/mitosis conversion after first meiosis [48]. Our present results support this proposed Mos/MAPK function.

In conclusion, although MAPK has a role in promoting MPF activation and in assisting meiotic resumption, MAPK activation is dispensable for GVBD in porcine oocytes. The major roles of MAPK activity during porcine oocyte maturation are to induce second meiosis and to arrest the oocytes at M2. The present results suggest that these roles of MAPK as shown in porcine oocytes may be general among vertebrates, because they are the same as those reported in other species except for the requirement for GVBD in Xenopus oocytes.

	FOOTNOTES

 
¹ Supported by Grants-in-Aid for Scientific Research (14360173 and 1465101 to K.N., 14360174 and 13876061 to H.T.) from the Ministry of Education, Science, Sports and Culture of Japan.

² Correspondence: Kunihiko Naito, Lab of Applied Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 5841 8191; aknaito{at}mail.ecc.u-tokyo.ac.jp

Received: 17 June 2002.

First decision: 11 July 2002.

Accepted: 3 September 2002.

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