CSIRO PUBLISHING
Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD15150
Mitogen-activated protein kinase-activated protein kinase 2 is a critical regulator of pig oocyte meiotic maturation Xiang-Hong Ou A,D,*, Sen Li A,*, Bao-Zeng Xu B,C,*, Lei-Ning Chen A, Man-Xi Jiang A, Shao-Qin Chen A and Nan-Qiao Chen A A
Reproductive Medicine Center, The 2nd People’s Hospital of Guangdong Province, #1 Shiliuguang RD, Haizhou, Guangzhou, 510317, China. B Institute of Special Wild Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Jilin, China. C Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. D Corresponding author. Email: [email protected]
Abstract. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple cellular processes. In the present study, we showed that MK2 affected not only cumulus expansion, but also the oocyte meiotic cell cycle in porcine oocytes. Inhibition of MK2 arrested oocytes at the germinal vesicle (GV) stage or the prometaphase I/metaphase I stage. Unlike in mouse oocytes, where phosphorylated (p-) MK2 was localised at the minus end of spindle microtubules and close to the spindle poles, in porcine oocytes p-MK2 was concentrated at the spindle equator and localised at the plus end of spindle microtubules. Knockdown or inhibition of MK2 resulted in spindle defects: spindles were surrounded by irregular chromosome non-disjunction or by chromosomes detached from the spindles. MK2 regulated spindle organisation and chromosome alignment by connecting microtubules with kinetochores. In addition, unlike in mitotic cells and meiotic mouse oocytes, the MK2–p38 MAPK pathway may not play an important role during meiotic cell cycle in porcine oocytes. In conclusion, MK2 is an important regulator of porcine oocyte meiotic maturation. Additional keywords: chromosome, meiosis, spindle.
Received 15 April 2015, accepted 16 June 2015, published online 21 July 2015
Introduction Mammalian oocytes are arrested at the prophase of the first meiosis in follicles at various stages, and fully grown oocytes in mature follicles acquire the ability to resume meiosis and complete maturation. The resumption of the first meiosis occurs in mammalian oocyte as a result of interactions between the oocyte and surrounding cumulus and granulosa cells in preovulatory follicles (Sun et al. 1999; Kahyaoglu et al. 2014). Thereafter, the first meiotic division progresses through MI and the oocyte completes maturation, manifested by the formation of the first polar body, before it is arrested again at MII until fertilisation (Goncharov and Skoblina 2014). During oocyte maturation, normal spindle assembly is the guarantee of correct chromosome segregation, a determining factor in female fertility. Abnormal spindle organisation and chromosome segregation errors lead to aneuploidy in oocytes, directly resulting in embryo developmental arrest or spontaneous abortion (Farghaly et al. 2015). Therefore, clarifying the mechanism of aneuploidy *
in female oocytes is important in reducing the generation of aneuploidy and improving the quality of oocytes. The processes of follicular development, oocyte meiotic resumption and subsequent maturation and ovulation in mammals are controlled by FSH and LH. Stimulation of meiotic resumption by gonadotrophins occurs via their actions on the surrounding somatic cells rather than on the oocyte itself, because oocytes lack gonadotrophin receptors. Cumulus expansion occurs along with oocyte maturation after germinal vesicle (GV) breakdown (GVBD; Sun et al. 2009). In an in vitro culture system, cumulus expansion and metabolic uncoupling can be induced by FSH (Salustri and Siracusa 1983), increasing cAMP concentrations in cumulus cells, which is required for oocyte maturation. Normal cumulus expansion in vitro is conducive to an increased maturation rate and a higher rate of male pronucleus formation after sperm penetration. Extracellular signal-regulated kinase (ERK) 1/2 controls granulosa cell fate decisions at the differentiation stage.
These authors contributed equally to this work.
Journal compilation Ó CSIRO 2015
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(Tosca et al. 2006). Cumulus cells lacking ERK1/2 fail to respond to epidermal growth factor (EGF)-like factors induced by FSH (Kawashima et al. 2012). LH also induces expression of EGF-like factors that, by activating the EGF receptor, reninangiotensin system (RAS) and ERKs, regulate oocyte maturation (Fan and Sun 2004). It has been shown that ERK1/2 and p38 mitogen-activated protein kinase (MAPK), which belong to the MAPK superfamily, play a role in oocyte maturation (Villa-Diaz and Miyano 2004; Fan et al. 2009) and that p38 MAPK may be involved in FSH-induced meiotic resumption of oocytes and participate in cumulus expansion (Villa-Diaz and Miyano 2004). MAPK-activated protein kinase 2 (MK2) is a protein kinase activated downstream of p38 MAPK that modulates the cellular distribution of p38 MAPK in mitosis (Reinhardt et al. 2007). Activated p38 MAPK phosphorylates nuclear MK2 and forms a complex whereby an MK2 nuclear export signal is unmasked, resulting in its rapid export from the nucleus. The p38 MAPK/MK2 signalling complex is considered to be a general stress response pathway that is activated in response to a variety of extrinsic and intrinsic stimuli, including osmotic stress, heat shock and various toxins, in somatic cells and mouse oocytes (Ashraf et al. 2014) and participates in centrosome maturation and chromosome alignment. In a previous study, we reported that phosphorylated (p-) p38a and p-MK2 were partially colocalised at the spindle poles in MI and MII mouse oocytes (Ou et al. 2010). Furthermore, using co-immunoprecipitation analysis of endogenous proteins, we demonstrated a direct interaction between p38a and MK2 in oocytes. The finding that p-MK2 protein levels were reduced following downregulation of p-p38a in p38a-targeting morpholino (p38a-MO)-injected oocytes implies that p38a may regulate MK2 in mouse oocyte meiotic maturation (Ou et al. 2010). Porcine oocytes differ from mouse oocytes in several ways. First, porcine oocyte meiotic resumption and maturation rely, to a large degree, on the surrounding cumulus cells, whereas mouse oocytes can complete maturation in the absence of cumulus cells (Jin et al. 2006). Second, the spindle morphology of porcine oocytes is more like that of human oocytes, but clearly different from that of mouse oocytes. Both porcine and human oocytes have a small and short barrel-shaped spindle and congressed chromosomes, whereas mouse oocytes have a large and long spindle and less congressed chromosomes. Finally, mouse oocyte contains many asters, which are required for spindle organisation in oocytes and fertilised eggs in the absence of a sperm centrosome, whereas asters are not evident in porcine oocytes and the microtubules may emanate from chromosomes and spermatozoa contribute a centrosome for spindle assembly in only fertilised eggs (Sun et al. 2001). In the present study, we explored the localisation and function of MK2 during porcine oocyte meiotic maturation. Materials and methods Chemicals and antibodies Unless noted otherwise, all chemicals used in the present study were purchased from Sigma Chemical (St Louis, MO, USA). A MK2 specific inhibitor, CMPD1, (Calbiochem) and SB203580
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were prepared as stock solutions (25 mM) in dimethyl sulfoxide (DMSO) and were stored at 208C in a dark box until use. The final working solutions were prepared by dilution of the stock solutions in culture medium just before use (final concentration of DMSO was 0.005%). Rabbit polyclonal anti-p-MK2 (Thr180/ Tyr182) and rabbit polyclonal anti-p38 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA); mouse monoclonal anti-a-tubulin-fluorescein isothiocyanate (FITC), mouse monoclonal anti-g-tubulin and mouse monoclonal anti-Polo-like kinase (PLK1) antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA). Human anti-Crest antibodies were obtained from Fitzgerald Co. Cy5conjugated goat anti-rabbit IgG (H þ L) and Cy5-conjugated goat anti-Human IgG (H þ L) were purchased from Jackson Co. FITC-conjugated goat anti-rabbit IgG (H þ L), tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (H þ L) and TRITC-conjugated goat anti-mouse IgG (H þ L) were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Oocyte collection and culture Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 1 h in Guangzhou. Oocytes were aspirated from medium-sized antral follicles using an 18-gauge needle fixed to a 20-mL disposable syringe. After three washes in maturation medium, oocytes with compact cumulus cells and an evenly granulated ooplasm were selected for maturation culture. The maturation medium was improved TCM199 (GIBCO BRL, Grand Island, NY, USA) supplemented with 75 mg mL1 potassium penicillin G, 50 mg mL1 streptomycin sulfate, 0.57 mM cysteine, 0.5 mg mL1 FSH, 0.5 mg mL1 LH and 10 ng mL1 EGF. A group of 25 oocytes was cultured in a 100-mL drop of maturation medium (with or without drugs) covered by liquid paraffin oil for up to 44 h at 398C in an atmosphere of 5% CO2 and saturated humidity. Oocytes were denuded after 0, 24, 30, 36 and 44 h culture by treatment with maturation medium containing 300 IU mL1 hyaluronidase (Sigma) and repeated pipetting. The cumulus-free oocytes were then washed twice in TCM-199 and used for either immunostaining or western blot analysis. Drug treatment To explore the role of MK2 in cumulus expansion, oocyte meiotic resumption and maturation, cumulus–oocyte complexes (COCs) were cultured for 24 or 44 h in maturation medium containing different concentrations of CMPD1. To investigate the effects of MK2 on meiotic progression from prometaphase I (pro-MI) to MII, COCs were cultured for 24 h in drug-free medium and subsequently treated with different concentrations of CMPD1 for a further 20 h (10, 20 and 30 mM). For taxol treatment, 5 mM taxol in DMSO stock was diluted in M2 medium to give a final concentration of 10 mM and oocytes at different stages were incubated for 45 min. After treatment, oocytes were washed thoroughly and used for immunofluorescence staining. For nocodazole treatment, 10 mg mL1 nocodazole in DMSO stock was diluted in M16 medium to give
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a final concentration of 20 mg mL1 and oocytes were incubated for 10 min. Control oocytes were treated with the same concentration of DMSO in the medium. Antibody injection Approximately 5–10 pL anti-MK2 antibody was microinjected into the cytoplasm of a fully grown GV oocyte using an inverted microscope (Nikon Diaphot ECLIPSE TE300; Nikon UK, Kingston-upon-Thames, Surrey, UK) equipped with hydraulic three-dimensional micromanipulators (Narishige MM0-202N; Narishige, Sea Cliff, NY, USA). After microinjection, oocytes were cultured in fresh M2 medium (HEPES) under paraffin oil at 378C in an atmosphere of 5% CO2 in air. Control oocytes were microinjected with 5–10 pL rabbit IgG at the same concentration as the anti-MK2 antibody (5 mg mL1). Finally, spindle phenotypes and chromosomal alignment were examined by confocal microscopy. Immunofluorescence and confocal microscopy To stain proteins, COCs or oocytes were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for at least 30 min at room temperature. After removing the zona pellucida in acidic Tyrode’s solution (pH 2.5), oocytes were fixed with 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. Oocytes were permeabilised with 1% Triton X-100 overnight at 378C in an incubator, followed by blocking in PBS containing 1% bovine serum albumin (BSA) for 1 h and incubation overnight at 48C with rabbit anti-MK2 antibody diluted 1 : 100 with blocking solution. After three 5-min washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100, oocytes were labelled with the primary antibody at 48C overnight, then with the secondary antibody for 1 h at room temperature and processed for indirect immunofluorescence microscopy as described previously (Yuan et al. 2010). Following extensive washing in PBS containing 0.1% Tween 20, immunostained oocytes were mounted on glass slides and examined under a confocal laser-scanning microscope (LSM 510 META; Zeiss, Oberkochen, Germany). The following primary antibodies were used: rabbit anti-pMK2 (Thr334; Cell Signaling Technology; 1 : 100 dilution), mouse anti-Plk1 antibody (Cell Signaling Technology; 1 : 50 dilution), mouse anti-g-tubulin antibody (Sigma; 1 : 200 dilution), mouse anti-a-tubulin-FITC antibody (Sigma; 1 : 200 dilution) and human anti-Crest antibody (Fitzgerald; 1 : 50 dilution). Accordingly, the following secondary antibodies were used: FITC- or TRITC-conjugated-anti-mouse IgG (Zhong Shan Jin Qiao; 1 : 100 dilution), FITC- or TRITC-conjugated-anti-rabbit IgG (Zhong Shan Jin Qiao; 1 : 100 dilution); FITC-conjugated anti-human IgG (Zhong Shan Jin Qiao; 1 : 100), Cy5-conjugated anti-human IgG (Jackson ImmunoResearch; 1 : 200 dilution) and Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch; 1 : 200). Each experiment was repeated at least three times. For double staining of MK2 and g-tubulin, after MK2 staining with TRITC-conjugated goat anti-rabbit IgG as described above, oocytes were blocked in blocking solution for 1 h at room temperature, incubated for 2 h at room temperature with FITC-conjugated phalloidin (1 ng mL1 in blocking
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solution; Sigma), washed three times with PBS containing 0.1% Tween 20 for 5 min each time and then stained with propidium iodide (PI; 10 ng mL1 in PBS) for 10 min at room temperature before being mounted on slides. Non-specific staining was determined by substituting the primary antibodies with normal rabbit IgG. Cells were observed under a confocal laser-scanning microscope (LSM 510 META; Zeiss) within 5 min. Each experiment was repeated three times and at least 30 oocytes were examined each time. In addition, the same instrument settings were used for each replicate. Western blot analysis For detection of MK2, proteins from 300 oocytes at the appropriate stage of maturation were collected in sodium dodecyl sulfate (SDS) sample buffer and heated for 4 min at 1008C. Centrifuged samples (1000 rpm for 1 min at 48C) were frozen at208C until use. Total proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) with a 4% stacking gel and a 10% separating gel before being transferred to polyvinylidene difluoride membranes, which were blocked in Tris-buffered saline Tween-20 (TBST) buffer containing 5% skimmed milk and then incubated with polyclonal rabbit antiMK2 antibody (diluted 1 : 500 in TBST containing 0.5% skimmed milk) overnight at 48C. After three washes, membranes were incubated for 1 h at 378C with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted 1 : 1000 in TBST. After three washes, membranes were processed using the enhanced chemiluminescence (ECL) detection system (Amersham, Piscataway, NJ, USA). Equal protein loading was confirmed by detection of b-actin. All experiments were repeated at least three times. Evaluation of nuclear status Nuclear status was evaluated using orcein staining, as described previously (Xu et al. 2009) with minor modifications. Briefly, denuded oocytes were mounted on slides, fixed in acetic acid : ethanol (1 : 3, v/v) for at least 48 h, stained with 1% orcein and examined under a phase contrast microscope. Statistical analysis Data from at least three repeated experiments are expressed as the mean s.e.m. with the number of oocytes (n) given in parentheses. Data were analysed by ANOVA, followed by Fisher’s least significant difference test. P , 0.05 was considered to be significant. Results Localisation of p-MK2 in cumulus cells and inhibition of cumulus cell expansion by an MK2 inhibitor in porcine COCs Immunofluorescent staining was used to examine the subcellular localisation of p-MK2 in porcine cumulus cells. As shown in Fig. 1a, p-MK2 was expressed in the cytoplasm and membrane of cumulus cells. CMPD1, an MK2-specific inhibitor that is not competitive with ATP and specifically prevents the phosphorylation and activation of MK2 by p38a (Davidson et al. 2004), was used to investigate the effect of MK2 on
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cumulus expansion during porcine oocyte maturation. Porcine COCs were cultured for 44 h in TCM-199 with FSH, in the presence (10, 20, 30 mM) or absence of CMPD1. The extent of cumulus expansion was evaluated under a dissecting microscope after 44 h of in vitro maturation (IVM). In the control group, 85.7% of COCs (n ¼ 328) showed cumulus expansion. At 0, 10, 20 and 30 mM, CMPD1 dose-dependently inhibited cumulus expansion, with only 24.39% (n ¼ 328), 12.5% (n ¼ 240) and 3.3% (n ¼ 242) of COCs in these groups showing cumulus expansion, respectively (P , 0.001; Fig. 1b, c).
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Effects of MK2 inhibition on oocyte meiotic progression To investigate the effect of MK2 on porcine oocyte meiotic maturation, porcine COCs were cultured for up to 44 h in TCM199 with FSH in the presence (10, 20, 30 mM) or absence of CMPD1. Progression of oocyte meiosis was evaluated by conventional light microscopy of oocytes stained with orcein after 22 and 44 h of IVM. In the absence of CMPD1, 63.16% of oocytes (n ¼ 95) reached MII after 44 h of IVM (Fig. 1d); CMPD1 inhibited meiotic resumption. In the presence of 30 mM CMPD1, all oocytes were arrested at the GV stage, even after 44 h IVM. In the presence of 20 and 10 mM CMPD1, first polar body extrusion was observed in 6.25% (n ¼ 176) and 37.9% (n ¼ 132) oocytes, respectively (P , 0.001 vs control for both; Fig. 1d). Of the oocytes that passed through GVBD, after 44 h in culture in the presence of CMPD1 most oocytes became arrested at the pro-MI or MI stage.
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CMPD1 (μM) Fig. 1. Effects of mitogen-activated protein kinase-activated protein kinase 2 (MK2) inhibition (CMPD1) on cumulus expansion of intact porcine cumulus–oocyte complexes (COCs). (a) Confocal microcopy showing immunostaining of phosphorylated (p-) MK2 in cumulus cells (purple), a-tubulin (green) and DNA (red). Scale bar ¼ 10 mm. (b) Representative photomicrographs of intact porcine expanded COCs cultured for 24 h in the presence of FSH (100 ng mL1) and FSHstimulated COCs treated with CMPD1 (10, 20 or 30 mM). Percentage of (c) expanded COCs and (d) polar body extrusion (PBE) from control oocytes and oocytes treated with 10, 20 or 30 mM CMPD1. CMPD1 inhibited the extrusion of the first polar body from porcine COC. Values are presented as mean s.e.m. Columns with different letters differ significantly (P , 0.05).
Expression and subcellular localisation of p-MK2 during porcine oocyte meiotic maturation To examine the subcellular localisation of p-MK2 in porcine oocytes, samples were collected after COCs had been cultured for 0, 24, 30, 36 and 44 h, corresponding to the GV, pro-MI, MI, telophase I (TI) and MII stages, respectively. Porcine oocytes were then processed for immunofluorescent staining. As shown in Fig. 2a, by Pro-MI, when chromosomes were aggregated and had started to migrate to the equator of the spindle, p-MK2 was partly colocalised with microtubules and concentrated on the chromosomes. After 30 h in vitro culture, when most porcine oocytes had developed to the MI stage and chromosomes were aligned on the equatorial plate, p-MK2 was specifically concentrated in the spindle equatorial region, like a drum (Fig. 2a). At anaphase/telophase, when homologous chromosomes were segregated and microtubules were distributed between segregated chromosomes, p-MK2 was localised between chromosomes (Fig. 2a). At this time point, p-MK2 was partly colocalised with microtubules. At MII, p-MK2 reappeared in the equatorial region and its distribution was similar to that at the MI stage (Fig. 2a). In mouse oocytes, p-MK2 was localised at the minus ends of microtubules and close to the spindle poles; in contrast, in porcine oocytes, p-MK2 was concentrated at the spindle equator and localised at the plus ends of microtubules at the MI, first meiosis anaphase in porcine oocytes (AI) and MII stages (Yuan et al. 2010). To further clarify the correlation between p-MK2 and microtubule dynamics, oocytes were treated with taxol, a microtubulestabilising drug. When spindle organisation started after GVBD, microtubule fibres in taxol-treated oocytes became excessively
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Fig. 2. Spatial relationship between phosphorylated (p-) mitogen-activated protein kinase-activated protein kinase 2 (MK2), a-tubulin and chromosomes during meiotic maturation of pig oocytes. (a) Oocytes at various stages were double stained with antibodies against p-MK2 (red) and a-tubulin (green). DNA is stained blue. Pro-MI, prometaphase I; AI/TI, first anaphase and telophase. Scale bar ¼ 10 mm. (b) Oocytes at the MI and MII stages were treated with 10 mM taxol for 180 min and then double stained with antibodies against p-MK2 (red), a-tubulin (green) and chromatin (blue). Scale bar ¼ 10 mm. (c) Localisation of p-MK2 in porcine MI and MII oocytes treated with 20 mg mL1 nocodazole for 120 min. After treatment, oocytes were double stained with antibodies against p-MK2 (red), a-tubulin (green) and chromatin (blue). Yellow indicates overlapping of green and red stains. Scale bar ¼ 10 mm. Each sample was counterstained with Hoechst 33258 to visualise DNA.
polymerised, leading to significantly enlarged spindles, together with numerous asters in the cytoplasm (Fig. 2b). The asters in porcine oocytes after taxol treatment took the shape of bamboo leaves and p-MK2 was localised at the apex and possibly at the plus ends of microtubules (Fig. 2b). To confirm the association of MK2 with spindle fibres, oocytes matured for 32 and 43 h were exposed to 10 mM nocodazole to depolymerise the microtubules. In nocodazole-treated MI and MII oocytes, p-MK2 was localised at the chromosomes (Fig. 2c). These data show that the association of MK2 with the chromosomes does not rely on the presence of intact microtubules. Thus, MK2 may participate in the connection between kinetochores and microtubules, as well as homologous chromosome separation. Spatial relationship between p-MK2 and kinetochores, g-tubulin or PLK1 during meiotic maturation of porcine oocytes Because the spindle in porcine oocytes is much smaller than in mouse oocytes, the localisation of protein staining in the former is not as clear as in mouse oocytes. To further examine the precise localisation of p-MK2, oocytes were stained for both pMK2 and Crest at the MI and MII stages. At Pro-MI, homologous chromosomes are formed as bivalents and p-MK2 was localised between the interstitial homologous chromosome arms and in the centromere region, whereas Crest signals appeared as dots on the kinetochores (Fig. 3a). At the MII stage, p-MK2 was localised as dots between the Crest signals on sister chromosomes (i.e. the inner centromere region of sister chromatids).
During mouse oocyte meiosis, g-tubulin is localised at the centre of microtubule-organizing centers (MTOC) and the poles of spindles, and plays an important role in spindle and spindle pole assembly (Ou et al. 2010). However, in porcine oocytes, g-tubulin was localised at the spindles and colocalised with a-tubulin. p-MK2 signals partly overlapped with g-tubulin near the centrosome and equator region (Fig. 3b). At AI, p-MK2 was localised between the chromosomes and partly overlapped with g-tubulin, but was not entirely colocalised with g-tubulin (Fig. 3b). After taxol treatment, p-MK2 was further verified to partly colocalise with g-tubulin on abnormal large spindles (Fig. 3c). These results show that MK2 is localised near the spindle equator and chromosome centrosome, and may be a plus-end microtubule protein. In mouse oocytes, PLK1 was localised at pole of the spindles and centromeres (Ou et al. 2010). However, in porcine oocytes PLK1 was localised across the whole spindle and centromere (Fig. 4a). MK2 was partly colocalised with PLK1 at the microtubules; the staining patterns for MK2 and PLK1 at the spindle were similar, but the area of MK2 staining was smaller than that of PLK1 (Fig. 4a). As shown in Fig. 4a, when p-MK2 was localised to the inner chromosome arms, PLK1 was colocalised with Crest at pro-MI and MI. At the same time, when taxol was used to decrease the tension between microtubules and chromosomes, PLK1 and p-MK2 were clearly localised in the centromere region (Fig. 4b). Further, the two proteins were colocalised partly in the centromere region, but the staining area of PLK1 was larger than that of p-MK2 (Fig. 4b). Thus, p-MK2 may contribute
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Fig. 3. Spatial relationship between phosphorylated (p-) mitogen-activated protein kinaseactivated protein kinase 2 (MK2) and Crest or g-tubulin during meiotic maturation of pig oocytes. (a) Oocytes at the prometaphase I (Pro-MI) and MII stages were stained with antibodies against p-MK2 (red), Crest (green) and DNA (blue). (b) Oocytes at various stages were fixed and stained for g-tubulin (green), p-MK2 (red) and DNA (blue), as visualised with Hoechst 33258 staining. Scale bar ¼ 10 mm. (c) Spatial relationship between p-MK2 and g-tubulin during meiotic maturation of pig oocytes treated with taxol. Green, a-tubulin; purple, p-MK2; red, g-tubulin; blue, chromatin. Scale bar ¼ 10 mm.
to the connection between microtubules and kinetochores. The special localisation indicates that p-MK2 may play an important role in spindle assembly and chromosome separation. Effects of MK2 on Plk1 localisation and p38 MAPK inhibition on MK2 localisation We next examined the localisation of centrosome-associated protein kinase Plk1 in CMPD1-treated oocytes. As shown in Fig. 4a, b, the centrosome-associated protein Plk1 was localised at the kinetochores and spindle in control oocytes, but PLK1 was dissociated from kinetochores and was scattered throughout the
cytoplasm in MK2-depleted oocytes (Fig. 4c). There was a significant decrease in the PLK1 signal in abnormal spindles (Fig. 4c). After p-p38 MAPK inhibition with SB203580 (0.3–0.5 mM for 30 min), there was no change in the localisation of MK2 in oocytes. Effects of MK2 inhibition on spindle organisation and chromosomes alignment In the present study, porcine oocytes were treated with 10 mM CMPD1, an MK2 inhibitor, to observe the effects of MK2 inhibition on spindle organisation and chromosome alignment.
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Fig. 4. Spatial relationship between phosphorylated (p-) mitogen-activated protein kinaseactivated protein kinase 2 (MK2) and Polo-like kinase (PLK1) during meiotic maturation of porcine oocytes. (a) Oocytes at the prometaphase I (Pro-MI), MI and MII stages were stained with antibodies against p-MK2 (red), PLK1 (green) and DNA (blue). (b) Spatial relationship between p-MK2 and PLK1 during meiotic maturation of pig oocytes treated with taxol (5 mM). Green, a-tubulin; purple, p-MK2; red, PLK1; blue, chromatin. Scale bar ¼ 10 mm. (c) PLK1 was dispersed from the chromosomes with CMPD1 treatment of porcine oocytes. Green, a-tubulin; purple, PLK1; red, DNA. Scale bar ¼ 10 mm.
As shown in Fig. 5a, the phosphorylation of MK2 was markedly reduced after CMPD1 treatment compared with control, indicating that CMPD1 successfully prevented phosphorylation of MK2. As stated above, after MK2 inhibition most oocytes were arrested at the Pro-MI stage. Downregulation of p-MK2 resulted in significant defects in spindle formation and chromosome alignment. In the control group, MI and MII oocytes that exhibited normal looking spindles and regular chromosomes (Fig. 5b), whereas most CMPD1-treated oocytes (Fig. 5b, c) cultured for 44 h had abnormal spindles. Some oocytes had irregularly scattered chromosomes with normal spindles, some had lagging chromosomes or scattered chromosomes surrounding shrinking or collapsed spindles and some had monopolar spindles surrounded by homologous chromosomes (Fig. 5b). Chromosomal p-MK2 staining was still observed along the centromere and arm regions of homologous chromosomes, whereas p-MK2 nearly disappeared from abnormal spindles (Fig. 5b). The rate of abnormal spindle formation in the CMPD1-treated group was
67.78% (n ¼ 90; Fig. 5c), which was considerably higher than that of the control DMSO-treated group 18.35% (n ¼ 109; P , 0.001). The incidence of misaligned chromosomes in the CMPD1-treated group was 63.33% (n ¼ 90; P , 0.001), much higher than in the control group (21.10%; n ¼ 109; Fig. 5d). These results suggest that MK2 is required for regulation of spindle organisation and chromosome alignment. Effects of anti-MK2 antibody on spindle organisation, chromosomes alignment and oocyte maturation To further elucidate the role of MK2 in porcine oocyte meiosis, we attempted to disrupt the functional roles of MK2 by antibody microinjection. Polar body extrusion (PBE) in antibody-injected oocytes was considerably lower than in the control DMSOtreated group (25.0% (n ¼ 118) vs 43.35% (n ¼ 200), respectively; P , 0.001; Fig. 6a). In the control group injected with rabbit IgG, spindle organisation was normal in most oocytes,
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Fig. 5. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) inhibition by CMPD1 (10 mM) leads to defective spindles and misaligned chromosomes. (a) There was a marked decrease in phosphorylated (p-) MK2 protein expression after CMPD1 treatment compared with control. (b) Spindle morphology and chromosome alignment after CMPD1 treatment of porcine oocytes. In the control group (n ¼ 109), most oocytes showed normal spindle morphology and chromosome alignment, whereas in the treatment group (n ¼ 90), most oocytes showed severely defective spindle and chromosome alignment. Green, a-tubulin; red, DNA; purple, MK2. Scale bar ¼ 10 mm. (c, d) Percentage of oocytes with abnormal spindles (c) and misaligned chromosomes (d) in the control and CMPD1 treatment groups. Values are presented as mean s.e.m. *P , 0.05 compared with the control group.
with only 21.0% (n ¼ 200) being morphologically abnormal (Fig. 6b). However, various spindle abnormalities and defects were observed in a considerable number of oocytes after antibody injection (66.9%; n ¼ 118), similar to findings after CMPD1 treatment, including irregularly dispersed spindle microtubules, monopolar spindles, round shape spindle, multiple spindle apparatuses and collapsed spindles (Fig. 6b). In addition, severe defects in chromosome alignment were seen in oocytes injected with the anti-MK2 antibody, including lagging chromosomes and irregularly scattered chromosomes (Fig. 6b). The incidence of abnormal spindles and misaligned chromosomes in the antibody-injected group was 66.9% (n ¼ 118) and 70.34% (n ¼ 118), respectively, much higher than corresponding values in the control group (23.5% (n ¼ 200) and 21.0% (n ¼ 200), respectively; P , 0.001; Fig. 6c, d). Discussion In the present study we showed that MK2 has a unique localisation pattern in porcine oocytes and that it is a critical regulator
of meiotic cell cycle progression. MK2 is localised in the regions where cohesion is found in porcine oocytes, as in mouse oocytes, but unlike mouse oocytes, MK2 localises at the plus end of spindle microtubules in porcine oocytes. In particular, MK2 regulates spindle assembly and chromosome alignment, and inhibition of MK2 impairs the connection of the kinetochore with microtubules and chromosome separation in meiotic oocytes by regulating the localisation of PLK1 on the kinetochores and microtubules. Unlike mouse oocytes, which can complete meiotic maturation in vitro in the absence of cumulus cells, the meiotic maturation of porcine oocytes relies on the function of these surrounding somatic cells. Treatment of porcine COCs with the MK2 inhibitor CMPD1 caused defects in cumulus cell expansion and oocyte meiotic maturation. In addition, microinjection of anti-MK2 antibody to deplete MK2 directly in oocytes decreased extrusion of the first polar body and caused defective spindle organisation and chromosome alignment. These results demonstrate that MK2 inhibition prevented porcine oocyte maturation, and this may be caused by the disruption of both cumulus cell function and the oocyte itself.
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Fig. 6. Injection of an anti-phosphorylated (p-) mitogen-activated protein kinase-activated protein kinase 2 (MK2) antibody led to defective spindles and misaligned chromosomes and affected extrusion of the first polar body. (a) Percentage polar body extrusion (PBE) in the control (IgG injected; n ¼ 200) and p-MK2 antibody-injected (n ¼ 118) groups. (b) Spindle morphology and chromosome alignment after microinjection of the anti-p-MK2 antibody and IgG in porcine oocytes. In the control group (n ¼ 200), most oocytes showed normal spindle morphology and chromosome alignment, whereas in the antibody injection group (n ¼ 118), most oocytes showed severely defective spindle and chromosome alignment. Green, a-tubulin; red, DNA. Scale bar ¼ 10 mm. (c, d) Percentage of oocytes with abnormal spindles (c) and misaligned chromosomes (d) in the control and antibody-injected groups. Values are presented as mean s.e.m. *P , 0.05 compared with the control group.
In somatic cells, MK2 is the substrate of p38a and exists in a stable complex with its upstream substrate (ter Haar et al. 2007). p38a phosphorylates and activates MK2, and the p38a/MK2 signalling complex is thought to participate in centrosome maturation and chromosome alignment (Ben-Levy et al. 1998). In a previous study, we showed that the p38a/MK2 signalling pathway may play an important role in mouse oocyte meiosis (Ou et al. 2010). However, it is not clear whether the
p38a/MK2 pathway plays a role in spindle assembly in porcine oocyte meiosis. In mitosis, p-p38a is colocalised with pMK2 at spindle poles (Tang et al. 2008). However, it was observed that p-p38a and pMK2 were only partially colocalised at the spindle poles of MI and MII mouse oocytes. MK2 localises at the meiotic bipolar spindle microtubule minus ends and in the regions where cohesion is found. p38a phosphorylates and activates MK2 and affects the localisation of MK2 in mouse
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oocytes. Thus, MK2 is involved in spindle assembly in mouse oocyte maturation and plays an important role in maintaining spindle stability and chromosome alignment (Yuan et al. 2010). Unexpectedly, in the present study, p-MK2 was localised at the plus ends of microtubules and near the equatorial region of the spindle in porcine oocytes. The p-MK2 was partially colocalised with microtubules and concentrated on the chromosomes, but the extent of p-MK2 staining on the chromosomes was less than that on microtubules. After 30 h culture in vitro, most porcine oocytes developed to the MI stage, chromosomes were aligned on the equatorial plate and p-MK2 was specifically concentrated in the spindle equatorial region, like a drum. At anaphase/ telophase, homologous chromosomes were segregated and microtubules were distributed between segregated chromosomes and p-MK2 was localised between chromosomes. At this time, p-MK2 was partly colocalised with microtubules and the level of p-MK2 staining between chromosomes was less than that on microtubules. At MII, p-MK2 reappeared in the equatorial region and its localisation was similar to that seen at the MI stage in porcine oocytes. Unlike in mouse oocytes, in which pMK2 was localised at the minus end of spindle microtubules and close to the spindle poles (Yuan et al. 2010), p-MK2 was concentrated at the spindle equator and localised at the plus end of microtubules at the MI, AI and MII stages in porcine oocytes. Microtubule fibres in taxol-treated oocytes became excessively polymerised, leading to significantly enlarged spindles, together with numerous asters in the cytoplasm. The asters in porcine oocytes after taxol treatment took the shape of bamboo leaves and p-MK2 was localised at the plus end of microtubules, indicating that MK2 is a plus-end microtubuleassociated protein. Unlike in mouse oocytes, there are no p38 MAPK signals on spindles (Villa-Diaz and Miyano 2004) and p38 MAPK did not affect the localisation of p-MK2 in porcine oocytes. Thus, p38 MAPK/MK2 pathway may not play functions in porcine oocytes. The MK2 inhibitor prevented porcine oocytes from developing GVBD in a dose-dependent manner, and this may have been caused by either the disruption of cumulus cell function, as indicated by cumulus expansion failure, by actions on the oocyte itself or both. After MK2 inhibition, oocytes showed abnormally organised spindles, such as a collapsed spindle surrounded by chromosomes or spherical monopolar spindles, as well as abnormal chromosome alignment. Disorganised spindles and misaligned chromosomes blocked the meiotic progression and anaphase transition, and thus extrusion of the first polar body. This indicates that MK2 may be involved in connecting the microtubules with kinetochores, and microtubule capturing of chromosomes. The results provide support for p-MK2 participating in the meiotic cell cycle, especially spindle assembly and chromosome alignment, in porcine oocytes, but the mechanisms involved may differ from those in mouse oocytes. Although the functions underlying the actions of MK2 in mitotic and meiotic spindles may vary between different cell types and different species, a conserved role for MK2 in stabilising meiotic chromosome alignment is observed. MK2 disappeared from chromosome arms and was concentrated as dots on the cohesion of chromosomes, colocalising with PLK1. Kinetochores can connect with microtubules and capture
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chromosome through microtubules, which plays an important role in spindle organisation and chromosome segregation (Sun et al. 2001; Deng et al. 2009; Kitajima et al. 2011). The special location pattern of MK2 on chromosomes is the almost same as that of meiotic recombination protein 8 (REC8) (Garcia-Cruz et al. 2010). At anaphase of the first meiosis, meiotic sister chromatids lose cohesin from their arms, as indicated by REC8 removal (Lee et al. 2006). Cohesin plays an important role in meiotic sister chromosome connection. Phosphorylation of cohesin depends on Plk1 (Sumara et al. 2002). MK2 has been proven to directly phosphorylate Ser326 of Plk1(Tang et al. 2008). In the present study, MK2 inhibition resulted in the dislocation of PLK1 from porcine oocytes. Thus, MK2 may participate in the regulation of chromosome alignment and segregation by affecting Plk1 in porcine oocytes. In conclusion, MK2 has a unique distribution pattern in porcine oocytes. It affects not only cumulus expansion, but also oocyte meiotic cell cycle progression. MK2 regulates spindle organisation and chromosome alignment by connecting microtubules with kinetochores in a different way from that in mouse oocyte. Unlike in mitotic cells and meiotic mouse oocytes, the MK2/p38 MAPK pathway may not play an important role in this process. Acknowledgement This study was supported by the National Natural Science Foundation of China (No. 81200421) to X-HO.
References Ashraf, M. I., Ebner, M., Wallner, C., Haller, M., Khalid, S., Schwelberger, H., Koziel, K., Enthammer, M., Hermann, M., Sickinger, S., Soleiman, A., Steger, C., Vallant, S., Sucher, R., Brandacher, G., Santer, P., Dragun, D., and Troppmair, J. (2014). A p38 MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury. Cell Commun. Signal. 12, 6. doi:10.1186/1478-811X-12-6 Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H. F., and Marshall, C. J. (1998). Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr. Biol. 8, 1049–1057. doi:10.1016/S0960-9822(98)70442-7 Davidson, W., Frego, L., Peet, G. W., Kroe, R. R., Labadia, M. E., Lukas, S. M., Snow, R. J., Jakes, S., Grygon, C. A., Pargellis, C., and Werneburg, B. G. (2004). Discovery and characterization of a substrate selective p38alpha inhibitor. Biochemistry 43, 11 658–11 671. doi:10.1021/BI0495073 Deng, M., Gao, J., Suraneni, P., and Li, R. (2009). Kinetochore-independent chromosome poleward movement during anaphase of meiosis II in mouse eggs. PLoS One 4, e5249. doi:10.1371/JOURNAL.PONE. 0005249 Fan, H. Y., and Sun, Q. Y. (2004). Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biol. Reprod. 70, 535–547. doi:10.1095/BIOLREPROD.103.022830 Fan, H. Y., Liu, Z., Shimada, M., Sterneck, E., Johnson, P. F., Hedrick, S. M., and Richards, J. S. (2009). MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science 324, 938–941. doi:10.1126/ SCIENCE.1171396 Farghaly, T., Khalifa, E., Mostafa, S., Hussein, M., Bedaiwy, M., and Ahmady, A. (2015). The effect of temporary meiotic attenuation on the in vitro maturation outcome of bovine oocytes. In Vitro Cell. Dev. Biol. Anim. . doi:10.1007/S11626-015-9878-Y Garcia-Cruz, R., Brieno, M. A., Roig, I., Grossmann, M., Velilla, E., Pujol, A., Cabero, L., Pessarrodona, A., Barbero, J. L., and Garcia Caldes, M. (2010). Dynamics of cohesin proteins REC8, STAG3, SMC1 beta and
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www.publish.csiro.au/journals/rfd
Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD15150
Mitogen-activated protein kinase-activated protein kinase 2 is a critical regulator of pig oocyte meiotic maturation Xiang-Hong Ou A,D,*, Sen Li A,*, Bao-Zeng Xu B,C,*, Lei-Ning Chen A, Man-Xi Jiang A, Shao-Qin Chen A and Nan-Qiao Chen A A
Reproductive Medicine Center, The 2nd People’s Hospital of Guangdong Province, #1 Shiliuguang RD, Haizhou, Guangzhou, 510317, China. B Institute of Special Wild Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Jilin, China. C Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. D Corresponding author. Email: [email protected]
Abstract. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple cellular processes. In the present study, we showed that MK2 affected not only cumulus expansion, but also the oocyte meiotic cell cycle in porcine oocytes. Inhibition of MK2 arrested oocytes at the germinal vesicle (GV) stage or the prometaphase I/metaphase I stage. Unlike in mouse oocytes, where phosphorylated (p-) MK2 was localised at the minus end of spindle microtubules and close to the spindle poles, in porcine oocytes p-MK2 was concentrated at the spindle equator and localised at the plus end of spindle microtubules. Knockdown or inhibition of MK2 resulted in spindle defects: spindles were surrounded by irregular chromosome non-disjunction or by chromosomes detached from the spindles. MK2 regulated spindle organisation and chromosome alignment by connecting microtubules with kinetochores. In addition, unlike in mitotic cells and meiotic mouse oocytes, the MK2–p38 MAPK pathway may not play an important role during meiotic cell cycle in porcine oocytes. In conclusion, MK2 is an important regulator of porcine oocyte meiotic maturation. Additional keywords: chromosome, meiosis, spindle.
Received 15 April 2015, accepted 16 June 2015, published online 21 July 2015
Introduction Mammalian oocytes are arrested at the prophase of the first meiosis in follicles at various stages, and fully grown oocytes in mature follicles acquire the ability to resume meiosis and complete maturation. The resumption of the first meiosis occurs in mammalian oocyte as a result of interactions between the oocyte and surrounding cumulus and granulosa cells in preovulatory follicles (Sun et al. 1999; Kahyaoglu et al. 2014). Thereafter, the first meiotic division progresses through MI and the oocyte completes maturation, manifested by the formation of the first polar body, before it is arrested again at MII until fertilisation (Goncharov and Skoblina 2014). During oocyte maturation, normal spindle assembly is the guarantee of correct chromosome segregation, a determining factor in female fertility. Abnormal spindle organisation and chromosome segregation errors lead to aneuploidy in oocytes, directly resulting in embryo developmental arrest or spontaneous abortion (Farghaly et al. 2015). Therefore, clarifying the mechanism of aneuploidy *
in female oocytes is important in reducing the generation of aneuploidy and improving the quality of oocytes. The processes of follicular development, oocyte meiotic resumption and subsequent maturation and ovulation in mammals are controlled by FSH and LH. Stimulation of meiotic resumption by gonadotrophins occurs via their actions on the surrounding somatic cells rather than on the oocyte itself, because oocytes lack gonadotrophin receptors. Cumulus expansion occurs along with oocyte maturation after germinal vesicle (GV) breakdown (GVBD; Sun et al. 2009). In an in vitro culture system, cumulus expansion and metabolic uncoupling can be induced by FSH (Salustri and Siracusa 1983), increasing cAMP concentrations in cumulus cells, which is required for oocyte maturation. Normal cumulus expansion in vitro is conducive to an increased maturation rate and a higher rate of male pronucleus formation after sperm penetration. Extracellular signal-regulated kinase (ERK) 1/2 controls granulosa cell fate decisions at the differentiation stage.
These authors contributed equally to this work.
Journal compilation Ó CSIRO 2015
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(Tosca et al. 2006). Cumulus cells lacking ERK1/2 fail to respond to epidermal growth factor (EGF)-like factors induced by FSH (Kawashima et al. 2012). LH also induces expression of EGF-like factors that, by activating the EGF receptor, reninangiotensin system (RAS) and ERKs, regulate oocyte maturation (Fan and Sun 2004). It has been shown that ERK1/2 and p38 mitogen-activated protein kinase (MAPK), which belong to the MAPK superfamily, play a role in oocyte maturation (Villa-Diaz and Miyano 2004; Fan et al. 2009) and that p38 MAPK may be involved in FSH-induced meiotic resumption of oocytes and participate in cumulus expansion (Villa-Diaz and Miyano 2004). MAPK-activated protein kinase 2 (MK2) is a protein kinase activated downstream of p38 MAPK that modulates the cellular distribution of p38 MAPK in mitosis (Reinhardt et al. 2007). Activated p38 MAPK phosphorylates nuclear MK2 and forms a complex whereby an MK2 nuclear export signal is unmasked, resulting in its rapid export from the nucleus. The p38 MAPK/MK2 signalling complex is considered to be a general stress response pathway that is activated in response to a variety of extrinsic and intrinsic stimuli, including osmotic stress, heat shock and various toxins, in somatic cells and mouse oocytes (Ashraf et al. 2014) and participates in centrosome maturation and chromosome alignment. In a previous study, we reported that phosphorylated (p-) p38a and p-MK2 were partially colocalised at the spindle poles in MI and MII mouse oocytes (Ou et al. 2010). Furthermore, using co-immunoprecipitation analysis of endogenous proteins, we demonstrated a direct interaction between p38a and MK2 in oocytes. The finding that p-MK2 protein levels were reduced following downregulation of p-p38a in p38a-targeting morpholino (p38a-MO)-injected oocytes implies that p38a may regulate MK2 in mouse oocyte meiotic maturation (Ou et al. 2010). Porcine oocytes differ from mouse oocytes in several ways. First, porcine oocyte meiotic resumption and maturation rely, to a large degree, on the surrounding cumulus cells, whereas mouse oocytes can complete maturation in the absence of cumulus cells (Jin et al. 2006). Second, the spindle morphology of porcine oocytes is more like that of human oocytes, but clearly different from that of mouse oocytes. Both porcine and human oocytes have a small and short barrel-shaped spindle and congressed chromosomes, whereas mouse oocytes have a large and long spindle and less congressed chromosomes. Finally, mouse oocyte contains many asters, which are required for spindle organisation in oocytes and fertilised eggs in the absence of a sperm centrosome, whereas asters are not evident in porcine oocytes and the microtubules may emanate from chromosomes and spermatozoa contribute a centrosome for spindle assembly in only fertilised eggs (Sun et al. 2001). In the present study, we explored the localisation and function of MK2 during porcine oocyte meiotic maturation. Materials and methods Chemicals and antibodies Unless noted otherwise, all chemicals used in the present study were purchased from Sigma Chemical (St Louis, MO, USA). A MK2 specific inhibitor, CMPD1, (Calbiochem) and SB203580
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were prepared as stock solutions (25 mM) in dimethyl sulfoxide (DMSO) and were stored at 208C in a dark box until use. The final working solutions were prepared by dilution of the stock solutions in culture medium just before use (final concentration of DMSO was 0.005%). Rabbit polyclonal anti-p-MK2 (Thr180/ Tyr182) and rabbit polyclonal anti-p38 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA); mouse monoclonal anti-a-tubulin-fluorescein isothiocyanate (FITC), mouse monoclonal anti-g-tubulin and mouse monoclonal anti-Polo-like kinase (PLK1) antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA). Human anti-Crest antibodies were obtained from Fitzgerald Co. Cy5conjugated goat anti-rabbit IgG (H þ L) and Cy5-conjugated goat anti-Human IgG (H þ L) were purchased from Jackson Co. FITC-conjugated goat anti-rabbit IgG (H þ L), tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (H þ L) and TRITC-conjugated goat anti-mouse IgG (H þ L) were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Oocyte collection and culture Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 1 h in Guangzhou. Oocytes were aspirated from medium-sized antral follicles using an 18-gauge needle fixed to a 20-mL disposable syringe. After three washes in maturation medium, oocytes with compact cumulus cells and an evenly granulated ooplasm were selected for maturation culture. The maturation medium was improved TCM199 (GIBCO BRL, Grand Island, NY, USA) supplemented with 75 mg mL1 potassium penicillin G, 50 mg mL1 streptomycin sulfate, 0.57 mM cysteine, 0.5 mg mL1 FSH, 0.5 mg mL1 LH and 10 ng mL1 EGF. A group of 25 oocytes was cultured in a 100-mL drop of maturation medium (with or without drugs) covered by liquid paraffin oil for up to 44 h at 398C in an atmosphere of 5% CO2 and saturated humidity. Oocytes were denuded after 0, 24, 30, 36 and 44 h culture by treatment with maturation medium containing 300 IU mL1 hyaluronidase (Sigma) and repeated pipetting. The cumulus-free oocytes were then washed twice in TCM-199 and used for either immunostaining or western blot analysis. Drug treatment To explore the role of MK2 in cumulus expansion, oocyte meiotic resumption and maturation, cumulus–oocyte complexes (COCs) were cultured for 24 or 44 h in maturation medium containing different concentrations of CMPD1. To investigate the effects of MK2 on meiotic progression from prometaphase I (pro-MI) to MII, COCs were cultured for 24 h in drug-free medium and subsequently treated with different concentrations of CMPD1 for a further 20 h (10, 20 and 30 mM). For taxol treatment, 5 mM taxol in DMSO stock was diluted in M2 medium to give a final concentration of 10 mM and oocytes at different stages were incubated for 45 min. After treatment, oocytes were washed thoroughly and used for immunofluorescence staining. For nocodazole treatment, 10 mg mL1 nocodazole in DMSO stock was diluted in M16 medium to give
MK2 regulates pig oocyte maturation
a final concentration of 20 mg mL1 and oocytes were incubated for 10 min. Control oocytes were treated with the same concentration of DMSO in the medium. Antibody injection Approximately 5–10 pL anti-MK2 antibody was microinjected into the cytoplasm of a fully grown GV oocyte using an inverted microscope (Nikon Diaphot ECLIPSE TE300; Nikon UK, Kingston-upon-Thames, Surrey, UK) equipped with hydraulic three-dimensional micromanipulators (Narishige MM0-202N; Narishige, Sea Cliff, NY, USA). After microinjection, oocytes were cultured in fresh M2 medium (HEPES) under paraffin oil at 378C in an atmosphere of 5% CO2 in air. Control oocytes were microinjected with 5–10 pL rabbit IgG at the same concentration as the anti-MK2 antibody (5 mg mL1). Finally, spindle phenotypes and chromosomal alignment were examined by confocal microscopy. Immunofluorescence and confocal microscopy To stain proteins, COCs or oocytes were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for at least 30 min at room temperature. After removing the zona pellucida in acidic Tyrode’s solution (pH 2.5), oocytes were fixed with 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. Oocytes were permeabilised with 1% Triton X-100 overnight at 378C in an incubator, followed by blocking in PBS containing 1% bovine serum albumin (BSA) for 1 h and incubation overnight at 48C with rabbit anti-MK2 antibody diluted 1 : 100 with blocking solution. After three 5-min washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100, oocytes were labelled with the primary antibody at 48C overnight, then with the secondary antibody for 1 h at room temperature and processed for indirect immunofluorescence microscopy as described previously (Yuan et al. 2010). Following extensive washing in PBS containing 0.1% Tween 20, immunostained oocytes were mounted on glass slides and examined under a confocal laser-scanning microscope (LSM 510 META; Zeiss, Oberkochen, Germany). The following primary antibodies were used: rabbit anti-pMK2 (Thr334; Cell Signaling Technology; 1 : 100 dilution), mouse anti-Plk1 antibody (Cell Signaling Technology; 1 : 50 dilution), mouse anti-g-tubulin antibody (Sigma; 1 : 200 dilution), mouse anti-a-tubulin-FITC antibody (Sigma; 1 : 200 dilution) and human anti-Crest antibody (Fitzgerald; 1 : 50 dilution). Accordingly, the following secondary antibodies were used: FITC- or TRITC-conjugated-anti-mouse IgG (Zhong Shan Jin Qiao; 1 : 100 dilution), FITC- or TRITC-conjugated-anti-rabbit IgG (Zhong Shan Jin Qiao; 1 : 100 dilution); FITC-conjugated anti-human IgG (Zhong Shan Jin Qiao; 1 : 100), Cy5-conjugated anti-human IgG (Jackson ImmunoResearch; 1 : 200 dilution) and Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch; 1 : 200). Each experiment was repeated at least three times. For double staining of MK2 and g-tubulin, after MK2 staining with TRITC-conjugated goat anti-rabbit IgG as described above, oocytes were blocked in blocking solution for 1 h at room temperature, incubated for 2 h at room temperature with FITC-conjugated phalloidin (1 ng mL1 in blocking
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solution; Sigma), washed three times with PBS containing 0.1% Tween 20 for 5 min each time and then stained with propidium iodide (PI; 10 ng mL1 in PBS) for 10 min at room temperature before being mounted on slides. Non-specific staining was determined by substituting the primary antibodies with normal rabbit IgG. Cells were observed under a confocal laser-scanning microscope (LSM 510 META; Zeiss) within 5 min. Each experiment was repeated three times and at least 30 oocytes were examined each time. In addition, the same instrument settings were used for each replicate. Western blot analysis For detection of MK2, proteins from 300 oocytes at the appropriate stage of maturation were collected in sodium dodecyl sulfate (SDS) sample buffer and heated for 4 min at 1008C. Centrifuged samples (1000 rpm for 1 min at 48C) were frozen at208C until use. Total proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) with a 4% stacking gel and a 10% separating gel before being transferred to polyvinylidene difluoride membranes, which were blocked in Tris-buffered saline Tween-20 (TBST) buffer containing 5% skimmed milk and then incubated with polyclonal rabbit antiMK2 antibody (diluted 1 : 500 in TBST containing 0.5% skimmed milk) overnight at 48C. After three washes, membranes were incubated for 1 h at 378C with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted 1 : 1000 in TBST. After three washes, membranes were processed using the enhanced chemiluminescence (ECL) detection system (Amersham, Piscataway, NJ, USA). Equal protein loading was confirmed by detection of b-actin. All experiments were repeated at least three times. Evaluation of nuclear status Nuclear status was evaluated using orcein staining, as described previously (Xu et al. 2009) with minor modifications. Briefly, denuded oocytes were mounted on slides, fixed in acetic acid : ethanol (1 : 3, v/v) for at least 48 h, stained with 1% orcein and examined under a phase contrast microscope. Statistical analysis Data from at least three repeated experiments are expressed as the mean s.e.m. with the number of oocytes (n) given in parentheses. Data were analysed by ANOVA, followed by Fisher’s least significant difference test. P , 0.05 was considered to be significant. Results Localisation of p-MK2 in cumulus cells and inhibition of cumulus cell expansion by an MK2 inhibitor in porcine COCs Immunofluorescent staining was used to examine the subcellular localisation of p-MK2 in porcine cumulus cells. As shown in Fig. 1a, p-MK2 was expressed in the cytoplasm and membrane of cumulus cells. CMPD1, an MK2-specific inhibitor that is not competitive with ATP and specifically prevents the phosphorylation and activation of MK2 by p38a (Davidson et al. 2004), was used to investigate the effect of MK2 on
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cumulus expansion during porcine oocyte maturation. Porcine COCs were cultured for 44 h in TCM-199 with FSH, in the presence (10, 20, 30 mM) or absence of CMPD1. The extent of cumulus expansion was evaluated under a dissecting microscope after 44 h of in vitro maturation (IVM). In the control group, 85.7% of COCs (n ¼ 328) showed cumulus expansion. At 0, 10, 20 and 30 mM, CMPD1 dose-dependently inhibited cumulus expansion, with only 24.39% (n ¼ 328), 12.5% (n ¼ 240) and 3.3% (n ¼ 242) of COCs in these groups showing cumulus expansion, respectively (P , 0.001; Fig. 1b, c).
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Effects of MK2 inhibition on oocyte meiotic progression To investigate the effect of MK2 on porcine oocyte meiotic maturation, porcine COCs were cultured for up to 44 h in TCM199 with FSH in the presence (10, 20, 30 mM) or absence of CMPD1. Progression of oocyte meiosis was evaluated by conventional light microscopy of oocytes stained with orcein after 22 and 44 h of IVM. In the absence of CMPD1, 63.16% of oocytes (n ¼ 95) reached MII after 44 h of IVM (Fig. 1d); CMPD1 inhibited meiotic resumption. In the presence of 30 mM CMPD1, all oocytes were arrested at the GV stage, even after 44 h IVM. In the presence of 20 and 10 mM CMPD1, first polar body extrusion was observed in 6.25% (n ¼ 176) and 37.9% (n ¼ 132) oocytes, respectively (P , 0.001 vs control for both; Fig. 1d). Of the oocytes that passed through GVBD, after 44 h in culture in the presence of CMPD1 most oocytes became arrested at the pro-MI or MI stage.
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CMPD1 (μM) Fig. 1. Effects of mitogen-activated protein kinase-activated protein kinase 2 (MK2) inhibition (CMPD1) on cumulus expansion of intact porcine cumulus–oocyte complexes (COCs). (a) Confocal microcopy showing immunostaining of phosphorylated (p-) MK2 in cumulus cells (purple), a-tubulin (green) and DNA (red). Scale bar ¼ 10 mm. (b) Representative photomicrographs of intact porcine expanded COCs cultured for 24 h in the presence of FSH (100 ng mL1) and FSHstimulated COCs treated with CMPD1 (10, 20 or 30 mM). Percentage of (c) expanded COCs and (d) polar body extrusion (PBE) from control oocytes and oocytes treated with 10, 20 or 30 mM CMPD1. CMPD1 inhibited the extrusion of the first polar body from porcine COC. Values are presented as mean s.e.m. Columns with different letters differ significantly (P , 0.05).
Expression and subcellular localisation of p-MK2 during porcine oocyte meiotic maturation To examine the subcellular localisation of p-MK2 in porcine oocytes, samples were collected after COCs had been cultured for 0, 24, 30, 36 and 44 h, corresponding to the GV, pro-MI, MI, telophase I (TI) and MII stages, respectively. Porcine oocytes were then processed for immunofluorescent staining. As shown in Fig. 2a, by Pro-MI, when chromosomes were aggregated and had started to migrate to the equator of the spindle, p-MK2 was partly colocalised with microtubules and concentrated on the chromosomes. After 30 h in vitro culture, when most porcine oocytes had developed to the MI stage and chromosomes were aligned on the equatorial plate, p-MK2 was specifically concentrated in the spindle equatorial region, like a drum (Fig. 2a). At anaphase/telophase, when homologous chromosomes were segregated and microtubules were distributed between segregated chromosomes, p-MK2 was localised between chromosomes (Fig. 2a). At this time point, p-MK2 was partly colocalised with microtubules. At MII, p-MK2 reappeared in the equatorial region and its distribution was similar to that at the MI stage (Fig. 2a). In mouse oocytes, p-MK2 was localised at the minus ends of microtubules and close to the spindle poles; in contrast, in porcine oocytes, p-MK2 was concentrated at the spindle equator and localised at the plus ends of microtubules at the MI, first meiosis anaphase in porcine oocytes (AI) and MII stages (Yuan et al. 2010). To further clarify the correlation between p-MK2 and microtubule dynamics, oocytes were treated with taxol, a microtubulestabilising drug. When spindle organisation started after GVBD, microtubule fibres in taxol-treated oocytes became excessively
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Fig. 2. Spatial relationship between phosphorylated (p-) mitogen-activated protein kinase-activated protein kinase 2 (MK2), a-tubulin and chromosomes during meiotic maturation of pig oocytes. (a) Oocytes at various stages were double stained with antibodies against p-MK2 (red) and a-tubulin (green). DNA is stained blue. Pro-MI, prometaphase I; AI/TI, first anaphase and telophase. Scale bar ¼ 10 mm. (b) Oocytes at the MI and MII stages were treated with 10 mM taxol for 180 min and then double stained with antibodies against p-MK2 (red), a-tubulin (green) and chromatin (blue). Scale bar ¼ 10 mm. (c) Localisation of p-MK2 in porcine MI and MII oocytes treated with 20 mg mL1 nocodazole for 120 min. After treatment, oocytes were double stained with antibodies against p-MK2 (red), a-tubulin (green) and chromatin (blue). Yellow indicates overlapping of green and red stains. Scale bar ¼ 10 mm. Each sample was counterstained with Hoechst 33258 to visualise DNA.
polymerised, leading to significantly enlarged spindles, together with numerous asters in the cytoplasm (Fig. 2b). The asters in porcine oocytes after taxol treatment took the shape of bamboo leaves and p-MK2 was localised at the apex and possibly at the plus ends of microtubules (Fig. 2b). To confirm the association of MK2 with spindle fibres, oocytes matured for 32 and 43 h were exposed to 10 mM nocodazole to depolymerise the microtubules. In nocodazole-treated MI and MII oocytes, p-MK2 was localised at the chromosomes (Fig. 2c). These data show that the association of MK2 with the chromosomes does not rely on the presence of intact microtubules. Thus, MK2 may participate in the connection between kinetochores and microtubules, as well as homologous chromosome separation. Spatial relationship between p-MK2 and kinetochores, g-tubulin or PLK1 during meiotic maturation of porcine oocytes Because the spindle in porcine oocytes is much smaller than in mouse oocytes, the localisation of protein staining in the former is not as clear as in mouse oocytes. To further examine the precise localisation of p-MK2, oocytes were stained for both pMK2 and Crest at the MI and MII stages. At Pro-MI, homologous chromosomes are formed as bivalents and p-MK2 was localised between the interstitial homologous chromosome arms and in the centromere region, whereas Crest signals appeared as dots on the kinetochores (Fig. 3a). At the MII stage, p-MK2 was localised as dots between the Crest signals on sister chromosomes (i.e. the inner centromere region of sister chromatids).
During mouse oocyte meiosis, g-tubulin is localised at the centre of microtubule-organizing centers (MTOC) and the poles of spindles, and plays an important role in spindle and spindle pole assembly (Ou et al. 2010). However, in porcine oocytes, g-tubulin was localised at the spindles and colocalised with a-tubulin. p-MK2 signals partly overlapped with g-tubulin near the centrosome and equator region (Fig. 3b). At AI, p-MK2 was localised between the chromosomes and partly overlapped with g-tubulin, but was not entirely colocalised with g-tubulin (Fig. 3b). After taxol treatment, p-MK2 was further verified to partly colocalise with g-tubulin on abnormal large spindles (Fig. 3c). These results show that MK2 is localised near the spindle equator and chromosome centrosome, and may be a plus-end microtubule protein. In mouse oocytes, PLK1 was localised at pole of the spindles and centromeres (Ou et al. 2010). However, in porcine oocytes PLK1 was localised across the whole spindle and centromere (Fig. 4a). MK2 was partly colocalised with PLK1 at the microtubules; the staining patterns for MK2 and PLK1 at the spindle were similar, but the area of MK2 staining was smaller than that of PLK1 (Fig. 4a). As shown in Fig. 4a, when p-MK2 was localised to the inner chromosome arms, PLK1 was colocalised with Crest at pro-MI and MI. At the same time, when taxol was used to decrease the tension between microtubules and chromosomes, PLK1 and p-MK2 were clearly localised in the centromere region (Fig. 4b). Further, the two proteins were colocalised partly in the centromere region, but the staining area of PLK1 was larger than that of p-MK2 (Fig. 4b). Thus, p-MK2 may contribute
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Fig. 3. Spatial relationship between phosphorylated (p-) mitogen-activated protein kinaseactivated protein kinase 2 (MK2) and Crest or g-tubulin during meiotic maturation of pig oocytes. (a) Oocytes at the prometaphase I (Pro-MI) and MII stages were stained with antibodies against p-MK2 (red), Crest (green) and DNA (blue). (b) Oocytes at various stages were fixed and stained for g-tubulin (green), p-MK2 (red) and DNA (blue), as visualised with Hoechst 33258 staining. Scale bar ¼ 10 mm. (c) Spatial relationship between p-MK2 and g-tubulin during meiotic maturation of pig oocytes treated with taxol. Green, a-tubulin; purple, p-MK2; red, g-tubulin; blue, chromatin. Scale bar ¼ 10 mm.
to the connection between microtubules and kinetochores. The special localisation indicates that p-MK2 may play an important role in spindle assembly and chromosome separation. Effects of MK2 on Plk1 localisation and p38 MAPK inhibition on MK2 localisation We next examined the localisation of centrosome-associated protein kinase Plk1 in CMPD1-treated oocytes. As shown in Fig. 4a, b, the centrosome-associated protein Plk1 was localised at the kinetochores and spindle in control oocytes, but PLK1 was dissociated from kinetochores and was scattered throughout the
cytoplasm in MK2-depleted oocytes (Fig. 4c). There was a significant decrease in the PLK1 signal in abnormal spindles (Fig. 4c). After p-p38 MAPK inhibition with SB203580 (0.3–0.5 mM for 30 min), there was no change in the localisation of MK2 in oocytes. Effects of MK2 inhibition on spindle organisation and chromosomes alignment In the present study, porcine oocytes were treated with 10 mM CMPD1, an MK2 inhibitor, to observe the effects of MK2 inhibition on spindle organisation and chromosome alignment.
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Fig. 4. Spatial relationship between phosphorylated (p-) mitogen-activated protein kinaseactivated protein kinase 2 (MK2) and Polo-like kinase (PLK1) during meiotic maturation of porcine oocytes. (a) Oocytes at the prometaphase I (Pro-MI), MI and MII stages were stained with antibodies against p-MK2 (red), PLK1 (green) and DNA (blue). (b) Spatial relationship between p-MK2 and PLK1 during meiotic maturation of pig oocytes treated with taxol (5 mM). Green, a-tubulin; purple, p-MK2; red, PLK1; blue, chromatin. Scale bar ¼ 10 mm. (c) PLK1 was dispersed from the chromosomes with CMPD1 treatment of porcine oocytes. Green, a-tubulin; purple, PLK1; red, DNA. Scale bar ¼ 10 mm.
As shown in Fig. 5a, the phosphorylation of MK2 was markedly reduced after CMPD1 treatment compared with control, indicating that CMPD1 successfully prevented phosphorylation of MK2. As stated above, after MK2 inhibition most oocytes were arrested at the Pro-MI stage. Downregulation of p-MK2 resulted in significant defects in spindle formation and chromosome alignment. In the control group, MI and MII oocytes that exhibited normal looking spindles and regular chromosomes (Fig. 5b), whereas most CMPD1-treated oocytes (Fig. 5b, c) cultured for 44 h had abnormal spindles. Some oocytes had irregularly scattered chromosomes with normal spindles, some had lagging chromosomes or scattered chromosomes surrounding shrinking or collapsed spindles and some had monopolar spindles surrounded by homologous chromosomes (Fig. 5b). Chromosomal p-MK2 staining was still observed along the centromere and arm regions of homologous chromosomes, whereas p-MK2 nearly disappeared from abnormal spindles (Fig. 5b). The rate of abnormal spindle formation in the CMPD1-treated group was
67.78% (n ¼ 90; Fig. 5c), which was considerably higher than that of the control DMSO-treated group 18.35% (n ¼ 109; P , 0.001). The incidence of misaligned chromosomes in the CMPD1-treated group was 63.33% (n ¼ 90; P , 0.001), much higher than in the control group (21.10%; n ¼ 109; Fig. 5d). These results suggest that MK2 is required for regulation of spindle organisation and chromosome alignment. Effects of anti-MK2 antibody on spindle organisation, chromosomes alignment and oocyte maturation To further elucidate the role of MK2 in porcine oocyte meiosis, we attempted to disrupt the functional roles of MK2 by antibody microinjection. Polar body extrusion (PBE) in antibody-injected oocytes was considerably lower than in the control DMSOtreated group (25.0% (n ¼ 118) vs 43.35% (n ¼ 200), respectively; P , 0.001; Fig. 6a). In the control group injected with rabbit IgG, spindle organisation was normal in most oocytes,
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with only 21.0% (n ¼ 200) being morphologically abnormal (Fig. 6b). However, various spindle abnormalities and defects were observed in a considerable number of oocytes after antibody injection (66.9%; n ¼ 118), similar to findings after CMPD1 treatment, including irregularly dispersed spindle microtubules, monopolar spindles, round shape spindle, multiple spindle apparatuses and collapsed spindles (Fig. 6b). In addition, severe defects in chromosome alignment were seen in oocytes injected with the anti-MK2 antibody, including lagging chromosomes and irregularly scattered chromosomes (Fig. 6b). The incidence of abnormal spindles and misaligned chromosomes in the antibody-injected group was 66.9% (n ¼ 118) and 70.34% (n ¼ 118), respectively, much higher than corresponding values in the control group (23.5% (n ¼ 200) and 21.0% (n ¼ 200), respectively; P , 0.001; Fig. 6c, d). Discussion In the present study we showed that MK2 has a unique localisation pattern in porcine oocytes and that it is a critical regulator
of meiotic cell cycle progression. MK2 is localised in the regions where cohesion is found in porcine oocytes, as in mouse oocytes, but unlike mouse oocytes, MK2 localises at the plus end of spindle microtubules in porcine oocytes. In particular, MK2 regulates spindle assembly and chromosome alignment, and inhibition of MK2 impairs the connection of the kinetochore with microtubules and chromosome separation in meiotic oocytes by regulating the localisation of PLK1 on the kinetochores and microtubules. Unlike mouse oocytes, which can complete meiotic maturation in vitro in the absence of cumulus cells, the meiotic maturation of porcine oocytes relies on the function of these surrounding somatic cells. Treatment of porcine COCs with the MK2 inhibitor CMPD1 caused defects in cumulus cell expansion and oocyte meiotic maturation. In addition, microinjection of anti-MK2 antibody to deplete MK2 directly in oocytes decreased extrusion of the first polar body and caused defective spindle organisation and chromosome alignment. These results demonstrate that MK2 inhibition prevented porcine oocyte maturation, and this may be caused by the disruption of both cumulus cell function and the oocyte itself.
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Fig. 6. Injection of an anti-phosphorylated (p-) mitogen-activated protein kinase-activated protein kinase 2 (MK2) antibody led to defective spindles and misaligned chromosomes and affected extrusion of the first polar body. (a) Percentage polar body extrusion (PBE) in the control (IgG injected; n ¼ 200) and p-MK2 antibody-injected (n ¼ 118) groups. (b) Spindle morphology and chromosome alignment after microinjection of the anti-p-MK2 antibody and IgG in porcine oocytes. In the control group (n ¼ 200), most oocytes showed normal spindle morphology and chromosome alignment, whereas in the antibody injection group (n ¼ 118), most oocytes showed severely defective spindle and chromosome alignment. Green, a-tubulin; red, DNA. Scale bar ¼ 10 mm. (c, d) Percentage of oocytes with abnormal spindles (c) and misaligned chromosomes (d) in the control and antibody-injected groups. Values are presented as mean s.e.m. *P , 0.05 compared with the control group.
In somatic cells, MK2 is the substrate of p38a and exists in a stable complex with its upstream substrate (ter Haar et al. 2007). p38a phosphorylates and activates MK2, and the p38a/MK2 signalling complex is thought to participate in centrosome maturation and chromosome alignment (Ben-Levy et al. 1998). In a previous study, we showed that the p38a/MK2 signalling pathway may play an important role in mouse oocyte meiosis (Ou et al. 2010). However, it is not clear whether the
p38a/MK2 pathway plays a role in spindle assembly in porcine oocyte meiosis. In mitosis, p-p38a is colocalised with pMK2 at spindle poles (Tang et al. 2008). However, it was observed that p-p38a and pMK2 were only partially colocalised at the spindle poles of MI and MII mouse oocytes. MK2 localises at the meiotic bipolar spindle microtubule minus ends and in the regions where cohesion is found. p38a phosphorylates and activates MK2 and affects the localisation of MK2 in mouse
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oocytes. Thus, MK2 is involved in spindle assembly in mouse oocyte maturation and plays an important role in maintaining spindle stability and chromosome alignment (Yuan et al. 2010). Unexpectedly, in the present study, p-MK2 was localised at the plus ends of microtubules and near the equatorial region of the spindle in porcine oocytes. The p-MK2 was partially colocalised with microtubules and concentrated on the chromosomes, but the extent of p-MK2 staining on the chromosomes was less than that on microtubules. After 30 h culture in vitro, most porcine oocytes developed to the MI stage, chromosomes were aligned on the equatorial plate and p-MK2 was specifically concentrated in the spindle equatorial region, like a drum. At anaphase/ telophase, homologous chromosomes were segregated and microtubules were distributed between segregated chromosomes and p-MK2 was localised between chromosomes. At this time, p-MK2 was partly colocalised with microtubules and the level of p-MK2 staining between chromosomes was less than that on microtubules. At MII, p-MK2 reappeared in the equatorial region and its localisation was similar to that seen at the MI stage in porcine oocytes. Unlike in mouse oocytes, in which pMK2 was localised at the minus end of spindle microtubules and close to the spindle poles (Yuan et al. 2010), p-MK2 was concentrated at the spindle equator and localised at the plus end of microtubules at the MI, AI and MII stages in porcine oocytes. Microtubule fibres in taxol-treated oocytes became excessively polymerised, leading to significantly enlarged spindles, together with numerous asters in the cytoplasm. The asters in porcine oocytes after taxol treatment took the shape of bamboo leaves and p-MK2 was localised at the plus end of microtubules, indicating that MK2 is a plus-end microtubuleassociated protein. Unlike in mouse oocytes, there are no p38 MAPK signals on spindles (Villa-Diaz and Miyano 2004) and p38 MAPK did not affect the localisation of p-MK2 in porcine oocytes. Thus, p38 MAPK/MK2 pathway may not play functions in porcine oocytes. The MK2 inhibitor prevented porcine oocytes from developing GVBD in a dose-dependent manner, and this may have been caused by either the disruption of cumulus cell function, as indicated by cumulus expansion failure, by actions on the oocyte itself or both. After MK2 inhibition, oocytes showed abnormally organised spindles, such as a collapsed spindle surrounded by chromosomes or spherical monopolar spindles, as well as abnormal chromosome alignment. Disorganised spindles and misaligned chromosomes blocked the meiotic progression and anaphase transition, and thus extrusion of the first polar body. This indicates that MK2 may be involved in connecting the microtubules with kinetochores, and microtubule capturing of chromosomes. The results provide support for p-MK2 participating in the meiotic cell cycle, especially spindle assembly and chromosome alignment, in porcine oocytes, but the mechanisms involved may differ from those in mouse oocytes. Although the functions underlying the actions of MK2 in mitotic and meiotic spindles may vary between different cell types and different species, a conserved role for MK2 in stabilising meiotic chromosome alignment is observed. MK2 disappeared from chromosome arms and was concentrated as dots on the cohesion of chromosomes, colocalising with PLK1. Kinetochores can connect with microtubules and capture
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chromosome through microtubules, which plays an important role in spindle organisation and chromosome segregation (Sun et al. 2001; Deng et al. 2009; Kitajima et al. 2011). The special location pattern of MK2 on chromosomes is the almost same as that of meiotic recombination protein 8 (REC8) (Garcia-Cruz et al. 2010). At anaphase of the first meiosis, meiotic sister chromatids lose cohesin from their arms, as indicated by REC8 removal (Lee et al. 2006). Cohesin plays an important role in meiotic sister chromosome connection. Phosphorylation of cohesin depends on Plk1 (Sumara et al. 2002). MK2 has been proven to directly phosphorylate Ser326 of Plk1(Tang et al. 2008). In the present study, MK2 inhibition resulted in the dislocation of PLK1 from porcine oocytes. Thus, MK2 may participate in the regulation of chromosome alignment and segregation by affecting Plk1 in porcine oocytes. In conclusion, MK2 has a unique distribution pattern in porcine oocytes. It affects not only cumulus expansion, but also oocyte meiotic cell cycle progression. MK2 regulates spindle organisation and chromosome alignment by connecting microtubules with kinetochores in a different way from that in mouse oocyte. Unlike in mitotic cells and meiotic mouse oocytes, the MK2/p38 MAPK pathway may not play an important role in this process. Acknowledgement This study was supported by the National Natural Science Foundation of China (No. 81200421) to X-HO.
References Ashraf, M. I., Ebner, M., Wallner, C., Haller, M., Khalid, S., Schwelberger, H., Koziel, K., Enthammer, M., Hermann, M., Sickinger, S., Soleiman, A., Steger, C., Vallant, S., Sucher, R., Brandacher, G., Santer, P., Dragun, D., and Troppmair, J. (2014). A p38 MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury. Cell Commun. Signal. 12, 6. doi:10.1186/1478-811X-12-6 Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H. F., and Marshall, C. J. (1998). Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr. Biol. 8, 1049–1057. doi:10.1016/S0960-9822(98)70442-7 Davidson, W., Frego, L., Peet, G. W., Kroe, R. R., Labadia, M. E., Lukas, S. M., Snow, R. J., Jakes, S., Grygon, C. A., Pargellis, C., and Werneburg, B. G. (2004). Discovery and characterization of a substrate selective p38alpha inhibitor. Biochemistry 43, 11 658–11 671. doi:10.1021/BI0495073 Deng, M., Gao, J., Suraneni, P., and Li, R. (2009). Kinetochore-independent chromosome poleward movement during anaphase of meiosis II in mouse eggs. PLoS One 4, e5249. doi:10.1371/JOURNAL.PONE. 0005249 Fan, H. Y., and Sun, Q. Y. (2004). Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biol. Reprod. 70, 535–547. doi:10.1095/BIOLREPROD.103.022830 Fan, H. Y., Liu, Z., Shimada, M., Sterneck, E., Johnson, P. F., Hedrick, S. M., and Richards, J. S. (2009). MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science 324, 938–941. doi:10.1126/ SCIENCE.1171396 Farghaly, T., Khalifa, E., Mostafa, S., Hussein, M., Bedaiwy, M., and Ahmady, A. (2015). The effect of temporary meiotic attenuation on the in vitro maturation outcome of bovine oocytes. In Vitro Cell. Dev. Biol. Anim. . doi:10.1007/S11626-015-9878-Y Garcia-Cruz, R., Brieno, M. A., Roig, I., Grossmann, M., Velilla, E., Pujol, A., Cabero, L., Pessarrodona, A., Barbero, J. L., and Garcia Caldes, M. (2010). Dynamics of cohesin proteins REC8, STAG3, SMC1 beta and
MK2 regulates pig oocyte maturation
Reproduction, Fertility and Development
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