To investigate the subcellular localization of p-MK2, mouse oocytes were processed for immunofluorescent staining at different stages of maturation. As shown in Figure 1B, in immature mouse oocytes classified as the germinal vesicle (GV) stage, p-MK2 signals appeared as numerous dots associated with chromatin in the nucleus (Fig. 1Ba). After germinal vesicle breakdown (GVBD), in prometaphase I, chromosomes began to migrate to the equator of the spindle, and p-MK2 was localized along the interstitial axes of homologous chromosomes extend- ing over centromere regions and arm regions, like ‘‘+’’ (Fig. 1Bb). When oocytes progressed to metaphase I, homolo- gous chromosomes aligned at the equatorial plate, and p-MK2 localized at the spindle minus ends and chromosomal axes (Fig. 1Bc). At anaphase I, the homologous chromosomes were segregated, and the p-MK2 signals disappeared from chromo- somes and localized to the equatorial region between the separating chromosomes (Fig. 1Bd). At telophase I, the p-MK2 signals were associated with the midbody between the oocyte and the first polar body (Fig. 1Be). At metaphase II, the p-MK2 signals again translocated to the spindle minus ends and the sister chromatids (Fig. 1Bf). For closer analysis of its localization on chromosomes (chromosome p-MK2), oocytes cultured to MI and MII stages were co-stained with p-MK2 and Crest antibodies (Fig. 1C). At the MI stage, p-MK2 localized along the interstitial axes of homologous chromosomes extending over centromere and arm regions, like ‘‘+’’, and the Crest signals appeared as dots on the kinetochores (Fig. 1Ca); at the MII stage, p-MK2 localized as dots at the center of two Crest signals, that is, the inner centromere region of sister chromatids (Fig. 1Cb). Notably, when p-MK2 and c-tubulin were co- stained at the MII stage, it was clearly observed that p-MK2 signals were not concentrated and overlapping with c-tubulin at the centrosome position (Fig. 1D). Furthermore, when the bipolar meiotic spindles were assembled during the MI and MII stages, the p-MK2 signals were detected at the spindle minus ends, but not spread over the entire bipolar spindles, just near the minus ends of spindles (cytoplasmic p-MK2) (Fig. 2Ba,Ca); therefore, we refer to the cytoplasmic p-MK2 signals as being localized to the spindle minus ends. In addition, p-MK2 localization at metaphse of meiosis II was similar to that of metaphase in mitosis (Fig. S1). These data imply that MAPKAP kinase 2 may be involved in bipolar spindle formation and chromosome segregation in mammalian meiosis. Localization of p-MK2 in mouse oocytes treated with spindle-perturbing drugs To further clarify the correlation between p-MK2 and microtubule dynamics, spindle-perturbing drugs were employed. First, taxol was used to treat oocytes, which stabilized microtubules and reduced the tension between microtubules and kinetochores [39,40]. When oocytes were cultured for 8.5 h and 12 h, corresponding to MI and MII stages, respectively, the microtubule fibers in taxol-treated oocytes became excessively polymerized, leading to significantly enlarged spindles and numerous asters in the cytoplasm. At MI and MII stages, p- MK2 signals were clearly detected on the chromosomes and dispersed at the two ends of stable bipolar spindles, however, p- MK2 signals were not detected in the cytoplasmic asters (Fig. 2A). These findings indicate that MK2 is required for bipolar spindle assembly but it is not a centrosome component. Moreover, the localization of p-MK2 on chromosomes was not affected by reducing the tension between microtubules and chromosomes (Fig. 2A). Roles of MK2 in Oocyte Meiosis When oocytes cultured to the MI or MII stage were treated with nocodazole, an inhibitor of tubulin polymerization, the microtubules were completely disassembled. Compared to control oocytes (Fig. 2Ba), the p-MK2 signals disappeared from the spindle and dispersed around the chromosomes at the MI stage (Fig. 2Bb,c). At the MII stage, the cytoplasmic p-MK2 signals also disappeared from the spindle and no visible signals were detected around the chromosomes (Fig. 2Cb), compared to control oocytes (Fig. 2Ca). Meanwhile, the chromosome p-MK2 signals were still detected on the chromosomes, when microtubule and chromosome attachments were completely destroyed (Fig. 2Bb,c, Cb). In addition, when the above-described nocodazole-treated oocytes were cultured in fresh medium for 30 min, the cytoplasmic p-MK2 relocated to the spindle minus ends when the bipolar spindle was re-assembled (Fig. 2Bd,Cc). We conclude that MK2 is involved in maintaining bipolar microtubule organization while microtubule disassembly does not affect MK2 function on chromosomes. Depletion of MK2 by morpholino injection leads to spindle defects, homologous chromosome nondisjunction and meiosis progression arrest To elucidate the physiological function of MK2 in oocyte maturation, we employed a morpholino-based gene silencing approach to deplete MK2 in oocytes. Negative control or MK2 morpholino was injected into GV stage oocytes and maintained in 2.5 mM milrinone for 24 h, and then transferred to fresh M2 medium for 6 h. As presented in Fig. 3A, compared to the control morpholino (MO) group, the MK2 expression in MK2 MO-injected oocytes was significantly reduced, revealing successful down-regulation of MK2. When the MK2 MO- injected oocytes were cultured for 12 h after release from the inhibitory environment, there was a marked reduction in the polar body extrusion (PBE) rate. Only 30% of MK2-depleted oocytes underwent PBE, which was significantly lower than that of the control group (65%) (p,0.05, Fig. 3B). Furthermore, immunofluorescent staining showed that most control MO- injected oocytes which extruded the PB (85.6%, n = 70) displayed normal-looking spindles and sister chromatids ar- ranged at the middle plate (Fig. 3Ca), however, most (90.4%, n = 89) MK2 MO-injected oocytes displayed abnormal spindles and homologous chromosome congression defects, such as irregularly scattered chromosomes surrounding shrinking or collapsed spindles (Fig. 3Cb), or normal-looking spindles with scattered misaligned chromosomes (Fig. 3Cc) or scattered chromosomes along the elongated spindle (Fig. 3Cd). Moreover, in the control MO-injected group, MK2 signals were observed at spindle ends and centromeres of sister chromatids, whereas in the MK2 MO-injected group, most of the MK2 signals could not be detected at the homologous chromosomes and microtubules. Notably, the morphology of the homologous chromosomes in MK2-deficient oocytes was different from the control (Fig. 3Cb1,c1–2,d1–3). These results revealed that down-regulation of MK2 results in spindle assembly defects, homologous chromosome nondisjunction and misalignment. We examined the localization of the centrosome protein c- tubulin and the centrosome-associated protein kinase Plk1 in MK2 MO-injected oocytes. As presented in Fig. 3D, the centrosome protein c- tubulin disappeared from the cytoplasm unlike in control oocytes in which c- tubulin was localized at the spindle poles, and the level of p-MK2 was significantly reduced in MK2 MO-injected oocytes. Fig. 3E shows that Plk1 was only detected at the kinetochores compared to control oocytes in which Plk1 was PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11247