Roles of MK2 in Oocyte Meiosis Figure 3. Depletion of MK2 by morpholino injection induced homologous chromosome misalignment and bipolar spindle assembly defects. (A) Samples from control and MK2 MO injection groups were collected to test the efficiency of MK2 depletion. A total of 200 oocytes were injected with control or MK2 morpholino and cultured for 6 h after arrest at the GV stage for 24 h in M2-containing 2.5 mM milrinone. (B) Percentage of polar body extrusion in the MK2 morpholino injected group (n = 196) and control morpholino injected group (n = 137). Data are presented as mean 6 SE. Different superscripts indicate statistical difference (p,0.05). (C) Immunostaining of oocytes injected with control and MK2 morpholino. After injection, oocytes were incubated for 12 h after arrest at the GV stage for 24 h, followed by immunostaining with a-tubulin antibody (green), p-MK2 (purple) and PI labeling for DNA (red). Bar 20 mm. (D) The above oocytes were immunostained with c- tubulin (green), p-MK2 (red) and labeled for DNA (blue). Bar 20 mm. (E) The above oocytes were immunostained with Plk1 (green) and labeled with PI (red). Bar 20 mm. doi:10.1371/journal.pone.0011247.g003 CMPD1 was used to prevent phosphorylation of MK2. GV oocytes were incubated in M16 medium containing 30 mM CMPD1 for 11 h, and then washed thoroughly for Western blot analysis. As shown in Figure 4A, the p-MK2 protein expression level was strikingly reduced compared to the control, which showed that CMPD1 had successfully prevented phosphoryla- tion of MK2. In addition, the cyclin B1 level in the CMPD1- treated oocyte group was not reduced compared to control oocytes, which showed that preventing the phosphorylation level of MK2 had impaired meiosis progression (metaphase-to- anaphase transition). Most oocytes of the CMPD1-treated group did not extrude the first polar body even if they were cultured for 12 hours, indicating that inhibition of MK2 had greatly impaired meiosis progression (Fig. 4B). Compared to control MI and MII oocytes which displayed normal looking spindles and regular chromosomes (Fig. 4Ca,b), almost all (86.4%, n = 217) of the CMPD1 treated oocytes cultured for 12 h displayed abnormal spindles with dispersed homologous chromosomes (Fig. 4C). Some oocytes showed lagging chromosomes or irregularly scattered chromosomes surround shrinking or collapsed spindles (Fig. 4Cc); some showed dished chromosomes surrounding the shrinking spindles (Fig. 4Cd); some showed irregularly scattered chromosomes with normal spindles (Fig. 4Ce); and some showed monopolar spindles surrounded by homologous chromosomes (Fig. 4Cf). The chromosome p- MK2 staining was still observed along the centromere and arm regions of homologous chromosomes, and the cytoplasmic p- MK2 had nearly disappeared from the abnormal spindles (Fig. 4Cc-f). However, the morphology of homologous chromo- somes was abnormal and different from each other as revealed by staining with p-MK2 and DNA labeling (Fig. 4Cc1-2,d1- 2,e1,f1-2). We next examined the localization of centrosome- associated protein kinase Plk1 in CMPD1-treated oocytes. As presented in Fig. 4D, the centrosome-associated protein Plk1 was only detected at the kinetochores compared to control oocytes in which Plk1 was localized at spindle poles and kinetochores, indicating that the CMPD1 treatment caused collapse of spindles with no apparent spindle poles. Notably, the MK2-deficient oocytes in both the inhibitor- treated group and the morpholino injection group showed various kinds of chromosome morphologies. To examine details in chromosome morphology, CMPD1-treated oocytes were used for chromosome spreading. In meiosis, the chromosomes of oocytes cultured for 5–8.5 hours displayed 20 bivalents, like‘‘+’’ PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11247