arrest at the metaphase I stage. In order to explore the MK2 function in second meiotic division, MK2 inhibition was used to treat oocytes cultured for 10 h when most oocytes had moved past the metaphase I stage. When the CMPD1-treated oocytes were further cultured for 5 h, the percentage of the first polar body extrusion was lower compared to that of the control group treated with DMSO, but higher than that of oocytes treated with CMPD1 at the GV stage (Fig. 5A, Fig. S2A). The staining showed that most CMPD1-treated oocytes with first polar body (78.1%, n = 128) displayed abnormal spindles and misaligned chromosomes com- pared to control oocytes (Fig. 5B), while some oocytes displayed normal looking spindles with scattered chromosomes (Fig. 5Bb), and some oocytes showed lagging chromosomes or irregularly scattered chromosomes surrounding shrinking or collapsed spindles (Fig. 5Bc,d). In addition, p-MK2 staining was detected at the centromeres of sister chromatids as dots and on the collapsed spindles (Fig. 5Bb-d). We further examined the localization of the centrosome-associated protein kinase Plk1 in CMPD1-treated oocytes. As presented in Fig. 5C, the centrosome- associated protein kinase Plk1 was only detected at the kinetochores compared to control oocytes in which Plk1 was localized to both spindle poles and kinetochores. Roles of MK2 in Oocyte Meiosis To examine details of sister chromatid segregation of CMPD1- treated oocytes at first meiosis, we employed chromosome spreading. Control oocytes cultured for 15 h contained 20 sister chromatids (84%, n = 50) (Fig. 5Da), however, most of the CMPD1-treated oocytes with the first polar body (65.2%, n = 63) showed incorrect numbers of sister chromatids with more or less than 20 (Fig. 5Db,c). Taken together, these results indicate that at the second meiotic division, CMPD1 treated oocytes displayed defective spindle assembly. The polar body extrusion was triggered despite the presence of unattached or improperly attached chromosomes, resulting in missing or extra chromosomes (aneuploidy) in the daughter cells. MK2 depletion impairs correct microtubule-kinetochore attachment As shown above, MK2 inhibition or depletion resulted in collapsed spindles, and chromosomes appeared to be excluded from the spindle structure or arranged in a dome-like configura- tion surrounding the spindle (Fig. 3C, Fig. 4C). These phenotypes suggest that MK2-depleted oocytes are unable to undergo proper chromosome alignment because of defects in spindles and/or kinetochore–microtubule attachments [42]. In order to confirm Figure 5. CMPD1 treatment leads to metaphase II spindle assembly defects, misaligned chromosomes and aneuploidy. (A) The rate of oocytes with first polar body in the control group and CMPD1-treated group. Control group (22): oocytes cultured with DMSO for 15 h (n = 100); CMPD1-treated group (2+): oocytes cultured with DMSO for 10 h, and then with 30 mM CMPD1 for 5 h (n = 265). Data are presented as mean percentage (mean 6 SEM) of at least three independent experiments. Different superscripts indicate statistical difference (p,0.05). (B) Spindle morphologies and chromosome alignment in the oocytes first cultured with DMSO for 10 h, and then with MK2 inhibitor CMPD1 for 5 h; then stained for p-MK2 (purple), a-tubulin (green) and DNA (red). Control group oocytes were cultured with DMSO for 15 h. Scale bar, 20 mm. (C) GV oocytes were treated as in (B), and then stained for p-MK2 (red), Plk1 (green) and DNA (blue). Bar 20 mm. (D) Chromosome spreading was performed in oocytes that had been treated as (B). Representative images for each sample are shown. Bar 10 mm. doi:10.1371/journal.pone.0011247.g005 PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11247