Roles of MK2 in Oocyte Meiosis Figure 4. MK2 inhibitor CMPD1 treatment impairs spindle organization and chromosome alignment in mouse oocytes. (A) Samples from control and CMPD1 groups were collected to test the efficiency of the MK2 inhibitor. CMPD1, 200 oocytes were cultured in M16 medium with 30 mM CMPD1 for 10 h; Control, 200 oocytes were cultured in M16 medium with DMSO for 10h. (B) The rate of oocytes with first polar body in the control group (n = 267) and CMPD1-treated group (n = 318). Data are presented as mean percentage (mean 6 SEM) of at least three independent experiments. Different superscripts indicate statistical difference (p,0.05). (C) Spindle morphologies and chromosome alignment in oocytes cultured with DMSO or MK2 inhibitor CMPD1. GV oocytes were cultured in M16 medium with DMSO or with 30 mM CMPD1 for 12 h and then stained for p- MK2 (purple), a-tubulin (green) and DNA (red). Bar 20 mm. (D) GV oocytes were cultured in M16 medium with DMSO or with 30 mM CMPD1 for 8.5 h and then stained for p-MK2 (red), Plk1 (green) and DNA (blue). Bar 20 mm. (E) Chromosome spreading was performed in oocytes that had been cultured for 8.5 h or 12 h of DMSO treatment (MI) (MII) or for 12 h of CMPD1 treatment. Representative images of each sample are shown. Bar 10 mm. doi:10.1371/journal.pone.0011247.g004 (Fig. 4Ea); when oocytes were cultured for 12 h, the bivalents separated and became 20 univalents (Fig. 4Eb). Notably, the CMPD1-treated oocytes cultured for 12 h showed abnormal 20 bivalents (Fig. 4Ec-e), and the morphology of chromosomes in most (75.4%, n = 57) oocytes was abnormal compared to the controls. The homologous chromosomes may not have been able to become segregated, perhaps due to errors in microtu- bule-kinetochore attachments and tension. However, meiosis progression of CMPD1-treated oocytes can be recovered by culture for 5 h in fresh M2 medium after CMPD1 treatment for 10 h. The recovered oocytes successfully extruded the first polar body (Fig. S3A), however, the recovered oocytes (81.6%, n = 98) displayed abnormal spindles with dispersed chromosomes compared to control MII oocytes (Fig. S3B). These results further show that MK2 is involved in microtubule dynamics, chromosome segregation and correct microtubule- kinetochore attachment. CMPD1 treatment leads to metaphase II spindle assembly defects, misaligned chromosomes and aneuploidy As shown above, MK2 morpholino injected oocytes or MK2 inhibitor CMPD1-treated oocytes showed meiosis progression PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11247