Roles of MK2 in Oocyte Meiosis of p38 MAPKs are coordinately activated in response to a wide range of extracellular stress stimuli, including cytokines and growth factors [26,27]. The biological functions of p38 include a role in inflammatory immune responses [28] and cell cycle checkpoint controls [29,30]. MAPK activated protein kinase 2 (MK2) is a direct substrate of p38 MAPK, and phosphorylation of MK2 by p38 MAPK results in the activation of MK2 kinase activity; in addition, MK2 determines the subcellular localization of p38 MAPK [31]. A wide variety of substrates has been described for MK2 including proteins interacting with the cytoskeleton, such as small heat shock protein Hsp25 [32]; mRNA-binding proteins, such as tristetraprolin (TTP) [33,34]; transcription factors, such as heat shock factor 1 [35]; and regulators of the cell cycle and apoptosis, such as Cdc25B/C [36]. Moreover, MAPKAP kinase-2, a direct downstream target of p38 SAPK, is directly responsible for phosphorylating Cdc25B and C and for maintaining the G1, S, and G2/M checkpoints in response to UV-induced DNA damage [37]. In this study, we analysed the roles of MK2 in mouse oocyte meiotic maturation. We show for the first time that MK2 may regulate bipolar spindle stability, microtubule-kinetochore attach- ments and chromosome segregation to participate in meiotic cell cycle regulation. Results Expression and subcellular localization of p-MK2 during mouse oocyte meiotic maturation MAPKAPK 2 (MK2) is a direct target of p38 MAPK. Multiple residues of MK2 are phosphorylated in vivo in response to stress. Phosphorylation at Thr222, Ser272 and Thr334 appears to be essential for the activity of MK2 [38]. Phospho-MAPKAPK- 2(Thr334) antibody (p-MK2) detects endogenous levels of MK2 protein only when phosphorylated at threoine 334. To examine the expression level of p-MK2 in mouse oocytes at different stages of meiotic maturation, samples were collected after oocytes had been cultured for 0, 4, 8, 9.5, and 12 h, corresponding to germinal vesicle (GV), prometaphase I (Pro-MI), metaphase I (MI), telophase I (TI), and metaphase II (MII) stages, respectively. The immunoblotting results showed that the expression level of p- MK2 was similar at all stages (Fig. 1A). Figure 1. Expression and subcellular localization of p-MK2 during mouse oocyte meiotic maturation. Samples were collected after oocytes had been cultured for 0, 4, 8, 9.5 and 12 h, corresponding to GV, Pro-MI, MI, AI to TI and MII stages, respectively. Proteins from a total of 200 oocytes were loaded for each sample. (B) Oocytes at various stages were double stained with antibodies against p-MK2 (green) and with propidium iodide (PI, red). GV, oocytes at germinal vesicle stage; Pro-MI, oocytes at first prometaphase; MI, oocytes at first metaphase; AI, oocytes at anaphase; TI, oocytes at telophase; MII, oocytes at second metaphase. Bar 20 mm. (C) Metaphase I and metaphase II oocytes were fixed and double labeled with rabbit p-MK2 antibody (red) and human Crest antibody (green). Each sample was counterstained with Hoechst 33258 to visualize DNA (blue). Bar 20 mm. (a) model chart for localization of p-MK2, Crest and chromosomes at MI stage oocytes. (b) model chart for localization of p-MK2, Crest and chromosomes at MII stage oocytes. (D) Oocytes cultured for 12h (MII) were fixed and stained for c- tubulin (green), p-MK2 (red) and DNA (blue) as visualized with Hoechst 33258 staining. Bar 20 mm. doi:10.1371/journal.pone.0011247.g001 PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11247