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Korean Journal of Reproductive Medicine 2009;36(3):187-198.
Published online September 1, 2009.
Simultaneous Detection of Seven Phosphoproteins in a Single Lysate Sample during Oocyte Maturation Process.
Se Jin Yoon, Yun Sun Kim, Kyeoung Hwa Kim, Tae Ki Yoon, Woo Sik Lee, Kyung Ah Lee
1Department of Genetics, Stanford University School of Medicine, CA 94305, USA
2College of Life Science, Department of Biomedical Science, CHA University, Seoul, Korea.
3Fertility Center, CHA General Hospital, Seoul, Korea.
Phosphorylation and dephosphorylation of proteins are important in regulating cellular signaling pathways. Bead-based multiplex phosphorylation assay was conducted to detect the phosphorylation of seven proteins to maximize the information obtained from a single lysate of stage-specific mouse oocytes at a time. METHODS: Cumulus-oocyte complexes (COCs) were cultured for 2 h, 8 h, and 16 h, respectively to address phosphorylation status of seven target proteins during oocyte maturation process. We analyzed the changes in phosphorylation at germinal vesicle (GV, 0 h), germinal vesicle breakdown (GVBD, 2 h), metaphase I (MI, 8 h), and metaphase II (MII, 16 h in vitro or in vivo) mouse oocytes by using Bio-Plex phosphoprotein assay system. We chose seven target proteins, namely, three mitogen-activated protein kinases (MAPKs), ERK1/2, JNK, and p38 MAPK, and other 4 well known signaling molecules, Akt, GSK-3alphabeta Ikappaalpha and STAT3 to measure their phosphorylation status. Western blot analysis and kinase inhibitor treatment for ERK1/2, JNK, and Akt during in vitro maturation of oocytes were conducted for the confirmation. RESULTS: Phosphorylation of ERK1/2, JNK, p38 MAPK and STAT3 was increased over 3 folds up to 20 folds, while phosphorylation of the other three signal molecules, Akt, GSK-3alphabeta and Ikappaalphawas less than 3 folds. All of these results except for Akt were statistically significant (p<0.05). CONCLUSION: This is the first report on the new and valuable method measuring many phosphoproteins simultaneously in one minute sample such as oocyte lysates. All of the three MAPKs, ERK1/2, JNK, and p38 MAPK are involved in the process of mouse oocyte maturation. In addition, STAT3 might be important regulator of oocyte maturation, while Akt phosphorylation at Serine 473 may not be involved in the regulation of oocyte maturation.
Key Words: Oocyte maturation; Protein phosphorylation; Mitogen-activated protein kinase (MAPK); Bio-Plex system
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