Korean J Reprod Med.  2009 Sep;36(3):187-198.

Simultaneous Detection of Seven Phosphoproteins in a Single Lysate Sample during Oocyte Maturation Process

• Yoon SJ
• Kim YS
• Kim KH
• Yoon TK
• Lee WS
• Lee KA

Abstract

OBJECTIVE
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.

Keyword

Oocyte maturation; Protein phosphorylation; Mitogen-activated protein kinase (MAPK); Bio-Plex system

MeSH Terms

Animals
Blotting, Western
In Vitro Oocyte Maturation Techniques
Metaphase
Mice
Mitogen-Activated Protein Kinases
Oocytes
p38 Mitogen-Activated Protein Kinases
Phosphoproteins
Phosphorylation
Phosphotransferases
Proteins
Serine
Mitogen-Activated Protein Kinases
Phosphoproteins
Phosphotransferases
Proteins
Serine
p38 Mitogen-Activated Protein Kinases