Effect of protein phosphatase inhibitors on the development of mouse embryos: protein phosphorylation is involved in the E-cadherin distribution in mouse two-cell embryos.

Abstract

Protein phosphorylation plays many important roles in cell functions and cell differentiation. To clarify the roles of protein phosphorylation in early embryonic development in mice, 2-cell embryos were cultured in the presence of various protein phosphatase inhibitors such as calyculin A, okadaic acid, cyclosporin A, tacrolimus (FK506) and benzyl-phosphonic acid. Calyculin A potently inhibited the 2-cell cleavage to the 4-cell stage. The concentration for 50% inhibition was 0.26 nM. At the same time, we found that calyculin A-treated 2-cell embryos showed a morula-like shape at a concentration of 2 nM in 24 h. It is well known that E-cadherin plays a key role in the compaction of late 8-cell stage embryos. In this report, we observed the distribution of E-cadherin protein using anti-E-cadherin antibody with a fluorescence microscope, and also evaluated the relative E-cadherin mRNA content at various stages of embryos by RT-PCR and ABI PRISM 7700 System (a real time PCR apparatus). The fluorescence intensity of E-cadherin increased along with the embryonic development. During the embryonic development from the 2-cell stage to the blastocyst stage, the relative E-cadherin mRNA content greatly increased in a time-dependent manner, while the mRNA did not increase with the addition of calyculin A at the 2-cell stage. Therefore, we observed the localization of the E-cadherin protein in calyculin A-treated embryos with a laser microscope. The distribution pattern of E-cadherin was altered by the addition of calyculin A from a scattered pattern throughout the embryos to a localized pattern at the cell-cell boundary region. These results strongly suggest that the distribution of E-cadherin protein is regulated by protein phosphorylation and/or dephosphorylation.