IMMUNOCYTOCHEMICAL DETECTION AND SPATIAL-DISTRIBUTION OF MYOSIN LIGHT-CHAIN KINASE IN PREIMPLANTATION MOUSE EMBRYOS

As a follow-up to our previous study on the role of myosin light-chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme, in the development of preimplantation mouse embryos, we examined the presence and patter n of distribution of MLCK during preimplantation development of the mo use by whole-mount, indirect immunocytochemistry and by Western blotti ng, using a monoclonal antibody against MLCK. At all stages of preimpl antation development, the nucleus was brightly stained with an unstain ed region around the nucleus, and regions near the cell membrane were also brightly stained. Using the optical sectioning capability of the confocal laser scanning microscope, we found that, up to the eight-cel l stage, the regions of cell contact were mostly unstained, but along with the process of compaction, cell contact regions showed a clear st aining pattern along with clearing of the cytoplasm. During formation of the blastocyst, a ring of immunofluorescence was found at the margi n of the blastocoel. In the blastocyst, cells of the inner cell mass w ere less immunofluorescent than trophectoderm cells. These staining re sults appear to be due to specific immunoreaction between MLCK and the antibody, because the staining patterns were abolished when the antib ody was preabsorbed by MLCK purified from chicken gizzard smooth muscl e. In Western blotting of blastocysts, we found a band at 130 kD. We a lso show by immunoblotting and immunohistochemistry of various mouse t issues that the antibody used in this study has cross-reactivity to ML CK of various muscle and non-muscle tissues of the mouse. The presence and spatial distribution of MLCK at various stages of preimplantation development of the mouse suggest that it could play a crucial role in the regulation of the contractile events involved in the initial diff erentiation that occurs during formation of the mouse blastocyst. (C) 1997 Wiley-Liss, Inc.