Regulation of preimplantation development of mouse embryos: effects of inhibition of myosin light-chain kinase, a Ca2+/calmodulin-dependent enzyme.

Abstract

We have examined the effects of ML-9 and wortmannin, which are, respectively, specific reversible and irreversible inhibitors of myosin light-chain kinase, a Ca2+/calmodulin-dependent enzyme, on preimplantation development of the mouse in an attempt to establish a regulatory role for this enzyme in preimplantation development. When late two-cell stage embryos were treated continuously with ML-9 or wortmannin at a concentration of 0, 1, 5, 10, or 15 microM, compaction and formation of the blastocyst were inhibited in a dose-dependent manner. Stage-specific treatment with ML-9 at 25 microM induced stage-specific responses of embryos after the eight-cell stage during the processes of compaction and cavitation. These morphological responses included aborted compaction, decompaction of compacted embryos, and the inability of embryos to form a cavity. These morphological effects were reversible, but, since cell proliferation was inhibited, the "recovered" embryos were small. Counting of cells on day 4 of culture, in both continuously treated and stage-specifically treated embryos, showed that the effect of ML-9 on cell proliferation was also dose-dependent. Wortmannin also had stage-specific effects at 15 microM, but these effects were irreversible and were more deleterious than those of ML-9. With neither inhibitor was there any apparent effect at the two-cell or the four-cell stage, although wortmannin inhibited cell division when applied stage-specifically at the four-cell stage. These results indicate that myosin light-chain kinase may be an important enzyme in the first steps of differentiation and in the maintenance of the differentiated state during preimplantation development of the mouse.