Role of actin filaments in the hatching process of mouse blastocyst

Hatching has been suggested to occur as a result of protease-mediated lysis and the blastocoele tension. However, even if rupturing is initiated at multiple sites, interestingly only a single site is used for escape. This implies that there are several mechanisms involved in hatching. In this study, the involvement of actin filaments in mouse embryo hatching was examined. We treated mouse embryos with cytochalasin B for 12 h or 24 h at the morula, middle blastocyst, expanded blastocyst, lobe-formed blastocyst and hatching blastocyst stages, and measured the amount and distribution of actin filaments using a confocal microscope. At morula, middle blastocyst, lobe-formed blastocyst and hatching blastocyst stages embryonic development was completely arrested by cytochalasin B. However, when transferred to cytochalasin-B-free medium, the embryos resumed development and escaped the zona pellucida. In the expanded blastocysts development was almost completely inhibited by cytochalasin B, but rupturing occurred in some embryos. However, development stopped completely at the ruptured stage. Distribution of actin filaments was prominent at rupturing and hatching sites regardless of cytochalasin B treatment. The amount of actin filaments was prominent at hatching embryos compared with other developmental stages of embryos. These actin filaments were distributed intensively between the trophectodermal cells, and formed locomotion patterns. Taken together, these results suggest that not only tension and lytic enzymes are required to rupture, but the activity of actin filaments may have a crucial role in the process of hatching.