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It has been reported that activity of Rho, one of the GTPases, is essential for division of nuclei and cytoplasm of fertilized mouse eggs. Since it has been reported that alteration of activities of GTPases modifies their ability to attach to each of their effector proteins in somatic cells, effector proteins seem to be able to control not only progression but also repression of cell division by changing their cellular localizations through activities of GTPases. For this reason, Rhophilin-2, one of the effector proteins of Rho, seems to be involved in the decision of progression of division of fertilized mouse eggs. To examine whether this involvement works in fertilized mouse eggs, cellular localization of Rho and Rhophilin-2 in fertilized mouse eggs that were treated with Rho inhibitor were analyzed. Moreover, cellular localization of GABA A receptor association protein (GABARAP), which was identified in our previous study (Matsuoka et al. 2006 Reprod. Fertil. Dev. 18, 176-177) as a protein that interacts with Rhophilin-2, was also analyzed. Fertilized mouse eggs were obtained from in vitro fertilization technique. One group of fertilized eggs was obtained at 24 h after insemination as experimental control. To obtain the mouse eggs in which Rho activities were inhibited, Clostridium botulinum C3 exoenzyme (C3-CB), an inhibitor of Rho activity, was injected into the other group of fertilized mouse eggs at 12 h after insemination, and were collected after 12 h of subsequent culture. Cellular localization of Rho (n = 100), Rhophilin-2 (n = 10). and GABARAP (n = 10) in the collected oocytes was analyzed by using immunofluorescence. Our results showed that Rho and Rhophilin-2 were co-localized at the midbody microtubule, which is an important device for cytoplasmic division in control eggs. However, the inhibition of Rho activity did not modify the co-localization of Rho and Rhophilin-2. On the other hand, localization of GABARAP was modified by the inhibition of Rho activity, and GABARAP was detected around the nuclei of fertilized eggs in which Rho activity was inhibited. In the next experiment, we examined whether interaction of Rhophilin-2 and GABARAP was modified by the inhibition of Rho activity by using a co-immunoprecipitation assay (co-IP) (n = 100). The interaction of Rhophilin-2 and GABARAP was found to disappear after inhibition of Rho activity. These results suggest that activity of Rho seems to regulate cytoplasmic division through Rhophilin-2 modification. Moreover, Rho seem to modulate the nuclear division of fertilized mouse eggs by regulating the interaction between Rhophilin-2 and GABARAP.<fn-group><fn>This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.</fn></fn-group> |
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