| Ezrin protein plays an important role in polarizing process of epithelial cells, and it regulates cell-cell adhesion, cell migration, cell signal transduction, maintenance of cell shape, and so on. Phosphorylate modification is the major mechanism on regulating the activity of Ezrin. So far, seven phosphorylation sites have been identified controlling ezrin functions. Existing research have shown that ezrin has the ability to express in oocytes and preimplantation embryos of mouse, and there are phosphorylation occurring on Tyr and some other amino acid residues. But what exactly sites phosphorylate, and what the significance of those sites in embryos are still unknown. We studied expression, modification and localization dynamic of Ezrin in mouse early embryos, explored the affects of ezrin on early mouse embryos development, provided some theory evidence for further study of preimplantation embryos.Our experiment studied the expression, modification and localization of ezrin in early mouse embryos by RT-PCR and indirect immunofluorenscece, and discussed the influence of ezrin on early mouse embryos development through siRNA interference. The study has three parts:1. the expression pattern of ezrin in mouse preimplantation embryosEmbryos from fertilized eggs to blastocyst were collected, and total RNA of each stage was extracted and reverse transcripted into cDNA. The expression pattern of ezrin was identified in early mouse embryos by RT-PCR with the reference gene actin. Results showes that ezrin in each stage of mouse embryos appeared a trend of increased first and than decreased. In 2-C, the relative expression of ezrin mRNA reach to the most, and significantly higher than other stage embryos(P<0.01), than fall along with development until 8-C the least, and rise after that. However, the relative expression of ezrin mRNA decline again.2. The modification and localization of ezrin in preimplantation mouse embryosDifferent stage embryos from pronuclear to blastocyst were colleted and determined by indirect immunofluorenscence with antibodies of ezrin, p-ezrin(Y354) and p-ezrin(T567), to observe phosphorylation and events and localization. Results showed that 1) ezrin is located in cytoplasm at stages of pronuclear and 2-C while in both cytoplasm and nuclei from 4-C to morula; however, p-ezrin(Tyr354) is rich in cell cortex in blastocyst. 2) phosphorylation on Tyr354 of ezrin can be occurred from pronuclear to blastocyst, and it has a sharp enrichment in nuclei of each stage except pronuclear.3) phosphorylation on Thr567 of ezrin can be occurred from pronuclear to blastocyst; p-ezrin(T567) locates in cell cortex in pronuclear and morula, especially in morula apical cortex; p-ezrin(T567) is distributed in cytoplasm and cell cortex of blastomeres in 2-C, while it is particulally rich in cell-cell contact; p-ezrin(T567) is not only enriched in cell-cell adhesion at the stages of 4-C and 8-C, but also enrich in nuclei, while concentrate in nuclei and cell cortex in blastocysts.3. Functions of ezrin in early development of mouse embryosPronuclear embryos were collected and divided into 2 groups, ezrin siRNA injected of experimental group and negative control siRNA injected of control grou, compared development efficiency and total number of the two groups. Results showed a similar development efficiency in 2-C,4-C and 8-C, while it obviously low in experiental group in morula and blastocyst, but there is no significant difference (p>0.05) between the two groups. In addition, both of the fluorescence and the total cell number in blastocyst of experiental group are lower compared to control group in ezrin knockdown embryo, and there is an extremely significant difference about each (P<0.01)Our study proved that phosphorylation on Tyr354 and Thr567 exist in primplantation mouse embryos, and ezrin is involved in regulating development of morula-blastocyst development. |
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