Effect Of Pin1 Antagonist On Mouse Preimplantation Embryo Development In Vitro And The Expression Of ERα Protein

Objective:To elucidate the further functions of Pin1 in the activation of zygotic genomes, We observed the localization and distribution of Pin1 in mouse preimplantation embryo and the affection of Pin1 antagonist(Juglone) on the development of mouse preimplantation embryo in vitro. Meanwhile, we analyzed the m RNA levels of four stem cell factors(Sox2, Oct4, c-Myc and klf4), and investigated the variation of the expression level of ERa protein after Pin1 inhibition. Methods:1. The different developmental stages of KM mouse preimplantation embryos were collected from hybrid(♀KM×♂KM), including oocytes, 1-cell embryos, 2-cell embryos, 4-cell embryos, 8-cell embryos, morula, blastocysts. The localization and distribution of Pin1 was observed by immunofluorescence technique.2. KM mouse 1-cell embryos were cultured in KSOM medium supplemented with different concentration of Juglone respectively. The different development potential was observed under an inverted microscope.3. 2-cell embryos were obtained from KSOM and Juglone(25μM) media, the different m RNA expression levels of Sox2, Oct4, c-Myc and klf4 were measured by Realtime-PCR.4. 1-cell embryos and 2-cell embryos remaining in interphase and mitotic phase were collected from hybrid(♀KM×♂KM), The localization and distribution of Pin1 and ERa were observed by immunofluorescence technique.5. 2-cell embryos were got from KSOM and Juglone(25μM), the variation of the expression level of ERa protein were detected by immunofluorescence technique. Results:1. The immunofluorescence results showed that Pin1 protein was distributed in both cytoplasm and nucleus of KM mouse preimplantation embryos. The immuneflurescence intensity in nucleus was more obvious.The 1-cell embryos treated with Juglone(10mM and 25mM) continuously in vitro were blocked at 2-cell stage, the very few could be developed to 4-cell embryos(P<0.01). The 1-cell embryos treated with Juglone(25mM) for 18h(from 27 h to 45 h post h CG) in vitro were also arrested at 2-cell stage, the few could be developed to 4-cell embryos(P<0.01).2. In Juglone(25mM) group, the Sox2 m RNA levels were significantly low compared with KSOM group(P<0.05). While the m RNA levels of Oct4, c-Myc and klf4 were no significant differences compared with KSOM group(P>0.05).3. In mouse preimplantation embryos, the changes of localization and distribution of Pin1 and ERa were consistent.4.Though the pan-ERarelative fluorescence intensity was not changed obviously in 2-cell embryos between Juglone(25mM)group and KSOM group(P>0.05),the p ERa-S118 relative fluorescence intensity was increased in Juglone巴斯德玻璃管美国Corning-Costar公司(25mM) group(P<0.01).Conclusion: The development of mouse preimplantatiom embryos from 2-cell to 4-cell are blocked and the m RNA expression levels of stem cell factor(Sox2) is decreased by Pin1 inhibitor(Juglone) treatment. The results suggest that Pin1 may play a important role in ZGA. At the same time, the Ser 118 phosphorylation level of ERa is up-regulated after Pin1 inhibition, revealing Pin1 may control ZGA by the influence of ERa activity.