RNAi Down Pyk2 Expression On Early Mouse Embryo Development

As a non-receptor cytoplasmic tyrosine kinase, Pyk2belongs to the focal adhesionkinase family and is an evolutionarily conserved protein. The activation of Pyk2requires phosphorylation, including auto-phosphorylation and phosphorylation withcorresponding kinase, which is of great importance to the activation and functionexertion of Pyk2. After being activated by other corresponding kinases, thephosphorylation of a variety of signal molecules which are related to Pyk2will befurther promoted，what’s more, there is possibility that the conformational changescaused by the phosphorylation of molecules may induce the combination betweenother protein molecules and Pyk2, which in turn affects the activity of Pyk2and itsrelated signaling pathway. Recently, research on Pyk2is mainly in somatic cells, andit has been found that there are diversities in its functions. As phosphorylated Pyk2takes part in the conduction of a variety of cell signals, it plays a crucial role in theprocesses of cell apoptosis, proliferation, adhesion, and migration. However, seldomcan researches in the area of reproduction be found. Many present studies found thatPyk2was involved in the proliferation and migration of cancer cells, besides, becausecell division in the process of mammalian early embryos is similar to the proliferationof cancer cells, Pyk2may also play an important role in this process, but no relatedreport has been found. In addition, it’s found that there is a large amount ofunactivated Pyk2protein in cells in the process of early embryos with large quantitiesof them gathering in the cell nucleus. In this study, we try to use methods such asQRT-PCR, immunocytochemistry and RNAi to research the role that phosphorylatedPyk2plays in mouse fertilization as well as in the early embryos, which is to lay afoundation for further research. The in vivo fertilized eggs were collected from the oviduct of the female mouse19hafter amphimixis and cultured in M16medium for about3days. We found p-Pyk2iscumulated in the nucleus apart from the nucleolus and distributed in the cytoplasm.The colocalization of p-Pyk2and the chromatin was also detected during the in vitroearly embryonic development. In blastomeres which entered M phase, thechromatin condensed as chromosomes and p-Pyk2around the distribution in thechromosomes. The2-cells,4-cells,6-cells,8-cells embryos and the blastulas wererespectively colleted from the oviduct or uterus of the mated female mouse and thelocolization of p-Pyk2was observed using confocal microscopy. The resultes showedthe distribution of p-Pyk2was same as the in vitro cultured embryos before theblastulas. However, along with the embryos development, p-Pyk2gradually disappearin the nucleus.More p-Pyk2gathered in the cortex of the trophoblast cells.Pyk2transcript expression was examined by QRT-PCR using oligo dT primer inoocytes and preimplantation. It decreased significantly at the2-cell stage, but theexpression level increased again at the4-cell stage stage. In order to explore Pyk2influence of early embryonic development in the mouse, we adopt the method of RNAinterference to reduce the by the expression of Pyk2. When Pyk2was knock out later,the ratio of fertilized eggs develop into2cells,4cell, and8cell decreased. Thisshows that Pyk2in early may play a role in the process of embryonic development.But the embryo of the experimental group is still has a lot of embryos could developto the blastocyst stage, this may be because the similarity between the tyrosine kinaseon the structure and function, a tyrosine kinase is missing, other tyrosine kinase mayhave the effect of cover.