Pyk2 In Mice Egg Fertilization And Early Embryo Development Orientation In The Process Of Research

Pyk2 (proline-rich tyrosine kinase 2), a non-receptor cytoplasmic tyrosine kinase,belongs to the focal adhesion kinase family. Pyk2 is an evolutionarily conserved protein andclosely related to focal adhesion kinase(FAK) . In different cells Pyk2 played different roles,which resulted from interact with different molecules. Recently, reseach on Pyk2 is mainly insomatic cells,in which its function was diversity.it can activated by the extracellularmatrix,including a variety of physical and chemical extracellular signal. So Pyk2 isinvolved in intracellular signaling and plays an important role in the process of cell adhesionmigration,survival,proliferation,and apoptosis.However,research on Pyk2 function in thegermce is very little.the licalizition and function of Pyk2 in ret oocyte meiotic maturation hadbenn studied in our laboratory.we found that Pyk2 may regulate microfilament polymerizationand assembly,which is important for the establishment of cell polarity in rat oocytes andemission of the first polar body. Another study found that Pyk2 was involved in theproliferation and migration of cancer cells, so it has been as a cancer treatment targets. Celldivision in the process of mammalian early embryonic development is similar to theproliferation of cancer cells, therefore, Pyk2 may also play an important role in this process.In thisreport, By using Western-blot analysis, confocal microscopy, inhibitors interference,werevealed the expression of Pyk2 in the matured oocytes and its localization in the mousefertilization eggs and the matured oocytes and its localization in the mouse fertilization eggsand the early embryos,which laied foundation for researching furtherly its functions.Pyk2 was specifically gathered in the sperm head, male and female nuclear in additionto its cytoplasmic localization during the process of in vivo or in vitro fertilization. The invivo fertilized eggs were collected from the oviduct of the female mouse 19h afteramphimixis and cultured in M16 medium for about 3days. We found Pyk2 is cumulated in thenucleus apart from the nucleolus and distributed in the cytoplasm. The colocalization of Pyk2and the chromatin was also detected during the in vitro early embryonic development. Inblastomeres which entered M phase, the chromatin condensed as chromosomes and Pyk2showed a punctate distribution on the chromosomes. The 2-cells, 4-cells, 6-cells, 8-cellsembryos and the blastulas were respectively colleted from the oviduct or uterus of the matedfemale mouse and the locolization of Pyk2 was observed using confocal microscopy. Theresultes showed the distribution of Pyk2 was same as the in vitro cultured embryos before theblastulas. However, In the blastocyst got from the uterus, Pyk2 disappeared from the nucleus and was discovered in cell-cell junctions and cytoplasm of only a small number of cells.MorePyk2 gathered in the space outside the trophoblast cells.In addition,we further studed the distribution of 402 tyrosine phosphorylation of Pyk2(P-Pyk2) in the early mouse embryo development and found that in early embryos collectedfrom in vivo, P-Pyk2 was almost undetectale. Only some point-like signal can be observed in3- cells, 4- cell embryos and morula. In the early embryo cultured in vitro P-Pyk2 wasactivated. Bright signal can be observed in cell junctions of the 2-cell embryos. In 4-cellembryo, morula and early blastocyst,a small amount of P-Pyk2 was star-sha[ped erpressed inthe cytoplasm.In order to study the function of Pyk2, The in vivo fertilized eggs were treated withdifferent concentrations (2.5μmol / L, 1.25μmol /L, 1μmol / L, 0.8μmol / L, 0.6μmol / L)of tyrosine kinase inhibitors Tyrphostin A9 and cultured in M16 medium. We found thatTyrphostin A9 dramatically inhibited the cleavage of fertilized eggs by characteristics of all ornone,but not by concentration-dependent manner. When the inhibitor concentration was morethan 1μmol / L, the cleavage was inhibited; When the inhibitor concentration was lessthan 0.8μmol / L, most of fertilized eggs cleaved although the rate of cleavage stillhad significant difference with the control group. In addition, we examined thedistribution of the Pyk2 in the fertilized eggs treated with tyrphostin A9 and found that Pyk2expression significantly increased. Surprisingly, the Tyrphostin A9 not only failed to inhibitPyk2 phosphorylation,but also significantly increased 402 tyrosine phosphorylation.We alsoexamined the distribution of the P-Pyk2 in 2 - cell embryos which were treated by theinhibitor but could normally cleave, and found the expression level of P-Pyk2 increasedsignificantly. P-Pyk2 is not only distributed throughout the cytoplasmand but also specificitygathered in the nucleus. These results indicated that the fertilized eggs in the environmentpresence of inhibitor Tyrphostin A9 could maintain their early embryonic developmentthrough the activation of Pyk2.