Research On Localization And Function Of FAK During Early Embryo Development Of Mouse

Focal adhesion kinase（FAK）,a non-receptor cytoplasmic tyrosine kinase,belongs to the focal adhesion kinase family which also includes PYK2／CAKβ.FAKis an evolutionarily conserved protein and is widespread in the body.FAK canlocalize at the cell cortex and in the nucleus.FAK is a key enzyme in the signalpathway introduced by integrin, a joint of a lot of signal pathway in the celland introduces the interaction between cell and extracellular matrix(ECM).FAK actswith corresponding functional proteins by it’s phosphorylation,facilitatingattachment,degradation and migration. FAK can regulateproliferation,survival,migration,invasion,metastasis,angiopoiesis, and soon.FAK is related with many signal pathway and can regulate many biologicalfunction.FAK is important for embryo development and regulateattachment,spreading, migration, proliferation and apotosis.One research showed that FAK expresses at the cortex of oocyte. Cumulus cellsacross ZP also express FAK and phosphorylated FAK involves signaling of PTK.Masahiro Sakurai et al revealed that total FAK and pY397FAK were highly expressedin the ovary and were localized to granulose cells of ovarian follicles,especiallypreantral follicles. In granulose cells of atretic follicles, pY397FAKdecreased.So far,researches which has carried out focus on expression and functionof FAK of follicle development,oocyte and late embryodevelopment.However,researches on localization and function of early embryodevelopment of mouse are not been seen.By using confocal microscopy,immunofluorescent staining and inhibitorsinterference, we revealed: (1) the cortex localization of FAK in the GV stage,GVBD，MI and MII stage ofoocyte;(2) the cortex localization and the nucleus localization of FAK in the fertilizedegg,2-cells,4-cells and8-cells embryo，and weak boundary distribution ofFAK in the2-cells,4-cells and8-cells embryo;the apical region distributionand the basal region distribution of superficial blastomeres and the boundarydistribution of the inside cells and the nucleus localization of FAK in themorula；the apical region distribution and the basal region distribution ofsuperficial blastomeres and the boundary distribution of the inside cells andthe nucleus distribution of FAK in the blastocyst;(3) We detected the cortex localization and the nucleus localization of pY397FAKin the fertilized egg,2-cells,4-cells and8-cells embryo，the weak boundarydistribution of pY397FAK in the2-cells,4-cells and8-cells embryo; the apicalregion distribution and the basal region distribution of superficial blastomeresand the boundary distribution of the inside cells of pY397FAK in the morula andthe blastocyst;(4) We also detected the weak cortex localization and the boundary localizationof pY576/577FAK in the fertilized egg and2-cells,4-cells and8-cells embryomorula; the apical region distribution and the basal region distribution ofsuperficial blastomeres and the boundary distribution of the inside cells ofpY576/577FAK in the morula and the blastocyst;(5) colocalization of FAK and F-actin at the cortex of the fertilized egg;(6) PF573228,the inhibitor of FAK,inhibited the cleavage of the fertilized egg;(7) Method1（PFA+BSA）could detect distinctly the cortex localization and theboundary localization of FAK in the2-cells、4-cells and8-cells embryo.Method2(PFA/Tritonx-100+BSA/Gly) could manifest distinctly the nucleus localizationof FAK at each stage and the cortex(apical) localization and theboundary(baselateral)localization of the morula and the blastocyst; In regardto the localization of pY397FAk and pY576/577FAK, the effect of method2(PFA/Tritonx-100+BSA/Gly) is better than method1（PFA+BSA）.Regardless of the morula or the blastocyst, method2(PFA/Tritonx-100+BSA/Gly) is significantlybetter than method1（PFA+BSA）for the localization detection of FAK or p-FKA。...