Involvement Of AKT And PIPP In Early Development Of Mouse Fertilized Eggs

ObjectivesProtein Kinase B(PKB/AKT)is a multifunctional serine-threonine protein kinase, which paly a key role in the process of cell growth,such as metabolism,reproducation, apoptosis,transcription and migration.In mammalian,AKT1,AKT2 and AKT3(also termed PKBα,PKBβand PKBγrespectively)have similar domain and can be activated by a large part of growth factors in a PI3K-dependent manner.AKT N-terminal(1～106) is its PH domain.The PH domain may mediate the interaction of PKB/AKT with other proteins.It can bind certain phospholipids such as PtdIns(3,4,5)P₃ and/or PtdIns (3,4)P₂ and meanwhile target AKT to plasma membrane.Plasma membrane localization cooperates with basal AKT activity to activate Ser473 phosphorylation of AKT C-termiinal.Therefore membrane recruitment of AKT is a hallmark of activation. Thr308 phosphorylation of AKT might promote a conformational change of a C-terminal hydrophobic motif(HM).And membrane localization of AKT is very critical for AKT activity and Ser 473 phosphorylation.Previous reports have indicated that AKT play an important role in the initiation of S and G₂/M transition.AKT1 can phosphorylate p21 T145 and p27 T157 and result in their cytoplasmic translocation and loss of function.However,when p21 binds with AKT2,it can no longer be phosphorylated by AKT1.AKT can promote CDC25B phosphorylation and regualte G₂/M transition.Proline-rich inositol polyphosphate 5-phosphatases(PIPP),also called PIB5PA,can dephosphorylate dextrorotation 5-position phosphorylation of PtsIns(3,4,5)P₃ and/or PtdIns(4,5)P₂ to form PtsIns (3,4)P₂ and/or PtdIns(4)P.PIPP expresses in multiple tissues such as brain,heart, kidney,stomach,small intestine and lung.Its special function remains to be established, especially there is still no report about the role of PIPP in mouse one-cell embryos.In this study,based on the expression and activity of AKT,we explore the effect of PIPP on AKT and its downstream substrates in mitotic division of mouse one-cell embryos in order to lay a important foundation to study the effect of PIPP on early development of mouse one-cell embryos.Materials and Methods1.AnimalKunming strain mice were obtained from the Department of Laboratory Animals in China Medical University.2.ReagentsThe pEFBOS-PIPP,pEFBOS-PIPP-H557A and pEFBOS vector plasmids were provided by Prof.Mitchell;Restriction endonuclease MluI were purchased from MBI. Antibodies were purchased from Santa-Cruz.Other reagents,unless otherwise specified, were purchased from Sigma(St.Louis,MO).3.Collection and Culture of Mouse fertilized Eggs and GV intact OocytesMouse one-cell embryos were collected from female mice with the method described by Hogan and Constantini.These eggs were then collected according to different cell cycles needed(G₁ phase:11～20 hr after hCG injection;S phase:21～26 hr after hCG injection;G₂ phase:27～29 hr after hCG injection;M phase:30～32 hr after hCa injection).As control group,mouse oocytes were collected and cultured from 20- to 28-day-old Kunming strain female mice according to the method described by Schultz and Jaroslav.The oocytes were matured in vitro for 0,24 h to reach the germinal vesicle(GV)and metaphaseâ…¡(Mâ…¡)stage respectively.4.RT-PCRTotal mRNA was extracted from mouse GV-intact oocytes and mouse fertilized eggs(200 eggs for each sample)at different phases by use of the QuickPrep^TM MicromRNA Purification Kit(Amersham Bioscience Co.)according to the manufacturers's instruction.RT-PCR reaction was carried out by RNA PCR Kit(AMV) Ver2.1(Takara Bio.,Inc.,Japan)according to its instruction.The sequences of the primers were designed by Primer 5.0 software and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co.,Ltd.5.Transfection of FLAG-PIPP,FLAG-PIPP-H557A and FLAG vectorMouse one-cell embryos were transfected with 0.2μg DNA(FLAG-PIPP, FLAG-PIPP-H557A,or FLAG vector,respectively)and 0.5μl Lipofectamine 2000 (Invitrogen,Carlsbad,CA)according to the manufacturer's instructions.After transfection,these eggs were cultured in M16 medium at 37℃24 hr and collected at required time.The supernatant was removed and then stored in -70℃until use.6.Immunoflueresence to analyse the distribution of phosphorylated AKT Ser473 in mouse fertilized eggsCollected mouse oocytes with GV and one-cell embryos at S phase,fixed in 4% polyformaldehyde,perforate with 0.1%Triton-X100;samples were incubated with the first antibody and the FITC conjugated second antibody,stain the DNA with PI; observed under the fluorescene microscope or confocal microscope.7.Effect of AKT inhibitors on division rate of mouse fertilized eggsEggs treated with AKT inhibitors were cultured in M16 medium,after 10 h,the division rate of eggs was observed,recorded and analyzed with SPSS 11.5 software. Compared with control group,it is significant for statistics if P＜0.05.8.Western BlotProtein extracts of mouse eggs were prepared with the similar method as previous reports.The primary antibodies against pAKT1/2/3(Ser 473),β-actin(Santa Cruz,CA, USA),pCDC2(Tyr15)(New England Biolabs)or CDC2(Neomarkers)were used at 1:500 dilution.HRP-conjugated secondary antibody was diluted 1:2000.The proteins were detected using an ECL detection system(Pierce Biotechnology Inc.,USA)9.Assays of AKT and MPF activitiesAKT or MPF kinase activity was measured by histone H2B kinase or histone H1 kinase assay.The frozen eggs were treated with the same method as we previously reported.The radioactivity on the filter paper was counted with a BECKMAN scintillation counter.10.Statisitical AnalysisMeasurements were expressed as mean±SD and statistical analysis was performed by one-way ANOVA test with SPSS 11.5 Software.P＜0.05 was regarded as a significant difference.Each experiment was repeated three times,and the value of three samples averaged was compared among groups.Results1.Expression and Activity of AKT in mouse fertilized eggsAKT1 transcripts showed detectable level in each phase of mouse one-cell embryos and GV-intact oocytes.However,AKT2 transcript was detected only in G₁ phase of mouse one cell embryos.And the protein levels of AKT(Ser473) phosphorylation were much higher in mouse one cell embryos than those in immature oocytes.AKT activity measurement indicated that the activity of AKT in one-cell embryos was higher than that in GV intact oocytes,suggesting that AKT may play a key role in mouse one cell embryos.Localization of AKT(Ser473)phosphorylation was regulated in an activity-dependent manner.2.Effect of PIPP on AKT and its G₂/M transition in mouse fertlilized eggsMouse fertilized eggs transfected with FLAG-PIPP were found decrease of AKT activity,compared with these eggs transfected with FLAG vector(P＜0.001). Meanwhile,eggs transfected with FLAG-PIPP or treated with LY294002 showed the change of AKT membrane localization,the decrease of cleavage rate,MPF activity and CDC2 Tyr15 dephosphorylation.3.Effect of PIPP-H557A on G2/M transition of mouse fertlilized eggsMouse fertilized eggs transfected with FLAG-PIPP-H557A showed that there were little effect on MPF activity and division rates and dephosphorylation of CDC2 Tyr15 in mouse fertilized eggs.DiscussionAs an important regulator to maintain the normal vital movement of organism, AKT is activated by the stimulation of many growth factors.AKT activity is regulated mainly by PI3K/AKT signaling pathway.Pleckstrin homology(PH)domain of AKT binds to PtdIns(3,4,5)P₃ and/or PtdIns(3,4)P₂ that is generated in response to PI3K activation.This binding alters AKT's conformation such that it becomes accessible to phosphorylation by its upstream kinases,such as phosphoinositide-dependent kinase 1. The Localization of AKT(Ser473)phosphorylation in mouse fertilized eggs of S and G₂ phase indicated that AKT was activated and translocated in an activity-dependent manner.During the mitosis of fertilized eggs,mRNA expression is distinct.It requires genome reprogramming and proper regulation of different gene expression patterns. Our data showed that AKT2 transcript fluctuates in mitotic cycle of mouse fertilized eggs,whereas AKT1 transcript level is unchanged,suggesting that AKT1 and AKT2 gene patterns might be properly regulated in mitotic cycle of mouse fertilized eggs.The apparent differences with respect to their expression suggest that their signal transduction pathways may differ,particularly with regard to their downstream targets or substrates.Our data would contribute to an understanding of relationship between embryogenesis and tumorigenesis.In dividing eukaryotic cells,entry into mitosis is governed by the MPF.Its activation is achieved mainly by phosphorylation of CDC2.Clearly,AKT activity is complex, exhibiting a series of spatial restrictions that limit phosphorylation by upstream activiators.It is very significant to explore the effect of AKT and PIPP on mitotic division of mouse fertilized eggs.Our data are rather compatible with previous reports. Potential inhibitors of AKT activity,Wortmannin or PIPP,distinctly decreased AKT activity.Moreover,the dramatic downregulation of AKT1 activity blocked the procedure of mitotic division of mouse fertilized eggs,such as decrease of MPF activity and increase of CDC2 Tyr15 phosphorylation.Meanwhile catalytic inactive PIPP-H557A was detected to loss the activity of PIPP and have little effect on AKT and its substrates.These data should be of great benefit for our understanding of AKT1 and PIPP sensitive signal cascades in mitotic cycle of mouse fertilized eggs.Taken together,our study indicated that AKT1 in mouse one-cell embryos may be located in cytomembrane and activated sufficiently.AKT1 activity may promote mitotic division of mouse fertilized eggs.However,PIPP paly a negative role to AKT1 and different with catalytic inactive PIPP-H557A.And whether AKT2 expression provides physiologic significance,or is predictive of response to a particular signaling pathway,needs a further study.Conclusion1.AKT1 and AKT2 mRNA expression were different in mitotic cycle of mouse fertilized eggs.AKT1 is the necessary for the development of mouse fertilized eggs.2.AKT1 loacted in cytomembrane of mouse fertilized eggs.3.AKT1 may regulate G₂/M transition of mouse fertilized eggs.4.PIPP regulates G₂/M transition by decreasing AKT activity,but inactive PIPP-H557A can not regulate AKT activity.