Involvement Of PLK1 And MTOR In Early Development Of Mouse Fertilized Egg

IntroductionFurther research was made about the fertilized eggs as a natural cell cycle mode, which was good for exploring the mechanism about the regulation of cell cycle,and made us understand the growth,development,the canceration and death.In the vertebrate,mouse fertilized egg is an intimate cell cycle mode comparing with human being.However,due to the limited material,the mechanism research about the early development of mouse fertilized egg becomes one of the international hot spot, especially the transformation from 1-cell stage to 2-cell stage.Plk1 kinase is a family of serine/threonine protein kinases in eukaryotic organism. The Plk family has many members.The polo gene of Plks family was discovered most early in drosophila melanogaster.Afterwards they were found,cdc5p kinase in saccharmyces cervisiae,polo1 kinase in shizosacchermyces pombe,Plx1 in xenopus, plkl in high vertebrate,Snk and Fnk in mouse as well as hSnk and prk in homo sapien.The function of Plk1 is important in different phases of mitotic division,which is supported by the time-dependent change of Plk1 activity.In the mammalian cells,Plk1 activity increases quickly at the phase of G2/M switch,almost at the meantime with the increase of CDK1 activity.However,Plk1 activity will keep a high level even if CDK1 activity decreases.Before thirty minutes of the GVBD in mouse,Plk1 activity increased and reached the peak at the GVBD,after that Plk1 activity decrease a little, but kept a high level at the whole process of oocyte maturation.After alcohol induced the parthenogenetic reproduction,Plk1 was phosphorylated and the activity decreased gradually.At the entry of M phase of the first mitosis,Plk1 was phosphorylated and the activity increased.Above all,the Plk1 function is related tightly with Plk1 location,the expression quantity and the activity change.At the phase of G2/M transformation,Plk1 was located around SPB,at the same time MPF complex also located at the same site,and it was found that Plk1 activity increased at the time of MPF activation,so it supposed that Plk1 may be involved in the MPF activation at the G2/M switch.After Plx1 was neutrality with antibody,cdc25 and MPF activities were inhibited.If the competent cdc25 may reverse the tendency,it indicated that Plx1 may be involved in the cdc25 activation and then activating MPF.It was also found that the Plx1 activation was later than that of MPF,and its activity could be inhibited by the specific CDK inhibitor.So it supposed that plx1 activation was dependent on the MPF activity.To conclude that MPF may activate plx1 to activate indirectly cdc25 and then activate more MPF,which is a self-catalytic loop and including Plx1.Simultaneously other research suggested that when plx1 was inactive,Myt1,the inhibitive kinase of MPF,may be activated and be overphosphorylated and cdc2 activation was suppressed. It was seen that at the phase of G2/M switch plx1 not only activated MPF activator, cdc25 but also inhibited MPF inhibitor,Myt1,and then promoted the MPF activation, which made the cell entry into M phase.Qian(1998)used the mutation technology to change the Ser125 and Thr201 residues to Asp(S128D/T201)and obtained high activative plx1 mutant,and microinjected the mRNA into xenopus oocytes and activated directly cdc25 and MPF.In addition,Roshak(2000)research discovered that plks protein can phosphorylate directly hominal cdc25c regardless of obtaining by co-immunoprecipitation or recombination.Recently,the reports about Plk1 signal transduction was increasing,but it was few that plk1 might play a role in early development of mammalian fertilized eggs.We observed the plk1 expression and activity in mouse 1-cell stage fertilized eggs,and found that there were some changes about plk1 expression and activity,therefore plk1 might be involved in the transformation of G2/M,for further research,we used plk1 shRNA and plk1 specific inhibitor,scytonemin,to inhibit plk1 expression and activity and observated plk1 function and signaling pathway in mouse fertilized eggs.Rapamycin is an antibiotic separated from soil moisture streptococcus,its structure is similar with macrolides antibiotic,FK506.After rapamycin entried into body,it combined with FKBP12 and then combined with another protein with big molecular weigh,which formed trilogy complex and named target of rapamycin,TOR. First TOR was discovered from yeast,and then in mammalian cells TOR homologous protein,mTOR,was detected,mTOR(mammalian target of rapamycin)protein is a conserved structure and function protein,and is a member of phosphatidylinositol kinase-like kinase family,and is a serine/threonine kinase,and is a core regulator to regulate the cell growth,mTOR is involved in a series of physiology and pathology process and there are two form complex:mTOR/Raptor complex and mTOR/Rictor complex,mTOR/Raptor complex was discovered earlier than the mTOR/Rictor complex.Its function was mainly effecting cell growth and proliferation by regulating protein synthesis,and its activity could be suppressed by rapamycin.The activity of mTOR/Rictor complex can't be inhibited by rapamycin and was involved in constructing the cellular skeleton protein.The concrete action is still be investigated.It is known to all that PKB is an important factor to regulate cell proliferation. There was a report that PKB could phosphorylate directly GSK-3 and inactivate it, thereby promoting the nucleic localization cyclin D1,at last activating Cdk4/cylcin D1 complex and impressing the G1 process.On the other hand,PKB phosphorylated the inhibitor p^21Wafl/Cip1and p^27Kip1,and made them locate in cytoplasm and inhibited their physiological function.The results showed that PKB activated positive regulator and inactivated negative regulator to promote G1 phase process and completed the switch of G1/S phase.At the meantime,PKB can also regulate M phase process.In the Rat-1 cells LY294002 was used to suppress the PI3K activity and then caused the blockage of G2/M phase.When constitutively active PKB was overexpressed or PTEN was deleted, the cells could overcome the blockage of G2/M which was caused byγ-rays exposure. Thus,PKB could promote effectively the G2/M transformation in mammalian cell cycle.In our group the former work determined that PKB especially constitutively active PKB promoted the MPF activation by effecting the phosphorylation status of Cdc2-Tyr 15,and was be involved in the regulatory process of the early development of fertilized eggs.However,in mouse fertilized eggs the reports were few about the function mechanism and signal pathway of mTOR/Rictor.In the experiment we observed the activity change of mTOR and PKB in mouse 1-cell stage fertilized eggs and then analyzed their interaction about their activities.Scansite software was used to analyze the protein sequence of PKB and combined the related reports,we ensured the phosphorylated site in PKB by mTOR.At the meantime,we constructed the wide-type and kinase-dead PKB expressive vector and the mutant,transcripted to mRNA in vitro, microinjected into mouse 1-cell stage fertilized eggs and approached the possible substrate and action site of mTORC2 in the early development of mouse fertilized eggs.Materials and methods1,Materials and reagentsFemales of 3-4 week-old Kunming strain mice were supplied by the Department of Laboratory of Animals,China Medical University.Scytonemin was kindly provided by Dr.Peter Richter at Friedrich-Alexander University in Germany.The plasmid of Plk shRNA and Plk scramble shRNA were kindly provided by Dr.Yves Matthess at Johann Wolfagang Goethe-University Medical School in Frankfurt.The plasmid of raptor shRNA and fictor shRNA were kindly provided by professor Estella Jacinto.The constructs encoding the wild type PKB(pcDNA-WT-PKB)was constructed by our group.E.coli JM109 and ampicillin was purchased from Takara company.Restriction endonuclease Hindâ…¢,BamHI,EoRI and AgeI were purchased from Fermentas. mMESSAGE mMACHINE kit from Ambion.QuickChange Site-directed Mutagenesis Kit from STRATAGENE.Anti-Cdc2-pTyr15 monoclonal antibody,anti-Plk1 antibody and anti-MYC antibody were purchased from Santa-Cruz.O-dianidine,β-naphthyl acid phosphate and reagents for Plk1,mTOR and PKB activity assay from Sigma.2,Superovulation and eggs collectionFemale mice 4-6 weeks old were abdominal injected with 10IU of pregnant mare's serum gonadotropin(PMSG),and 48h later with 10IU human chorionic gonadotropin(hCG).A single female was placed with a single male for fertilization. One-cell embryos were collected with M2 medium the next day(24h after hCG injection)from oviduct of females possessing a vaginal plug.After injected with specific shRNA or different kinds of mRNA,embryos were transferred to culture in a drop of M16 medium,at 37℃,in a humidified atmosphere of 5%CO₂ in air,under paraffin oil.3,Construction of mRNA expression vectorsThe pBSK-PKB-WT were subcloned into pcDNA3.0(+)vector using Hindâ…¢and BamHI.The recombinant was named pcDNA-PKB-WT.Using Site-directed Mutagenesis Kit to mutate Serine 473 to Alanine of PKB and the mutant was called pcDNA-PKB-S473A.All the recombinant plasmids were sequenced to verify the inserted gene correct and mutation successful.4,In vitro transcriptionAll the constructs in pcDNA were cut singly with AgeI and in vitro transcribed into 5'-capped mRNA for microinjection by using mMESSAGE mMACHINE kit. After a transcription reaction was done,the reaction mixture was treated with DNase I to remove the DNA template.Then the mixture was extracted with phenol/chloroform, and RNA was precipitated with LiCl.5,mRNA microinjectionMouse fertilized eggs were microinjected using a micropipette and Eppendorf transferman manipulators mounted on a Olympusâ…¨-70 inverted microscope with DIC optics.Eggs were placed in a drop of M2 medium under paraffin oil in a lid of 3cm Falcon culture dish.Typical injection volume was 5%of total cell volume or 10pl per egg.mRNA was diluted to various concentrations in 5mmol/L Tris and 0.5mmol/L EDTA(pH7.4)without nuclease contaminant.Eggs in control groups were either not microinjected,or microinjected with TE buffer.6,shRNA microinjectionMouse fertilized eggs were microinjected using a micropipette and Eppendorf transferman manipulators mounted on a Olympusâ…¨-70 inverted microscope with DIC optics.Eggs were placed in a drop of M2 medium under paraffin oil in a lid of 3cm Falcon culture dish.Typical injection volume was 5%of total cell volume or 10pl per egg.shRNA was diluted to various concentrations in 5mmol/L Tris and 0.5mmol/L EDTA(pH7.4)without nuclease contaminant.Eggs in control groups were either not microinjected,or microinjected with TE buffer.The percentages of cell division and cell survival were counted under a dissecting microscope 30hr and 35hr after injection of hCG,and the results were analyzed statistically.7,Examination of rnRNA level by RT-PCR100 eggs were collected into 2ml eppendorf tube.The experiment was undertaken by the instruction of AuichPrep micro mRNA Purification Kit and it included that the extraction of samples,the separation of mRNA,washing the fiber particles combined with mRNA by the high salt buffer and low salt buffer and the elution of mRNA.The TaKaRa RNA PCR Ver3.0 Kit was used and we carried out to do the RT-PCR.The primers of Plk1 were 5'-TGACGAGTTCTTCACTTCTGGCT A-3' and 5'-ATTGCGGAAATAGTTGAGGAGAGT-3',and the product was designed for 548bp.The primers of raptor were 5'-GGCGGACCTTACAGATTGG-3' and 5'-TTGGCTGCCAGTTCTCATAC-3',and the product was designed for 230bp.The primers of rictor were 5'-CGAAGCATTTCCTGTCCC-3' and 5'-ACGGCTCCTGGTGACTTG-3',and the product was designed for 1347bp.The PCR reaction system was 25μl.The reaction condition wasâ‘ 94℃,3min;â‘¡94℃, 30sec;â‘¢50℃,30s ec;â‘£72℃,1min;⑤72℃,10min;â‘¡-â‘£30 cycles.Theβ-actin was used as the control.The primers ofβ-actin were 5'-TCCTATCGGCCTCCTCCTAC-3' and 5'-AGAATGTAGTCCGCCAGGTC-3', the product was designed for 337bp.The product was imaged by the agrose electrophoreses.8,Westem-blotting200 mouse 1-cell stage fertilized eggs were collected and transferred into 1.5ml Eppendorf tube.Centrifuging 10 minutes at 3000rpm,4℃and removal the culture solution.Adding the appropriable lysate(20mmle/L,Tris-HCl,PH7.5,2mmol/L EDTA, 10mmlo/L EGTA,0.25mmol/L Glucose),oscillating sufficiently and then freezing and trawing 3-4 cycles and making the eggs disruption,and putting into -20℃for use. Before electrophoreses,adding the SDS sample buffer,and boiling 5 minutes at 100℃, and centrifugating.10%SDS-PAGE electrophoreses was used for protein separation, and then protein was transferred into the NC filter.After that,5%BSA was used to incubate the NC filter 1 hour and then incubating the NC filter by the specific antibody at 4℃overnight.After washing by TTBS,incubating by HRP-couplant-secondary antibody 2 hours,washing the NC filter and then using the ECL luminescent technique to colourate.9,Plk1 activity assay Plk1 kinase activity was measured using histone H1 kinase assay.Five oocytes cultured in M16 medium were collected and subjected to freezing and thawing three times.A total of 25ul of Plk1 buffer was then added to the disrupted cells.The mixture was incubated at 30℃for 7min.After that,25ul aliquots were spotted on Whatman p81 paper.The radioactivity on the filter paper was counted with a Beckman scintillation counter.10,mTOR activity assay by radioautographyProtein extract from 20 oocytes was incubated with 50μl of roTOR buffer containing 50μCi/ml[γ-³²p]ATP at 37℃for 30 minutes,and the reaction was stopped by adding equal amount of 2×sodium dodecyl sulfate(SDS)buffer.The reaction was then resolved on a 12%SDS polyacrylamide gel electrophoresis(SDS-PAGE)gel,and the incorporation of ³²p into histone H1 was visualized by autoradiography.11,PKB activity assay by radio autographyProtein extract from 20 oocytes was incubated with 50μl of PKB buffer containing 50μCi/ml[γ-³²p]ATP at 37℃for 30 minutes,and the reaction was stopped by adding equal amount of 2×sodium dodecyl sulfate(SDS)buffer.The reaction was then resolved on a 12%SDS polyacrylamide gel electrophoresis(SDS-PAGE)gel,and the incorporation of ³²p into histone H2B was visualized by autoradiography.Result1,Expression of Plk1 in Mouse One-Cell Stage EmbryosIn order to detect Plk1 protein expression in mouse one-cell stage embryos during G1,S,G2,and M phase,samples were taken from four stages.Western blot analysis showed that Plk1 protein was expressed in mouse one-cell stage embryos and its quantity seemed unchanged at different phases.Furthermore,RT-PCR was used to examine the mRNA level in mouse one-cell stage embryos.The result suggested that the mRNA levels of Plk1 at G1,S,G2 and Mphase were not significantly different. These results were compared to the quantity of b-tubulin,whose expression remained contant during the four phases.2,Activity of Plk1 in Mouse One-Cell Stage Embryos In order to observe whether Plk1 plays a role during early development of mouse one-cell stage embryos,the Plk1 activity was assayed by using dephosphorylated casein as a substrate.The outcome revealed that the activity of Plk1 was stable in G1 and S phase.However,in G2 and M phase the activity increased and decreased at the end of M phase(P＜0.005 statistics analysis by SPSS12.0).This suggested that Plk1 may play a role during the early development of mouse one-cell stage embryos.3,Scytonemin and Plk1 shRNA Inhibits G2/M Transition of Mouse One-Cell Stage EmbryosTo examine the biological function of Plk1 on the early development of mouse fertilized eggs,we used scytonemin,the inhibitor of Plk1,to cultivate mouse one-cell stage fertilized eggs to observe its effect on egg cleavage.DMSO vehicle increasing the dissolubility of scytonemin was added(DMSO final concentration＜0.5%)to culture the mouse one-cell stage embryos.After 24 hr incubation,we examined the percentage of the egg cleavage at different scytonemin concentrations(0,1,2,3,and 4 mM).The result showed that the egg cleavage could be inhibited significantly by 2 mM of scytonemin(Fig.4A)(P＜0.005 statistics analysis by SPSS12.0).The result agreed with the point of view that scytonemin inhibited Plk1 activity in a concentration-dependent manner with an IC50 of 2 mM.We observed the eggs with or without the treatment of 2 mM scytonemin and discovered that the eggs with the treatment were blocked at G2 orM phase and those without the treatment were divided into two-cell stage embryos. These demonstrated that scytonemin interfered with G2/M transition of mouse one-cell stage embryos.Furthermore,it also proved that Plk1 regulated cell cycle of mouse fertilized eggs.4,Elevation of the Phosphorylation of Tyr15 of Cdc2 With Treatment of Scytonemin and Plk1 shRNATo investigate whether Plk1 may suppress the phosphorylation of Cdc2 at Tyr15 in early development of mouse fertilized eggs,we used pTyr15 of Cdc2 antibody to detect the phosphorylation of Tyr15 of Cdc2 inMphase of mouse one-cell stage embryos under the treatment of scytonemin.The experiment was repeated in mouse one-cell stage embryos with the microinjection of Plk1 shRNA or Plk1 scramble shRNA.The data showed that when the activity or expression of Plk1 was inhibited either by scytonemin or by Plk1 shRNA,the phosphorylation of Tyr15 of Cdc2 increased at M phase.The result suggested that Plk1 inhibited the phosphorylation of Tyr15 of Cdc2,which maybe one of the reasonswhyPlk1 could be important to G2/M transition of mouse fertilized eggs.5,The mRNA level of raptor and rictor in mouse fertilized eggsTo examine the protein expression of Plk1 in mouse 1-cell stage fertilized eggs, we used specific primers to do the PCR.The results showed that the raptor expression was not obviously different,but the rictor expression was high level in S and G2 phase and was low level in G1 and M phase.6,Microinjection of raptor shRNA/rictor shRNA to effect the development of mouse fertilized eggsWe microinjected raptor shRNA/rictor shRNA into G1 phase fertilized eggs.After 35 hours with the culture in M16,we observed the green fluorescent protein expression with the fluorescent microscope,and fotmd that the fertilized eggs with the microinjection of raptor shRNA can entry into 2 cell stage but the fertilized eggs with the microinjectin of rictor shRNA can not normally entry into 2 cell stage and was blocked at G2 phase or abnormal division.1-cell stage fertilized eggs were microinjected with 5pg raptor shRNA and rictor shRNA respectively,after 35 hours with the injection of HGC we observed the cleavage rate.In control group the cleavage rate was about 73.43%,in the group with the microinjection of raptor shRNA the cleavage rate was 74.17%,however the cleavage rate in the group with the microinjection of rictor shRNA was 17.07%.Comparing with the control group,the cleavage rate in the group with the microinjection of raptor was not obviously changed (P＜0.05);but comparing with the control group and the raptor shRNA microinjection group,the cleavage rate with the microinjection of rictor shRNA decreased significantly(P＜0.05).7,The activity change of mTOR and PKB in mouse fertilized eggs4EBP1 was used as the substrate to examine the mTOR activity in the different phase of mouse 1-cell stage fertilized eggs by the radioautography.The results showed that at G1 phase mTOR activity was high,and then mTOR activity increased further with the S phase entry and reached the peak in the whole cell cycle and at G2 phase the activity decreased sharply.At the same time H2B was used as the substrate to examine the PKB activity in the different phase of mouse 1-cell stage fertilized eggs by radioautography.The results showed that at G1 phase PKB had a low activity level, and the PKB activity increased at S phase.At G2 phase PKB activity increased further and reached the highest level in the whole cell cycle.Until M phase in the first mitosis PKB activity decreased sharply.8,Microinjection PKB-WT mRNA,PKB-S473A mRNA and rictor shRNA/PKB-WT mRNA,rictor shRNA/PKB-S473A into mouse 1-cell stage fertilized eggs to effect the cleavage rateWe microinjected 0.01ng PKB-WT mRNA and PKB-S473A mRNA into 1-cell stage fertilized eggs.After 30-33 hours with the HCG injection the cleavage rate was observated.In the control group the cleavage rate was about 73.43%,in the group of PKB-WT mRNA microinjection the cleavage rate was 73.5%and compared with the control group there was not obviously different.However the cleavage rate in the group with the PKB-S473A mRNA microinjection was 16.93%and comparing with the control group and PKB-WT mRNA microinjection there was significantly decreased (P＜0.05%).For further researching the interaction between mTOR/Rictor and PKB,we microinjected 0.05ng rictor shRNA into the nucleus.After fertilized eggs was cultured in M16 medium one hour,we microinjected 0.01ng PKB-WT or PKB-S473A mRNA into the cytoplasm again.The cleavage rate was investigated after 30-33 hours with HCG injection.The cleavage rate was 21.60%in the group of co-microinjection of rictor shRNA/PKB-WT and compared with the control group and PKB-WT mRNA microinjection group,the cleavage rate was significantly decreased(P＜0.05).However comparing with the cleavage rate in PKB-S473A mRNA microinjection group,there was not different.The results showed that the microinjection of PKB-S473A mRNA could suppress effectively the development of mouse 1-cell stage fertilized eggs.9,Microinjection PKB-WT mRNA,PKB-S473A mRNA and rictor shRNA/PKB-WT mRNA,rictor shRNA/PKB-S473A into mouse 1-cell stage fertilized eggs to effect PKB activityThe fertilized eggs at G1 phase were microinjected 0.01ng PKB-WT and PKB-S473A mRNA into.After 27-30 hours with HCG injection the eggs were collected and PKB activity was assayed.In the group of TE injection PKB activity was high level.Compared with the group of PKB-WT microinjection and the control group, PKB activity was obviously changed,PKB activity in the groups of PKB-S473A,rictor shRNA/PKB-WT and rictor shRNA/PKB-S473A mRNA microinjection respectively was significantly low level with contrast to that of the PKB-WT mRNA microinjection group,but there were not obviously different in three groups.The data showed that mTOR/rictor regulated PKB activity by PKB serine 473 phosphorylation and then regulated the early development of mouse fertilized eggs.DiscussionPlk1 is an important regulator in mitosis and plays a very important role in cell division.In the eucaryotic organism,Plk1 is involved in the regulation of cell cycle and is related with mitosis including the centrosome replication in the G1/S switch and the formation of spindle pole.Recently some researches showed that the protein expression of Plk1 kept a constant level in mitosis.This was agreement with our experiment about the Plk1 expression level.We examined the Plk1 protein and mRNA level by western blotting and RT-PCR and the results indicated that it will keep a constant level in cell cycle. However,we used 32P to assay Plk1 kinase activity and the results indicated that Plk1 activity would increase at G2/M switch,and then decreased obviously at the end of M phase.The data determined that Plk1 played an important role in G2/M switch of mouse fertilized eggs.Following,we used the Plk1 specific inhibitor,scytonemin that is a small molecular compound and can suppress the Plk1 activity specifically and its inhibiting is concentration dependent.After 24 hours with the treatment of scytonemin,80% fertilized eggs was blocked in G2/M phase in invert microscope and could not entry into two cell stage.Simultaneously we examined the phosphorylation level of cdc2 15 Tyrosine and found the phosphorylation level was significantly increasing with contrast to the group without treatment of inhibitor.The data was agreement with the former research,Plk1 is very important to the transition of G2/M phase in mouse fertilized eggs. For further determining our view,we used Plk1 shRNA to suppress Plk1 expression.When we microinjected Plk1 shRNA into the cytoplasm of fertilized eggs at G1 phase and which can inhibit Plk1 expression.Simultaneously the phosphorylation level of CDC2 15 tyrosine was elevated with the microinjection of Plk1 shRNA at M phase in mouse fertilized eggs,which is accordance with the treatment of scytonemin. This determined thoroughly that Plk1 was involved in regulating the mitosis of mouse fertilized eggs by dephosphorylating CDC2 15 tyrosine phosphorylation.Currently it is still hot spot about the research on the early development mechanism of mouse fertilized eggs.In the experiment we explored preliminarily the early development of mouse fertilized eggs.We thought that Plk1 could dephosphorylate CDC2 15 tyrosine phosphorylation to regulate the mitosis of mouse fertilized eggs.However,how dephosphorylated CDC2 15 tyrosine is the research trend in future.mTOR is an important regulator in the process of cell growth.mTOR has two complex,including mTOR-raptor(mTORC1)and mTOR-rictor(mTORC2).mTORC1 can phosphorylate 389 threonine of P70S6K,and then mTORC2 can phosphorylate 473 serine of PKB.In the lastest research it was found that mTORC2 was very important factor for activating PKB-FOXO and PKC signal pathway.In the former experiments we microinjected rictor shRNA into mouse 1-cell stage fertilized eggs and observed that mTOR/rictor could promote the development of mouse fertilized eggs,and when rictor was suppressed the PKB activity decreased.On the basis we applied the bioinformatics software to ensure the mTOR/rictor phosphorylative target site,Serine 473,in mouse PKB protein sequence.We microinjected rictor shRNA and PKB-WT mRNA into fertilized eggs and found that rictor shRNA could suppress the promotion to the development of fertilized eggs of PKB-WT mRNA microinjection.It suggested that PKB might be the substrate of mTOR/rictor and be involved in regulation process of early development in mouse fertilized eggs.At the same time with the microinjection of PKB-S473A mutant into 1-cell stage fertilized eggs,the development almost suppressed and the cleavage rate was only 16.93%.The results indicated that mTOR/rictor might effect the development of mouse fertilized eggs by the phosphorylation of 473 serine of PKB.Thus mTOR/rictor/PKB pathway played an important role in the development of fertilized eggs.It needs to determined by the experiments in vitro whether the action is direct.The research introduces first that in the early development of mouse fertilized eggs mTOR activates PKB by phosphorylating 473 serine of PKB and then promotes the development of 1-cell stage fertilized eggs.This finding provides the evidence and new clue to elucidate the concrete mechanism of action of mTOR/rictor. Simultaneously for further determining the direct phosphorylation of PKB from mTOR/rictor,we will carry out the kinase-substrate phosphorylation in vitro.Conclusion1,The Plk1 expression didn't change in the first mitosis of mouse fertilized eggs. The Plk1 activity changed by the process of cell cycle,and reached the peak at M phase.2,Plk1 can promote the development of mouse 1-cell stage fertilized eggs and the activation of Plk1 is necessary to the development of fertilized eggs.Plk1 regulates the mitosis of mouse fertilized eggs by dephosphorylating the 15 tyrosine of CDC2.3,Raptor expression is not different at four phases in mouse 1-cell stage fertilized eggs,but rictor expression is high level at S/G2 phase and is low at G1 and M phase,mTOR combined with rictor to regulate the development of mouse 1-cell stage fertilized eggs and their combination is necessary for the development of mouse fertilized eggs.4,PKB might be the substrate of mTOR/rictor in mouse 1-cell stage fertilized eggs and be activated by the phosphorylation of 473 serine to promote the early development of fertilized eggs.mTOR/rictor/PKB pathway plays an important role in the early development of mouse fertilized eggs.