Involvement Of The Catalytic Subunit Of PI3K P110α And S6K1 In Early Development Of Fertilized Mouse Egg

IntroductionThe PI3Ks are subclassified into three distinct groups based on their substrate specificity,primary sequences and mode of regulation.PI3Kα,which is studied most frequently,is a heterodimeric lipid kinase,consisting of a p 110a catalytic subunit and a regulatory subunit p85a.The PI3K signal transduction pathway is a well-known mediator of cell growth,proliferation,survival and tumorigenesis.Furthermore,a body of evidence has been accumulated to indicate a pivotal role for the PI3K pathway in cell cycle progression.The PI3K pathway is activated during G1/S transition,and may be required for G2/M transition.But the unique role of PI3K isoforms remains unclear. The activation and function of PI3K during the early development of mammalian embryos have not been addressed clearly until now.PI3K regulates cellular glucose metabolism in the preimplantation blastocyst.Nevertheless,the physiological roles of the different PI3Ks isozymes during embryonic development are not well elucidated. Previous studies also demonstrated that p110a-KD/KD(kinase dead) blocks early embryonic development,indicating a widespread role of p110a in the signaling pathways with several receptor types.We speculated that p110a might play a potential role in the regulation of mouse early embryo development.The regulating role and time of mouse early embryo development remain unknown.Over the past decade,the PI3K/Akt pathway has been extensively studied and implicated as a key regulator of a number of cellular functions,including cellular migration,mitogenesis,differentiation and cell survival.Some results show that Akt promotes G2/M transition in mammalian cell cycle progression.However,the direct connection between the PI3K/Akt signaling pathway and their regulation on G2/M transition is not fully understood.In dividing eukaryotic cells,entry into mitosis is governed by M-phase promoting factor(MPF). MPF undergoes tight regulation during the G2/M transition by phosphorylation and dephosphorylation of Cdc2. Further 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,fertilized mouse 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 fertilized mouse egg becomes one of the international hot spots, especially the transformation from 1-cell stage to 2-cell stage.Previous studies also demonstrated that p110a-KD/KD(kinase dead) blocked early embryonic development, indicating a widespread role of p110a in the signaling pathways with several receptor types.We speculated that p110a may play a potential role in the regulation of mouse early embryo development.Our experimental group has explored the expression and localization of p110a,and furthermore,p110a shRNA and three kinds of mRNA construction were used for studying the biological function to examine the role and signaling passway ofp 110a during fertilized mouse eggs early development.Our experimental group has found that mTOR is involved in the transition of G2/M in first mitosis,and mTORC2 may regulate the development of mouse embryos by phosphorylating Akt on S473.S6K1 is one of important downstream targets of mTOR and PI3K in cell and organism growth control.Recent studies with cultured cell lines indicate that mTORC1 phosphorylates the rapamycin-sensitive forms of S6K1.To date,the cell-cycle effects of S6K1 are not fully elucidated in the mouse early embryo development.In order to better understand a more complete picture of S6K1 functions, we undertook detailed studies on expression and subcellular localization of S6K1 and the phosphorylated S6K1 on Thr389 during cell cycle progression.Mechanisms linking S6K1 and mTORC1/mTORC2 in mammalian embryo development have not been fully understood.Thus,to determine the role of each branch of the mTOR signaling network in regulating S6K1 phosphorylation during mouse early embryo development, RNAi-mediated depletion of mTOR,raptor,and rictor was exploited in our experiments.Materials and methods1,Materials and reagentsKunming genealogy mice were provided by the Department of Laboratory Animals,China Medical University(CMU).Anti-p110αantibody,anti-S6K1 antibody and anti-Thr389-phosphorylated S6K1 antibody were purchased from Santa-Cruz. The plasmids of wild type p110α(pBJ-p110α-WT),constitutively active p110α(pBJ-p110α-AC),and kinase deficient p110α(pBJ-p110α-KD) were gifts from Anke Klippel.PHI-p110αshRNA/control shRNA were gifts from Osamu Hazeki(Hiroshima University).The plasmid of mTOR shRNA,raptor shRNA and rictor shRNA were kindly provided by professor Estella Jacinto.E.coli DH5α,RNA PCR Kit(AMV) Ver 2.1 and ampicillin were purchased from Takara company.Restriction endonucleases were purchased from Fermentas and NEB.Ellustra^TM QuickPrep Micro mRNA Purification Kit was from GE Healthcare UK Limited.O-dianidine,β-naphthyl acid phosphate and reagents for MPF and PKB activity assay were 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 for ferilization. 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 coding sequences of p110α-WT,p110α-AC and p110α-KD were amplified by PCR using the pBJ-p110α-WT,pBJ-p110-AC and pBJ-p110α-KD as templates and introduced the enzyme-incision site of BamHI and XbaI into 5' and 3' end respectively. The products were cloned into the mRNA expression vector pcDNA3.1.All the above recombinant plasmids were sequenced to verify the correct gene insertion.4,In vitro transcriptionAll the constructs in pcDNA3.1 were cut singly with AgeI and in vitro transcribed into 5'-capped mRNA for microinjection by using mMESSAGE mMACHINE T7 Ultra kit.After a transcription reaction was done,the reaction mixture was treated with 2U TURBO DNase to remove the DNA template.Then the mixture was extracted with phenol/chloroform,and RNA was precipitated with LiCl.5,mRNA mieroinjeetionMouse fertilized eggs were microinjected using a micropipette and Eppendorf transferman manipulators mounted on a Olympus IX-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.Smmol/L EDTA(pH7.4) without nuclease contaminant.Eggs in control groups were microinjected with TE buffer,or not.6,shRNA microinjectionFertilized mouse eggs were microinjected using a micropipette and Eppendorf transferman manipulators mounted on a Olympus IX-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.shRNA(1 mg/ml) was microinjected into the pronucleus of one-cell embryos at G1 phase.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 microinjected with control shRNA,or microinjected with TE buffer.7,Examination of mRNA level by RT-PCR100 eggs were collected into 2ml eppendorf tube.The experiment was unde rtaken by the instruction of Ellustra^TM QuickPrep 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 ancl low salt buffer and the elution of mRNA.The RNA PCR Kit(AMV) Ver 2.1 Kit was used and we carried out to do the RT-PCR.For p110α(NM₀08839),the sense 5'-GAACCAGTAGGCAACCGTGAA-3' and antisense 5'-GCATCCTCCCCAG CATTAT-3' generated a 541-bp fragment.For S6K1(NM₀28259.4),the sense 5'-AGGGACTGGAGAGATGGGTC-3' and antisense 5'-GATCTGGGCAGGAGA CAGAA-3' generated a 644-bp fragment.For mTOR(NM₀20009.1),the sense 5'-GCACATTGACTTTGGGGACT-3'and antisense 5'- TCATGAGAGAAATCCC GACC-3' generated a 486-bp fragment.The primers of raptor(NM₀28898.2) w ere 5'-GGCGGACCTTACAGATTGG-3' and 5'-TTGGCTGCCAGTTCTCATAC-3', and the product was designed for 293bp.The primers of rictor(NM030168. 3) were 5'-CGAAGCATTTCCTGTCCC-3' and 5'-ACGGCTCCTGGTGACTTG-3', and the product was designed for 1348bp.Forβ-actin(NM₀07393),the sen se 5'-ACACTGTGCCCATCTACG-3' and antisense 5'-CAGGATTCCATACCCA AG-3' generated a 334-bp fragment.The PCR reaction system was 25μl.The re action condition wasâ‘ 94℃,3min;â‘¡94℃,30sec;â‘¢p110α55℃,S6K1 58℃,mTOR 57℃,raptor 54℃,rictor 50℃,β-actin 54℃,30sec;â‘£72℃,1min;⑤72℃,10 min;②—④30 -31cycles.The products were analysed by electrophoresis on agar ose gel stained with ethidium bromide to visualize PCR products on a UV trans illuminator.8,Western-blotting150 mouse 1-cell stage fertilized eggs were collected and transferred into 1.5ml Eppendorf tube.The extracts were briefly vortexed,quickly frozen on dry ice,and stored at -20℃until used.Laemmli sample buffer was added to the protein extracts, and the mixture was boiled for 5min and resolved on a SDS-PAGE gel.For immunoblotting,the fractionated proteins were transferred to a nitro-cellulose membrane.After that,5%BSA was used to incubate the nitrocellulose membrane 1 hour and then the nitrocellulose membrane was incubated by the specific antibody at 4℃overnight.The alkaline phosphatase-conjugated IgG was used as secondary antibody.The proteins were detected by using O-dianidine andβ-naphthyl acid phosphate as the substrates of alkaline phosphatase.The intensity of the signal was quantitated by computerized densitometry.9,ImmunofluorescenceEmbryos were washed in PBS with 0.1%BSA,and fixed in 4%paraformaldehyde in PBS(pH 7.4) for 1 h at room temperature.After being permeabilized with 0.2% Triton X-100 at room temperature for 30 min,embryos were blocked in 5%BSA in PBS for 1 h and incubated overnight at 4℃with 1:100 primary antibody.After being washed three times in PBS with 0.1%BSA,embryos were incubated with FITC-conjugated secondary antibody(dilution 1:50 in PBS).Then,embryos were washed again and counterstained with 10μg/ml PI for 5 min. For double staining of S6K1 and raptor,after staining with S6K1 overnight at 4℃and with second antibody FITC-conjugated donkey anti-goat IgG,the embryos were washed 3 times in washing buffer,and then stained with raptor for 3 h,followed by incubation with 1:100 TRITC-conjugated goat anti-rabbit IgG.After being washed three times in washing buffer,the embryos were stained with Hoechst 33258(Sigma; 10μg/ml in PBS) for 20 min.Finally,fluorescence was detected with Laser Confocal Scanning Microscope(Leica,Germany,400×magnification).As controls,fixed embryos were stained with affinity-purified IgG.Each experiment was repeated three times,and at least 50 embryos were examined in each group.The same instrument settings were used for each replicate.10,Activity assay of Akt and MPF by autoradiographyProtein extract from 20 embryos was incubated with 45μl of MPF/Akt buffer containing[γ-³²p]ATP at 37℃for 30 min,and the reaction was stopped by adding an equal amount of 2×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 or histone H2B was visualized by autoradiography.Result1,Expression of p110αin Mouse One-Cell Stage EmbryosIn order to detect p110αprotein 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 p110αprotein 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 p110αat G1,S,G2 and M phase were not significantly different. These results were compared to the quantity ofβ-actin,whose expression remained constant during the four phases.2,Subcellular Distribution of p110αin Mouse One- and Two-cell EmbryosImmunofluorescent staining was used to analyze the subcellular localization of endogenous p110α.Immunofluorescent staining of one- and two-cell embryos showed that p110αwas predominantly localized to the plasma membrane of embryos. Subcellular localization of protein can restrict its function to a precise cellular location.3,p110αshRNA—Mediated Inhibition of p110αExpressionTo explore a functional role for p110αin one-cell embryos,short hairpin interference RNA(p110αshRNA) was used to knock down the expression of endogenous p110α.The effect of p110αshRNA on p110αmRNA and protein expression was evaluated by RT-PCR and Western blot analysis,respectively.The knockdown was specific for p110α,given that control levels ofβ-actin did not change substantially.4,p110αshRNA and mRNA Regulate the Cleavage of One-cell EmbryosThe percentages of cell cleavage in each group were calculated after counting under a dissecting microscope 27 h and 32 h after injection of hCG.In the control groups,embryos remained at the one-cell stage 27 h after hCG injection,but around 68%of embryos reached two-cell stage 5 h later(total 32 h after the injection of hCG), and there was no significant difference among the three control groups.Compared with the control shRNA-treated embryos,p110αknockdown significantly inhibited one-cell embryo cleavage(about 11%).The embryos treated with p110αshRNA were blocked at G2 or M phase.This demonstrated that p110αshRNA interfered with G2/M transition of mouse one-cell stage embryos.Embryos microinjected with mRNA of p110α-WT remained in one-cell stage at 27 h after hCG injection.However,approximately 79%of embryos reached two-cell stage at 32 h after the hCG injection.For embryos microinjected with mRNA of p110α-AC,the percentage of cleavage reached nearly 90%at 32 h.The cleavage was also accelerated,as indicated by almost 24%of embryos having reached the two-cell stage at 27 h after the injection of hCG in this group.Irregular cleavage was observed at 32 h after the hCG injection.On the other hand,only about 38%of erabryos that were microinjected with p110a-KD mRNA reached two-cell stage 32 h after the hCG injection.As shown in result,microinjection of different kinds of mRNA of p110αcould regulate not only the time course of embryo cleavage,but also the rate of embryo cleavage. 5,p110αshRNA and mRNA Microinjection Regulate Akt and MPF ActivityThe regulation of the development of the one-cell stage by p110αshRNA and p110αmRNA microinjection into embryos suggests a role of p110αin regulating mouse embryonic cleavage.To investigate a potential mechanism by which p110αregulates one-cell embryo development,the effects of endogenous p 110a depletion as wells as the overexpression of three kinds of p110a were explored on Akt and MPF signaling.First,the time course for the activation of the endogenous Akt in one-cell embryos was probed.The Akt activity reached a maximum at 27 h after the hCG injection,whereas a substantial decrease of Akt activity was detected between 28 h and 30 h after the hCG injection.Therefore,the change in Akt activity at 26-27 h after the hCG injection was chosen to test in the following experiments.Akt activity was found to decrease significantly in the p 110αshRNA-microinjected group,compared to that of control shRNA group.Akt activity in p110α-WT mRNA-microinjected group was roughly twice as high as that in the control groups,and Akt activity in the p110α-AC mRNA-microinjected group was around 3 times higher than that of control groups. However,Akt activity in the p110α-KD mRNA-microinjected group was lower than in the control groups.Then,MPF activity was measured at different time points in each group.The results showed that in control groups,the peak of MPF activity occurred during 28 h to 29 h after hCG injection,lagging behind Akt activation around 1 h.In contrast,MPF activity of p110αshRNA group was far lower than that of the control groups.In the p110α-WT group,MPF activity reached its peak value at 27.5 h after hCG injection, about 30 min earlier than the control groups.In the p110α-AC group,the peak of MPF activity occurred at 27 h after hCG injection,about 1 h earlier than the control groups. In the p110α-KD group,MPF activity reached its peak value at 29 h after hCG injection,which was lower than that of the control groups.Here,we present data to show that knockdown of p110αand p110αconstructs regulated both activity value and time course of Akt and MPF in mouse one-cell embryos.6,Expression and Subeellular Localization of S6K1It was observed that mRNA levels of S6K1 seemed unchanged at G1,S,G2 and M phase.Immunoblot of S6K1 also showed that the 70-kDa band did not vary in either intensity or mobility at different phases of mouse one-cell embryos withβ-actin as an internal control.Immunofluorescent staining was used to analyze the subcellular localization of endogenous S6K1 in mouse one-cell embryos.Green fluorescent signals of S6K1 mainly distributed in the cytoplasm of one-cell embryos.7,Expression and Subcellular Distribution of Thr389 phospho -S6K1 in Mouse One-cell EmbryosSince phosphorylation of S6K1 Thr389 can indicate the activity of S6K1,we explored the expression of Thr389 phospho-S6K1 in mouse one-cell embryos by Western blot.It is interesting that S6K1 Thr389 appeared from G1 to M phase,and furthermore,the expression of Thr389 phospho-S6K1 increased in G2 and M phases, suggesting that S6K1 is active during the whole one-cell cycle.Since compartmentation is known to be an important mechanism that can regulate protein function,the subcellular localization of phospho-S6K1 was examined with phospho-specific Thr389 antibody.A strong immunofluorent staining of phospho-S6K1 was observed in the region of cortex,but the cytoplasm of one-cell embryos was still faintly stained.8,Co-localization of S6K1 and raptor during One-cell EmbryosThe correlated localization of S6K1 and mTORC 1 in mouse one-cell emabryos was also examined by double staining with anti-S6K1 and anti-raptor antibodies.The distribution of S6K1 was coincident with that of raptor in mouse embryos.Both S6K1 and raptor were localized in the cytoplasm of the embryo.9,mTORC1 is Necessary for S6K1 Thr389 PhosphorylationTo testify which mTOR complex is essential for the phosphorylation of S6K1,the expression of mTOR and its associated proteins were inhibited by microinjection of short hairpin interference RNAs(shRNAs).The effect of knockdown on mTOR and its associated proteins mRNA and protein expression was evaluated by RT-PCR and Western blot respectively.The results showed that the microinjection of mTOR shRNA could suppress the transcription of mTOR,and raptor shRNA could decrease the mRNA level of raptor,as well as rictor shRNA.β-actin was also detected as an intrinsic control.The data showed that there were the same protein quantity in each group and shRNA could inhibit specially the substrate mRNA.The reduced expression of mTOR, or raptor,but not rictor,strongly inhibit the phosphorylation of S6K1 Thr389.Conclusion1,The p110αexpression did not change in the first mitosis of mouse fertilized eggs.p110αwas predominantly localized to the plasma membrane of embryos.2,Silencing of p110αprevented the activation of Akt and MPF and resulted in a G2/M arrest.The synthesized mRNA coding for a constitutively active p110αinduced cell cleavage more effectively than wild-type p110αmRNA,whereas mRNA of kinase-deficient p110αdelayed the first mitotic cleavage,p110αis significant for G2/M transition of mouse one-cell embryos and p110αcould regulate the activity of MPF through PI3K p110α/Akt signaling.3,S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases.When S6K1 is phosphorylated on Thr389 site,its subcellular distribution translocated from cytoplasm to embryo cortical,mTORC1 is necessary for the phosphorylation of S6K1 on Thr389 of its hydrophobic motif.