Invetigation Of Cdc25B Regulating Mouse Oocytes Maturation

IntroductionMammalian animal oocytes are naturally arrested at late G2 stage of meiosis Iwhich is also named GV stage because the typical morphologic character at this stage isa big nucleus called germinal vesicle(GV). After reinitiation of meiosis, germinalvesicle breaksdown, chrosome condense, this first body forms and the second meiosisimmediately continues until the oocyte become a mature, fertilizable egg. During theresumption of meiosis, the activation of maturation promoting factor (MPF) is thecentral step MPF is a heterogeneous dimmer, which is composed of cyclin-dependentkinase (CDK1) and cydinB. Phosphorylation and dephosphorylation of Thr-14 andThr-15 in CDK1 is the important regulation of MPF activition.cdc25 family is a dual specific protein phosphatase, which can dephosphorylate theThr-14 and Thr-15 in CDK1 and activated MPF at the key cell cycle transition. Thephosphorylation and dephosphorylation of cdc25 also regulate its activation, theinteraction with 14-3-3, subcellular localization are involved in the kinase activity. TheSer-321 is a novel phosphorylation site discovered by our laboratory, the mutation ofSer-321 changed the distribution of cdc25B, simultaneously pretreated mouse oocyteswith Leptomycin B (LMB) suggested that there existed a protein export from nucleusto cytoplasm though GV stage oocytes seemed to be quilt.In this study, in order to detect the involvement of cdc25B with oocytes GVBD, Weconstructed several mutants microinjected into GV stage oocytes to observe theinformation about resumption of meiosis and the proteins distribution. Materials and methods1,Materials and reagentsKunming strain mice were obtained from the Department of Laboratory Anim-als,China Medical University; mMESSAGE mMACHINE kit (Ambion); The PBSK-cdc25B, PEGFP-G3, pBluescriptâ…¡/SK vector, and JM109 were previously reserved inour lab. Restriction endonuclease XhoI, BamHI, XboaI and DNA polymerase from M-BI; DNA Fragment Recovery Kit from Tiangene Anti-cdc2-pTyr15 monoclonal antibo-dy, FITC labed goat-anti-rabbit IgG From Santa-Cruz.2,Collection and Culture of Mouse OocytesImmature germinal vesicle (GV) stage oocytes were collected from 20- to 28-day-old Kun- ming strain mice that had been injected with 5 IU pregnant mare.s serumgonadotrophin 48 hours before as previously described. Mice were killed by cervicaldislocation and the ovaries were placed in M₂ medium containing 200μM dibutyryl cy-clic adenosinemonophosphate (dbcAMP). The folic-les were punctured with a fineneedle to release the cumulus-enclosed oocytes or naturally denuded oocytes. Oocyteswere freed of attached follicular cells by repeated pipetting with a mouth-operatedmicropipette. The released oocytes were collected and washed three times in M₂contains 200μM dbcAMP. They were cultured in a drop of medium, at 37℃,in ahumidified atmosphere of 5% CO₂ in air, under paraffin oil. Incubation wasperformed in Waymouths MB752/1 Media (Invitro- gen) supplemented with 100μg/mLsodium pyruvate, 50 U/mL penicillin, 50μg/mL streptomycin sulfate, 3 mg/mL bovi-ne serum albumin (BSA,), which was named MB medium.3,Preparation of recombinant plasmidspBSK-cdc25B and pEGFP-cdc25B was constructed by Zhang Yang, 1-52 and1-65 amino acids deletion of cdc25B N-terminal mutants by PCR were amplified bypolymerase chain reaction (PCR) using primers. Leucine 62 to Alanine (L62A) and Serine 149 to Alanine(S149A) mutants,PBSK-cdc25B-L62A/S 149A was constructed by a single-site mutantgenesis kit (Merk).All the mutants were then subcloned into the pEGFP-C3 vector to express thepEGFP-cdc25B-S149A, pEGFP-cdc25B-Δ51, pEGFP-cdc25B-Δ65, pEGFP-cdc 25B-L62A and mutants. Fused plasmids were for subcellular localization analysis.4,In vitro transcriptionAll the constructs in pBSK were cut singly with XbaI and in vitro transcribedinto 5'-capped mRNA for microinjection by using mMESSAGE mMACHINE kit(Ambion). The in vitro synthesized mRNA was dissolved in nuclease-free 5 mM Trisand 0.5 mM EDTA (TE), pH 7.4. We determined mRNA yield by measuringabsorbance at 260 nm and by carrying out modified nondenaturing gels loaded withRNA.5,Microinjection and observation of the mouse oocytesVarious mRNAs were microinjected into GV-stage oocytes using a micropipetteand Eppendorf manipulators mounted on a Olympus inverted microscope as previouslydescribed). Oocytes were placed in a drop of M₂ contained 200μM dbcAMP underparaffin oil in a lid of a 3 cm Falcon culture dish. Typical injection volume was 5% ofthe total cell volume, or 10 pL. They were subsequently scored for the continuedpresence of the GV, germinal vesicle breakdown(GVBD), or death and abnormalitywith an inverted microscope fitted with a DIC contrast lens, and the results wereanalyzed statistically.6,Western blottingProtein extracts of mouse oocytes were prepared by adding 200 oocytes in aminimal volume of collection medium to 20μl of protein extraction buffer and themixture was boiled for 5 minutes and resolved on a 12% SDS-PAGE gel. Forimmunoblotting, the fractionated proteins were transferred to a nitro-cellulosemembrane, and blocked with 3% BSA in Tris-buffered saline containing 0.05%Tween-20(TBST). The membrane was incubated overnight at 4℃with a 1:500 dilution of primary antibody against pTyrl5 of Cdc2 antibody in TBST (1% BSA),washed three times with TBST, incubated for 2 hours at room temperature with a 1:2,000 dilution of HRP-conjugated anti-rabbit IgG secondary antibody in TBST(1%BSA), and finally washed three times with TBST. The proteins were detected byenhanced chemical luminescence, exposed with X-ray film in the dark room.7,Analysis of cdc25B subcellular localizationVarious plasmids were diluted to a desired concentration and injected into the GV.Typical injection volume was 1-2 pl. After microinjection the oocytes were transferredfrom M₂ medium to MB medium containing 500μM dbcAMP. The injected oocytewere observed and photographed with Leica DMIRE2 fluorescence microscope at theindicated time.8,Immunoflueresence to analyse the distribution of cdc25B andecotic in mouse oocytesCollect oocytes at GV stage and G₂/M transition, fixed in 4% polyformaldehyde,perforate with 0.1% Triton-X100; immunoreaction; after block for an hour, the oocyteswere incubating with the first antibody and the FITC conjugated second antibody, stainthe DNA with PI; observed under the fluorescene microscope and take picture9,The expression of protein in BL₂₁cdc25B and cdc25B-S321A were subcloned to PEGX-4T2-cdc25B andPEGX-4T2-cdc25B-S321A, which were transformed BL₂₁, cultured 6-8 h at 37℃,collected and performed SDS-PAGE electrophoresis to identify the expressed proteins.Results1,The influence of 149 serine on the mouse oocytes maturationWild type cdc25B promoted the meiosis progression. The mutation of Serine 149to Alanine lead to the abrogation of inducing function of cdc25B; the distribution alsowas changed, obviously nuclear detention was observed. 2,The identification of NES in cdc25B in mouse oocytes.The deletion mutatants 1-51 and 1-65 amino acid, cdc25B-Δ51 andcdc25B-Δ65 all made the cdc25B abolish the role as a meiosis inducer. Thedistribution analysis demonstrated cdc25B-Δ51 appeared in cytoplasm and nuclei,cdc25B-Δ65 mainly in nuclear. The results of microinjection and immunofluoresencewere consistent that WT-cdc25B was mainly in cytoplasm. However, during G₂/Mtransition, We observed obviously nuclear accumulation of cdc25B-WT in mouseoocytes.3,Expression ofcdc25B-WT and cdc25B-S321A in BL₂₁The result of western-Blot showed that approximately 27KD of GST protein and60KD objective protein were detected.DiscussionThe activation of MPF is the trigger of meiosis resumption, cdc25 plays an veryimportant role during this process, cdc25 activated at G₂/M transition anddephosphorylated Thr-14 and Thr-15 in CDC2, then activated MPF, promoted cellcycle progression. There are three members in cdc25: A,B and C. They share about50% similarity at the protein levels so they are involved in specific regulatory process.More evidences support that cdc25B plays a essential role during cell cycle. Mammaliaoocytes are naturally arrested at G2 stage, meiosis can be resumpted by activated MPF,wild type cdc25B can promote cell cycle progress in a dosen dependent manner.In this study, Serine 149 to Alanine mutation was constructed, the results indicatedthat Serine 149 mutation made cdc25 abolished inducing meiosis function and thesubcellular localization showed that protein was retended in the nuclei while wild typecdc25B was mainly in the cytoplasm at G₂ stage. A markly nuclear accumulation ofcdc25B at G₂/M transition was observed which suggested that there was a shufflingbetween nuclear and cytoplasm at G₂ stage oocytes though they seemed very quilt apparently. The movement of cdc25B associated with cell cycle progressionclosely.Several evidences provided in this study assured that there surely was afunctional NES in N-terminal of cdc25B. The crucial amino acid Leucine 62 mutant toAlanine also abrogated the normal NES function include export protein and the meiosisreinitiation.In conclusion, We have shown for the first time that Serine 149 was a activatedphosphorylation site, the mutation of it influenced not only the protein function but alsothe distribution. We identified that there was surely a functional NES between 51-65amino acids, whose crucial amino acids mutation resulted in the loss of normal nuclearexport and meiosis inducer. All above will help us understand better meiosis reinitiationand cell cycle regulation in development.Conclusion1,Serine 146 phosphorylation associated with the meiosis inducing function andinfluenced the protein subcellular localization2,A functional NES was between 52-65 amino acids in cdc25B and involvedproper distribution and normal function. Leucine 62 is a crucial amino acid in the NES.