The Variety Of The Cdc25B Activity At Different Cell Stage In The Early Development Of Mouse Fertilized Egg

IntroductionThe cell cycle is a very important process in cell development and differentiation. The initiation and process of cell cycle is under restrictive regulation by many factors. Among these maturation -promoting factor (MPF) is a very important one involved in the key cell cycle transitions. It is a heterogeneous di-mer, which is composed of cyclin - dependent kinase - 1 ( CDK - 1) and cy-clinB.Recently Cdc25 family, which is the upstream phosphatase of CDK1 is significantly noticed by many scientists. The Cdc25 phosphatases are involved in the dephosphorylation and activation of CDK - 1 at the key cell cycle transitions. There are three Cdc25 phosphatases in mammals;Cdc25A, B and C. The specific role of these enzymes in the control of the cel cycle is still not clear. It was initially proposed that Cdc25A acted at the G1/S transition, and Cdc25C at mitosis. However, recent reports have demonstrated that Cdc25A is also involved during mitosis and that Cdc25C may be required during S - phase. But Cdc25B is the most important one to regulate cell cycle transition. It is not only involved in the control of entry and progression into mitosis, but also in Gl/S transition. At the same time Cdc25B is the central factor in regulating the oocyte meiosis reinitiation. As the CDK1/cyclinB complex accumulates in the S and G2 phases of the cell cycle, certain kinase keeps CDK1 inactive by phos-phorylating two residues, Thr -14 and Tyr -15, in CDK1. The Cdc25B can remove of these two inhibitory phosphates to active CDKl/cyclinB. The Cdc25B will be activated after phosphorylation by certain kind of protein kinase. The Ser -321 is a novel phosphorylation site discovered by our laboratory. My experiment is to detect the amount of the phosphorylated Cdc25B using the specific Ser-321 phosphorylated Cdc25B antibody. With these we will observe the role of Cdc25B in controlling the cell cycle progression.Materials1. KUNMING mice were supplied from the Department of Laboratory Animals, China Medical University2. Pregnant mare serum gonadotropin(PMSG) and human chorionic gona-dotropin (hCG) were obtained from Tianjin Huafu Biological Products Research Institute and Shanghai Products Research Insititute, respectively.Anti - cyclinB mouse monoclonal antibody from Wuhan Shanying Biotechnology CompanyAnti - Cdc25B Ser - 321 phosphorylation mouse monoclonal antibody from Wuhan Shanying Biotechnology CompanyAnti - mouse and anti - goat IgG secondary antibody from Santa Cruz BiotechnologyTritonX -100 from BOEHRINGER MANNHEIN GmbHDL -2000 DNA marker from TaKaRa CompanyThe primers for RT - PCR were kindly, provided by the Institute of Molecular Biology and Tumor Research, Philipps University.Methods1. Collection and culture of mouse GV stage oocytesMouse GV stage oocytes were collected from 3 ~ 5 - week - old Kunming strain mice that had been injected 48 h previously with 5 IU pregnant mare' s serum gonadotrophin. Mice were killed by cervical dislocation and the ovaries placed in M2 medium containing 125jxmol/L dibutyryl cyclic adenosine mono-phosphate (dbcAMP). The follicles were punctured with fine needle and let release the oocytes. Only oocytes with easily removable cumulus cells and a germinal vesicle were selected for incubation. The released oocytes were collected andwashed three times in M2medium. They were cultured in a drop of medium, at 37 °C , in a humidified atmosphere of 5% C02 in air, under paraffin oil. Incubation was performed in MB medium (Waymouth's Media MB752/1 supplemented with 100 g/ml sodium pyruvate, 50U/ml Penicillin, 50ug/ml streptomycin sulfate, 3mg/ml bovine serum albumin).2. Superovulation and in vivo fertilizationFor superovulation, female KUNMING mice 3-5 week - old were injected with pregnant mare serum gonadotropin (PMSG) , and after 46 ~48 hours with human chorionic ginadotropin ( hCG). Then one female mouse with one male mouse in one cage overnight.3. Collection and culture oocytes at different cell stageAfter injection hCG 10 hours, Gl stage fertilized eggs were collected from oviduct of females. Then half of the Gl stage fertilized eggs were transferred to M16 for culture in incubator with an atmosphere of 5% CO2. According to the time point, collected S or G2/M phase eggs after injection hCG 20 or 28 hours. The M II stage oocytes were collected from unfertilized mouse oviduct.4. Western Blot analysisWe treated 200 eggs in a series of steps including lysing them in lOul of lysis buffer(50mM Tris - HC1 pH 7.5 , 250mM NaCl, 5mM EDTA,lmM DTT , 0.1% Triton ,50mM sodium orthovanadate, lOOug/mg PMSF and TPCK, 50ug/ ml TLCK, lug/ml leupeptin pepstatin and aprotinin) , frozing them below -70 Xl, and then thawing them at room temperature for 10 min. We did the same steps for three times, so we could get the extraction using the steps mentioned. Laemmli sample buffer was added to the extracted protein, which were then boiled for 5 min. The protein samples were separated by 10% SDS - PAGE and transferred to nitrocellulose membrane. The membranes were blocked in 5% BSA for 2 ~ 3 h at 37 °C. Later the Membranes were incubated overnight at 4 *€ with mouse cyclinB monoclonal antibody 1:300 (Cdc25B Ser-321 phosphorylation monoclonal antibody 1:250) in TBS/milk with 0. 1% Tween 20, followed by washing the membrane in TBS/milk for 5 min three times. Blots were then incubated for 1 h at room temperature in secondary antibody {1:2000). The proteins were detected by using O - dianidine and (3 - naphthyl acid phosphate as thesubstrates of alkaline phosphatase.5.RT-PCRWe treated 200 eggs at different cell stages, then isolated and analyzed the total RNA.Primerl:5'-TCTAGCTAGCCTTTGCCCGC -3'Primer2:5'-GGTCATTCAAAATGAGCAGT -3'Condition of RT-PCR: 94X 2min, 1 cycle;95 X 15sec, 55 <€ 30sec, 72X1 30sec,28 cycles;final elongation at 72X1 5min. The product was stored atAnalysis of the product: Use IOjjJ of the product to be analyzed with 8% PAGE.Condition of electrophoresis: 150V,30mA. After EB stained, the gel was photographed.Result1. The quantity of the phosphorylated Cdc25B has not much difference between the GV and MII stage of oocyte.Western blot analysis of Cdc25B in GV and M II stage of oocyte with the Cdc25B monoclonal antibody against the phosphorylated Ser 321, using murine spleen and lung as normal control, reveals that there is no obvious difference between these two cell stages.2. The quantity of the cyclinB is a little lower in Gl and S phase fertilized eggs than in G2/M transition stage.Western blot analysis of cyclinB in Gl and S, G2/M transition stage fertilized eggs with the cyclinB specific monoclonal antibody. This study reveals the quantity of the cyclinB is a little lower in Gl and S transition phase fertilized eggs than in G2/M transition stage.3. The quantity of the phosphorylated Cdc25B is much higher in G2/M transition stage fertilized eggs than in Gl and S phase fertilized eggs.I analyzed the phosphorylation form of Cdc25B in Gl, S and G2/M transition stage of zygote with the Cdc25B monoclonal antibody against the phosphoryl-ated Ser 321. This Western blot result reveals the quantity of the phosphorylated Cdc25B is much higher in G2/M transition stage fertilized eggs than in Gl and S phase fertilized eggs. This shows us that the activity of Cdc25B is necessary for G2/M transition stage.4. The quantity of Cdc25B mRNA fluctuates not much at different cell cycle stage.Reverse PCR analysis of the Cdc25B mRNA at different cell stage. The result shows that there is not much difference among GV, M H stage of oocyte, Gl, S and G2/M stage of zygote. This reveals that the transcription of Cdc25B is constant at different cell stage.Conclusion1. The transcription of Cdc25 B mRNA has not much difference among oocyte and different cell cycle stages of fertilized eggs.2. The activation of Cdc25B is essential to promote the G2/M transition in fertilized egg.3. The Ser -321 of Cdc25B is an effective phosphorylation site..