Involvement Of 14-3-3 And Cdc25B In Early Development Of Mouse Fertilized Eggs

IntroductionThe cell cycle consists of G1 phase, S phase, G2 phase and M phase. The mousefertilized eggs is the most simple and natural cell cycle model in vertebrate which is closerto human. But the mechanism on the regulation of early development of mouse fertilizedeggs is unclear. The 14-3-3 proteins are a family of highly conserved 28-31 KDa acidicadapter proteins. In mammals, there are at least seven isoforms includingβ,ε,η,ζ,Ï„,γ,σ. Up to 15 isoforms are present in plants and two isoforms have been identified inyeast, Drosophila melanogaster and Caenorhabditis elegans. The seven highly conserved14-3-3 proteins expressed in mammalian cells form a complex pattern of homo- andhetero-dimers. They play a important role in a variety of cellular processes including cellcycle control, signal transduction, apoptosis, cell grow, tumor formation, stress response,pathogenesis and progression of diseases. 14-3-3 acts as an adaptor or "chaperonemolecule", which is able to move freely from cytoplasm to nucleus and vise-versa.In mammalian cells, there are three Cdc25 isoforms—Cdc25A, -B, and -C—all ofwhich have been implicated in the regulation of mitosis. Cdc25A was originally thought tobe active only at the G1/S transition, but has recently been implicated as a mitotic regulatoras well. Cdc25B's role in the initiation of mitosis is also suggested by the findings thatCdc25B can active cyclinB1-Cdk1 and produces G2/M transition. Mammalian Cdc25C isinactive during most of the cell cycle but is activated in mitosis by hyperphosphorylationmediated by cyclinB1-Cdk1, suggesting a positive feedback loop. Mice with Cdc25Bdisrupted are also viable and healthy, although females are sterile due to a meiotic defectduring oogenesis. The meiosis arrest is restored by microinjecting Cdc25B mRNA. So, Cdc25B is required for the mouse oocyte maturation. The phosphatase activity and thelocalization of Cdc25B are also regulated by14-3-3 proteins. Recent reports provideevidence that Cdc25 in the G2-arrested Xenopus oocytes is bound to 14-3-3 proteins. Littleis known about the effect of 14-3-3 and Cdc25B on the regulation of early development ofmouse fertilized eggs. Here we report that the localization of Cdc25B is regulated by14-3-3 proteins in mouse fertilized eggs. Cdc25B protein is required for the developmentof mouse fertilized eggs. Our findings identify Cdc25B as a target of PKA and providenew insight into the effect of PKA on the regulation of the development of mouse fertilizedeggs.MaterialsFemale mice (KUMING strain, 4-5 weeks old) were supplied by the Department ofLaboratory Animals, China Medical University. The full length Cdc25B cDNA clone waskindly provided by Prof.Tony Hunter at The Salk Institute. pEGFP-C3 vector is a gift fromDr. Liou Yu in FuDan university, pGEM-T Easy vector was purchased from Promega.pBluescriptâ…¡/SK vector was purchased from Stratagene. E.coli JM109 was purchasedfrom TaKaRa. mMESSAGE mMACHINE kit was purchased from Ambion. QuickChangeSite-Directed Mutagenesis Kit was purchased from Stratagene. QIAprepSpinMiniprep Kitwas purchased from Qiagen.Restriction endonuclease XhoI, BamHI and Xbal werepurchased from MBI. Anti-cdc2 pTyr15 monoclonal antibody, anti-14-3-3 polyclonalantibody and anti-Cdc25B monoclonal antibody were purchased from Santa Cruz.M-P-Cdc25B-S321 antibody was purchased from Proteintech Group Inc. M2, M16 andHoechst 33258 from Sigma. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbitIgG and rhodamine (TRITC)-conjugated rabbit anti-goat IgG were purchased from ZSGB.Methods 1,Super-ovulation and eggs colletionFemale mice were injected intraperitoneally with 10IU pregnant mare serumgonadotropin (PMSG) for Super-ovulation. After 48h, the human chorionic gonadotropin(hCG) was injected to the mice. A single female was placed with a single male of the samestrain, and one-cell embryos were collected with M2 medium the next day from theoviducts of females possessing a vaginal plug. The embryoswere then cultured in M16medium at 37℃in a humidified atmosphere of 5% CO₂ in air.2,Indirect immunofluorescence microscopyMouse fertilized eggs at various phases were fixed with 2% paraformaldehyde andpermeabilised with 0.1%Triton X-100. Fertilized eggs were stained overnight withanti-14-3-3 polyclonal antibody and anti-Cdc25B monoclonal antibody at 4℃. Afterwashing three times with PBS, the fertilized eggs were incubated 1 hour with FITC-goatanti-rabbit lgG or TRITC rabbit anti- goat lgG at 37℃. The DNA was stained with Hoechst33258 for 30 min at 37℃. Signals were detected using fluorescence microscope.3,Construction of Cdc25B wild type and mutant expressionvectorsUsing Site-directed Mutagenesis Kit to mutate serine 229 to alanine of Cdc25B and thetwo mutants were called pBSK-Cdc25B-S229A and pBSK-Cdc25B-S229A/S321A. All therecombinant plasmids were sequenced to verify the mutation successful.4,Construction of pEGFP-Cdc25B plasmidsThe four types of pBSK-Cdc25B plasmids were subcloned into pEGFP-C3 vectorafter digestion of XhoI and BamHI. The recombinants were namedpEGFP-Cdc25B-S229A/S321A, pEGFP-Cdc25B-S321A, pEGFP-Cdc25B-S229A andpEGFP-Cdc25B-WT respectively.5,In vitro transcriptionAll the constructs in pBluescriptâ…¡/SK were cut with XbaI and in vitro transcribed into5'-capped mRNA for microinjection by using mMESSAGE mMACHINE kit.After atranscription reaction was done, the reaction mixture was treated with DNaseâ… to remove the DNA template. Then the mixture was extracted with phenol/chloroform, and RNA wasprecipitated with LiCl. DNA was resuspended in TE buffer at a concentration of0.5-3μg/μl.6,Fertilized eggs microinjectionMouse fertilized eggs were microinjected using micropipette and Eppendorftransferman manipulators mounted on a Olympusâ…¨-70 inverted microscope with DICoptics. Eggs were placed in a drop of M2 medium under paraffin oil in a lid of 3cm Falconculture dish. Typical injection volume was 5% of the total cell volume, or 10 pl. Eggs incontrol groups were either not microinjected, or microinjected with the same amount of TEbuffer. The percentages of cell division and cell survival were counted under a dissectingmicroscope 25-35 hr after injection of hCG, and the results were analyzed statistically.7,Western-blottingProtein extracts of mouse embryos were prepared by adding appropriate number ofembryos in a minimal volume of collection medium to 20μl of protein extraction buffer.The extracts were briefly vortexed, quickly frozen on dry ice, and stored at -20℃untilused. Laemmli sample buffer was added to the protein extracts, and the mixture was boiledfor 5 min and resolved on a 10% or 12% SDS-PAGE gel. For immunoblotting, thefractionated proteins were transferred to a nitrocellulose membrane. The primary antibodyagainst 14-3-3,β-Actin, Cdc25B, pTyr15 of Cdc2 and M-P-Cdc25B-S321 antibody wereused and HRP conjugated lgG was as secondary antibody. The proteins were detected byusing an enhanced chemiluminescence detection system.8,Production of glutathione S-transferase (GST) fusionproteinsA 360 bp section of a mouse Cdc25B cDNA, encoding amino acides 212-330 wasamplified by PCR and cloned into the pGEX-4T-2 vector. GST fusion proteins wereexpressed in E.coli BL21 (DE3) cells transformed with the plasmids ofpGEX-4T-2-Cdc25B-WT, pGEX-4T-2-Cdc25B-S321A, pGEX-4T-2-Cdc25B-S229A andpGEX-4T-2-Cdc25B-S229A/S321A. The corresponding proteins were expressed in the presence of isopropyl-β-D -thiogalactopyranoside (IPTG).Results1,The expression and localization of 14-3-3 and Cdc25BThere is one subtype of 14-3-3 proteins at all phases in mouse fertilized eggs. Theexpression of 14-3-3 proteins was identical in mouse fertilized eggs. Western blotsrevealed that Cdc25B protein was phosphorylated at G1 and S phase, dephosphorylated atG2 and M phase. The expression of Cdc25B protein increased at G2 phase and decreasedat M phase. At various phases of fertilized eggs investigated, 14-3-3 proteins had a mainlycytoplasmic distribution and only low level of fluorescence were observed in the nucleus.The localization of Cdc25B proteins was mainly in cytoplasm and nucleus at G1, but incytoplasm cortex at S and G2 phases whereas in whole cell at M. The 14-3-3 wasco-localized to cytoplasm cortex immunolabed for Cdc25B at S and G2 phases. To furtherinvestigate the sub-cellular localisation of the mouse Cdc25B phosphatase, we made afusion protein, tagging GFP to the N-terminus of Cdc25B, and analysed its distributionvariation at M phase in mouse fertilized eggs. We found that Cdc25B protein transfersfrom cytoplasm to nucleus in part at M phase in mouse fertilized eggs.2,The detection of phosphorylation status of cdc2-Tyr15 inmouse fertilized eggsThere was strong phosphorylation signal of cdc2-Tyr15 detected at G1 and S whereasonly slight phosphorylation signal of cdc2-Tyr15 found at G2 and no phosphorylationsignal of cdc2-Tyr15 was identified at M phase in mouse fertilized eggs.3,Effect of Cdc25B mRNA microinjection on the development ofmouse fertilized eggs and detection of the phosphorylation statusof cdc2-Tyr15To determine if Cdc25B protein is important for the mitotic cell cycle of mousefertilized eggs, we created a series of Cdc25B mutants and assessed their effects on development of mouse fertilized eggs. We found that the G1 phase of mouse fertilized eggsinjected with mRNA of Cdc25B-S229A/S321A and Cdc25B-S321A enter mitosis 26.5hrand 27hr after the hCG injection respectively, and the percentage of cleavage was up to90% and 80.2% respectively. Embryos microinjected with mRNA of Cdc25B-S229Aentered mitosis 27.5hr after the hCG injection while the eggs entered mitosis with mRNAof Cdc25B-WT 28hr after the hCG injection. Only 75.2% and 75.5% of embryos reachedtwo-cell stage in Cdc25B-S229A and Cdc25B-WT group respectively. Thephosphorylation status of cdc2-Tyr15 after microinjection of mRNA ofCdc25B-S229A/S321A, Cdc25B-S321A, Cdc25B-S229A and Cdc25B-WT was not found26.5hr, 27hr, 27.5hr and 28hr after the hCG injection respectively indicating that MPF hasbeen activated.4,Effect of Cdc25B antibody microinjection on cleavage of mousefertilized eggsEmbryos microinjected Cdc25B antibody at G1 phase remained at one-cell stage28.5hr after the hCG injection, but there was about 10.9 per cent fertilized eggs enteringtwo-cell stage 35hr after the hCG injection.5,Effect of PKA/Cdc25B signal path on the development of mousefertilized eggsTo further explore the relationship between the PKA activity and Cdc25B proteins, weexamined the effect of microinjecting diffent kinds of Cdc25B mRNA on the developmentof mouse fertilized eggs under the condition of PKA activated successively. We thereforeinjected 1-cell stage embryos with Cdc25B-S229/S321A mRNA, Cdc25B-S321A mRNA,Cdc25B-S229A mRNA and Cdc25B-WT mRNA respectively. In the control groups,embryos remained at the one-cell stage 28.5 hr after the hCG injection (G2 phase), butabout 10% of embryos reached two-cell stage 35 hr after the injection of hCG and therewas no significant difference between the two control group. On the other hand, theembryos of G1 phase microinjected with Cdc25B-S229/S321A mRNA entered the mitosis30 hr after the hCG injection with the cleavage rate at 57.4%. The embryos with Cdc25B-S321A mRNA injection entered M phase 30.5 hr after the hCG injection andabout 43.2% of embryos reached two-cell stage 32.5 hr after the injection of hCG. Thepercentage of cleavage was up to 28.1% and 28.6% 31 hr and 31.5 hr after the hCGinjection in Cdc25B-S229A mRNA injection group and Cdc25B-WT mRNA injectiongroup respectively. There was a strong signal of cdc2-Tyr15phosphorylation detected 31 hrafter the hCG injection in control group whereas a slight signal of cdc2-Tyr15phosphorylation found in the Cdc25B-S229A/S321A group as well as Cdc25B-S321Agroup 29 hr and 29.5 hr after the hCG injection respectively. We detected a slight signal ofcdc2-Tyr15 phosphorylation 30 hr and 30.5 hr after the hCG injection in Cdc25B-S229Agroup and Cdc25B-WT group respectively. The results showed that not only the Cdc25Bmutant mRNA but also the Cdc25B wide type mRNA can restore the mouse fertilized eggsmitosis arrest caused by the PKA partly.6,The phosphorylation status of Cdc25B protein serine 321In order to identify if the serine 321 of Cdc25B was phosphorylated, we detected thephosphorylation status of Cdc25B protein serine 321 in mouse fertilized eggs usingphospho-specific antibody against Cdc25B serine 321 that primarily recognise thephosphorylation form of Cdc25B serine 321. We found that the S321 was phosphorylatedat G1 and S whereas the S321 was dephosphorylated at G2 and M in mouse fertilized eggs.7,Overexpression of Cdc25B protein disturbs the two-cell stagearrest in mouse embryosThe Cdc25B proteins were expressed 42 hr after the hCG injection. The mouseembryos with Cdc25B proteins expressed reached four-cell stage 48 hr after the hCGinjection with the percentage of cleavage over 30% compared with the embryos in controlgroup which still remained two-cell stage at that time. Irreguar division of minorityembryos occurred 48 hr after the hCG injection in Cdc25B overexpression groups. So,Cdc25B overexpression in early two-cell stage eggs promoted their development into thefour-cell stage.8,Cloning of mouse Cdc25B cDNA specific fragment and expression in Escherichia coliThe prokaryotic expression plasmids were successfully constructed with GST tag,SDS-PAGE assays yielded four roughly Mr 40000 expressed proteins. Western blotsrevealed that the expressed proteins corresponding to 40kDa could react with GSTpolyclonal antibody.DiscussionNucleocytoplasmic transport of proteins plays an important role in the regulation ofmany cellular processes. It is reported that 14-3-3 proteins rapidly shuttle in and out of thenucleus through active transport and that the distinct subcellular distributions of 14-3-3proteins are caused by differences in nuclear export. In this study, the immunofluorescencestaining revealed that 14-3-3 proteins locate mainly in the cytoplasm and only low levelsare presented in the nucleus. These data suggest that 14-3-3 proteins perform the effects ofregulating cell cycle of mouse fertilized eggs through its localization. In addition, Cdc25Bproteins expression was tightly regulated in our report, accumulated rapidly as fertilizedeggs entered G2 phase and then decreased as fertilized eggs exited mitosis. Thephosphorylation status of Cdc25B in cell cycle indicated that Cdc25B proteins have rolesin regulating S/G2 and G2/M progression in our present study. Cdc25B2 fused to greenfluorescent protein, partitioned primarily in the cytoplasm during G1 and progressivelymigrates to the nucleus as cells transites from S to G2/M phase in Elizabeth's test. Thesubcellular redistribution of Cdc25B2 could be functionally important for G2/Mcheckpoint regulation. Additionally, the binding of 14-3-3βdroves Cdc25B to thecytoplasm and that mutation of serine-309 to alanine completely abolishes the cytoplasmiclocalization of Cdc25B. Nuclear exclusion of Cdc25B is required for promoting mitosis. Itis well known that the master regulator of mitosis in mammalian cells is the proteincomplex consisting of cyclin B1 and cdk1(cdc2). It was reported that Cdc25B is able toactive MPF efficiently and promote mitosis. Moreover, the complex of cyclin B1-cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integrationfor the proteins that trigger mitosis. Recently it has been shown that Cdc25B specificallyactivates cyclin B1-cdk1 on centrosomes prior to cdc25A. In conclusion, we suggest thatCdc25B proteins are phosphorylated when it transfers from nucleus to cytoplasm entirelyas the mouse fertilized eggs transites from G1 to S phase according to our results. Themouse fertilized eggs remain at S phase with the binding of Cdc25B proteinsphosphorylated to 14-3-3 proteins. The mouse fertilized eggs enter mitosis on account ofthe activation of MPF in the cytoplasm when the Cdc25B proteins is dephosphorylated atG2. The activating of MPF in nucleus region by Cdc25B induced the completion ofmitosis.Cdc25 phosphatases family which including Cdc25A, Cdc25B and Cdc25C controlcell cycle progression through dephosphorylating and activating cyclin-dependentkinase/cyclin complexes. In order to identify the effect of Cdc25B on the development ofmouse fertilized eggs, we constructed a variety of Cdc25B mutants includingCdc25B-S229A and the double mutant of Cdc25B-S321A/S229A.The mouse fertilizedeggs took place G2/M transition with Cdc25B-S321A, Cdc25B-S229A/S321A andCdc25B-S229A mRNA microinjection in advance compared with Cdc25B-WT group. TheCdc25B-S229A/S321A mRNA microinjection group increased the percentage of cleavagesignificantly. In contrast to the Cdc25B-WT mRNA microinjection group, the fertilizedeggs injected Cdc25B-S229A mRNA entered mitosis in advance. We also found that theMPF in Cdc25B-S229A/S321A group was activated prior to the other groups by detectingthe cdc2-Tyr15 phosphorylation status. These data indicated that the serine 321 is the mainsite involved in mouse fertilized eggs mitosis. Additionally, the percentage of cleavage offertilized eggs decreased significantly as well as the G2 phase delaying after Cdc25Bantibody performed. So, it is indicated that Cdc25B protein is required in the developmentof mouse fertilized eggs.Protein kinase A is a cAMP-dependent serine/threonine kinase and one of the mostimportant components in signal transduction pathway that plays a pivotal role in cell growth, differentiation, proliferation, and cell cycle control. The purified PKA catalyticsubunit and cAMP, all inhibit germinal vesicle breakdown and meiotic maturation both inmouse oocytes. Using an RNA interference approach to disrupt the role of the predominantregulatory subunit, RIalpha of PKA during mouse oocyte maturation find that theseoocytes resume meiosis, but the resulting eggs display meiotic spindle abnormalities andabnormal cleavage planes leading to extrusion of large polar bodies. In conclusion, inmammalian oocytes, cyclic AMP-dependent protein kinase (PKA) is responsible formaintaining meiotic arrest. As shown that PKA, by inhibiting MPF, regulated cell cycleprogression of fertilized eggs in our previous results. But little knowledge is available onthe target of PKA in mitosis in mouse fertilized eggs. Based on the analysis result ofScansite software, two potential PKA phosphorylation sites including serine 321 and serine229 were predicted. We hypothesized that S321is the main target of PKA and a lesser siteof S229. To test this, we injected a variety of Cdc25B mutants into mouse fertilized eggsincubated in the present or absence of dbcAMP. Strong overexpression of either S321A ordouble mutant overcame the G2 arrest induced by dbcAMP (PKA). On the other hand, weexamined the phosphorylation status of cdc2-Tyr15. We found that the MPF inCdc25B-S229A/S321A group was activated precede the other groups. Taken together, wedemonstrated in mouse fertilized eggs that PKA plays a critical, regulatory role in cellcycle progression, by modulating the activity of Cdc25B. These data implied that PKAshould directly phosphorylate Cdc25B on Ser321. The double mutant of Cdc25B exerts theeffect on the development of mouse fertilized eggs significantly. So, the PKA/Cdc25Bpathway can operate in the development of mouse fertilized eggs. Moreover, the S321 wasphosphorylated at G1 phase and S phase detected using specific mouse Cdc25B S321phosphorylation antibody but no phosphorylation signal found at G2 phase as well as Mphase. It has been shown that G2 arrest in Xenopus oocytes depends on phosphorylation ofcdc25 S287 by protein kinase A and the serine 287phosphorylation and 14-3-3 bindingnegatively regulate the cell cycle. So we deduced that Cdc25B protein S321 binding to14-3-3 facilitated the S229 binding to 14-3-3, thereby forming a 14-3-3/Cdc25B-S321/S229 protein complex. Here, we conclude that S321 and S229 arephosphorylated by PKA and creating the binding site for a 14-3-3 protein and restrainsactivity during interphase, whereas the inhibitory phosphorylation site S321and S229 aredephosphorylated by some phosphatases and the mouse fertilized eggs entering mitosis.The overexpression of Cdc25B protein disrupts two-cell arrest and promotes thedevelopment of mouse embryos including Cdc25B- WT and the mutant forms of Cdc25B.The overexpression of Cdc25B protein results in irregular cleavage 47 hr after the hCGinjection. It has been identified that the 2-cell embryos from the most mouse strainsproduced in vitro are arrested and not degenerating in normal condition. There is a reportshows that the phenomenon of two-cell block in vitro is a specific functional state ofembryos. Our results indicate that Cdc25B proteins have role in the early development ofmouse embryos.To investigate whether PKA could effectively phosphorylate S321or S229 on Cdc25B,some fragments derived from residues 212-330 of mouse Cdc25B were expressed asGST-fusion proteins in E.coli BL21. The mouse Cdc25B specific proteins were expressedsuccessfully in E.coli BL21.Conelusion1,Cdc25B protein can shuttles between cytoplasm and nucleus in mouse fertilizedeggs. The nucleus export of Cdc25B is required for the initiating the mitosis of mousefertilized eggs. 14-3-3 protein regulates the development of mouse fertilized eggs bycontrolling the localization of Cdc25B protein.2,Cdc25B protein is required for the development of mouse fertilized eggs.3,PKA plays a critical, regulatory role in mouse fertilized eggs cell cycle progression,by phosphorylating the Cdc25B S321 mainly.4,To construct the prokaryotic expression vectors of mouse Cdc25B specific fragment,and to express GST fusion proteins in Escherichia coli.