Involvement Of PKA/Cdc25B Pathway In G₂-arrested Mouse Oocytes

IntroductionVertebrate oocytes are naturally arrested at late G₂ stage of meiosis I which is also named GV stage because the typical morphologic character at this stage is a big nucleus called germinal vesicle ( GV ). Exposure to progesterone, their natural mitogen, breaks this arrest and induces resumption of the two meiotic division cycles and maturation of the oocyte into a mature, fertilizable egg. Studies in species as diverse as starfish, Xenopus, and mammals have reached the identical conclusion that the decreased level of cAMP and inhibition of protein kinase A(PKA) can break this arrest. But the physiological substrates of PKA and molecular mechanisms regulating the arrest are still unknown in mammalian oocytes.Mouse is a simple and natural cell cycle model in vertebrate which is closer to human. We sought to identify physiologically relevant targets that contained PKA phosphorylation consensus sites. All the mouse proteins from Swiss - prot database were examined with a sequence - scanning algorithm to identify motifs likely to be phosphorylated by PKA. Then programs in PERL ( Practical Extraction and Reporting Language) were used to extract information. According to the prediction, we screened the relevant target sites of PKA through constructing various mutants and tried to explore the mechanism of G₂ arrest by PKA in mouse oocytes. It will be of great importance for the study of mechanisms regulating the oocyte maturation of human and other mammals.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. The full - length cdc25B cDNA clone (pBSK -cdc25B) was kindly provided by Dr. Tony Hunter at Salk Institute. Plasmid pEGFP - C3 were kindly provided by Dr. Liuyu at Fudan University. Leptomycin B(LMB) was kindly provided by professor Mino-ru Yoshida at Tokyo University. pGEM - T vector was purchased from Promega. pBluescript II/SK vector and E. coli DH5a were previous kept in our lab. Restriction endonuclease XhoI^Bamfll^Xbal and pfu DNA polymerase from MBI. DNA Fragment Recovery Kit from Dingguo. Plasmid DNA purification kit from Promega. mMESSAGE mMACHINE kit from Ambion. Anti - Cdc2 - pTyrl5 monoclonal antibody%alkaline phosphatase -conjugated anti -goat IgG secondary antibody and protein kinase A inhibitor (PKI) from Santa - Cruz. 0 - diani-dine>,p - naphthyl acid phosphate and reagents for M - phase promoting factor ( MPF) and PKA activity assay from Sigma.2. prediction of PKA relevant substratesAll mouse protein sequences from Swiss - prot database were scanned with Scansite software to predict the relevant substrates of PKA which contained the PKA phosphorylation consensus sites. Then the programs in PERL were used for information extraction. The key words used for extraction were on cell proliferation and development. At last 14-3 - 3 conservative binding motif was used for further screening.3. collection and culture of mouse oocytesImmature GV stage oocytes were collected from 20 - to 28 - day - old Kunming strain mice that had been injected 48 h previously with 5 IU pregnant mare's serum gonadotrophin(PMSG). Mice were killed by cervical dislocation and the ovaries were placed in M2 medium containing 125jxmol/L dibutyryl cyclic adenosine monophosphate (dbcAMP). The follicles were punctured with fine needle and let release the oocytes. Only oocytes with easily removable cu-mulus cells and a germinal vesicle were selected for incubation. They were cultured in a drop of medium, at 37X., in a humidified atmosphere of 5% C02 in air, under paraffin oil. Incubation was performed in MB medium( Waymouth's MB752/1 Media supplemented with 100 |xg/ml sodium pyruvate, 50U/ml Penicillin, 50ug/ml streptomycin sulfate, 3mg/ml bovine serum albumin).4. preparation of recombinant plasmidsThe coding sequence of cdc25b was amplified by PCR using the pBSK -cdc25B as template and introduced the enzyme - incision site of Xhol and Bam-HI into 5'and 3'end respectively. The product was cloned into the vector pBlue-script II/SK and named pBSK - cdc25B - WT. Using the same template to construct the 288 amino acids of N — terminus and 251 amino acids of C - terminus deletion mutants which were called pBSK - cdc25B - AN288 and pBSK -cdc25B-AC251.Using the overlapping PCR to mutate the serine 321 to nonphosphorylatable alanine of Cdc25B and recombinant mutations of N - terminus deletion and S321A.All the recombinant plasmids were sequenced to verify the inserted gene correct and mutation successful.5. In vitro transcriptionAll the constructs in pBluescript were cut singly with Xbal and in vitro transcribed into 5'- capped mRNA for microinjection by using mMESSAGE mMA-CHINE 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 IiCl.6. mRNA and PKI microinjectionOocytes were microinjected using a micropipette and Eppendorf transferman manipulators mounted on a Olympus IX - 70 inverted microscope with DIC optics. Oocytes were placed in a drop of M2 medium containing 125u,mol/L db-cAMP under paraffin oil in a lid of 3 cm Falcon culture dish. Typical injection volume was 5% of total cell volume or lOpl. mRNA was diluted to various concentrations in 5mmol/L Tris and 0.5mmol/L EDTA (pH7.4) without nuclease contaminant. PKI was diluted in 5 mmol/L 2 - [ N - morpholino] - ethanesulfo-nic acid( MES, pH6. 5) . Oocytes in control groups were either not microinject-ed, or microinjected with the same amount of mRNA of phosphatase - deficient mutant of cdc25B or MES.7. MPF and PKA activity assayMPF kinase activity was measured using histone HI kinase assays. Five oocytes cultured in MB medium were collected and subjected to freezing and thawing three times. A total of 25ul of MPF 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.The PKA activity was determined by using the MPF buffer, except that no PKA inhibitor was included and histone HI was replaced by 0. lmmol/L Kemp-tide, which is a specific synthetic substrate peptide for PKA.8. Western blottingProtein extracts of mouse oocytes were prepared by adding appropriate number of oocytes in a minimal volume of collection medium to 20 ul of protein extraction buffer. The extracts were briefly vortexed, quickly frozen on dry ice, and stored at - 20TI until used. Laemmli sample buffer was added to the protein extracts, and the mixture was boiled for 5min and resolved on a 12% SDS - PAGE gel. For immunoblotting, the fractionated proteins were transferred to a nitro - cellulose membrane. The primary antibody against pTyrl5 of Cdc2 was used and alkaline phosphatase - conjugated anti - goat IgG was as secondary antibody. The proteins were detected by using 0 - dianidine and {3 - naphthyl acid phosphate as the substrates of alkaline phosphatase.9. Subcellular localizationThe pBSK - cdc25B - WT or pBSK - cdc25B - S321A and pEGFP - C3 vector were digested with Xhol and BamHI and then ligated to construct the re-combinant plasmid pEGFP - cdc25B - WT and pEGFP - cdc25B - S321 A. Various plasmids were injected into the germinal vesicle of mouse oocytes pretrea-ted with or without LMB and the pEGFP - C3 vector was as controls. The green fluorescent signals were observed under fluorescence microscope. The Psort II and Target P software were also used to predict the subcellular localization ofCdc25B.Results1. Prediction of physiological substrates of PKA during meiosis maturation of mouse oocytesIn order to seek the relevant targets of PKA, we used mouse as models and analyzed all the proteins in Swiss - prot database. We sought to identify physiologically relevant targets that contained PKA phosphorylation consensus sites with Scansite software by high - stringency sequence - scanning. 120 proteins were picked out. Key words search program was then used to extract information from the abstracts in pubmed and Swiss - prot original record of the 120 proteins. We finally got 17 proteins that were relevant with cell proliferation or development. Among those 5 were in the progesterone - activated signaling pathway. Using 14-3-3 binding motif for further screening showed that Cdc25B was a candidate PKA substrate and SI49 and S321 were the relevant target sites.2. Truncated versions of Cdc25B do not promote germinal vesicle breakdown (GVBD)mRNAs encoding WT or AN288 or AC251 mutant Cdc25B were injected into G2 - arrested mouse oocytes and assayed for their ability to induce entry into M phase, which was scored by GVBD or MPF activity or phosphorylation status of Cdc2 -Tyrl5. Cdc25B - WT induced GVBD in a dose - dependent manner. The AC251 mutant as the phosphatase - deficient mutant had essentially no effects on the meiotic maturation of oocytes and can be used as a good control for mRNA injection. The AN288 mutant of Cdc25B did not induce GVBD either.3. Constitutive inhibition of Cdc25B by PKA during G2 arrestmRNAs encoding Cdc25B - WT or Cdc25B -S321A were injected into G2?-arrested mouse oocytes. The S321A mutant was considerably more potent.Despite the same levels of overexpression, Cdc25B -S321A could induce Cdc2- Tyrl5 dephosphorylation and maturation much more rapidly and efficientlythan Cdc25B - WT. When treated with dbcAMP, Cdc25B - S321A mRNAcould bypass the inhibitory effect of PKA while Cdc25B - WT mRNA could not. To test more directly whether Cdc25B inhibition in G2 - arrested oocytes was indeed due to the activity of endogenous PKA, finally we coinjected PKI and Cdc25B - WT or Cdc25B -S321A mRNA into oocytes incubated in MB medium containing 200jxmol/L dbcAMP. Injection of Cdc25B - S321A mRNA induced GVBD very rapidly and essentially with the same kinetics in both PKI - and control MES - injected oocytes. By contrast, while injection of Cdc25B - WT mRNA induced GVBD only inefficiently in MES - injected oocytes as in unin-jected oocytes, it did induce maturation very rapidly and efficiently in PKI - injected oocytes. Thus, without endogenous PKA function, wild -type Cdc25B had a strong maturation — inducing activity, close to that of the PKA - nonphos-phorylatable Cdc25B mutant.4. Subcellular localization of the ectopic expressed Cdc25B pEGFP - cdc25B - WT or pEGFP - cdc25B - S321A or pEGFP - C3 were microinjected into the germinal vesicle of mouse oocytes. In oocytes microinject-ed with pEGFP - cdc25B - WT, the dotted green fluorescent signals were detected only in cytoplasm but not in nucleus. In contrast, strong green fluorescent signals were detected evenly in nucleus in oocytes microinjected with pEGFP -cdc25B - S321A. However, the fluorescent signals of pEGFP - C3 alone was distributed throughout the nucleus and cytoplasm. The green fluorescent signals of pEGFP - cdc25B - WT transferred from cytoplasm to nucleus when preincu-bated with LMB. The prediction of subcellular localization with Psort II and Target P software indicated that 52 - 65 and 343 - 350 residues of Cdc25B represented nuclear export sequence ( NES) and nuclear localization sequence (NLS) respectively. It seemed that the NES and NLS were interactional in determining the location of Cdc25B in mouse oocytes.DiscussionArrest of immature oocytes in late G2 phase is universal in the animal kingdom and PKA seems to play essential role during this arrest. Elevation of cAMP or injection of PKA blocks progesterone - induced entry into M phase. Injectionof PKI induces M - phase entry in the absence of progesterone. High PKA activity inhibits Mos accumulation and inhibits the ability of injected Mos, cyclinB, or Cdc25 to induce GVBD, suggesting that PKA inhibits oocyte maturation at several steps. More recently it has been found that injection of catalytically inactive PKA can also block progesterone - induced GVBD as well as GVBD induced by Mos and partially reduces GVBD induced by cyclin B. By contrast, inactive PKA is completely unable to block Cdc25 - induced GVBD, leading to the conclusion that the kinase activity of PKA is required to inhibit activation of cyclin B/cdc2 by Cdc25.Although increases in cAMP and activation of PKA are crucial in the induction and maintenance of meiotic arrest, the precise targets and downstream effectors of PKA signalling have not been completely defined in mammalian oocytes. In this work we examined the role of PKA/Cdc25B pathway in G2 arrest of mouse oocytes. The results indicated that PKA/Cdc25B pathway can operate in meiosis resumption. Moreover, we showed that PKA function in oocytes was accomplished via a direct inhibition of Cdc25B, a dual - specific phosphatase and inducer of M phase entry .To understand how PKA blocked the G2/M transition in mouse oocytes, we first sought to identify physiologically relevant targets that contained PKA phos-phorylation consensus sites. Further scanning was combined with 14-3-3 binding motif due to its highly conservative role in the G2 arrest of eukaryotes. The results indicated that Cdc25B was a candidate target and S321 was more likely to function in G2 arrest of mouse oocytes when compared with the study in Xenopus oocytes.We constructed the N - terminal deletion mutant of Cdc25B and tried to explore its role in G2 arrest. The present study provided evidence that truncated version of Cdc25B containing the catalytic domain was insufficient for inducing GVBD. This was surprising, considering that the truncated Cdc25B protein retained phosphatase activity and activated para -nitrophenyl phosphate (PNPP) in vitro. Since the removal of the N - terminus of Cdc25B results in loss of GVBD - inducing activity, it is unlikely that it plays an inhibitory role in this system. However, the mechanism of the N - terminus of Cdc25B regulating mei-osis resumption remains largely unknown.The serine321 of Cdc25B was mutated into nonphosphorylatable alanine. We indeed showed that overexpression in oocytes of Cdc25B -S321A alone can induce Cdc2 - Tyrl5 dephosphorylation and maturation much more efficiently than overexpression of Cdc25B - WT. More specifically, we demonstrated that, in oocytes lacking endogenous PKA function, even overexpression of wild - type Cdc25B could induce maturation very efficiently, if not fully comparably to over-expression of Cdc25B -S321A. Thus, it appears certain that PKA directly acts on and inhibits Cdc25B in immature oocytes, to ensure G2 arrest of the oocytes until the oocytes meet with the maturation inducer.The functions of proteins are firmly correlated with their subcellular localization. However, the subcellular distribution of Cdc25B during meiosis resumption is still poorly understood. In our present study, we analyzed the subcellular localization of Cdc25B by constructing EGFP recombinant plasmids for wild type and S321A mutant of Cdc25B. The data strongly suggested that Cdc25B could shuttle in and out of the nucleus and export of Cdc25B from oocyte nuclei was inhibited by LMB, suggesting the involvement of NES. Indeed, we found that an N -terminal fragment of Cdc25B containing a putative NES. The subcellular localization of Cdc25B - S321A demonstrated that mutagenesis was critical for the export of Cdc25B. Considering the presence of NLS in 343 - 350 of Cdc25B, we thought that Cdc25B - S321 phosphorylation may cause consequent 14-3—3 binding, which masked the NLS and selectively reduced the rate of Cdc25B nuclear import while leaving its rate of export unaffected, presumably resulting in more efficient exclusion of Cdc25B from the nucleus. When Cdc25B -S321 was mutated to Ala, the 14-3-3 binding was abrogated and the homeostasis was recovered.In conclusion, we have shown for the first time that Cdc25B - S321A can bypass the inhibitory effect of PKA during meiosis resumption in mouse oocytes. These observations lead to the proposal of a model for the regulation of meiotic maturation and arrest in mammalian oocytes that is based on the fact that Cdc25B functions in mouse oocytes as the relevant substrate of PKA. PKA can phosphorylate Cdc25B -S321 and make consequent 14-3-3 binding, which...