| INTRODUCTIONA rapid succession of interphase and mitotic states characterizes the early embryonic divisions of many species. The onset of mitosis is induced by activation of MPF, a highly conserved complex consisting of a kinase, Cdc2, and an activating subunit, cyclin B. During the cell cycle the concentration of the catalytic subunit Cdc2 remains constant while cyclin accumulates from interphase to mitosis and is degraded at each metaphase - anaphase transition. In interphase, association with cyclin induces phosphorylation of Cdc2 at key tyrosine and thre-onine residues. In this state the complex is enzymatically inactive. At mitosis, the complex is dephosphorylated and activated by Cdc25.Protein kinase A is cAMP - dependent kinase, one of the most important signal transduction pathways, plays a pivotal role in growth, differentiation, tumor occur, cell cycle control, etc. PKA activators such as cAMP, 8 - Br -cAMP or phosphodiesterase(PDE) inhibitor isomethyl butyl xanthine(IMBX) or purified PKA catalytic subunit all can inhibit germinal vesicle breakdown (GVBD) and meiotic maturation in mouse oocytes, also in Xenopus oocytes. In Xenopus embryonic extracts cAMP concentration and PKA activity also oscillations accompany with the cell cycle, low in mitosis and increased during M/G1 transition, keep high until next mitosis, and the mechanism concern with the Cdc25 phosphorylation state and cyclin B concentration. Mouse fertilized egg is the most simple and natural model for cell cycle that near to human, but little is know about PKA on MPF also PKA on mitosis. In this article, we microinjected cAMP (as activator of PKA) and protein kinase inhibitor (PKI) (as inhibitor ofPKA) into mouse 1-cell stage fertilized eggs, the cAMP concentration, PKA and MPF activaty were detected, also the Cdc25C, Cdc2 phosphorylated state and the concentration of pTyr15 for Cdc2, cyclin B1.MATERIALSFemales of 4-5 week-old KUMING mice and males of 8 week-old KUMING mice were supplied from the Department of Laboratory Animals, China Medical University. cAMP, PKA inhibitor peptide (PKI) , Kemptide, histone H1 (type III-S), polyvinyl alcohol, Na3VO4, NaF, B-glycerophosphate, p- nitrophenylphosphate, 3-[N-morpholino]-propanesulfonic acid (MOPS), genistein, ML-9, leupeptin, aprotonin, pepstatin, chymostatin, trypsin-chymotrypsin inhibitor were purchased from Sigma Chemical Co. [r-32 P] ATP was obtained from Yahui biomedical Enginnering co. Anti -Cdc25C rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology, Anti-Cdc2 and anti - cyclinBl mouse monoclonal antibody were from Neo-markers, Anti - Cdc2 pTyrlS monoclonal antibody was obtained from New England biolabs. HRP - conjugated anti - mouse or anti - rabbit IgG secondary antibody, purchased from Beijing Zhongshan Biotechnology.METHODS1. Superovulation, natural mating, collection of fertilized eggs;2. Microinjection: Microinjection was performed using NARISHIGE micro-injection system ( JAPAN) , Olypus Model EX - 70 inverted microscope with Hoffmann optics. Typical injection volume was 5% of total cell volume, or 10 pl, one - cell stage embryos in G2 were microinjected with cAMP, 10 mmol/L, 40 mmol/L, 100 mmol/L solution in water, or PKI, 200nmol/L, 800mol/ L, 2 mmol/L soluted in 5 mmol/L 2 - [ N - morpholino ] ethane - sulfonic acid (MES), pH6.5; no microinjection or microinjected with M2 medium or 5 mmol/L MES as control.3. Observed under a dissecting microscope and registered the percentage of division and death at 30 or 35 hours after HCG, respectively.4. Histone HI kinase assay; Three eggs were removed from M16 medium, washed in collection buffer and transferred to a 0. 5 ml Eppendorf tube in 5 l of collection buffer. The eppendorf tube was immediately stored at - 80 C until the kinase assay was performed. The frozen eggs were frozen and thawed for three times, then added into 25l MPF buffer. The reaction was allowed to proceed for 7 min at 30C , then 25 L aliquots was removed and spotted on whatman p81 paper, counted t...
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