CAMP As The Trigger Inducing Mouse Zygote To Develop In The First Cell Cycle

PrefaceOur lab group previous work has demonstrated that both protein kinase A (PKA) and prtein kinase C (PKC) are important elements to regulate the activity of M - phase promoting factor (MPF) in signal transduction pathway of mouse zygote early development. When the mouse zygote entrys into M phase of the first cell cycle, both the adenosine 35'- monophosphate (cAMP) concentration and the PKA activity decrease, whereas the MPF activity increases.All the reported data have pointed out that MPF is playing a central role in signal transduction pathway of mouse zygote first cell cycle. First, MPF, Plk1 (polo like kinase 1) and Cdc25 form a positive feedback loop. Secondly, the Mitogen - activated protein kinase kinase kinase (Mos) activity follows the MPF activity pattern, and Mitogen - activated protein kinase (MAPK) is activated through the Mos - MEK - MAPK cascade (MEK: Mitogen - activated protein kinase) Thirdly, Myt1 and Wee1 can inhibit the MPF activity by phosphoryla-tion Cdc2 on Thr14 and Tyr15 site, but the Mytl activity can be inhibited through a phosphorylation by Plk1. Once the MPF activity reaches the maximum , APC begins to degrade CyclinB, thus reducing the MPF activity. In the later period of the M phase, all the active Cdc25, Mos, MEK, MAPK become inactive due to the dephosphorylation by protein phosphatase 2A (PP_2A). At this point, the cAMP concentration and the PKA activity increase again, and the mouse zytoge divides into two cells. Overall, cAMP acts as a trigger to induce mouse zygote to development in this whole process.The protein interactive network is dynamic in essentially. The dynamic relationship between a serial of protein effection can be converted into a serial ofmath equation to describe system space - time evolution. The equation type is decided by biology question to be treated. To cell cycle control system, it is suitable to use ordinary differential equatins ( ODEs). In terms of mathematical, the ordinary differential equatins used can assist us capture the intuitive gnosia about protein synthesis and degradation, phosphorylation and dephosphorylation. And, these equations can permit us to calculate the concentration change of each protein. We used diffferent rate laws to different reaction types, and we get different ordinary differential equatins: Gepasi jxgeneral pathway simulator) , Gepasi is a software package for modeling biochemical systems. It simulates the kinetics of systems of biochemical reactions and provides a number of tools to fit models to data, optimize any function of the model, perform metabolic control a-nalysis and linear stability analysis. We just constructed a math model by using gepasi and simulated the signal transduction in the first cell cycle about mouse zytoge early development.Materials and MethodsMaterials:1. experimental animal: Kunming mouse supply by CMU experimental animal department2. instrumenttation: a Personal computer (Intel Pentium 2. 4G CPU, HY 512M Memory, MicroStar 845E MainBoard, MicroSoft?XP System).3. Software for math model. Gepasijxgeneral pathway simulator), free download from www. gepasi. org for Windows,version is 3. 30,ZIP format.Methods:1. The ovaries were removed from Kunming pregnant female mice (5 - 6w) and transferred to prewarmed (37T! ) M2 medium. Cumulus cells were removed by 300|xg/ml hyaluronidase. Zygote were cultured in M16. Zygotes were collected at appointed time.2. Collect experimental data about mouse zytoge early development.3. Construct quantitative model by using ordinary differential equations. We use Gepasi to simulate this math model on PC. That is, We write outreaction formula, then Gepasi can automatic convert reaction formula to ordinary differential equations automaticly and can plot simulation graph.Results1. The cAMP concentration and the PKA activity lower at mitosis, higher after M/Gl transition point until the next mitosis. When the cAMP concentration and the PKA activity decrease, the MPF activity rises. Our simulated results u-sing Gepai approach exhibit the similarities of The cAMP concentration , the active PKA and MPF to that of the experimental results.2. The quantity of Plkl protein fluctuates slightly during mitotic after fertilization. However, the activity of Plkl after phosphorylation has a remarkable change during the first cell cycle in mouse zygotes, which reaches the maximum level at the M phase. Gepai simulation also shows a similar change of Plkl activity from Gl to M phase.3. When mouse oocytes are arrested at Mil, both MAPK activity and MPF activity stayed at a higher level. After fertilization, both activities begin to decrease and reach the minimal level after 7 hours. The simulation results show MAPK activity are higher at Mil phase and decreases to a minimal level after fertilization. At mitsis of first cell cycle, MAPK activity rise, but it's activity could not get to Mil level.4. CyclinB continues to be accumulated before metaphase, and start to degrade near the metaphase in the case that no activators and inhibitors are added. In PKI - microinjected embryo, CyclinB maintains a higher quantity level even after exiting the M phase. CyclinB simulation result;CyclinB accumulate to top at M phase and degradate when exit M phase. This is in coincidence with experimental data.DiscussinEarly zygote division is characterized by a rapid succession of interphase and mitotic state in many species including mouse. In our study, all the experi-mental evidence has shown that cAMP is an upstream signal substance whose concentration influences the activity of downstream kinases such as PKA, MPF, Plkl, Cdc25 , Weel et al. PKA as a negative regulator whose activity changes is opposite to MPF activity, mediates cAMP signal pathway to downstream. It only in xenopus ocytes that PKA can phosphorylate Cdc25 protein, and cause meio-genic Gil phase block. In mouse zygotes, it unclear about the relation between PKA and Cdc25. Cdc25a, Cdc25b and Cdc25c are all activators of cyclin - dependent kinases,but maybe Cdc25b plays chief effect in mouse zygotes early development.In the signal transduction pathway of first mouse embryo mitosis, cAMP acts as a trigger. Namely, cAMP concentration drops first, then a serial of ki-nase change its activity,mouse zygotes entry intocell cycle and complete mitosis. We just construct a math model byusing Gepasi algorithm has been used to explore this process, and the simultion results demonstrated a greemnet with the experimental data.The model may not answer all the question we posed, but it can integrate our knowledge to a system. It is a start point to ask questious regarding the system as a whole, with the help of more experimental results, the mode may be verified for reliability. And the simulation process may help us to have a good comprehension about experimental phenomenon.Based on the complexity in signal transduction pathway of mouse zygotes early development, our model is far from complete and accurate in details and need to be expanded and corrected in future. But, such a model can be extended in continuely till we have a complete mode to reveal the cell cycle regualte mechanism overall in mouse zygotes early development.Conclusion1. The math model can rudiment reflect the basic state about the first cell cycles signal transduction pathway in mouse zygotes early development.2. We think cAMP is the trigger that induce mouse zygote to develop in the first cell cycle.