Study Of The Variation Of Expression And Activity Of Plk1 During Early Development Of Mouse Zygote And Its Relation With MPF

INTRODUCTIONPolo - like kinase (Plks) are serine/threonine protein kinase. The founding member of this family, polo, was originally identified in Drosophila. Having been identified: Cdc5p in Saccharomyces cerevisiae plol in Schizosaccharrmyces pombe, plxl in amphibians and plkl in mammals. Plkl has a conserved kinase domain in N terminal and a conserved domain in C terminal, termed the polo -box,which has been proposed to target plk to its subcellular substrates.Plk is implicated in the regulation of multiple aspects of mitosis, including the activation of Cdc25c, formation of spindle, entry into and exist from M phase. The variation of plka activity and sucellular localization suggest its function. MPF controls both the entry and exist from M - phase and is a complex of Cdc2 and cyclinB. The activity of MPF is regulated not only by formation of the complex but also by phosphatization and de phosphatization of the Cdc2 subunit. In Xenopus the activity of Cdc25c and MPF was depressed when plxl was immuno precipitated by its antibody. This suggests that plxl participate in the the positive feedback loop that allows MPF autoamplification. A report said that when lost the activity of plxl ,Mytl ,a negative regulator of MPF,will be activated by super phosphatization. So,plxl not only activates Cdc25c ,but also restrains the activity of Mytl and promote mitotic to M - phase.Mouse is a good mammalian model. The study of the activity and expression of plkl during early development of mouse zygote is very important for we to grasp the mechanism of signal transduction of mammalian zygote. plkl play a very important role in early development of zygote. We detected the activity of Plklafter fertilization- And at the same time in this experiment, the changes in Plkl expression were, for the first time, detected during first mitosis of mouse zygote through Western blotting.MATERIALS AND METHODS1. The ovaries were removed from Kunming pregnant female mice (5 -6w) and transferred to prewarmed (37*^0 ) M2 medium. Cumulus cellswere removed by 300jxg/ml hyaluronidase. Zygotes were cultured in M16 . Zygotes were collected at appointed time.2. Add sample buffer to the collected zygotes and heat them to lOOTl for 5 minutes. The ptoteins were separated by SDS - PAGE with 10% separating gel and transferred to the NC membrane. The membrane was incubated with anti - plkl antibody for 2h and with 2n antibody for lh. After washed by TTBS the membrane was detected.3. Add lysis buffer to the collected zygotes and frozed them for 3 times. The reaction was started by adding 30jxl assay buffer to the samples and the samples were incubated for 30min at 30T!. Quantity of plkl or MPF activity was detected by Phoshorimager.ResultsThe quantity of Plkl protein remained stable during early mitotic after fertilization. The activity of Plkl was fluctuated during the mtosic cycle. The plkl activity increases at the beginning of the first mitosis , peaks at M - phase and decreases rapidly on existing from M - phase, the activity of MPF did not increase in M - phase after treating sample with roscovitine, the specific inhibitor of MPF. the activy of plkl also did not increase in M - phase.DiscutionPolo - like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells, such as the activation of Cdc25c, formation of spindle, entry into and exist from M phase. Mouse is a good mammalian model. The study of the activity and expression of plkl during early development of mouse zygote is very important for us to grasp the mechanism of signal transduction of mammalian zygote. Plkl expression during the early deveopment of mouse zygote is still not reported . In this experiment, the changes in Plkl expression were, for the first time , detected during first mitosis of mouse zygote through Western blotting. The result showed that the plkl was present in mouse zygote and the quantity of Plkl protein remained stable during early mitotic after fertilization. At the same time we detected the activity of Plkl after fertilization. The result showed that the activity of Plkl was fluctuated during the mtosic cycle. The plkl activity increases at the beginning of the first mitosis , peaks at M - phase and decreases rapidly on existing from M - phase. The Western Blots suggests that the quantity of Plkl protein does not affect the activity of Plkl and activity of Plkl is regulated through phosphatization. The above results are the same as that gained from abstract of Xenopus. the results in this experiment indicate that plkl play the same role in the M - phase of mouse zygote as plxl does in the M - phase of Xenopus zygote and provide a foundation for farther study of plkl.MPF controls both the entry and exist from M - phase and is a complex of Cdc2 and cyclinB. The activity of MPF is regulated not only by formation of the complex but also by phosphatization and dephosphatization of the Cdc2 subunit. The result from abstract of Xenopus suggest that plxl participate in the the positive feedback loop that allows MPF autoamplification which is that plxl phospho-rylate Cdc25 and activate Cdc25 , and the active Cdc25 will phosphorylate Thrl4 and Tyrl5 of Cdc2 ,the subunit of MPF,which consequently results in the activation of more MPF and also the activity of MPF is required for the activation of...