Study On The Function Of MAPK And MPF In The Early Development Of Mouse Fertilized Eggs And The Relationship Between Them

PrefaceOvulated eggs of many species, including mammals, are arrested at meta-phase II of meiosis (MII)until they are fertilized. MII eggs are characterized by high activity of mitogen - activated protein kinase ( MAPK ) and maturation promoting factor( MPF). The MOS - MEK - MAPK pathway in oocytes is thought to be a required component of the cytostatic factor( CSF) activity of eggs. Castro reported that the high activity of MPF can maintain the activation of MOS. Some people established Mos-/- mouse oocytes,which fail to arrest in MII,thus establishing a requirement for MOS activity in the maintenance of MII arrest in the mouse.Thomas study the function of MAPK in mitosis of Xenopus eggs. Xenopus eggs can complete mitosis and MPF can be activated normally in the absence of MAPK activity. But the Xenopus eggs extract in which MAPK was inhibited exited mitosis prematurely. If we sustained activation of MAPK in a cycling extract at about the time when transient MAPK activation normally occurs, the extract maintain the mitotic state in the absence of active MPF and intact nuclear envelopes can not be formed normally. Another experiment indicate MAPK play an important role in nuclear envelope breakdown( NEBD). My article mainly study the function of MAPK and MPF in the early development of mouse fertilized eggs and the relationship between them.Materials and Methods1. ChemicalsHyaluronidase(Sigma) ,MBP (substrate of MAPK,Sigma) , Histone III -s (substrate of MPF, Sigma) , U0126 ( inhibitor of MEK, Sigma), roscovitine (inhibitor of MPF, Sigma )2. Egg and Zygote CollectionSuperovulation: the times that the PMSG and HCG are administered relative to each other and to the light cycle of the mouse room will affect both the developmental uniformity and the number of eggs that are recovered from superovu-lated female mice. For most strains, a 46 - to 48 - hour interval between the PMSG injection and the HCG injection has been found to be optimal in terms of egg yield.Collection of Zygote: after injecting HCG female mice mated with male mice 8 weeks old. Kill the mated female and dissect out the oviduct. Transfer one oviduct at a time into another 35 - mm petri dish containing M2 plus hyaluronidase (about 300 micro g/ml) at room temperature and view though the stereo-microscope at 20x or 40x magnification. Using one watchmakers forceps, grasp the oviduct next to the swollen infundibulum and hold it finnly on the bottom of the dish. Use the other forceps to tear the oviduct close to where the eggs are located , releasing the clutch of eggs. Allow the eggs to incubate in the enzyme for several minutes until the cumulus cells fall off. Using transfer pipets pick up the eggs and transfer them to a fresh dish of M2 to rinse off the hyaluronidase and then transfer the eggs to CZB for culture at 37 °C until needed.3. Kinase AssaysMPF kinase assay: five frozen eggs were thawed and subjected to freezing and thawing three times. A total of 25 ul of MPF buffer was then added to the disrupted cells. The histone HI kinase reaction was started by adding 25 ul of 20 uCi/ml'y32P - ATP,incubated at 30^ for 7 min. After that,25 ul aliquots were spotted on whatman p81 paper and the reaction was stopped with 5% H3PO4 solution. After thorough washing,the radioactivity on the filter paper was counted with a BECKMAN scintillation counter.MAPK kinase assay: five frozen eggs were thawed and subjected to freezing and thawing three times. A total of 5 ul of MAPK buffer was then added to the disrupted cells. The samples were incubated for 30X. for 30 min and the reactionwas then stoped by addition of 15ul of 20% TCA. BSA(5ul of 10 -mg/ml solution) was added. Protein was precipitated for 10 min on ice,and the sample then centrifuged at lOOOOXg for 6 min. 15 ul supernatant were spotted on whatman p81 paper and paper was immersed in 0. 75 mM H3PO4 solution. After thorough washing,the radioactivity on the filter paper was counted with a BECKMAN scintillation counter.Results1. The active change of MAPK and MPF in mouse fertilized egg Fertilization of mouse oocytes resulted in a decrease of both MPF andMAPK activities, although the decrease in MPF was virtually undetectable by 3h after insemination,50% of the MAPK was still present at 5h and could not be detected for up to 7h after insemination. During the first mitosis, MAPK and MPF can be activated transiently.2. The decrease of MAPK activity parthenogenetically activates unfertilized eggs and sequentially result in MPF inactivation.Eggs were cultured in the presence of the MEK inhibitor U0126( 100 u.M) for up to 8h. A much higher proportion of unfertilized Mil eggs treated with U0126(85%) was parthenogenetically activated compared with control group (4.4%). The treated oocyte yields the same phenotypes as Mos~ ~ parthenog-enotes and the decrease of MAPK activity can sequentially result in MPF inactivation.3. The decrease of MPF activity parthenogenetically activates unfertilized eggs and sequentially result in MAPK inactivation.Eggs were cultured in the presence of the MPF inhibitor roscovitine (100|xM)for up to 8h. A much higher proportion of unfertilized Mil eggs treated with roscovitine (92% ) was parthenogenetically activated compared with control group(4. 4% ). The treated oocyte yields the same phenotypes as Mos" " par-thenogenotes and the decrease of MPF activity can sequentially result in MAPK inactivation.4. Continuous exposure to U0126 or roscovitine is required to induce par-thenogenetic activation sufficientlyActivated rate of MII eggs treated by either U0126 or roscovitine in 8h group > 2h group > 1h group. Eggs activation was dependent on duration of U0126 or roscovitine exposure. Inhibition of eggs activation of U0126 and roscovitine was reversible.5. In metaphase the inactive state of MAPK induce that fertilized eggs can not complete mitosis, but have no influence on MPF activationInhibiting MAPK activation in metaphase induce zygote can not cleave,but MPF still was activated normally. If we remove the inhibition of U0126 to MAPK activation, fertilized eggs can normally complete the first mitosis.6. In metaphase the inactive state of MPF induce that fertilized eggs can not complete mitosis and MAPK maintain inactive stateInhibiting MPF activation in metaphase induce zygote can not cleave and MAPK can not be activated . If we remove the inhibition of roscovitine to MPF activation,fertilized eggs can normally complete the first mitosis.DiscussionIf eggs were cultured in the presence of the MAPK inhibitor U0126 (100μM) ,the activated oocyte yields the same phenotypes as Mos~ ~ parthe-nogenotes. It indicate that Mos -/- and U0126 result in MII oocyte activation by the same pathway - inhibition MAPK eventually and it is an important function for MAPK to regulate polar body emission. The oocyte treated by roscovitine can be activated and the activated oocyte yields the same phenotypes as Mos ~ ~ par-thenogenotes, which show that MPF may play an important role in the formation of spindle and the emission of polar body.Some paper reported that promoting nulear envelope breakdown ( NEBD) and mitosis completion require both MPF and MAPK. In view of result 5, If we inhibited MAPK activation, zygote can not complete mitosis. on the contrary, if we release MAPK from U0126's inhibition, zygote can enter two - cell stage successfully, so the active MAPK is required for mitosis and MAPK do not participate in MPF activation.