Roles Of The MAPK And MPF Activities In Fusion Of Nucleolar Precursor Bodies Following Activation Of Mouse Oocytes

Nucleoli are the structures in which ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins to form ribosomes. After fertilization, nucleolus precursor bodies (NPBs) are established in the pronuclei. Although studies indicated that the assembly, growth and mutual fusion of NPBs were dependent on an early wave of pronuclear transcriptional activity and on the presence of ooplasmic factors produced during oocyte maturation, the mechanism for the movement and fusion of nucleolar precursor bodies during the M/G1 transition was not known. In this study, the roles of the MAP kinase and MPF activities in fusion of NPBs following activation of mouse oocytes were studied by using signalling pathways' inhibitor/activator treatment, observation under a light microscope, protein kinase assay after activation. The results were as follows:1. First, there existed numerous discrete and small NPBs in the pronuclei following activation of mouse oocytes, then these NPBs fused gradually. Finally, a large NPB could be found in the pronuclei.2.3.9 h after activating by ethanol, there appeared numerous discrete and small NPBs in the pronuclei, then these NPBs fused to form a large NPB 5.5h after activating. Finally, the NPB would disappear 15.1h after activating. When oocytes were activated by SrCl2 (6h), there existed numerous discrete and small NPBs in the pronuclei 3.8 h after initiating the activation, then these NPBs fused to form a large NPB 4.5h after activating. Finally, the NPB would disappear 18.7h after activating. 3. MPF activity was reduced to the lowest level one hour after oocytes were activated by ethanol, however MAP kinase activity didn't decline until 3h after activation. When oocytes were activated by SrCl2 (6h), there were similar changes about MPF activity and MAP kinase activity. Nevertheless, it was notable that MAP kinase would persist for more than 3 hours by SrCl2.4. After activating by ethanol, MAPK inhibitor (U0126) treatment and MPF activitor (MG132) treatment both hampered the fusion of NPBs. However, MPF inhibitor (ROS) treatment had no influence on the fusion. U0126 and MG132 treatment after activating by SrCl2 (5min or 6h) also had obtained the same results.5. In the experiment,, the activated oocytes were treated using U0126 and MG132 in different time periods following activation. The obtained results indicated that the maintenance of the first two hours' high MAP kinase activity following. activation were necessary for the fusion of NPBs. However, the longer MPF activity was maintained, the worse the capacity of fusion was.6. During the newly ovulated oocytes were activated for 6 hours by SrCl2, treatments by signalling pathway inhibitor/activator had the same effect as the aged oocytes(18h after hCG injection) for the fusion of NPBs.7. During aging in vivo, MPF and MAP kinase activity declined gradually with the increase of oocyte age. At the same time, the capacity of the fusion of NPBs also declined accordingly.In conclusion, The high MAP kinase activity at least needed to be maintained for 2 hours, and MPF activity must decline immediately after activation, only in this way could NPBs fuse.