Study Of Function And Molecular Mechanism Of SUMO-1during Mouse Oocyte Meiosis

As an important posttranslational modification of proteins, SUMOylation have been reported involved in regulating a range of intracellular processes. To date, four mammalian SUMO proteins have been identified, including SUMO-1,2,3and4. Due to96%similarity between SUMO-2and SUMO-3, they are commonly known as SUMO-2/3. During mitosis, growing evidences indicated that SUMO pathway induced by SUMO-1have multiple biological function, including regulated the stability, transcriptional activity and localization of substrates, in addition to chromosome condensation and segregation, DNA repair, nucleo-cytoplasmic transport and the signal transduction in cells, but there are limited studies about its function and the molecular mechanism during mouse oocyte meiosis.Oocytes from Kunming mouse were studied in this experiment, we employed oocyte in vitro culture, microinjection, in vitro transcription, Western blot, immunofluorescence and chromosome spread to uncovered these problems:(1) to find out whether SUMO-1was involved in oocyte meiotic maturation process;(2) the temporal role of SUMO-1during oocyte meiotic maturation;(3) the impact of SUMO-1on aneuploidy formation;(4) the influence of SUMO-1on spindle organization and chromosome alignment;(5) the effect of SUMO-1on chromosome separation and condensation;(6) the affect of SUMO-1on kinetochore-microtubule attachment;(7) the affect of SUMO-1on proteins which involved in spindle organization and chromosome alignment;(8) to explore whether the expression and localization of SUMO-1owned age difference. Base on these researches, we can further comprehend the regulating mechanism of mouse oocyte meiosis, support more theoretical principle to better understand mammalian oocyte meiotic processes. The contents and results of these studies were indicated as following:1. The study of oocyte meiotic process after SUMO-1inhibition or depletion: specific antibody or siRNA of SUMO-1/UBC9was microinjected into GV stage oocytes to inhibit or deplete SUMO-1function. After microinjection, the oocytes were in vitro cultured for14h, the percentage of GVBD and PB1extrusion were checked at2.5h and14h, respectively. The results indicated that the percentage of GVBD and PB1extrusion were significant decreased after inhibition or depletion of SUMO-1.2. The study of oocyte meiotic process after SUMO-1overexpression: SUMO-1mRNA was in vitro synthesized and microinjected into GV stage oocytes. After microinjection, the oocytes were in vitro cultured for14h, the percentage of GVBD and PB1extrusion were checked at2.5h and14h, respectively. The results indicated that overexpression of SUMO-1has no significant effect on the percentage of GVBD and PB1extrusion when compared to the control group.3. The study of temporal role of SUMO-1during oocyte meiotic process:to study the temporal role of SUMO-1during oocyte maturation, GV stage oocytes were in vitro cultured for0,6, or9.5h, corresponding to GV, pro-metaphase, and anaphase I stages, respectively. After SUMO-1antibody microinjection and cultured for total14h (before and after antibody injection), the percentage of PB1extrusion was checked. The results indicated when SUMO-1antibody injected at GV and pro-MI stage, the percentage of PB1extrusion was significant decreased, but there was no significant difference when SUMO-1injected at AI stage, these results indicated SUMO-1function before AI during PB1extrusion.4. The study of aneuploidy formation after SUMO-1inhibition: after inhibition of SUMO-1in GV stage oocytes and cultured in vitro for14h, chromosome spread was employed to check the number of chromosome in matured oocytes. The results indicated aneuploidy appeared after SUMO-1inhibition but no aneuploidy was observed in the control group.5. The study of spindle organization and chromosome alignment after SUMO-1inhibition or depletion: after inhibition or depletion of SUMO-1in GV stage oocytes and cultured in vitro for14h, immunofluorescence was employed to stain the a-tubulin while used PI to stain the chromosome in matured and immatured oocytes. The results indicated the percentage of abnormal spindle organization were significant increased in both matured and immatured oocytes after SUMO-1inhibition or depletion, the chromosome alignment were also abnormal.6. The study of y-tubulin localization after SUMO-1inhibition: after inhibition of SUMO-1in GV stage oocytes and cultured in vitro for14h, immuno fluorescence was employed to stain the y-tubulin in matured and immatured oocytes. The results indicated in matured and immatured oocytes, the localization of y-tubulin was at the spindle poles in the control group. After SUMO-1inhibition, a small portion of γ-tubulin was dissociated from the poles in the immatured oocytes while the signal of γ-tubulin was disappeared at the poles in some matured oocytes. 7. The study of chromosome morphology after SUMO-1inhibition: after inhibition of SUMO-1in GV stage oocytes and cultured in vitro for8h, chromosome spread was employed to study the chromosome morphology. The results indicated74.2%of the oocytes exhibited less condensed chromosome after SUMO-1inhibition.8. The study of kinetochore-microtubule attachment after SUMO-1depletion: after depletion of SUMO-1in GV stage oocytes and cultured in vitro for8h, the oocytes were processed at4℃for10min and then immunofluorescence were employed to stain the a-tubulin and kinetochore. The results indicated the kinetochore-microtubule attachment was unstable and the kinetochore was disorder when compared with the control group.9. The study of protein involved in spindle organization and chromosome segregation after SUMO-1inhibition: after inhibition of SUMO-1in GV stage oocytes and cultured in vitro for8h. Immunofluorescence and chromosome spread were employed to check the expression and localization of proteins involved in spindle organization and chromosome segregation. The results indicated the centromere localization of BubRl was reduced, the accumulation of securin was decreased on spindles and REC8was almost disappeared in the chromosomes after SUMO-1inhibition.10. The study of expression and localization of SUMO-1in age difference mouse GV stage oocytes:oocytes were obtained from3weeks and12months old female mouse, immunofluorescence and western blot were employed to study the localization and expression of SUMO-1in age difference oocytes. The results indicated the expression of SUMO-1in12months old mouse was weaker than3weeks old mouse in GV stage oocyes, the signal of SUMO-1in GV was also decreased.In short, these results indicated SUMO-1played an important role during mouse oocyte meiotic maturation, was involved in spindle assembly and chromosome behavior through regulation of the attachment of K-MT and the localization of y-tubulin, BubRl, REC8, and securin, finally, its expression and localization owned age difference. These results can provide reference to understand the mechanism of oocyte meiosis, as well as lay foundation for improving domestic animal fertility.