Study Of Roles And Regulatory Mechanism Of Aurora B And Its SUMO Modification During Follicular And Granulosa Cells' Growth In Mouse

Experiment 1.Impact of Aurora B on Follicular and Granulosa cells' Development and Its regulatory Mechanism.1.The localization and protein expression of Aurora B in different stages of follicles and granulosa cells(GCs).The result showed that Aurora B was localized in the nucleus,and it was trans-located into midbody and cytoplasm during cytokinesis.Furthermore,following follicular development,protein expression level was increased(p<0.01),and reached the maximum at the antral stage.2.Effect of Aurora B on follicular development.The secondary follicles were co-cultured with Aurora B's inhibitor and the development was observed in vitro.The result indicated that follicular growth and atresia rate were decreased and increased respectively after inhibiting Aurora B phosphorylation(p<0.05).3.Effect of Aurora B on GCs' growth.The antral stage of GCs(pre-GCs)were co-cultured with Aurora B's inhibitor and the growth was observed in vitro.Our finding revealed that cell cycle was arrested at G1-S phase,proliferation rate was decreased and apoptosis rate was increased after inhibiting Aurora B phosphorylation(p<0.05).4.Regulatory mechanism of Aurora B on GCs' growth.The pre-GCs were co-cultured with Aurora B's inhibitor,then mRNA and protein expression levels of growth and apoptosis related gene were analyzed.The present result showed the mRNA and protein expression of CDK4,PCNA,and Bcl-2 expression were down-regulated,while caspase-8 and caspase-3 were up-regulated.Finally,we observed that Aurora B regulated the growth of GCs through p38 MAPK and Fas/FasL dependent pathway.Experiment 2.Verification of SUMOylation of Aurora B and its effects on GCs.1.Verification of SUMOylation.Eukaryotic expression plasmids was Successfully constructed.The pre-GCs were extracted and SUMOylation was tested by co-IP.The results showed that Aurora B could be modified by SUMO2 and Lys207 as major modification site.2.The effect of SUMO2 modification on Aurora B's function.The localization of Aurora B was performed by ICC in pre-GCs.The results showed that Aurora B was localized in the nucleus,but a litter Aurora BK207 R was localized in the cytoplasm.The protein stability of Aurora B was tested by western blot and found that SUMO2 modification accelerated Aurora B's protein expression level,which indicated SUMO2 modification could keeping localization and protein stability of Aurora B in GCs.3.Effect of SUMO2 modification on GCs' growth.The pre-GCs were transfected by control(HA),wild type(HA-Aurora B)and mutant type(HA-Aurora BK207R),and cell cycle,proliferation and apoptosis were performed.The results showed that the cell cycle was arrested at G1-S phase,proliferation was reduced,apoptosis rate was induced(p<0.01),finally,GCs growth was blocked.In summary,Aurora B localized in the nucleus,and trans-located into midbody during cytokinesis.As follicular development,the expression of Aurora B was increased in follicles and GCs,and reached maximum in the antral stage.Inactivity Aurora B regulated follicular and GCs' growth via down-regulated p-p38 MAPK,CDK4,PCNA and Bcl-2 and up-regulated caspase-8 and caspase-3.Furthermore,Aurora B could be modificated by SUMO2 at Lys207,SUMOylation keeping the localization and protein stability of Aurora B in GCs and influenced GCs' growth and follicular development.This study enhance theoretical basis of our understanding of follicular development and atresia as well as treatment of ovarian disorder.