The Study Of Improving The Quality Of In Vitro Matured Oocytes:Carrying-Over Effects Of GVBD Blocking AND The Optimizationin Of In Vitro Maturation System

Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD) and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on postblocking meiotic progression (PBMP). Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a) whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b) when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c) whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF) in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor) to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity.Many studies have failed to get rat offspring by somatic cell nuclear transfer. The most important reason is the spontaneous activation of rat oocytes. Spontaneous activation rate of in vitro matured oocytes was significantly lower than in vivo matured oocytes. There were many researches about development of in vivo matured oocytes no matter by parthenogenetic activation or in vitro fertilization, but few about in vitro matured oocytes not to mention live birth after fertilization. Oocytes were cultured in TCM-199 supplemented with cysteamine/cystine and coculture with cumulus can improve cytoplasmic maturation of in vitro matured oocytes. After PA or IVF, the developmental capacity of oocytes in our culture system did not have any difference with in vivo matured oocytes, also including the live birth. After improved the in vitro culture condition, the oocytes nuclear progression and Cortical granule (CG) migration were accelerated; the intracellular glutathione level were increased and the ROS level was reduced; the mRNA about cytoplasmic maturation expression pattern analysis in matured oocytes showed a significant upregulation. so, we have built a better in vitro maturation system of rat oocytes, and laid a good foundation for the further research in the future.