The Effect Of EGF And Coculture With GCs To GSH Concentration And Ultrastructure Of Goats Oocyte In Vitro Maturation

The aim was to study how to inhibit apoptosist of goats oocyte and to study intracellular GSH concentration including the effect of EGF and coculture with granulosa cells(GCs); Compared with the difference of GSH concentration in pregnancy goats and unpregnancy goats. The effect of EGF and coculture with GCs to Ultrastructure changes of goats oocyte such as lipid droplets, microvillus and mitochondria were also studied. It is the basic research to oocyte and embryo Engineering.Cumulus-oocyte complexes were collected from goats at an abattoir by aspiration from antral follicles. Oocytes were matured in TCM199 medium supplemented with 15% bovine fetal serum, 1 mg/L 17β-estradiol, 10 IU/ml PMSG, 20 IU/ml hCG, 0.146 mg/ml L- glutamine, 0.1 mg/ml Cys, 50ng/ml EGF, 75.0mg/L penicilin, 50.0mg/L streptomyci. Oocytes were matured 24 h at 38.5℃, in 5% CO₂, in air, the saturated humidity. The intracellular GSH leVel was evaluated with spectrophotometric methodology. Result as follows: GSH concentration in pregnancy aboriginal goats oocyte and unpregnancy milk goats were 2.38 pmol/oocyte, 3.52 pmol/oocyte respectively. GSH concentration of oocyte matured in EGF-free medium in pregnancy aboriginal goats oocyte and unpregnancy milk goats are 5.25 pmol/oocyte, 6.68 pmol/oocyte respectively. GSH concentration in mature oocyte was about double than one in immature oocyte. GSH concentration of oocyte matured in EGF-added medium in pregnancy aboriginal goats oocyte and unpregnancy milk goats were 6.63 pmol/oocyte, 8.14 pmol/oocyte respectively. The effect of EGF on intracellular glutathione concentration were remarkable(P＜0.05). GSH concentration of oocyte matured in EGF-added and coclture medium in pregnancy aboriginal goats and unpregnancy milk were 8.32 pmol/oocyte, 10.24 pmol/oocyte respectively. The effect of coculture on intracellular glutathione concentration were remarkable(P＜0.05). GSH concentration in pregnancy goats and unpregnancy milk goats are 6.57 pmol/oocyte, 6.63 pmol/oocyte respectively. There is not remarkable difference between them(P＞0.05). GSH concentration of oocyte in pregnancy goats were lower than oocyte in unpregnancy goats(P＜0.05).After the completion of culture of oocytes for IVM, cumulus cells were removed. Ultrastructure was studied under electron microscope. Result as follows: the majority of microvillus fell off from the plasmalemma, vanished, there were big lipid droplets in the cytoplasm of oocyte matured in EGF-free medium. Mitochondria approximately assumed the circular and majority of presents the vacuole. The majority of microvillus were attached to the plasmalemma, mitochondria were tending to the ellipse in the cytoplasm of oocyte matured in EGF-added medium. Cortical granule (CGs) clustered under serried microvillus. The majority of microvillus were attached to the plasmalemma, rough endoplasmic reticulum was seen, mitochondria were rod-shaped or elongated in the cytoplasm of oocyte matured in EGF-added and coculture medium, there was healthy ultrastructure in it. There were high GSH concentration and healthy ultrastructure in EGF-added and coculture medium. GSH concentration could estimate cuclutre condition.