The Influence Of Bovine Oocytes In Vitro Maturation And Apoptosis By Stress-induced And The Preliminary Research On Metallothionein Genes

Cell apoptosis is one of the main ways for early embryonic cells to remove bad cells which produced by its adverse or external environment. Oocytes in vitro is easily affected by some of the environmental and exogenous stimuli, such as oxidants, mechanical stimulation, heavy metals, biological toxins, ultraviolet ,which produce a series of irritation, including cell transduction path, gene expression and protein synthesis, cell division and and proliferation,cell apoptosis, or even necrosis; Metallothionein has the effects on cells to clear free radicals and inhibit the cells apoptosis. Due to the influence of the environment, the cells will inevitably get the outside environment influence.Through in vitro oocytes, adding exogenous stress induced factor further discusses the influence of development and apoptosis of oocytes, which can improve the mature rate, be helpful for in vitro fertilization, improve the development rate of early embryo implanted rate, reduce with attach the infertility rates or embryos aberrtion rate and abortion rate.The test materials of this study was bovine oocytes,using the method of confocal microscopy, Annexin V - FITC/PI double dye test, Fluo3 / AM and RH123 dyeing, RT– PCR,which can be applied in the research of the influence of bovine oocytes in vitro maturation and apoptosis by CdCl₂,ultraviolet,H2O2. The eukaryotic expression vectors of MT were completed. The results were following:1. In the experiment, it's the first time we studied the influence of bovine oocytes in vitro maturation and apoptosis by CdCl₂. Experiments show that the time effect on oocytes of CdCl₂ is dose increased effect.With its concentration increased, oocytes apoptosis reach intensifies. Compared with control oocytes apoptosis rate,1.6μg/ml and 2.4μg/ml CdCl₂ treatment groups have obvious difference (P < 0.05). As CdCl₂ doses increased, its Ca2+ fluorescence intensity and the fluorescent intensity of the mitochondrial membrane potential are increasing., the ratio of Bcl-2 / Bax has obvious changes.2. In the experiment, it's the first time we studied the influence of bovine oocytes in vitro maturation and apoptosis by ultraviolet. Experiments show that the effect on oocytes of ultraviolet is time increased effect.With its concentration increased, oocytes apoptosis reach intensifies. Compared with control oocytes apoptosis rate,20min and 30min ultraviolet treatment groups have obvious difference (P < 0.05). As the time of exposuring under ultraviolet extending , its Ca2+ fluorescence intensity and the fluorescent intensity of the mitochondrial membrane potential are increasing., the ratio of Bcl-2 / Bax has obvious changes.3. In the experiment, it's the first time we studied the influence of bovine oocytes in vitro maturation and apoptosis by H2O2. Experiments show that the doses effect on oocytes of H2O2 is dose increased effect.With its concentration increased, oocytes apoptosis reach intensifies. Compared with the oocytes apoptosis rate of control,100μmol/L and 200μmol/L treatment groups, 300μmol/L and 400μmol/L treatment groups have obvious difference (P < 0.05). As H2O2 doses increased, its Ca2+ fluorescence intensity and the fluorescent intensity of the mitochondrial membrane potential are increasing., the ratio of Bcl-2 / Bax has obvious changes.4. This experiment is constructed of metallothionein gene expression vector,which can do further preparation for the subsequent carrying experiments and MT of gene expression detection.