Study On Oxidative Damage Of Embryos Cloned From Somatic Cell In Yanbian Cattle

Yanbian cattle as one of top five local varieties cattle possess meat tender, nutrient-rich, excellent meat potential and broad prospects of products for commercial development. With the development of animal husbandry and "East West, Hong Huang," Beef Cattle Development Strategy in Jilin Province of China, the development of Yanbian cattle breeding industry has been coming faster and faster, so it is necessary to expand our industrialization scale and improve the additional value of science and technology. Somatic cell cloning technology considered as an emerging scientific and technological means, not only support embryonic stem cell technology, transgenic technology and medical and other scientific researchs, but also can be used to produce a large number of livestock in animal husbandry that have the same genotype and have a profound significance in stock breeding, but so far studies have shown that there is low efficiency of the technology produced more offspring with birth defects, etc., therefore, it does not adapt to the actual production needs. Numerous researches on oxidative damage of gametes in vitro production and storage have been reported in artificial insemination, in vitro fertilization, embryo transfer and other reproductive biotechnologies, and there have been lots of studies on micro-manipulation method, fusion activation method, the nuclear re-programming mechanism, DNA methylation etc. in the somatic cell cloning technology, now, it is a new hotspot to research the molecular mechanism of physical defects of offspring in somatic cell cloning, but the research on oxidative damage of cloned embryos of somatic cell cloning has not been reported.Objective:(1) To observe the effect of oxidant hydrogen peroxide (H2O2) on the development of cloned embryos from Yanbian cattle in vitro and explore the oxidative damage of H2O2 on the reconstructed embryos; (2) To observe the effect of anti-oxidant glutathione (GSH) on the development of cloned embryos from Yanbian cattle in vitro and explore the protective role of GSH to the oxidative damage; (3) To quantify the H2O2 and superoxide anion (O2-) two kinds of reactive oxygen species (ROS) levels within embryos cloned from Yanbian cattle in contrast to parthenogenetic embryos of Yanbian cattle and explore the change law of them at different stages of development in vitro; (4) To detect mRNA expression levels of encoding catalase (CAT), manganese super-oxide enzymes (Mn-SOD) and glutathione peroxidase (GPx) at different developmental phase of reconstructed embryos from Yanbian cattle in contrast to- parthenogenetic embryos and explore the relationship between the expression levels and oxidative damage.Methods and contents:(1) Adding exogenous H2O2 to in vitro culture medium, observe the development efficiency and development speed of reconstructed embryos from somatic cell cloning in Yanbian cattle in vitro and test the quality of the impact caused by oxidative damage to determine the critical H2O2 concentration of impact their development and the stage of impact greatest; (2) Adding exogenous GSH in vitro culture medium, observe the development efficiency of reconstructed embryos from somatic cell cloning in Yanbian cattle to determine the appropriate concentration and the required suitable time relieving oxidative damage; (3)Detect content of ROS (H2O2 and O2·- within reconstructed embryos at different developmental stages using dihydro-Ethidium and 2',7'-dichlorofluorescein diacetate, respectively, and using fluorescent microscope camera and image processing software for quantitative analysis and explore the internal change law of ROS; (4) The mRNA of encoding CAT, Mn-SOD and GPx of reconstructed embryos were amplified using specific primers and reverse transcription-polymerase chain reaction and detected by agarose gel electrophoresis, and then determine the expression law of reconstructed embryos using gel image analysis software and semi-quantitative analysis.Results:(1) the cleavage rate of adding 0μMol/L,60μMol/L,80 uMol/LH2O2 was significantly higher than that of 100μMol/L and 120μMol/L group (P<0.05), the development rate of all of the treatment group adding H2O2 was significantly lower than the non-adding treatment group (P<0.05); the cleavage rate of the treatment group adding H2O2 at 0-24h was significantly higher than the other treatment groups (P<0.05), the development rate more than 16 cells of 48-72h treatment group was significantly lower than the other treatment groups (P<0.05); there were no significant differences in different periods of time without adding H2O2 (P> 0.05), but the development rate more than 16 cells of different periods of time non-adding H2O2 was significantly lower than that the control group (all-time do not add, P<0.05); the reconstructed embryos treated with H2O2 displayed rough blastomere surface, uneven cytoplasm, (small and loose) and occasionally saw little or no cytoplasm of the blastomere, the gap between blastomeres and zona pellucida was large, and blastomere sizes developed irregularly and differently in scale, and there were a large number of cell debris. (2) the cleavage rate of 1 mMol/L group adding exogenous GSH was significantly higher than that of 7 mMol/L group (P<0.05), the cleavage rate of 1 mMol/L and 7 mMol/L group was not significantly higher than that of 0 mMol/L and 4 mMol/L group (P> 0.05), the blastocysts rate of 1 mMol/L group was significantly higher than that of the other treatment groups (P<0.05); the cleavage rate of 0-24 h group and 24-48 h group adding exogenous GSH was significantly higher than that of 48-72 h group and 72-96h group (P<0.05), did not differ significantly with the other treatment groups (P> 0.05), the development rated of more than 16 cell of the control group was significantly higher than that of the 48-72 h group and 72-96h group (P<0.05). (3) there was no significant difference on O2-levels at 2-cell stage between parthenogenetic activation and somatic cell cloning groups (P> 0.05), the O2-levels at 4-cell stage,8-cell stage,16-cell stage and blastocyst stage of somatic cell cloning groups was significantly higher that of the parthenogenetic activation group (P<0.05), O2-level of parthenogenetic embryos from Yanbian cattle was up to the highest at 2-cell stage, down to the lowest at 4-cell stage, that of 8-cell stage has increased, decreased at 16-cell stage,and increased again at blastocyst stage. The O2-of reconstructed embryos from Yanbian cattle was lowest at 2-cell stage, increased sharply at 4-cell stage and decreased at 8-cell stage, began to rise at 16-cell stage and was basically the same to the blastocyst stage; the H2O2 level of reconstructed embryos was significantly higher than that of parthenogenetic embryos at any cell stage (P<0.05), the H2O2 level of parthenogenetic embryos continueed to rise gradually at 2-cell stage to the blastocyst stage, that of reconstructed embryos was lowest at 2-cell stage, increased at 4-cell stage sharply, reduced after the 8-cell stage and continued to rise.to the blastocyst stage. (4) there was no expression of encoding CAT, Mn-SOD and GPx genes in oocytes, there was expression of encoding CAT genes of reconstructed embryos at 2-cell stage, expression of encoding CAT and GPx genes of reconstructed embryos and encoding CAT genes of parthenogenetic embryos at 4-cell stage, expression of encoding CAT, Mn-SOD and GPx genes of reconstructed embryos and parthenogenetic embryos at 8-cell stage and more than 16-cell stage; abundance of CAT gene expression of reconstructed embryos and parthenogenetic embryos was significantly different at 2-cell stage and latter development stage (P<0.05), abundance of Mn-SOD gene expression of reconstructed embryos and parthenogenetic embryos was not significantly different at 8-cell stage (P> 0.05), but that was significantly different at 16-cell stage and latter development stage (P<0.05), abundance of GPx gene expression of reconstructed embryos and parthenogenetic embryos was significantly different at 4-cell stage, but that was not significantly different at 8-cell stage and latter development stage (P> 0.05).Conclusions:(1) H2O2 affecting on reconstructed embryos from somatic cell cloning in Yanbian cattle can result in oxidative damage to the embryos of reconstructed, and aslo accelerating the development speed. GSH has an anti-oxidant protective role to the reconstructed embryos. (2) The ROS level within the reconstructed embryos is higher than the parthenogenetic embryos, a period of great change almost focus on the chromosome genome activation period. (3) The mRNA of encoding CAT, Mn-SOD and GPx of reconstructed embryos expressed with the genomic activation and periods of expression a lot was later than the period of embryonic ROS generated within expression a lot.