The Artificial Interfering Of Antiapoptosis In Embryonic Development

The consenescence and death of oocytes and embryos is the key factor whichlimited us to get ideal outcome of assisted reproductive techniques (ART) includingin vitro fertilization and embryo transfer (IVF-ET). Apoptosis is one of the leadingdeath modes of the oocyte and embryo as well as the main cause of fertility failure,embryo developmental retardation and blastocyst implantation dysfunction. Maybewe can get higher quality embryos and better outcomes of ART by controllingapoptosis. As we know, both promoting factors and antiapoptotic molecules playimportant roles in regulating the apoptosis of the cell. The death of the oocyte andembryo is also determined by the balance of the two kinds of contrary moleculers.Considered as one of the sub-family members of Bcl-2 gene family, Bax and Bakcan promote apoptosis. We brought forward a new artificial antiapoptotic interferedmethod in promoting the development of embryos in the first time. By transfectingsmall interfering RNA(siRNA) of Bax and/or Bak into the human granulosa cellsand Kunming mouse embryos, we interfered the expression of the Bax and/or Bakin these objects. The experimental results suggested a new approach on improving the quality of oocytes and embryos, established the basement of improving theblastocyst forming rate in the culturing of embryonic stem cells, and provided thetheory and experimentation bases of apoptosis intervention in sterility treatment inclinic.Part OneThe Apoptosis of Human Granulosa CellsObjectiveTo investigate the relationships between the apoptosis of human granulosa cellsand Bax-Bak by interfering the expression of the Bax/Bak by transfecting Bax/BaksiRNA into human granulosa cells. Try to explore a new approach of improving thequality of oocyte and embryo and promoting the successful rate of IVF-ET.Methods1.Detected the Bax-siRNA and Bak-siRNA with Westernblot.2.Separately or contemporarily transfected Bax-siRNA and Bak-siRNA intohuman granulosa cells, and observed the modality, detected the apoptosis of humangranulosa cells with flow cytometry.3. The experiment were divided into blank control group, positive control group,negative control group, Bax interfered group, Bak interfered group and Bax-Bakinterfered group. Results1. Western blot: the Bax-siRNA and Bak-siRNA are in effect.2. Modality observed:●The modality of cells in blank control group, Bak interfered group andBax-Bak interfered group was normal, the shape of them mostly waspolygon and shuttle, and most of them sticked to the wall.●The modality of cells in Bax interfered group mostly was normal, but therewere some black dot, the shape of the cells mostly was polygon and shuttle,and most of them sticked to the wall.●The modality of cells in positive and negative control group partly changedto round and shrinked, the space between cells increased, and there weremany black dot.●3. Results of flow cytometry:●The apoptosis of human granulosa cells of the groups of Bak and Bax-Bakinterfered was 3.44% and 3.97%. Compared to the negative group, theywere lower in evidence.●The apoptosis of human granulosa cells of the groups of Bax interfered was19.98%, conform to the negative group.Conclusions1. The apoptosis of human granulosa cells can be restrained by interfering theexpression of Bak gene.2. The effect of Bak orBak-bax interfered in antiapoptosis was remarkable.3. The effect of Bax disturbed in antiapoptosis of human granulosa cells was notremarkable. Part TwoThe Artificial Interfering inAntiapoptosis of Mouse EmbryosObjectiveTo investigate the apoptosis by transfecting Bax/Bak siRNA into mouseembryos, study a new approach of improving the quality of embryo and promotingthe successful rate of IVF-ET.Methods1. The negative siRNA labelled with FAM was transfected into mouse embryosthrough the stiletto by laser, then examined the efficiency by fluorescence.2. Separately or contemporarily transfected Bax-siRNA and Bak-siRNA intomouse embryos through the stiletto by laser, and observed the modality, calculatedthe rate of blastocyst formed.3. Separately or contemporarily transfected Bax-siRNA and Bak-siRNA intomouse embryos through the stiletto by laser. To detect the apoptosis of mouse embryos by fluorescence and calculated the apoptotic cells in mouse embryos, afterembryos were dyed by Hoechst33342 or PI.4. Detected the expression of Bak and Bax by immunofluorescence, andcompared to negative control group with light density.5. Detected the expression of precaspase-3 in Bak and Bax interfered groupseparately by immunofluorescenceResults1. There is fluorescence in the mouse embryos, which were transfected withnegative siRNA labelled by FAM, so we can conclude the method of transfectionmay be efficient.2. The rate of blastocyst formed: blank control group81.0%, negative controlgroup69.5%, Bax interfered group74.0%, Bak interfered group 92.5%and Bax-Bakinterfered group89.5%. There was statistically significant between the negativecontrol group and Bak interfered group or Bax-Bak interfered group, p＜0.001;There was no statistically significant between the negative control group and Baxinterfered group, p=0.318; and there was statistically significant between thenegative control group and blank control group, p=0.011.3. Detected the number of apoptotic cells of mouse embryos dyed byHoechst33342 and PI: blank control group26.0%, negative control group33.0%,Baxinterfered group30.5%, Bak interfered group 17.6%and Bax-Bak interferedgroupl9.1%. There was statistically significant between the negative control groupand Bak interfered group or Bax-Bak interfered group, p＜0.001; There was nostatistically significant between the negative control group and Bax disturbed group,p=0.470; 4. Detecting the expression of Bak and Bax by immunofluorescence: Thelight density of Bax interfered group and negative control group was 25.39±4.73,52.89±3.91, it weakened significantly in the former, p＜0.001. The light density ofBak interfered group and negative control group was 17.10±3.79, 35.26±4.88, itweakened significantly in the former, p＜0.001. The light density of Bak-Baxinterfered group and negative control group was: TRITC labelled group: 19.68±4.86,35.26±4.88, FITC labelled group: 32.78±3.73,52.89±3.91, all weakenedsignificantly in the former, p＜0.001.5. Detecting the expression of precaspase-3: The light density of Bax interferedgroup and negative control group was 31.48±2.65,30.24±3.40, no statisticallysignificant between them, p=0.318. The light density of Bak interfered group andnegative control group was 65.68±4.79,30.24±3.40, there was statisticallysignificant between them p＜0.001.Conclusions1. We first apply the method of stiletto by laser to transfect siRNA into mouseembryos.2. The expression of Bak and Bax was weakened by Bax-siRNA andBak-siRNA interfered into the mouse embryos.3. In Bak and Bak-Bax interfered group, the number of apoptotic cells inmouse embryos had significantly decreased, and the rate of blastocyst formed hadsignificantly increased; but in Bax interfered group, there was no significantlychanges. So the apoptosis of mouse embryos could be restrained by interfering theexpression of Bak and Bak-Bax.4. The expression of precaspase-3 in Bak interfered group enhanced, but no obviously changed in Bax interfered group.Part ThreeMouse Blastocyst Transfer andInspection of ChromosomeObjective1. To investigate whether the mouse blastocyst could develop to mature unit.2. To observe whether the born mice have physiological vice and abnormalchromosome.Methods:1. 10 blastocysts were choosed from each interfered group, and were transferredinto the mouse uterus; observe the basic complexion of the offspringmice.2. Inspect the chromosome of the offspringmice.Results1. 6mice were born in Bak group, and 5 mice were born in the other two group,there were no obviously malformation and action abnormity in all the offsprings.2. There were no obviously abnormity in the chromosome of the offspring mice. Conclusions1. The mouse embryos treated with antiapoptotic could develop to mature unit.2. The treatment of antiapoptotic has no significant effect on offspring mice.3. The treatment of antiapoptotic has no significant effect on the chromosomeof the offspring mice.