Expression And Activation Of JNKs Protein In Mouse Preimplatation Embryos And The Relationship To Apoptosis

PART I. Expression of JNKs Proteins in Different Stages of Preimplantation Mouse EmbryosObjective: To investigate the expression of JNKs proteins in different stages of preimplantation mouse embryos.Materials and Methods:E0.5,1.5,2.5and 3.5 mouse embryos were gathered after PMSG and HCG administration. Immunofluorescence and laser confocal microcopy technique were applied to detect the expression of JNKs protein in different stage embryos.Result and Conclusion:1. JNKs protein were expressed in all stages of preimplantation mouse embryos;2. Expression of the protein were increased with the embryos stage progressed., and this increase was significant in E1.5-E2.5(P<0.05);PART II. The Influence of In-Vitro Environment and Stress from the Culture System to the Activation of JNKs ProteinObjective: To investigate the influence of In-Vitro environment and stress from the culture system to the activation of JNKs protein;Materials and Methods: Immunofluorescence and Wester-Blotting technique were used to compared the activation (phosphorylation) of JNKs protein in mouse embryos grow in vitro or in vivo; Immunofluorescencewas used to compared the activated level of JNKs protein in different culture contion as following: (1)Culture in different temperature (35°c,37 °c and 39 °c);(2)Culture in different media(CZB,G-l and G-2);(3)Cultured I different density(<3 embryos/25ul, 15-25 embryos/25ul and >50 embryo/25ul)Result: (1)Embryo grew in vitro seemed in a higher level of JNKs activation than the ones grew in vivo;(2) Embryo cultured in 39c seemed in a higher level of JNKs activation than the ones grew in other temperatures;(3) Embryo cultured in CZB seemed in a higher level of JNKs activation than the ones grew in other media; (4) Embryo cultured in a density of >50/ul showed a higher level of JNKs activation than the ones cultured in other density.Conclusion: (1)JNKs protein were more easily activated in embryos grew in vitro than in vivo;(2)Stress from culture system had a influence in activation of JNKs proteins. PART III. Relationship of JNKs Activation and Apoptosis of Mouse Embryo cellObjective: To investigate weather and how JNKs activation contributed to thedevelopment and apoptosis of mouse embryo cell.Materials and Methods: (1)Compare the addition of SP600125,a specific inhibitor of JNKs activation, in different concentration or different time to the development of mouse embryo development; (2)Detect the influence of Sp600125 to the apoptosis rate of blastocyst cells suffered in a heat shock of 41c ,6h.;(3)Detect the influence of SP600125 to the mitochondrial membrane potential of the E1.5 mouse embryos suffer a heat shock of 41c ,lh;(4)Detect the influence of SP600125 to Caspase-3 level in E1.5 mouse embryos suffer a heat shock of 41c ,3h;Result: (1) Embryos treated bySP600125 in a concentration of 20uM and in E1.5-2.5 show a better blastcyst formation rate than others; (2)Blastcyst treated by SP600125 showed a significant lower apoptosis rate than positive control after a heat shock of 6h; (3) E1.5 mouse embryos treated by SP600125 have more high membrane potential mitochondrial than positive control after heat shock of 1h; (4) E1.5 mouse embryos treated by SP600125 showed a higher expressed level of Caspase-3 than positive control after heat shock of 3h;Conclusion: Activation of JNKs protein perhaps negatively influence the development potential of preimplantation mouse embryos and can promote the apoptosis program , which had a relationship with the mitochondrial-dependent parthway.