Protective Effect Of Icariin On The Development Of Preimplantation Mouse Embryos Against Hydrogen Peroxide-induced Oxidative Injury

Objectives: 1.To observe the effects of transient exposure of different concentrations of hydrogen peroxide（H₂O₂）on the development of kunming（KM）mouse 1-cell embryos.2.To observe whether icariin（ICA）supplement will reverse the developmental blockage of 1-cell embryos caused by H₂O₂ pre-treatment.3.To study the effects of ICA treatment on the expression levels of ROS,mitochondrial membrane potential,and the four zygotic marker genes in the KM mouse 1-cell embryos after pretreated with hydrogen peroxide,in order to understand the protective mechanisms of ICA on the mouse preimplantation embryos that are injured by oxidative stress,and to lay foundations for further investigations about improving the in vitro culture system of mammalian preimplantation embryos.Methods: 1.One-cell embryos of KM mice were collected and cultured in KSOM medium supplemented with different concentrations of H₂O₂ for 30 mins,and moved into fresh KSOM medium,respectively,for further cultivation.The development ratios of the preimplantation embryos in each group was recorded,and the concentration of H₂O₂ for further investigation was determined.2.One-cell embryos of KM mice were collected and cultured in KSOM medium supplemented with the proper concentration of H₂O₂ for 30 mins,and then moved into KSOM medium supplemented with different concentrations of ICA.The development ratios of the preimplantation embryos in each group was recorded.The protective effects of ICA on the oxidative stress injured 1-cell embryos after H₂O₂ pre-treatment was investigated,and the proper protective concentration of ICA was determined for further investigation.3.One-cell embryos of KM mice were collected and cultured in the following groups: KSOM,H₂O₂ and H₂O₂+ICA.At 30 h post-h CG injection,1-cell embryos were recovered and stained,respectively,with DCFH-DA and JC-1,and observed under an immunofluorescence microscope,to detect the expression levels of ROS and mitochondrial membrane potential.Furthermore,Real Time-PCR was performed to detect the m RNA expression levels of the four zygotic gene activation marker genes,Zscan4 d,Mu ERV-L,Hsp70.1 and e IF1 A.Results: 1.After KM mouse 1-cell embryos were pretreated with H₂O₂ for 30 mins,lower developmental ratios of 2-cells,4-cells and blastocysts were observed comparing with the control group.Among these groups,embryos cultured in 40 and 60 μmol/L H₂O₂ showed significant lowered developmental ratios（P<0.01）;and the embryos treated with 80 μmol/L H₂O₂ blocked totally at 1-cell stage.2.ICA was able to remit the oxidative stress injury,caused by 60 μmol/L H₂O₂ treatment,on the 1-cell embryos of KM mouse.The most significant protective effect of ICA was observed in the 40 μmol/L group;comparing the the 60 μmol/L H₂O₂ treatment group,the developmental ratios of 2-cells,4-cells and blastocysts were significantly elevated（P<0.01）.3.Results of immunofluorescence staining showed that,comparing with KSOM group,ROS expression levels of the 1-cell embryos in H₂O₂ group was significantly elevated（P<0.01）,and with lowered mitochondrial membrane potential（P<0.01）.Comparing with H₂O₂ group,ROS expression levels of the 1-cell embryos in ICA group was significantly decreased（P<0.01）,and with significant higher mitochondrial membrane potential（P<0.01）.4.Results of Real Time-PCR showed that,comparing with KSOM group,e IF1A mRNA expression levels of the 1-cell embryos in H₂O₂ group was significantly lowered（P<0.01）.Comparing with H₂O₂ group,e IF1 A m RNA expression levels of the 1-cell embryos in ICA group was significantly elevated（P<0.01）,while that of the other three zygotic marker genes showed no significant difference（P>0.05）.Conclusion: 1.Proper concentration（40μmol/L in the resent study）of ICA is able to remit the oxidative stress injury on the KM mouse 1-cell embryos induced by high concentration（60μmol/L）H₂O₂ pre-treatment,and significantly elevated the developmental ratios of 2-cells,4-cells and blastocysts;2.ICA is able to down-regulate the ROS expression levels of the KM mouse 1-cell embryos that are pre-treated with H₂O₂,and thus protects the preimplantation embryos from oxidative stress.Meanwhile,ICA is able to elevate the viability of the mitochondria,and thus takes roles in ensuring normal cellular biological activities by protecting mitochondria.3.ICA elevates the down-regulated m RNA expression levels of zygotic activation marker gene e IF1 A,caused by oxidative stress,in KM mouse 1-cell embryos,indicating that,other functional mechanisms may also exist in the regulation of ICA on mouse preimplantation embryo development.