Effects Of Sphingosine 1-phosphate, Broad-spectrum Caspase Inhibitor, Stem Cell Factor On Mouse Early Embryonic Development

Apoptosis is the main form of cell death of oocytes and embryos, and it can cause failure of oocyte fertilization, arrest of embryonic development and failure of in vitro fertilization and embryo transfer(IVF-ET). The mechanism of molecular regulation of apoptosis may be very complicated and has not yet been clear. The current study suggests that both proapoptotic factors and antiapoptotic factors play important roles in regulating embryonic development and cell apoptosis, and the balance of the two contrary factors can determine cell survival or death of oocytes and embryos. As we know, Bcl-2 family and Caspase are the two main apoptotic regulary genes expressed in early embryos and have important significance. Antiapoptotic artificial interfering technique may be a new approach of improving embryonic quality and promoting the successful rate of IVF-ET. Therefore, we study mouse early embryo to evaluate the effects of antiapoptotic factors such as Sphingosine 1-phosphate(S1P), broad-spectrum Caspase inhibitor(CAI), stem cell factor(SCF) on early mouse embryonic development and to determine effective concentrations of these antiapoptotic factors, and to further investigate effects of them on suppressing apoptosis of early mouse embryos induced by ceramide(CED) or hydrogen peroxide(H2O2). Experimental evidence can be provided for application of antiapoptotic artificial interfering technique to treatment of IVF-ET in future.PartⅠRelationships between quality and apoptosis of early mouse embryosOBJECTIVETo investigate the relationships between quality and apoptosis of early mouse embryos. Make profound understanding of the significance of apoptosis regulatory genes in early embryonic development.Try to explore a new approach of improving the embryonic quality.MATERIALS AND METHODSThe levels of apoptosis and proliferation of early mouse embryos were detected with several important methods, including the apoptotic assays for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), Caspase in situ fluorescence and Bcl-2 immunofluorescence, and proliferative detection of proliferating cell nuclear antigen (PCNA) immunofluorescence. The levels of apoptosis and proliferation were detected for morphologically normal embryos, arrested embryos and fragmented embryos.RESULTS1. TUNEL fluorescences were enhanced for arrested embryonic cells and embryonic fragments.2. Caspase fluorescences were enhanced for arrested embryonic cells and embryonic fragments.3. Fluorescences of Bcl-2 and PCNA were attenuated for arrested embryonic cells and embryonic fragments.CONCLUSIONSFor mouse early arrested embryonic cells and embryonic fragments, the incidence of DNA fragmentation and the activity of Caspase were increased, the expression of Bcl-2 and PCNA were decreased. It is indicated that for early embryos, apoptosis may cause embryonic arrest and fragments, and may be closely related to early embryonic development. PartⅡEffects of Sphingosine 1-phosphate, broad-spectrum Caspase inhibitor, Stem cell factor on early embryonic developmentOBJECTIVETo evaluate the effects of antiapoptotic factors such as SIP, CAI, and SCF on early mouse embryonic development and to determine effective concentrations of these antiapoptotic factors. Experimental evidence is provided for application of antiapoptotic artificial interfering technique to improving embryonic quality and promoting the successful rate of IVF-ET in future.MATERIALS AND METHODS1. Zygotes obtained from the female mice after superovulation and mating were cultured in vitro for 120 hours in different groups of HTF media. The number and morphous of proliferating embryonic cells were regularly observed. Blastocyst development rates,2- to 4-cell arrested embryo rates and fragmented embryo rates were counted.2. SIP experiment was divided into blank control group(BC group), negative control group(NC group), S1P100nM group, S1P50nM group, S1P25nM group and S1P12.5nM group according to different concentrations of SIP.3. CAI experiment was divided into BC group, NC group, CAI20μM group, CAI10μM group, CAI5μM group and CAI2.5μM group according to different concentrations of CAI.4. SCF experiment was divided into BC group, NC group, SCF200ng/ml group, SCF100ng/ml group, and SCF50ng/ml group according to different concentrations of SCF.RESULTS1. S1P experiment:(1) Blastocyst development rates:BC group 71.6%, NC group 68.6%, S1P100nM group 62.5%, S1P50nM group 75.0%, S1P25nM group 92.3% and S1P12.5nM group 72.6%. Blastocyst development rate of S1P25nM group increased significantly compared with NC group and BC group (P<0.001 and P<0.01); there was no statistically significant difference between other S1P concentration groups and NC or BC group(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).(2) 2- to 4-cell arrested embryo rates:BC group 24.7%, NC group 25.7%, S1P100nM group 27.8%, S1P50nM group 20.8%, S1P25nM group 7.7% and S1P12.5nM group 26.0%.2- to 4-cell arrested embryo rate of S1P25nM group decreased significantly compared with NC group and BC group (P<0.01); there was no statistically significant difference between other SIP concentration groups and NC or BC group(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).(3) Fragmented embryo rates:BC group 22.2%, NC group 25.7%, S1P100nM group 30.6%, S1P50nM group 19.4%, S1P25nM group 7.7% and S1P12.5nM group 21.9%. Fragmented embryo rate of S1P25nM group decreased significantly compared with NC group and BC group (P<0.01 and P<0.05); there was no statistically significant difference between other SIP concentration groups and NC or BC group(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).2. CAI experiment:(1) Blastocyst development rates:BC group 71.8%, NC group 68.9%, CAI20μM group 35.5%, CAI10μM group 76.2%, CAI5μM group 86.9% and CAI2.5μM group 71.4%. Blastocyst development rate of CAI5μM group increased significantly compared with NC group and BC group (P<0.01 and P<0.05); blastocyst development rate of CAI20μM group decreased significantly compared with NC group and BC group (P<0.001); there was no statistically significant difference between other CAI concentration groups and NC or BC group(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).(2) 2- to 4-cell arrested embryo rates:BC group 25.4%, NC group 29.7%, CAI20μM group 54.8%, CAI10μM group 20.2%, CAI5μM group 13.1% and CAI2.5μM group 28.6%. 2- to 4-cell arrested embryo rate of CAI5μM group decreased significantly compared with NC group(P<0.05); 2- to 4-cell arrested embryo rate of CAI5μM group was lower than BC group but their differences were not statistically significant (P=0.051); 2- to 4-cell arrested embryo rate of CAI20μM group increased significantly compared with NC group and BC group (P<0.01); there was no statistically significant difference between other CAI concentration groups and NC or BC group(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).(3) Fragmented embryo rates:BC group 22.5%, NC group 25.7%, CAI20μM group 41.9%, CAI10μM group 23.8%, CAI5μM group 10.7% and CAI2.5μM group 22.9%. Fragmented embryo rate of CAI5μM group decreased significantly compared with NC group and BC group(P<0.05); fragmented embryo rate of CAI20μM group increased significantly compared with NC group and BC group (P<0.05); there was no statistically significant difference between other CAI concentration groups and NC or BC group(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).3. SCF experiment:(1) Blastocyst development rates:BC group 71.2%, NC group 69.6%, SCF200ng/ml group 62.5%, SCF100ng/ml group 77.8%, and SCF50ng/ml group 77.8%. Blastocyst development rates of SCF100ng/ml group and SCF50ng/ml group were separately higher than NC group and BC group but their differences were not statistically significant(P>0.05); blastocyst development rate of SCF200ng/ml group was lower than NC group and BC group but their differences were not statistically significant(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).(2) 2- to 4-cell arrested embryo rates:BC group 25.8%, NC group 28.6%, SCF200ng/ml group 29.2%, SCF100ng/ml group 20.6%, and SCF50ng/ml group 20.4%.2- to 4-cell arrested embryo rates of SCF100ng/ml group and SCF50ng/ml group were separately lower than NC group and BC group but their differences were not statistically significant(P>0.05); 2- to 4-cell arrested embryo rate of SCF200ng/ml group was higher than NC group and BC group but their differences were not statistically significant(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).(3) Fragmented embryo rates:BC group 22.7%, NC group 25.0%, SCF200ng/ml group 26.4%, SCF100ng/ml group 22.2%, and SCF50ng/ml group 18.5%. Fragmented embryo rates of SCF100ng/ml group and SCF50ng/ml group were separately lower than NC group and BC group but their differences were not statistically significant(P>0.05); fragmented embryo rate of SCF200ng/ml group was higher than NC group and BC group but their differences were not statistically significant(P>0.05); there was no statistically significant difference between NC group and BC group(P>0.05).CONCLUSIONS1. Both SIP and CAI can effectively inhibit apoptosis of mouse embryos and so promote development of early mouse embryos.2. SCF have no significant effect on suppressing apoptosis of mouse embryos and promoting development of early mouse embryos.PartⅢEffects of Sphingosine 1-phosphate, broad-spectrum Caspase inhibitor on suppressing apoptosis of early mouse embryos induced by ceramideOBJECTIVETo investigate effects of SIP and CAI on suppressing apoptosis of early mouse embryos induced by ceramide(CED). It aims to study the modular mechanism of antiapoptotic factors in inhibiting apoptosis and promoting development of early embryos.MATERIALS AND METHODS1. Zygotes obtained from the female mice after superovulation and mating were cultured in vitro for 120 hours in different groups of HTF media. The number and morphous of proliferating embryonic cells were regularly observed. Blastocyst development rates,2- to 4-cell arrested embryo rates and fragmented embryo rates were counted. 2. The experiment was divided into BC group, CED group, CED+S1P50nM group, CED+S1P25nM group, CED+S1P12.5nM group, CED+CAI10μM group, CED+CAI5μM group.RESULTS1. Blastocyst development rates:BC group 71.3%, CED group 32.9%, CED+S1P50nM group 42.7%, CED+S1P25nM group 60.5%, CED+S1P12.5nM group 51.4%, CED+CAI10μM group 32.5%, CED+CAI5μM group 44.2%. Blastocyst development rate of CED group decreased significantly compared with BC group (P<0.001). Blastocyst development rate of CED+S1P25nM group and CED+S1P12.5nM group increased significantly compared with CED group (P<0.001 and P<0.05); Blastocyst development rates of CED+S1P50nM group and CED+CAI5μM group were higher than CED group but their differences were not statistically significant(P>0.05); there was no significant difference between CED+CAI10μM group and CED group(P>0.05). Blastocyst development rate of CED+S1P25nM group was lower than BC group but their differences were not statistically significant(P>0.05); blastocyst development rates of CED+S1P12.5nM group, CED+S1P50nM group, CED+CAI5μM group and CED+CAI10μM group decreased significantly compared with BC group (P<0.01).(2) 2- to 4-cell arrested embryo rates:BC group 25.3%, CED group 55.7%, CED+S1P50nM group 46.7%, CED+S1P25nM group 32.6%, CED+S1P12.5nM group 40.5%, CED+CAI10μM group 43.8%, CED+CAI5μM group 37.7%.2- to 4-cell arrested embryo rate of CED group increased significantly compared with BC group (P<0.001).2- to 4-cell arrested embryo rates of CED+S1P25nM group and CED+CAI5μM group decreased significantly compared with CED group (P<0.01 and P<0.05); 2- to 4-cell arrested embryo rates of CED+S1P12.5nM group, CED+S1P50nM group and CED+CAI10μM were lower than CED group but their differences were not statistically significant(P>0.05).2- to 4-cell arrested embryo rates of CED+S1P25nM group and CED+CAI5μM group were higher than BC group but their differences were not statistically significant(P>0.05); 2- to 4-cell arrested embryo rates of CED+S1P12.5nM group, CED+S1P50nM group and CED+CAI10μM group increased significantly compared with BC group (P<0.05).(3) Fragmented embryo rates:BC group 21.8%, CED group 50.6%, CED+S1P50nM group 44.0%, CED+S1P25nM group 29.1%, CED+S1P12.5nM group 35.1%, CED+CAI10μM group 51.3%, CED+CAI5μM group 45.5%. Fragmented embryo rate of CED group increased significantly compared with BC group (P<0.001). Fragmented embryo rate of CED+S1P25nM group decreased significantly compared with CED group(P<0.01); fragmented embryo rates of CED+S1P12.5nM group, CED+S1P50nM group and CED+CAI5μM were lower than CED group but their differences were not statistically significant(P>0.05); there was no statistically significant difference between CED+CAI10μM group and CED group(P>0.05). Fragmented embryo rates of CED+S1P25nM group and CED+S1P12.5nM group were higher than BC group but their differences were not statistically significant(P>0.05); fragmented embryo rates of CED+SlP50nM group, CED+CAI5μM group and CED+CAI10μM group increased significantly compared with BC group (P<0.01).CONCLUSIONS1. CED can induce apoptosis of early mouse embryos.2. S1P can suppress apoptosis of early mouse embryos induced by CED.3. CAI can suppress apoptosis of early mouse embryos induced by CED.PartⅣEffects of Sphingosine 1-phosphate, broad-spectrum Caspase inhibitor on suppressing apoptosis of early mouse embryos induced by H2O2OBJECTIVETo investigate effects of S1P and CAI on suppressing apoptosis of early mouse embryos induced by H2O2. It aims to observe whether antiapoptotic factors can protect early embryos from oxidative stress. MATERIALS AND METHODS1. Zygotes obtained from the female mice after superovulation and mating were cultured in vitro for 120 hours in different groups of HTF media. The number and morphous of proliferating embryonic cells were regularly observed. Blastocyst development rates,2- to 4-cell arrested embryo rates and fragmented embryo rates were counted.2. The experiment was divided into BC group, H2O2 group, H2O2+S1P50nM group, H2O2+S1P25nM group, H2O2+CAI10μM group, H2O2+CAI5μM group.RESULTS1. Blastocyst development rates:BC group 72.1%, H2O2 group 31.0%, H2O2+S1P50nM group 32.6%, H2O2+S1P25nM group 51.1%, H2O2+CAI10μM group 38.5%, H2O2+CAI5μM group 50.0%. Blastocyst development rate of H2O2 group was significantly lower than BC group(P<0.001). Blastocyst development rates of H2O2+S1P25nM group, H2O2+S1P50nM group, H2O2+CAI5μM group and H2O2+CAI10μM group were all higher than H2O2 group but their differences were not statistically significant(P>0.05). Blastocyst development rates of H2O2+S1P25nM group, H2O2+S1P50nM group, H2O2+CAI5μM group and H2O2+CAI10μM group were all lower than BC group(P<0.05).2.2- to 4-cell arrested embryo rates:BC group 25.6%, H2O2 group 59.5%, H2O2+S1P50nM group 51.2%, H2O2+S1P25nM group 31.1%, H2O2+CAI10μM group 56.4%, H2O2+CAI5μM group 44.7%.2- to 4-cell arrested embryo rate of H2O2 group was significantly higher than BC group(P<0.01).2- to 4-cell arrested embryo rate of H2O2+S1P25nM group was significantly lower than H2O2 group(P<0.01); 2-to 4-cell arrested embryo rates of H2O2+S1P50nM group, H2O2+CAI5μM group and H2O2+CAI10μM group were lower than H2O2 group but their differences were not statistically significant (P>0.05).2- to 4-cell arrested embryo rates of H2O2+S1P25nM group and H2O2+CAI5μM group were both lower than BC group but their differences were not statistically significant (P>0.05);H2O2+S1P50nM group and H2O2+CAI10μM group were significantly higher than BC group(P<0.05). 3. Fragmented embryo rates:BC group 23.3%, H2O2 group 52.4%, H2O2+S1P50nM group 55.8%, H2O2+S1P25nM group 33.3%, H2O2+CAI10μM group 53.8%, H2O2+CAI5μM group 31.6%. Fragmented embryo rate of H2O2 group was significantly higher than BC group(P<0.01). Fragmented embryo rates of H2O2+S1P25nM group and H2O2+CAI5μM group were lower than H2O2 group but their differences were not statistically significant (P>0.05); There were no statistically significant differeces between H2O2+S1P50nM group or H2O2+CAI10μM group and H2O2 group(P>0.05). Fragmented embryo rates of H2O2+S1P25nM group and H2O2+CAI5μM group were higher than BC group but their differences were not statistically significant (P>0.05); H2O2+S1P50nM group and H2O2+CAI10μM group were significantly higher than BC group(P<0.01).CONCLUSIONS1. H2O2 can induce apoptosis of early mouse embryos.2. SIP can suppress apoptosis of early mouse embryos induced by oxidative stress.3. CAI have no significant effect on suppressing apoptosis of early mouse embryos induced by oxidative stress.