Regulation Of NO/cGMP Pathway In Mouse Blastocyst Hatching

Part I: The effect of nitric oxide of different concentrations on blastocyst formation and hatchingObjective: To investigate the effect of NO of different concentrations on blastocyst formation and hatching.Method:(1)The morula obtained were randomly divided into five groups supplemented with different concentrations(125 ?M,250 ?M,500 ?M,5 m M)of NOS inhibitor(L-NAME).The development and hatching of blastocysts were observed and statistically analyzed.(2)All blastocysts that collected on D5 from these 5 groups were treated with eNOS immunofluorescence staining.The intensity of eNOS fluorescence expression was statistical analysted.And the mRNA level of eNOS of those different groups were detected respectively.(3)The morula obtained were randomly divided into five groups supplemented with different concentrations(0 n M,10 n M,100 n M,500 n M and 2 ?M)of SNP for continuing blastocyst culture.The development and hatching of blastocysts were observed and statistically analyzed.(4)The morula obtained were randomly divided into five groups supplemented with 5m M L-NAME and different concentrations of(10 n M,500 n M,and 2 ?M).The development and hatching of blastocysts were observed and statistically analyzed.Results:(1)The results showed that comparing with the control group,125 ?M L-NAME had no significant effect(P> 0.05)on blastocyst development and hatching.250 ?M,500 ?M and 5 m M L-NAME significantly reduced blastocyst development and hatching rate(P <0.05),and with increasing doses of L-NAME significantly reduced blastocyst development and hatching.Blastocyst hatching was completely inhibited with 5 m M L-NAME.(2)The results showed that eNOS was expressed in the cytoplasm of cells in the blastocyst.As the concentration increasing of L-NAME(0 ?M 125 ?M,250 ?M,500 ?M,5 m M),decreased eNOS expression was observed,and hatched cells associated with hatching blastocyst had strong fluorescence.Similarly,eNOS mRNA levels was also dose-dependent decreased with increasing concentration of L-NAME.(3)Compared with the control group,10 n M SNP had no significant effect on the blastocyst hatching rate(P> 0.05).However,100 n M,500 n M and 2 ?M SNP significantly reduced the blastocyst hatching.With the SNP doses increasing,blastocyst hatching was significantly reduced and 2 ?M SNP completely inhibited blastocyst hatching.(4)10 n M SNP reversed the inhibition effect of 5 m M L-NAME for D4,D5 blastocyst hatching,and significantly promoted the hatching blastocyst(P <0.05).However,with the increasing concentration of SNP,blastocyst development and hatchability significantly decreased(P <0.05).Conclusion: NO of appropriate concentration was the indispensable condition of blastocyst hatching,NO of low or high concentration would reduce the rate of blastocyst hatching.Part II: NO / cGMP pathway in blastocysts hatchingObjective: To study whether NO regulate blastocyst hatching through cGMP singal transduction pathway.Method:(1)Randomized obtained morula were divided into different groups.The culture media were supplemented with different concentrations(10 n M,100 n M,500 n M and 2 ?M)of NO-sensitive guanylyl cyclase inhibitor(8-Br-cGMP).The development and hatching of blastocysts were observed and statistically analyzed.(2)The blastocyst culture media were added with different concentrations(10 n M,100 n M,500 n M and 2 ?M)of 8-Br-cGMP along with 5 m M L-NAME.The development and hatching of blastocysts were observed and statistically analyzed.(3)The harvested blastocysts were collected,the RNA level of cathepsin L and cathepsin P were detected using real-time quantitative PCR(SYBR Green method).Results:(1)Compared with the control group,10 nM,100 nM,500 nM and 2 ?M 8-Br-cGMP significantly decreased blastocyst hatching rate(P <0.05).With the increasing of 8-Br-cGMP concentration,blastocyst hatching rate was significantly reduced,and 2 ?M 8-Br-cGMP completely inhibited blastocyst hatching.(2)10 n M and 500 n M 8-Br-cGMP significantly promoted blastocyst hatching,partially reversed the inhibitory effect of 5 m M L-NAME on blastocyst hatching(P <0.05).However,no blastocysts hatching phenomenon was found in 2 ?M 8-Br-cGMP and 5 m M L-NAME co-added groups.(3)L-NAME reduced cathepsin L and cathepsin P mRNA expression levels,and resulted in reduced hatching of blastocysts.Conclusion:(1)Blastocyst hatching was regulated through NO/cGMP signal transduction pathway.(2)L-NAME reduced the expression levels of cathepsin L and cathepsin P mRNA,and ultimately lead to lower rate.Part III: NO effect on apoptosis in blastocystsObjective: To study the effect of NO on blastocyst apoptosisMethod:(1)In this study,5 m M L-NAME,2 ?M 8-Br-cGMP,10 nm SNP,500 nm SNP,2 ?M SNP was added alone and applied immunological techniques to detect fluorescent blastocysts in different groups for the actived caspase 3 expression level,and analyzed the impact of different additions on blastocyst apoptosis.(2)In order to further study the impact of NO generation on D5 blastocyst apoptosis,this experiment added separately of 5 m M L-NAME,2 ?M 8-Br-cGMP,10 nm SNP,500 nm SNP,2 ?M SNP,and using 5 ?g/ml propidium iodide(PI)staining necrotic blastocyst cells.Flow cytometry analysis was applied to statistical detect cell viability,and detect the effect of different additions on blastocysts cell apoptosis.Results: Compared with the control group,5 m M L-NAME,2 ?M 8-Br-cGMP and 10 nm SNP had no significant effect on caspase 3 levels in blastocyst cells,but 500 nm SNP and 2 ?M SNP were significantly increased the levels of caspase 3(P <0.05)in blastocysts,and showed a dose-dependent manner.With the increasing concentration of SNP,caspase 3 expression was significantly enhanced and promoted apoptosis of blastocyst cells.(2)Flow cytometry showed that distribution of cells in the control group and 5m M L-NAME,2u M cGMP,and 10 n M SNP group were no significant difference,but obviously necrotic cell population were observed in 500 n M SNP and 2u M SNP groups.Conclusion: Blastocyst apoptosis seems not caused by an excessive NO through cGMP pathway,there were other pathways involved in the regulation of blastocyst cells apoptosis through Caspase 3 activation.