Effects Of Pyruvate On Zygote Genome Activation In Early Porcine Embryos

In mammals,the activation of the zygotic genome is an important process in early embryonic development,which occurs during the transfer of the embryo from the fallopian tube to the uterine horn.Failure of the activation of the zygote genome will lead to the arrest of early embryonic development.Pyruvate can provide energy for embryos and inhibit the production of reactive oxygen species during the development of early embryos in mammals,but its effects on the genomic activation of porcine early embryo zygote have not been reported in detail.Therefore,this study aims to explore the effects of pyruvate on the genomic activation of porcine early embryo zygote and its mechanism.This experiment was divided into three groups: PZM-3 group(control group),-P group(without pyruvate group,removing all substances that interfere with the formation of pyruvate in PZM-3 medium),and +P group(pyruvate was added on the basis of-P group with the same amount of addition as PZM-3 group).There were 50 fertilized eggs in each group,and each experiment was repeated at least 3 times.To explore the pyruvate influence on pigs early embryos zygote genome activation,adopt the method of in vitro fertilization(ivf),will develop mature oocytes put eggs respectively in three groups after fertilization in vitro embryo culture,after 48 h observed number of cleavage,determination of 4-cell zygotic genome activation marker gene Eif1 a and maternal GDF9 gene m RNA expression quantity relatively,120 h after statistical number 4-cell block.After in vitro culture for 48 h,4-cells were collected,and the levels of reactive oxygen species,glutathione,mitochondrial membrane potential and ATP in each group were detected,and embryo culture medium was collected for the determination ofα-ketoglutaric acid,amino acid content and calcium ion concentration.The effects of pyruvate on genomic activation of porcine early embryonic zygotes were investigated from three aspects:oxidative stress,energy metabolism and signal regulation.The results showed as follows:(1)compared with PZM-3 group,the cleavage rate of-P group was significantly decreased(P < 0.05),but the cleavage rate of +P group was not significantly changed(P > 0.05),and the cleavage rate of +P group was significantly higher than that of-P group(P < 0.05);Compared with PZM-3 group,the 4-cell block rate in-P group was significantly increased(P < 0.05),the 4-cell block rate in +P group was not significantly changed(P>0.05),and the 4-cell block rate in +P group was significantly lower than that in-P group(P < 0.05).Compared with PZM-3 group,the m RNA relative expression level of Eif1 a in-P and +P groups was significantly decreased(P < 0.05),but the relative expression level of Eif1 a in +P group was significantly higher than that in-P group(P < 0.05).Compared with PZM-3 group,the relative expression level of maternal gene GDF9 in +P group was not significantly changed(P > 0.05),the relative expression level of maternal gene GDF9 in-P group was significantly increased(P < 0.05),and the relative expression level of maternal gene GDF9 in +P group was significantly lower than that in-P group(P < 0.05).The results showed that pyruvate could promote the activation of genomes in porcine early embryo zygotes.(2)Compared with PZM-3 group,the level of reactive oxygen species in-P group and +P group was significantly increased(P < 0.05),but the level of +P group was significantly lower than that of-P group(P < 0.05).The level of glutathione in-P group was significantly lower than that in PZM-3 group(P < 0.05),but the level of glutathione in +P group was not significantly changed compared with that in PZM-3 group(P > 0.05),and the level of glutathione in +P group was significantly higher than that in-P group(P < 0.05).Compared with PZM-3 group,the ATP level in-P group and +P group was significantly decreased(P <0.05),and the ATP level in +P group was significantly higher than that in-P group(P < 0.05).The level of mitochondrial membrane potential in-P group was significantly lower than that in PZM-3 group(P < 0.05),but the level of mitochondrial membrane potential in +P group had no significant change(P >0.05),and the level of mitochondrial membrane potential in +P group was significantly higher than that in-P group(P < 0.05).Compared with PZM-3 group,the content of α-ketoglutarate in medium of-P group and +P group was significantly decreased(P <0.05),and the content of α-ketoglutarate in +P group was significantly higher than that in-P group(P < 0.05).There was no significant difference in the content of amino acids between +P group and-P group(P >0 0.05),but the content of amino acids in-P group decreased.Compared with PZM-3 group,the calcium ion concentration in-P group was significantly decreased(P < 0.05),the calcium ion concentration in +P group had no significant change(P >0.05),and the calcium ion concentration in +P group was significantly higher than that in-P group(P < 0.05).In conclusion,pyruvate is the main energy donor before genomic activation of porcine early embryonic zygote,which may promote genomic activation of porcine early embryonic zygote by alleviating oxidative stress of embryonic cells and reducing reactive oxygen species.At the same time,pyruvate can produce α-ketoglutarate through the tricarboxylic acid cycle and then affect the production of ATP,which provides a safeguard for energy metabolism during genomic activation of pig early embryo zygote.In addition,pyruvate may also promote the activation of zygote genomes by regulating the content of intracellular calcium ions through calcium channels.