Regulatory Mechanism Of Arginine On Zygotic Genome Activation In Pigs

Embryonic loss is a significant problem for sow reproduction.Early embryo loss accounts for more than 60% of the total embryo loss,which is a sensitive period for embryo loss.Studies have reported that arginine plays an important role in early embryonic development and can reduce embryo loss.Early mammalian embryonic development is composed of a series of highly conserved,regulated,and predictable cell division events,including: sperm-egg fusion to form a zygote,initial embryonic cell division(cleavage),and zygotic genome activation(ZGA),densification of morula and formation of implantation-competent blastocysts.Therefore,this study aimed to explore the mechanism of arginine during early embryonic development in pigs.The test is divided into four parts:1.Single-cell transcriptome sequencing analysis of porcine in vivo early embryoFirst,the test sows were naturally mated after estrus.The breeding sows were surgically flushed 3-7 days after estrus to collect in vivo zygote,2-cell,4-cell,8-cell and morula stage embryos.Subsequently,embryos at each developmental stage were isolated into single blastomeres for single-cell transcriptome sequencing.The results showed that the number of differential genes was the largest at the 4-cell to 2-cell developmental stage in pigs,indicating that the main ZGA in pigs occurred in the 4-cell stage.KEGG enrichment showed that the differential genes at the 4-cell and 2-cell stage were mainly enriched in signaling pathways such as RNA transport,ribosome biosynthesis,protein processing in the endoplasmic reticulum,and arginine and proline metabolism.This result suggests that arginine metabolism may play an important role during the ZGA period.The results of clustering heatmap analysis of arginine metabolism pathways showed that genes related to arginine metabolism began to be expressed at the 4-cell stage during early embryonic development in pigs.In addition,correlation analysis showed that arginine metabolism genes were correlated with ZGA genes.2.Effects of arginine on zygotic genome activation in pigsTo verify whether arginine affects ZGA,in vitro fertilization(IVF)pig embryos were cultured in normal PZM-3 medium(control group),modified PZM-3 medium(block group,lack of arginine and other amino acids,pyruvate and taurine,0 m M arginine)and modified PZM-3 medium supplemented with arginine(arginine group,0.12 m M arginine).The effect of arginine on ZGA was evaluated by observing the embryonic development and counting the 4-cell arrest rate after 72 h of culture,and detecting the expression of porcine ZGA marker genes and early transcription product SIRT1 protein in 4-cell embryos.The results showed that compared with the control group,the arrest rate of 4-cell embryos in the block group was significantly increased after 72 h of culture(P < 0.05).Supplementation of 0.12 m M arginine allowed embryos to continue to develop beyond 4-cell arrest,with a significantly lower rate of 4-cell arrest in the arginine group compared with the block group(P < 0.05).Previous sequencing results confirmed that the major ZGA in pigs is at the 4-cell stage.This is the stage at which embryos are developmentally arrested in modified PZM-3 medium.Compared with the control group,the expressions of the porcine zygotic genome activation marker genes EIF1 A,DPPA2 and ZSCAN4 and the early transcription product SIRT1 protein of the 4-cell embryos in the block group were significantly decreased(P < 0.05).Compared with the block group,the porcine zygotic genome activation marker genes EIF1 A,DPPA2 and ZSCAN4 and the early transcript SIRT1 protein were significantly increased of the 4-cell embryos in the arginine group(P <0.05).3.The effect of arginine on embryonic metabolism during the zygotic genome activationAfter 48 h of in vitro culture,4-cell embryos in the control group,the block group and the arginine group were collected respectively.The 4-cell embryos of each group were stained with fluorescent probes to detect reactive oxygen species(ROS),glutathione(GSH)and ATP levels,mitochondrial membrane potential changes,and lipid droplet content,respectively,to assess the effect of arginine on the embryonic metabolic status during ZGA stage.The results showed that compared with the control group,the content of ROS in 4-cell embryos in the block group was significantly increased(P < 0.05),but the content of GSH was significantly decreased(P < 0.05).Compared with the block group,the content of ROS in 4-cell embryos in the arginine group was significantly decreased(P < 0.05),and the content of GSH was significantly increased(P < 0.05).The ATP levels were significantly lower in 4-cell embryos in the arrest group compared with the control group(P < 0.05).The ATP levels of 4-cell embryos were significantly increased in the arginine group compared with the block group(P < 0.05).Compared with the control group,the lipid droplet content of the 4-cell embryos in the block group was significantly decreased(P < 0.05).The lipid droplet content of 4-cell embryos was significantly increased in the arginine group compared with the block group(P < 0.05).However,the changes in mitochondrial membrane potential were not significantly different between the control,block and arginine groups(P > 0.05).4.The metabolic pathway of arginine affects porcine zygotic genome activationAfter 48 h of in vitro culture,the 4-cell embryos of the control group,the block group and the arginine group were collected respectively to detect the NO content,NOS and ODC1 gene expression in the embryos,and the embryo culture medium of each group was collected to determine the contents of polyamine(agmatine,S-adenosyl-Lmethionine,putrescine,cadaverine,spermidine and spermine)in the embryo culture medium.The NO content of 4-cell embryos in the arginine group was slightly increased compared with that in the block group,but the difference among the three groups was not significant(P > 0.05).Next,we detected the NOS gene expression of 4-cell embryos in the three groups and found that the NOS gene expression in the block and the arginine groups was significantly decreased(P < 0.05)compared with that in control group,but the difference between the block and arginine groups was not significant(P > 0.05).It was found that only spermine could be detected,other polyamines were not present in the medium.The spermine content of the culture medium in the block group was significantly decreased(P < 0.05)compared with that in the control group.The spermine content of the culture medium in the arginine group was significantly increased(P < 0.05)compared with that in the block group.In addition,the m RNA expression of ODC1,the key enzyme of polyamine synthesis,in the block and the arginine groups was significantly decreased(P < 0.05)compared with that in control group,and the m RNA expression of ODC1 in the arginine group was significantly increased(P < 0.05)compared with that in the block group.The above results indicate that arginine may affect ZGA through the polyamine pathway.To test our hypothesis,zygotes were cultured in the modified PZM-3 medium supplemented with DFMO and arginine(DFMO group).The spermine content of medium and the m RNA expression of ODC1 of 4-cell embryos in the DFMO group were significantly decreased(P < 0.05)compared with those in the arginine group.The expression levels of SIRT1 protein and the zygotic genes of 4-cell embryos in the DFMO group were significantly decreased(P < 0.05)compared with those in the arginine group.It was found that ROS contents in the DFMO group were significantly increased(P < 0.05)compared with those in the arginine group,whereas the GSH,ATP and lipid droplet contents were significantly decreased.In conclusion,single-cell transcriptome sequencing revealed that the main ZGA in pig embryos occurred at the 4-cell stage,and arginine metabolism was correlated with porcine ZGA.Arginine promotes ZGA in pig embryos under nutrient restriction.Arginine can reduce embryonic oxidative stress and increase embryonic energy levels under nutrient restriction.Arginine may regulate ZGA in porcine embryos by promoting polyamine synthesis under nutrition restriction.