Analysis Of Fatty Acid Metabolism In Porcine In Vitro Preimplantation Embryos And Naive-like ESCs

Lipids act as an energy source during preimplantation embryos development to affect the properties and functions of biofilms,which play an important role in cell-cell interactions and cell proliferation.Moreover,lipids play an important role in oocyte maturation,embryonic development and stem cell proliferation.Pigs have important research value in agricultural production and biomedical research.There are large amounts of lipids in porcine oocytes and preimplantation embryos compared to other cells.However,the mechanism of fatty acid metabolism in porcine PED is poorly understood.The mechanism of fatty acid metabolism in pig PDE and naive-like Embryonic stem cells(nlESCs)is investigated in this study.At first,we established two oocyte in vitro maturation culture systems.By comparing the maturation rate,blastocyst rate and cell morphology,the system containing 10%porcine follicular fluid was more suitable for oocyte maturation.After maturation,oocytes were activated by 5 different ways,which are electroactivation,electroactivation binding CB and 9% ethanol for 3 min,8 min and 11 min,respectively.By comparing the cleavage and blastocyst rate,the results showed that the electrical activation(the cleavage rate was 81.69±0.41%,and the blastocyst rate was 26.45±0.28%)was the best method to obtain parthenogenetic embryos.Subsequently,the lipid droplet content of parthenogenetic embryos was detected by lipid droplet staining,and the results showed that the lipid droplet content in morula was the highest,and the diameter of lipid droplet reached the maximum in the 8-cell period.The lipid droplets content in PA embryos is higher than that in IVF embryos.Secondly,the parthenogenetic 8-cell embryo,parthenogenetic blastocyst,in vitrofertilized 8-cell embryo,in vitro fertilized blastocyst and porcine fetal fibroblast were collected for single cell transcriptome sequencing.The results showed that the maximum differential genes existed between the in vitro fertilized 8-cell to blastocyst development stage,and the in vitro fertilized embryo and parthenogenetic embryo shared 1.56% of maternal mRNA.GO and KEGG enrichment showed that parthenogenetic embryo and in vitro fertilized embryo share some signaling pathways,which are mainly enriched in biological processes related to cytoplasmic translation,cellular metabolism,organelle organization,cell cycle and ribosome in the 8-cell stage.During the development of blastocyst,the differential genes are mainly enriched in biological processes such as cell metabolism,fatty acid metabolism,fatty acid ?-oxidation,and cellular lipid catabolism.Clustering heat map analysis of fatty acid metabolism pathways revealed that even though the same pathway is shared at the same stage in the PED process specific members of the highly expressed specific signaling pathway are different between the porcine parthenogenetic embryos and the in vitro fertilized embryos.Fatty acid-related enzymes are activated at different levels at development stages and in different ways.It is indicated that fatty acid metabolism plays an important role in PED processes between different embryos.Thirdly,RT-PCR results showed that the expression of FAS was increased during each period in the fatty acid synthesis pathway compared with oocytes.The expression of ACACA was increased in 4-cell and morula stage,and decreased in blastocyst stage.The expression of ELOVL6 upreguled in 4-cell and morula stage and decreased in the blastocyst stage.The expression of ME1 was decreased at the morula and blastocyst stage.The expression of SCD1 decreased in 4-cell stage,and gradually increased during the morula and blastocyst stage,the expression level of DGAT increased during the morula stage and decreased in the blastocyst stage.Compared with the oocyte,the expression of FABP5 was increased in all stages during fatty acid transport and degradation pathways,while the expression of ACADM,ECHS1 and PPAR? were decreased in all stages.Finally,pluripotent stem cells(PSCs)can be classified into two distinct and stable pluripotent states: the naive and the primed pluripotent states.Naive PSCs correspond to the preimplantation of the inner cell mass cells of blastocysts,whereas primed PSCs correspond to the post-implantation of epiblasts pluripotent stem cells.nlESCs can be cultured in large quantities for a long time in vitro,which can be used as a high-quality resource for PED research.In order to clarify the role of fatty acid metabolism in PED,the lipid additives was added to the nlESCs culture system.The results showed that the lipid additive had no significant effect on the cell morphology of nlESCs,and SOX2,OCT4 and NANOG were expressed in both cultures by immunofluorescence staining,but the expression level of SOX2 was significantly higher than OCT4 and NANOG.RT-PCR results showed that lipid-additive(100×CDLC)-treated nlESCs could promote the expression of pluripotency gene SOX2(P<0.05),reduced the expression of pluripotency genes OCT4 and NANOG(P<0.05).In the fatty acid synthesis pathway,the expression of ACACA was decreased,while FAS,ME1 and SCD1 were not significantly changed;DGAT,PLIN1 and PLIN2 were not significantly changed in the lipid droplets synthesis pathway;FABP5 and PPAR? were significantly decreased in the fatty acid transport and degradation pathway(P<0.05),the expression of ACADM and ECHS1 were decreased,but not significant(P>0.05).In summary,the system containing 10% porcine follicular fluid was more suitable for oocyte maturation,the electrical activation was the best method to obtain parthenogenetic embryos,the fatty acid metabolism plays an important role in PED and the lipid additives is affecting the nlESCs state.This study laid a foundation for further understanding the mechanism of fatty acid metabolism in PED and nlESCs.