The Effect Of Mogroside V On Porcine Oocyte Development And Embryonic Development And Mechanism Study

Oocyte development and embryo in vitro production(IVP)are key techniques in the field of animal reproduction.Oocyte quality determines the fate of the embryo,and which is closely related to the health of the offspring.Improving the quality of oocytes and embryos has important theoretical and practical significance for improving livestock reproductive efficiency.Lots of reactive oxygen accumulated in cytoplasm is an important cause for the lowered oocyte and embryo quality.As one of the main pharmacodynamic components of Siraitia grosvenorii,mogroside V(MV)has abundant biological characteristics,among which it plays a significant role in anti-oxidation.To explore the effect of MV on oocyte and embryo development of livestock,this article evaluated roles of MV on the in vitro maturation(IVM)of porcine oocyte and its related molecular mechanism.The macroscopic transcriptome changes of oocyte exposed to MV was revealed by Single-cell transcriptome sequencing(RNA-seq).In additional,the molecular mechanism of MV delaying egg aging is preliminary explored in this paper,and the effect of MV on the development of pig somatic cell nuclear transfer embryos and parthenogenetic activated embryos were also evaluated.Results are as follows:1.Effects of MV on in vitro maturation of pig oocytesTo optimized the concentration of MV,different dose of MV(1,5,10,20 and 40 μM)were added to IVM medium.Cumulus oocyte complexs(COCs)were maturated for 40-44 h,5,10,20 and 40 μM MV can increase the first polar body excretion(PBE)rate of oocytes.There is no significant difference between 20 μM and 40 μM,so 20 μM was selected for subsequent experiments.After addition of MV,the cleavage rate and blastocyst rate of PA embryo were significantly increased,however MV has no significant effect on SCNT embryo development.Furthermore,MV significantly promoted the generation of cortical granules(CGs),and improved the transcriptional level of mitochondrial related gene(PGC-1α and TFAM),thus enhancing mitochondrial activity and function.MV decreased cytoplasm ROS by improving the transcription level of antioxidant enzyme(SOD,CAT and PRDX2).Specific inhibitor EX-527 was further used to confirm that MV regulated ROS and mitochondria by activating SIRT1,thus improving oocyte quality.The luciferase reporting system determined that MV increased the activity of SIRT1 promoter,laying the foundation for further studying the relationship of MV with SIRT1 in oocyte IVM in the future.2.Revealing the changes at the transcriptional level in pig IVM oocytes by single cell RNA-seqThe matured oocytes were collected,which were cultured in mature medium supplemented with 20 μM MV for 40-44 h,and each group had three repetitions.RNA was extracted from oocytes and c DNA library was constructed using Illumina Hiseq sequencing platform.The obtained data were processed by quality control,database search,differentially expressed genes(DEGs)analysis,Gene Ontology(GO)enrichment analysis,KEGG analysis and results verification.A total of 10631 co-expressed genes and 440 DEGs were detected,of which 216 were up-regulated and 224 downregulated.In addition,GO analysis found that 30 genes were upregulated of stress response,and 31 were downregulated;the upregulated genes of DNA repair were 9,and downregulated genes were 5.In addition,KEGG analysis showed that the mode of MV action was closely related to the WNT signaling pathway,pyruvate metabolism and the synthesis of fatty acid in IVM oocytes.Based on the results of KEGG analysis,fluorescent staining and TG enzyme reaction tests were conducted.The results showed that MV can increase the lipid droplet synthesis and triglyceride content of mature oocytes in vitro.The results of real time fluorescence quantitative PCR(SOD,PRDX2 and TFAM)were consistent with the results of singlecell RNA-seq,suggesting that the results of RNA-seq were reliable.3.The effect of MV on oocytes aging and its molecular mechanism44 h after IVM(fresh oocytes),oocytes were transferred to PZM-3 medium with MV(25,50 and 100 μM)or without MV(aging group)for another 24 h.A weak electrical stimulation(0.6 k V/cm,2DC,10 μs)was applied to oocytes,and the activation rate(division or fragmentation)was examined after 8 h.Compared with the fresh group,the activation rate of aged oocytes increased significantly,however addition of 50 and 100 μM MV significantly reduced the activation rate of aged oocytes.50 μM MV was selected for subsequent test.After 24 h of oocyte aging,PA embryos were made,results showed that the blastocyst development rate of the aged oocytes decreased from 31.1% to 2.4%,while the blastocyst rate of the aged oocytes treated by MV increased to 14.3%.In addition,MV also stabilized aged oocyte spindle and chromosome morphology,reduced apoptosis formation,promoted mitochondrial activity and function,decreased ROS levels and increased SIRT1 transcription level.When the expression of SIRT1 was suppressed by EX-527,the antioxidant effect of MV and the improvement role of MV on spindle and chromosome disappeared.The results suggested that MV can reduce ROS level and delay oocyte aging by upregulation of SIRT1.4.The effect of MV on the development of porcine early embryoTo evaluate the effect of MV on embryo cleavage,blastocyst formation,blastocyst cell number and cell activity,20 μM MV was added to PZM-3 culture medium for PA and SCNT embryos.For PA embryos,there was no statistical difference on cleavage rate,blastocyst rate,blastocyst cell number and cell activity,but MV increased the blastocyst rate from 36.3% to 43.8% and blastocyst cells number from 43.8 to 50.0.Moreover,MV also increased the Ed U positive rate of blastocyst cells from 27.8% to 33.1%,indicating that MV increased the blastocyst activity of PA embryo.And the results showed that MV increased the blastocyst rate from 9.8% to 15.0% of SCNT embryos.In the current system,MV has no significant effect on early embryonic development.In summary,this study demonstrated that MV improved oocyte IVM,delayed oocyte aging and had no significant effect on early embryonic development using porcine oocyte and embryo model.The results lay the foundation for the further study of MV on improving the quality of pig oocytes and embryos.Moreover,this article provides new ideas for the research on the improvement of livestock reproduction.