Expression Analysis Of LncRNA Associated With Antioxidantion In Bovine Embryos

It is well known that the developmental rate of in vitro embryos in mammalian is much lower than that of in vivo.And its low pregnancy and high fetal abortion rate are main factors to restrict its wide application in cattle husbandry,even affect the income of herdsmen and the healthy,fast and sustainable development of our animal husbandry economy.In order to improve the quality of in vitro embryos,glutathione(GSH),an antioxidant,is often used to scavage oxygen free radicals in embryos and plays a crucial role in subsequent embryo development.However,its effect on embryos in the level of the whole transcriptome is unclear.Recently,with the discovery of long non-coding RNA(lnc RNA),its regulatory role has become a new research focus.In present study,in vitro fertilized(IVF)embryos treated with glutathione were collected for RNA sequencing using single-cell sequencing technique.The lncRNA was analyzed with bioinformatics to reveal its antioxidant function in early embryo and find some critical regulatory molecules or sequences.Experiment 1,bovine embryos were prepared by in vitro fertilization and cultured in medium containing 3 mM glutathione.Embryos were collected at 8~16-cell stage and mulberry,respectively,and non-added group was used as control.20 embryos of each group were collected as a sample and made two biologically repeat.The sequence of lncRNA was obtained by Hi Seq2000 sequencing platform,and its bioinformatics analysis was carried out.A total of 4273 lncRNAs were obtained,of which 62% were located at intergenic regions,and their distribution in each chromosome was not obvious,indicating that the transcription of lncRNAs was genome-wide.The mean expression,average length and mean exons number of lncRNAs were consistent with the known lncRNAs,demonstrating the reliability of lncRNA sequencing.23 lncRNAs were up-regulated in the control group while down-regulated in the treated group,and 36 lncRNAs were down-regulated in the control group while up-regulated in the treated group.In experiment 2,23 DElnc RNAs were co-expressed with 599 and 2554 DEGs,which were involved in 25 and 71 GO terms,enriched in 6 and 8 pathways in control and treated group respectively.36 DElncRNAs were co-expressed with 483 and 609 DEGs,which were involved in 18 and 33 GO terms,enriched in 5 and 10 pathways in control and treated group respectively.In addition,14 differentially expressed genes co-expressed with lncRNAs were found to be involved in the glutathione metabolism pathway.Neighbor genes of 23 and 36 DElncRNAs were participated in 35 and 43 GO terms respectively.Real-time quantitative PCR was performed on GSH-treated 8~16-cell and morula embryos to verify the transcript data.Results showed that4 lncRNAs were down-regulated in the treatment group while the other lncRNAs were up-regulated,which was consistent with the sequencing results,indicating the structural reliability of sequencing.In conclusion,GSH treatment in vitro culture medium could change the lncRNA profiling of cattle early embryos,but its effect and mechanism needed further study.Experiment three,Bull granulosa cell with 3 mM and 5 mM GSH added in the medium were cultured.The qPCR was used to detect the expression level of 4 lncRNAs and 6 genes associated with GSH synthesis and metabolism.Results showed that the addition of GSH with two concentrations decreased significantly the expression of CUFF.152963.1and CUFF.30537.1 in granulosa cells,5 mM GSH increased significantly the expression of CUFF.33095.2 and had no effect on CUFF.42178.2 expression,3 mM GSH had no effect on CUFF.33095.2 and CUFF.42178.2.Additionally,significant effects of GSH with 3 mM and 5 mM on the expression level of GCLM,GCLC,GSTA1,and GSTM genes were observed in granulosa cells.However,there was no effects of GSH on the GSTP1 and GGCT expression.