| The Yak is a unique type of livestock associated with the Qinghai Tibetan Plateau region.This region is a core area for animal production in China but the production systems are dependent on animals being able to survive in the natural environment and receiving adequate nutrition for growth and reproduction.The Yak has a low rate of reproduction and survival and the mating and reproductive cycle is very seasonal.Therefore,it is valuable to explore the use of in vitro embryo production technology and somatic cell nuclear transfer and other modern biotechnology applications for Yak.These technologies are likely to improve individual propagation speed,reduce the cost of breeding,preservation of the yak genetic resources.In this study,we optimized a culture system for in vitro fertilization and somatic cell nuclear transfer of embryos of Yak,using RNA-seq biotechnology screening of differentially expressed genes in vitro fertilization and somatic cell nuclear transfer of blastocysts.The study also explored methods to improve the efficiency of yak cloning by histone methyltransferase inhibitor and effects of sodium fluoride on the apoptosis and the development of preimplantation bovine embryos.The main results are as follows:1.Optimization of culture system for in vitro fertilization and somatic cell nuclear transfer of Yak(1)In vitro maturation of cumulus-oocyte complexes cultured for 24 h,the in vitro maturation rate,cleavage rate and blastocyst rate of Grade A COCs was significantly higher than that of Grade B COCs and grade C COCs.Separation of yak semen by Percoll gradient centrifugation method and BO method,and Yak oocyte was fertilized with sperm at concentration in the 1-5×106﹒m L-1 range no significant difference in 3 mg/m L BSA and 2.5 m M BO Theophylline fertilization system.The effect of in vitro fertilization embryo cultured with TCM199+cumulus cells or TCM199+oviduct epithelial cell co-culture system was significantly higher than that of G1/G2 solution and m SOF solution culture system.(2)The optimized Yak somatic cell nuclear transfer system: Yak oocytes in vitro maturation for 20 h as receptor cell,Yak ear fibroblast cells as donor cells for V passages within 6 generations and serum starvation 2 d for nuclear transfer;Fusion parameters for nuclear transfer reconstructed embryos is 1.4-1.6 kv/cm,two pulse,pulse duration of 20 s and pulse interval of 10 s;ionomycin united 6-DMAP activated 4 h after embryos fused and cultured using G1/G2 culture system.2.Screening of differentially expressed genes in vitro fertilization and somatic cell nuclear transfer blastocysts by RNA-seq(1)IVF-Blastocyst library and SCNT-Blastocyst library were sequenced by RNA-seq,a total of 52365530 and 52365908 raw reads obtained respectively,and 48810404(93.21%)and 49328624(94.20%)clean reads obtained respectively.Compared with the yak genome,genome map rate is 74.18% and 74.54%,respectively.Compared with the yak gene,gene map rate is 52.36% and 51.00%,respectively.(2)Through the comparison,there were 14352 genes,of which 12395 genes were co expressed,837 genes and 1120 genes were specifically expressed in IVF-Blastocyst and SCNT-Blastocyst respectively.The differentially expressed genes were selected based on the expression profiles and the following criteria: if the change in gene expression levels between IVF-Blastocyst and SCNT-Blastocyst was greater than or equal to two-fold(|log2Ratio|≥1)and if the false discovery rate(FDR)was less than or equal to 0.001.we restricted 576 genes that exhibited differential expression between IVF-Blastocyst and SCNT-Blastocyst,IVF-Blastocyst as a control,of these,the expression levels of 342 genes were up-regulated in SCNT-Blastocyst,the remaining 234 genes were down-regulated in IVF-Blastocyst.(3)The results of GO functional annotations are revealed that 150 terms corresponding to cellular components,218 terms corresponding to molecular functions and 1042 terms corresponding to biological processes.There are 4 terms corresponding to cellular components and 2 terms corresponding to biological processes were significant enrichment by significant analysis.The annotations indicated that these genes mainly involved in the regulation of extracellular domain(GO:0044421 and GO:0005576),lipoprotein complexes(GO:0032994 and GO:0034358)as well as inflammation and trauma reaction(GO:0006954 and VI GO:0009611).(4)KEGG pathway analysis showed that 218 pathways enriched for 576 differentially expressed genes.218 pathways involved in metabolic pathways,transcriptional misregulation in cancer,steroid hormone biosynthesis,neurotrophin signaling pathway,complement and coagulation cascade and cell adhesion molecules.Only pentose and glucuronate interconversions pathway(ko00040)were significantly enriched by significant analysis(Qvalue<0.05).(5)Randomly selected 20 differentially expressed genes were verified by q PCR.The results showed that the expression levels of EGR1,CPEB1,FGFR2 and SLC2A4 in the SCNT embryos were significantly up-regulated and HSPB1,WNT2 B,LOC102279723,CDCA7 L,POLR2A,CRABP1,KIFC3,CDC25 A,DDIT3,SDC1 and LOC102282421 gene expression in the IVF embryos was significantly increased.The q PCR validation results are basically consistent with RNA-seq data.(6)In total,we obtained 1435 and 1499 novel transcript units in IVF-Blastocyst library and SCNT-Blastocyst library,respectively.It was found that 23.62% and 22.28% novel transcript units were predicted to be encoded by coding potential calculator.3.Effects of histone methyltransferase inhibitor on the development of SCNT embryo in Yak(1)There was no significant change in cell morphology and cell viability of yak fibroblasts treated with low concentration of GSK126(10 μM and 20 μM),while the high concentration of GSK126(40 μM and 80 μM)treatment group significant decreased cell viability,increased G0/G1 ratio,decreased S and G2/M ratio and increased the number of apoptotic cells.(2)Blastocyst rate,TE cells and ICM cells of nuclear transfer reconstructed embryos treated with low concentration of GSK126 was significantly higher than that of high concentration of GSK126 treatment group,while the apoptotic cells in blastocysts were the opposite.Based on the cell viability,blastocyst development rate and blastocyst development quality,the 20 μM GSK126 treatment of yak fibroblast 20 h was used as donor cells for nuclear transfer.(3)Analysis of histone methylation level in IVF blastocysts,and 20 μM GSK126 treated SCNT blastocysts,we found that histone H3K27me2 and histone H3K27me3 methylation level were significantly decreased in 20 μM GSK126 treated SCNT blastocysts,but no significant effect on histone H3K4me2 methylation level.(4)Analysis of embryo development related genes and apoptosis related genes,we found that apoptosis related genes BCL-2 m RNA expression level decreased and Caspase-9 m RNA expression level increased in SCNT blastocysts treatment with 20μM GSK126,while the expression level of embryo development related genes NANOG,OCT4 and GATA3 up regulation in SCNT blastocysts treatment with 20μM GSK126.4.Effects of sodium fluoride on the apoptosis and the development of preimplantation bovine embryos(1)Na F in a dose-dependent manner decreased the developmental potential of oocyte,as indicated by the maturation rate,or 2-cell rate,and the blastocyst rate.High concentration of Na F treatment significantly increased the content of ROS in oocytes and inhibited the activity of T-AOC,GSH-Px,CAT and GSH in oocytes,which induced the oxidative stress of oocytes.(2)Na F exposure increased levels of the apoptotic protein Bax in addition to activities of caspase-3 and caspase-9,and reduced anti-apoptotic protein Bcl-2 in oocytes.Moreover,increased apoptosis rates were further analyzed in blastocysts developed from the Na F-treated oocytes.(3)Na F exposure decreased the numbers of total,ICM cells,and TE cells of blastocyst,while the apoptotic cells in blastocysts was increased.In addition,it decreased the relative expression levels of the development related genes(Oct4,Nanog,Sox2,and Cdx2). |
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