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Epigenetic Reprogramming Of Histone Modifications In Bovine IVF And Cloned Embryos Development

Posted on:2009-09-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:X WuFull Text:PDF
GTID:1100360245987022Subject:Zoology
Abstract/Summary:PDF Full Text Request
Since the birth of Dolly, the first somatic cell nuclear transfer (SCNT) mammal, a number of species such as cattle, mouse, goat, pig, cat, mule, horse, rat, and ferret were produced. However, the low pregnant rate, high abortion occurrence, placental abnormalities, increased birth weight, and perinatal death resulted in a very low cloning efficiency. Differentiated somatic nuclei can be dedifferentiated in oocyte cytoplasm, converte to totipotent, and induce nuclear reprogramming, although the mechanisms involved are not clear. DNA methylation and histone modifications are considered to be importance in nuclear reprogramming. During natural reproduction, asymmetry demethylation, remethylaiton and histone modifications are observed between fertilization and blastocyst formation. Recently, more and more studies show that SCNT embryos exhibit defects during nuclear reprogramming. In this study, changes in histone modifications during bovine oocytes meiosis, fertilization, and embryos development in vitro were examined; histone modifications of embryos derived from IVF and SCNT were compared; Using trichostatin A (TSA), a histone deactylase inhibitor, to treat donor cells and recipient oocytes, change of DNA methylation and histone modifications of the resultant embryos were also investigated.1. Dynamic Changes in Histone Modifications during Bovine Oocytes Meiosis, in vitro Fertilization, and Embryo DevelopmentThis experiment was designed to investigate changes of histone methylation and acetylation during bovine oocytes meiosis, IVF, and embryo development. The results showed that histone acetylations were gradually disappeared from germinal vesicle (GV) stage to MII stage, histone methylations, however, did not change. At the time of sperm chromosome decondensation acetyl-histone H4 appeared. When sperm chromosome was recondensation, acetylation of histone H3 was observed. After male pronuclear formation, intensive histone acetylation signals were observed. When oocyte meiosis was activated, no histone acetylation signals were found, except for H4K5ac which expressed in AII-TII female chromosome. Intensive histone acetylation fluorescence signals were examined in female pronuclear. Methyl-histone H3 was not examined during male chromosome condensation and recondensation. Male pronuclear formed, and the histone methylation was examined. In female chromosome, intensive histone methylation fluorescence signals were examined from oocyte activation to pronuclear formation. When embryos cleaved, acetyl-histone H3 lysine 9 and lysine 18 (H3K9ac, H3K18ac) significantly reduced at 8-cell stage, and increased at morula stage. However, the fluorescence signals of acetyl-histone H4 lysine 8 and lysine 5 (H4K8ac, H4K5ac) did not change. Before zygotic genome activation, the enhanced staining for H4K8ac and H4K5ac were observed at the nuclear periphery. At mitosis, H4K8ac was located at condensing chromosomes, and acetylation signals of H4K5ac significantly reduced. At blastocyst stage, the H3K18ac, H4K8ac and H4K5ac signals were less intense in the inner cell mass (ICM) when compared to the trophectoderm cells (TE). During embryo development, fluorescence signals of trimethyl-histone H3 lysine 4 (H3K4me3) reduced or disappeared. However, the fluorescence intensity of dimethyl-histone H3 lysine 9 (H3K9me2) was gradually increasing during embryos development. At blastocyst stage, the levels of histone methylation between ICM and TE were with no difference. In conclusion, histone deacetylation was a meiotic-stage dependent and lysine residue-specific processes; During fertilization and embryo development, various lysine residues at the same histone had a similar dynamic changes (H3K9ac vs H3K18ac; H4K8ac vs H4K5ac); The similar dynamic changes of lysine residues were functional correlation (H3K9ac, H3K18ac vs H3K4me3); localizations of histone acetylation at nuclear stage was relative with zygotic genome activation (H4K8ac, H4K5ac), and changes of histone modifications were relative with embryonic DNA methylation (H3K9ac, H3K18ac, H3K4me3 vs H3K9me2).2. Comparative Study of Histone Modifications in Bovine IVF and SCNT EmbryosThe distribution patterns of acetylation on histone H3, H4, methylation on histone H3 lysine 9 and lysine 4 were examined in bovine preimplantation IVF and cloned embryos by using indirect immunofluorescence and scanning confocal microscopy. As results, high levels of histone acetylation and methylation were located in cloned embryos before donor genome activation (H3K9ac, H3K18ac, H4K5ac, H4K8ac, H3K4me3), and abnormal nuclear localizations of H4K8ac and H4K5ac were observed. Compared IVF with SCNT embryos, high level of H3K9me2 was examined during cloned embryos preimplantation development. When donor genome activation, all histone modifications were similar, except for H3K9me2, between IVF and cloned embryos. Therefore, somatic cells genome was widely epigenetic reprogrmming after somatic cell genome activation.3. Effect of Trichostatin A on Epigenetic Modifications of eGFP Transfected Cells and Subsequent Cloned Embryo DevelopmentBovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was in a dose dependent. When the TSA concentration was over 5ng/mL, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100ng/mL (P<0.01). Number of cells in S phase was significantly decreased in the 5 to 50ng/mL TSA-treated groups, while the majority of the cells were at G0/G1 phases. The number of eGFP expressed cells was approximately two-fold higher in 25ng/mL (30.5%) and in 50ng/mL (29.5%) TSA treatment groups when compared to the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50ng/mL TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50ng/mL TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, Treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation and eGFP gene expression was activated. The donor cells with reduced DNA methylation did not improve subsequent cloned embryo development. However, transgene expression was improved in cloned embryos.4. Treatment of oocyts with Trichostatin A, Resulted in Improved Nuclear Reprogramming and Cloned Embryo DevelopmentTrichostatin A (TSA), a histone deacetylase inhibitor, was used to treat bovine oocytes during in vitro maturation (IVM). Change of oocytes histone acetylation, cloned embryos development, and donor chromosome epigenetic reprogramming in enucleated oocytes was examined. The results showed that the effect of TSA on bovine oocytes was in a dose dependent. When the TSA concentration was over 2.5ng/mL, oocytes IVM were significantly inhibited. The majority of the oocytes inhibited at MI stage when TSA reached 10ng/mL (10ng/mL TSA, 61.9% vs control, 31.4%, P<0.05). However, effect of TSA on oocytes karyotype was not found (1ng/mL 85.5%, 10ng/mL 85.9% TSA vs 81.8% control, P>0.05, respectively). Histone acetylation levels of oocytes from TSA treatment were significantly increased. TSA treated oocytes were used to parthenogenetic development, and high development rates was achieved (0.5ng/mL 28.7%, 1ng/mL 36.4%, 2.5ng/mL 25.9%, 5ng/mL 27.1% vs controls 19.0%, respectively). Meanwhile, Transfer of donor cells to enucleated TSA-treated recipient oocytes resulted in a higher blastocyst development in lng/mL TSA (1ng/mL TSA 39.3% vs control 25.7%, P<0.05). However, similar total cell number per-blastocyst was observed.Recently, dynamic reprogramming of histone acetylation and methylation was investigated in the first cell cycle of cloned and TSA-treated cloned embryos. As results, when donor nuclear was induced premature chromosome condensation (PCC), part of somatic lysine acetylation on core histones (H3K9ac, H3K18ac) were quickly deacetylated in cloned and TSA-treated cloned embryos. The fluorescence intensity of H4K8ac was significantly decreased in control embryos, but the signals disappeared in cloned embryos from recipient oocytes treated by TSA. The H4K5ac fluorescence signals were not change in control embryos, and were significantly decreased in TSA treated group. Stability of donor chromosome was significantly increased when TSA treated oocytes were used as cytoplasm recipient (TSA-treated group 74.2% vscontrol 39.6%, P<0.01). In conclusion, bovine oocytes treated with TSA inceased global histione acetylation. Cloned embryos development and epigenetic reprogramming from TSA-treated oocytes were improved, and stability of donor chromosome in TSA-treated oocytes was increased.
Keywords/Search Tags:SCNT, IVF, embryo, Trichostatin A, histone methylation, histone acetylation
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