Function Of HDAC1 Gene During The Development Of Bovine Oocytes And Cloned Embryos

Within the past decade, mammalian nuclear transfer (NT) cloning has progressed remarkably in technology improving and success rates. Bovine nuclear transfer technology is limited by the expensive recipients, long time gestation period, and high abortion rates. Therefore studies on improving bovine nuclear transfer efficient are still necessary. In order to enhance the bovine nuclear transfer efficient and reveal the mechanism of reprogramming in this process, we performed the present study to optimize the procedure of bovine nuclear transfer and detect the function of HDAC1 gene during the development of bovine oocytes and cloned embryos.Bovine nuclear transfer procedure was systematically examined and optimized:(1) Enucleation of bovine MⅡstage oocytes was performed at different time point of in vitro maturarion (IVM). The enucleation rates and the maturation rates were higher than other groups when oocytes were cultured for 18-20 hours. (2) The electric fusion parameter was optimize as follow,180v/mm-250v/mm,10μs, two current (DC)-pulses. The fusion rates and developmental capability was much higher in the performed fusion parameter. The developmental capability of clone embryos impaired significantly when electric intensity was higher than 250v/mm in the fusion process. Significant lower fusion rates were obtained when electric intensity was 150v/mm. (3) Enucleated MⅡstage oocytes were treated with 0-600mg/ml PHA. The electric fusion rates was significantly enhanced when the enucleated MⅡstage oocytes were treated with 200mg/ml PHA for 30min. Oocytes treated with 100mg/ml PHA for 30min shown no significantly effect in improving fusion rates. When the oocytes were treated with 400mg/ml and 600mg/ml PHA for 30min the survival rates of clone embryos decreased significantly after electric fusion. (4) Manipulation speed and fusion rates were enhanced when nuclear donor cells were treated with 5mg/ml pronase for 5min. Fusion rates did not significantly different when electric fusion were performed in the mannitol and sorbierite. (6) Feeder layer of bovine fetal fibroblast cells or granular cells were added to improve bovine embryo culture system. (7) Embryo transfer was performed when the clone embryos were developed to blastocyst in day7. The results indicated that multi-embryo implantation leads to hydroperitonia in recipients. Embryo transfer experiments were performed in autumn and winter. The group of embryos transfered in autumn obtain higher pregnancy rate than the winter group.Developmental capability and H3K14ac level was detected in parthenogenetic embryos when HDAC1 gene was down regulated.(1)GV stage oocytes were treated by 20μg/ml IBMX for 2 to 3 hours to produce a perivitelline space between zona pellucida and cytomembrane of oocytes. Therefore. The survival rates significantly enhanced in the oocytes with zona pellucida after microinjection. (2)Three siRNAs (si299, si672 and si 1272) that correspond to HDAC1 mRNA were designed. siRNAs were microinjected into bovine GV stage oocytes to identify which was most effective for HDAC1 knockdown. Results of Real-time PCR indicated that si299 was the most efficient siRNA in HDAC1 downregulation at 24h after microinjection. Then, the most effective siRNA, si299. was microinjected into bovine germinal vesicle (GV) stage oocytes to determine the functions of HDACl in the maturation of bovine oocytes and H3k14 acetylation. The maturation rates and H3K14 acetylation level of oocytes did not influenced by HDAC1 down regulation. (3) Immunofluorescence experiments were performed on oocytes at the GV and MⅡstages and on parthenogenetic embryos at the two-cell, four-cell, eight-cell and blastocyst stages, following parthenogenetic activation. HDAC1 protein was detected in the nuclei of oocytes and parthenogenetic embryos. HDAC1 fluorescent signal appeared in the nuclei of GV-stage oocytes but disappeared in MⅡstage oocytes. HDAC1 fluorescence signals once again emerged in the two-cell stage of parthenogenetic embryos, but the intensity was quite weak. Then. HDAC1 fluorescence intensity enhanced in the four-cell and eight-cell stages but diminished during the blastocyst stage.(4) Si299 was microinjected into mature oocytes, which were then parthenogenetically activated and cultured in vitro until the blastocyst stage. Cleavage rates, blastocyst rates and histone H3K14 acetylation state were examined in bovine parthenogenetic embryos. HDAC1 knockdown in oocytes did not influence maturation rates or cleavage rates of parthenogenetic embryos. However, the blastocyst rates decreased after siRNA microinjection. Furthermore, histone H3K14 acetylation increased after siRNA microinjection into parthenogenetic embryos. (3)Maturation rates of oocytes are affected by adding no more than 50nM TSA or 2mM VPA in to the IVM media. H3K14 deacetylation in oocytes maturation was inhibited when GV stage oocytes were treated by 50nM TSA for 5 hours.Bovine fetal fibroblast cells were transfected by si299 and shRNA vector. At 96h after the si299 or shRNA vector transfection. the cells were used as nuclear transfer donor cells to construct clone embryos. Developmental capability and apoptosis of blastocyst cells were detcted in HDAC1 knockdown clone embryos. (1)At first, a fluorescein-labelled non-sense control siRNA was used to optimize the transfection efficiency of bovine fibroblast cells. The 1.5μg/ml siRNA concentration at a 2:6 ratio with the transfection agent FuGENE HD was used for all the transfection experiments. (2)HDAC1 down regulation in donor cells have no effect in improving developmental capability of clone embryos. Embryo developmental capability decreased in the shRNA vector transfection group compared to the control group and siRNA transfection group. Cell apoptosis in blastocyst were compared in control group, siRNA transfection group and shRNA vector transfection group by TUNEL cell death detection kit. More cell apoptosis in blastocyst of the shRNA vector transfection group were detected compared to the other groups. (4)TSA(ClassⅠandⅡa/b HDACs inhibitor) and VPA (ClassⅠandⅡa HDACs inhibitor) were added in to embryo culture media for 12h to detect their function in embryo development. Bovine clone embryo treated by 50nM TSA for 12h obtained higher blastocyst rates compared to the control group.2mM VPA treated group have no function in improving clone embryo development. So we speculate that the ClassⅡb HDACs might be the functional HDACs in improving bovine clone embryo development.