| 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 by this technology. However, the low pregnant rate, high abortion occurrence, placental abnormalities, increased birth weight, and perinatal death resulted in a very low cloning efficiency, which impeded the extensive application of SCNT. Abnormal development in cloned embryos was partially due to incomplete or abnormal epigenetic reprogramming of donor somatic cell nuclear in the oocytes cytoplasm. Differentiated somatic nuclei can be dedifferentiated in oocyte cytoplasm, convert to totipotent, and induce nuclear reprogramming, although the mechanisms involved are not clear. Valproic Acid (VPA), a histone deacetylase inhibitor, could specifically inhibit the histone deacetylase activity, increase histone acetylation, and active gene expression in somatic cells. However, the effect of VPA on fetal fibroblast, oocytes maturation and embryonic development are not resolved.In this report, the effect of VPA on bovine fetal fibroblast cell growth, cell cycle and gene expression were studied, which could accelerate researches on bovine SCNT embryo epigenetic reprogramming and development. The effect of VPA on bovine oocytes maturation, early embryo development from in vitro fertilization (IVF) or parthenogenesis were also studied. This research will benefit for improving the efficiency of assisted reproductive technologies and cloning.1. Effect of Valproic Acid on cell cycle in bovine fetal fibroblast cellsThe fetal bovine fibroblast cells were treated with different concentrations of VPA, then cell cycle changes were examine by flow cytometry and the expression of transcriptional factors such as Oct4, Nanog and Cdx2were detected by Real-time PCR. The results showed that cell growth gradually decelerated with the increase of VPA concentration, cells were mostly arrested at G0/G1phase and the gene expression of Oct4and Nanog were increased while Cdx2gene expression was reduced. The results suggested that VPA-treated bovine fetal fibroblast cells may benefit the epigenetic reprogramming and the cloned embryos development when it was used as donor cells in somatic cell nuclear transfer.2. The effect of Valproic acid treatment on bovine oocytes maturation and embryo development.In this experiment, firstly, the effect of VPA on in vitro maturation of bovine oocytes, the development of embryos from in vitro fertilization and parthenogenesis were detected. Secondly, the effect of VPA on the embryonic development and expression of Oct4, Nanog, and Cdx2genes at blastocyst stage were examined. Compared with control group, the maturation rate of oocytes had no significant differences when oocytes were cuitured wuih0.03mM or0.3mM VPA for24h, the maturation rate of oocytes in3mM and6mM group were significantly lower than that of control group (p<0.001); After maturation cultured, the treated oocytes were fertilized or used for parthenogenesis, the blastocysts rate in0.3mM group and in3mM group were significantly lower than that of control group (p<0.001). For embryos cultured in0.3mM VPA group, there were no remarkable differences on the cleavage rates, but the expression of Oct4, Nanog, and Cdx2expression in blastocysts was significantly improved(p<0.005). When embryos from IVF or parthenogenesis were treatment with0.3mM VPA, there are no significant effects on cleavage rate or blastocyst rate, but the expression of oct4, Nanog, and Cdx2in bastocyst was significantly increased (p<0.005) However, the3mM VPA treatment significantly decrease the cleavage rate and blastocyst rates (p<0.01). In conclusion, small dose of VPA (0.3mM) had no significant effects on blastocyst rate of IVF or parthenogenesis embryos, but the expression of several transcription factors was significantly changed, which may benefit to blastocyst quantity or the following development after implantation. |
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