Optimization Of In Vitro Production And Immuno-Fluorescence Staining Against DNA Methylation Of Parthenotes And Cloned Embryos In Pigs

Pigs are increasingly regarded as ideal model for human medicine due to anatomical and physiological similarity between pigs and mankind. Somatic cloning in pigs is still limited inside labotoray because of low efficiency, high cost and demanding skills. In orde to optimize and simplify pig cloning procedures, in this study, some key aspects such as activation, in vitro culture of porcine reconstructed embryos were exmined, and some key points engaged in protocols of indirect immuno fluorescence staining against whole genome DNA methylation were also modified prior to examine the DNA methylation reprogramming dynamics in preimplantational embryos in pigs. The experiments were as following.Experiment 1. Effect of valproic acid (VPA), a histone deacetylase inhibitor, treatment after nuclear transfer on in vitro development of pig cloned embryos was investigated. No significant difference between VPA treatment and the control group was found in the cleavage rate (86.67﹪vs. 85.00﹪, P>0.05), or the blastocyst rate (21.33﹪vs. 13.00﹪,P>0.05). The results indicated that addition of 200μM VPA to embryo culture medium for 36 h immediately after activation is not necessary for pig cloned embryos.Experiment 2. The effect of myoinositol (Myo) addition into PZM3 on development of porcine parthenogenetic activated (PA) and somatic cloned embryos. For PA embryos, the blastocyst formation rate (76.1% vs 42.7%,P<0.05) was enhanced by Myo addition; while for SCNT embryos, no difference between the treatment and the control group was observed (27.8 vs 30.9,P>0.05). The results demostrate that embryos from cloning and parthnogentic activation prefere to different nutrient requirement.Experiment 3. In the present study, in order to explore the possibilties of simplify activation manipulations engaged in somatic cloning, the reconstructed oocytes were divided randomly into four groups: electrical activation followed by exposure to cycloheximide (CHX, 10μg/mL) for 4h (ELE+CHX4h, traditional activation), exposure to 10μg/mL CHX for 10min prior to electrical activation (CHX10min+ELE), exposure to 10μg/mL CHX for 10min after electrical activation (ELE+CHX10min), and electrical activation alone (ELE). For PA embryos, similar cleavage rate, blastocyst rate were observed among the four groups. For cloned embryos, however, we found that the CHX10min+ELE group significantly more effective than the ELE group and ELE+CHX10min group, while resulted in similar blastocyst rate to the traditional group. These results suggested that CHX10min+ELE treatment could repace the traditional activation methods without compromizing preimplantation development of cloned embryos, and is an efficient, stable, and simple protocol for activating cloned embryos.Experiment 4. The present experiment was designed to establish and optimize procedures of the indirect immunofluorecence staining against whole genome DNA methylation in porcine PA embryos and cloned embryos. Dilution ratio of primary antibody, and choice of secondary antibodies were selected. The results reveal that if the domestic or imported secondary antibody diluted by the ratio of 1:200, and the primary antibody diluted at 1:300 or 1:200, and the embryos permeabilized by 0.5% Triton-X 100 for 30min, staining of the whole genome DNA methylation will be significantly improved.Experiment 5. Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of the present study was to establish the optimal TSA treatment time in order to examine the effect of TSA combined with CHX10min+ELE activation protocol on the cloned embryos. The blastocyst percentage of cloned embryos treated with 50 nM TSA for 30-35 h immediately after activation climbed up to 33.6% (control group: 17.0%; P<0.05). We found that treatment with 50 nM TSA for 36 h after activation notably increased the blastocyst rate compared to the control group. Our data demonstrate that TSA treatment could significantly improve pig nuclear transfer embryos produced by a simple activation method combined with TSA treatment.Experiment 6. TSA-mediated reduction in DNA methyaltion was found by immunofluorescence analysis. Blastocyst stage embryos derived from TSA treatment displayed DNA hypomethylation compared to control embryos, although no significant differences were observed (P>0.05). The results suggest that high blastocyst formation is associated closely with low DNA methylation level, although the exact contribution of inner cell mass or trophoblast should be figure out in future studies.Taken together, the protocol for in vitro production of pig cloned embryos was simplified and improved by using CHX10min+ELE and TSA treatment, and the immunofluorescence staining against 5MeC was improved, which finally help us found that DNA methylation is postively correlated with improved preimplantation development.