Abstract
Synchronization of cell cycles at GO/G1 phases is very important for the development of nuclear transfer reconstructed embryos. Four different studies were conducted on interspecies cloning and in vitro embryo production in porcine.
The first study was conducted to examine the effect of cell culture conditions, antioxidants, protease inhibitors, and different levels of dimethylsulphoxide (DMSO) for the promotion of synchronization of different cell cycles of Siberian tiger skin fibroblasts and compared the ability of somatic cell nuclei of the Siberian tiger in pig cytoplasts and to support early development after reconstruction. Experiment I focused in the effects of cycling, serum starved and fully confluent stages of Siberian tiger cells on different cell cycles. In Experiment II, the effects of different antioxidants such as B-Mercaptoethanol (B-ME, 10μM), cysteine (2 mM), and glutathione (2 mM) were examined on the synchronization of cell cycles after cells were fully confluent without serum starvation for 4 h treatment. In Experiment III, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 μg/ml) and cytochalasin B (7.5 μg/ml) were used in the same manner as in experiment II. In Experiment IV, different levels of DMSO at 0%, 0.5%, 1.0% and 2.5% were tested their effects on cell cycle stages of Siberian tiger examined by Flowcytometry (FACS). In experiment I, 67.2% of the Siberian tiger skin fibroblasts reached the Go/G1 stage (2C DNA content) in fully confluent conditions which was more than the cycling (49.8%) and serum starved (SS) medium (65.5%) (p<0.05). Among the chemically treated group, glutathione (72.6%) and cycloheximide (71.3%) had little bit better results for the synchronization of Go+G1 phases than serum starved and fully confluent. After nuclear transfer there were no significant differences in the development of tiger-porcine reconstructed embryos at cycling, SS and fully confluent. Data indicate that prolonged culture of cells in the absence of serum as well as using different chemicals for this experiment does not imply a shift in the percentage of cells that enter Go/G1 and that confluency is sufficient to induce quiescence.
The second study was undertaken to examine cell cycle characteristics of endangered Goral (CITES Appendix I) adult skin fibroblasts. As the best condition for nuclear transfer is known to be GO/G1 phase, here this experiment study the optimal conditions for the better synchronization of in vitro Goral skin fibroblast in these cell cycle phases, Cycling, serum starvation, fully confluent and chemical inhibitors like protease and antioxidants were applied and the positions of cells in the cell cycle were estimated by DNA flow cytometry. Statistical analysis was applied. The obtained results show that beta mercaptoethanol, cysteine, and DMSO are better synchronizers of Goral fibroblasts than serum deprivation, culture confluence and protein synthesis inhibitors.
For interspecies cloning the present study was conducted by using the porcine oocytes as cytoplast. The last two studies were conducted with a view to improve the basic culture media NCSU-23 used for porcine embryo with the supplementation of amino acids and antioxidants and the possibility to use the improved supplemented group as a culture medium for interspecies NT reconstructed embryos in future. The third study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryos. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and 20 μL/mL MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and 10 μL/mL MEM) and essential amino acids (0, 5, 10 and 20 μL/mL MEM) in NCSU-23 medium (Exp. 3), first 72 h with non- essential amino acids, and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased, cleavage rate, rate of morula and blastocyst development and the number of ICM. In the case of combined addition significantly better results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the stage specific effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst
stage.
The fourth study was conducted to examine the comparative efficacy of potassium simplex optimization medium (KSOM) and North Carolina State University (NCSU)-23 medium supplemented with beta mercaptoethanol (B-ME) and amino acids (AA) on the developmental competence of porcine in vitro fertilized (IVF) embryos. Four experiments were conducted. KSOM and NCSU-23 medium were used to culture porcine parthenogenetic (Exp. 1) and IVF (Exp. 2) embryos. KSOM and NCSU-23 were equally effective to support porcine parthenogenetic and IVF embryo development from 1-cell to blastocysts. The NCSU-23 medium (Exp. 3) and KSOM (Exp. 4) were supplemented with amino acid (AA; 5 μL/mL non-essential amino acids + 10 μL/mL essential amino acids) and/or 10 μM beta mercaptoethanol (B-ME). Supplementing with AA and B-ME or B-ME alone in NCSU-23 had significant (p<0.05) differences in the rate of cleavage to 2- 4 cell (85.5 to 86.5% vs. 73.8%) and blastocyst (22.3 to 27.1% vs. 17.2%) over control group. On the contrary, supplementing KSOM with AA and/or B-ME had significant (p<0.05) difference in the rate of cleavage to 2-4 cell (70 to 79.8% vs. 67.7%) and blastocyst (19.2% to 24.4% vs. 16%) over control group. In conclusion, this study demonstrate that both KSOM and NCSU-23 medium supplemented with AA and B-ME or only ẞ-ME are superior to normal KSOM and NCSU-23 for porcine IVF embryo culture in terms of embryo developmental competence and quality.
In general conclusion, confluency is sufficient to synchronize the cell cycles for Siberian tiger skin fibroblasts and porcine oocytes can be used as a cytoplast for interspecies cloning. Beta mercaptoethanol, cysteine and DMSO at certain concentration can be used for the synchronization of goral cell cycles at GO/G1 phases effectively. NCSU-23 and KSOM medium can be improved with the supplementation of essential and nonessential amino acids and beta mercaptoethanol which can be used as a culture medium for interspecies nuclear transfer reconstructed embryos in future.
Key Words: Interspecies cloning, cell cycle, synchronization, amino acids, antioxidants.
Student Number: 2004-30860