Development to blastocyst following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to that of a zygote. This reprogramming step is inefficient and may be dependent on a number of factors, including chromatin organization. Trichostatin A (TSA; 0-5 microM), a histone deacetylase inhibitor, was used to increase histone acetylation and 5-aza-2'-deoxycytidine (5-aza-dC; 0-5 microM), a DNA methyl-transferase inhibitor, was used to decrease methylation of chromatin in donor cells in an attempt to improve their reprogrammability. Adult fibroblast cells treated with 1.25 or 5 microM TSA had elevated histone H3 acetylation compared to untreated controls. Cells treated with 0.3 microM 5-aza-dC had decreased methylation compared to untreated controls. Both drugs at 0.08 microM caused morphological changes of the donor cells. Development to blastocysts by embryos cloned from donor cells after 0.08 or 0.3 microM 5-aza-dC treatments was lower than in embryos cloned from untreated control cells (9.7% and 4.2%, respectively, vs. 25.1%), whereas 0.08 microM TSA treatment of donor cells increased blastocyst development compared to controls (35.1% vs. 25.1%). These results indicate that partial erasure of preexisting epigenetic marks of donor cells improves subsequent in vitro development of cloned embryos.

No abstract

Differentiated somatic cells and embryos cloned from somatic cells by nuclear transfer (NT) have higher levels of DNA methylation than gametes and early embryos produced in vivo. Reducing DNA methylation in donor cells before NT by treating them with chemicals such as the DNA methyl-transferase inhibitor (5-aza-2'-deoxycytidine; 5-aza-dC) may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Previously, high levels of this reagent were used to treat donor cells, and decreased development of cloned embryos was observed. In this study, we tested a lower range (0.005 to 0.08 microM) of this drug and used cell cycle distribution changes as an indicator of changes in the characteristics of donor cells. We found that at 0.01 microM 5-aza-dC induced changes in the cycle stage distribution of donor cells, increased the fusion rate of NT embryos, and had no deleterious effect on the percentage of blastocyst development. Levels of 5-aza-dC greater than 0.01 microM significantly decreased embryo development. Embryos cloned from donor cells treated with a low dose of 5-aza-dC had higher levels of DNA methylation than embryos produced by in vitro fertilization, but they also had higher levels of histone acetylation. Although 5-aza-dC at 0.04 microM or higher reduced DNA methylation and histone acetylation levels to those of in vitro-fertilized embryos, development to blastocyst was reduced, suggesting that this concentration of the drug was detrimental. In summary, 5-aza-dC at 0.01 microM altered donor cell characteristics while showing no deleterious effects on embryos cloned from treated cells.

No abstract

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100μM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000μM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies.