330 TRICHOSTATIN A IMPROVES THE IN VITRO DEVELOPMENT OF CLONED BOVINE EMBRYOS RECONSTRUCTED WITH TRANSGENIC DONOR CELLS

Abstract

Trichostatin A (TSA), a histone deacetylase inhibitor, has been described as a potential modulator of nuclear reprogramming in bovine zygotes reconstructed by somatic cell nuclear transfer (SCNT), but with controversial results (Lee et al. 2011 J. Reprod. Dev. 57, 34–42; Sangalli et al. 2012 Cell Reprogramming 14, 1–13). The effect of TSA in zygotes reconstructed with transgenic cells cultured for long periods is not known. This study aimed to evaluate the effect of TSA on development of bovine embryos reconstructed with donor cells transfected with a green fluorescent protein (GFP)-reporter transgene. Bovine fibroblasts at second passage were transfected with lentiviral vectors carrying the GFP transgene and cultured at 37.5°C under 5% CO₂ in air. Transfected cells were cultured for additional 10 passages to establish a cell lineage expressing the protein. In the 12th passage, the cells were frozen in 10% dimethyl sulfoxide plus FCS (Nutricell, Campinas, Brazil) and frozen–thawed cells expressing GFP were used as nucleus donors. In vitro-matured oocytes were enucleated, fused to GFP positive fibroblasts, and activated with ionomycin. Putative zygotes were randomly distributed into 2 groups: SCNT-CONT (n = 55): zygotes were cultured for 4 h in CR₂aa medium plus BSA with 6-DMAP followed by 7 h in CR₂aa medium plus 2.5% FCS; SCNT-TSA (n = 49): zygotes were cultured in the same conditions described above, but supplemented with 50 nM TSA (Sigma-Aldrich, St Louis, MO). Then, embryos from all groups were cultured in CR₂aa supplemented with 2.5% FCS under 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C. Evaluations of cleavage and blastocyst percentages were performed at 72 and 168 h post-activation, respectively, and 4 replicates were carried out. Expression of GFP in embryos at blastocyst stage was visualised using an epifluorescence microscope. Statistical analysis was performed by ANOVA and data are shown as mean ± SEM. No difference (P > 0.05) on cleavage percentage was found between groups (72.9 ± 11.3% and 66.1 ± 14.4% for SCNT-CONT and SCNT-TSA, respectively). The blastocyst percentage calculated based on putative zygotes tended (P = 0.077) to be higher for SCNT-TSA (16.7 ± 4.0%) than for SCNT-CONT (6.8 ± 2.3%). When the blastocyst percentage was calculated based on cleaved embryos, a higher rate (P < 0.05) was achieved in SCNT-TSA (26.7 ± 3.8%) than in SCNT-CONT (10.3 ± 3.6%) group. Blastocysts of both groups expressed GFP, with no difference among embryos. In a previous study, we reported that TSA had no positive effect on in vitro embryo development or gene expression, despite the reduction on apoptosis index [Camargo et al. 2011 Acta Sci. Vet. 39(Suppl.), S442; Camargo et al. 2012 Reprod. Fert. Dev. 24, 121–122). In the present study, however, the treatment with TSA of zygotes reconstructed with transgenic cells cultured for a long time improved embryo development without impairing GFP expression. This result suggests that TSA may be effective in clones reconstructed with transgenic cells.

Supported by Embrapa 01.07.01.002, CBAB/CNPq, CAPES and Fapemig.