6 EFFECT OF TRICHOSTATIN A ON HISTONE ACETYLATION AND METHYLATION, EMBRYO DEVELOPMENT, AND IN VIVO VIABILITY OF INTER-SPECIES BLACK FOOTED CAT CLONED EMBRYOS
Authors: Ricks D. M. , Reiser J. , Pope C. E. , Dresser B. L. , Gómez M. C. 
Published: December 2008

Aberrant DNA methylation and histone acetylation patterns following somatic cell nuclear transfer (SCNT) may disrupt the expression of imprinted and pluripotency-related genes, resulting in improper embryo development and fetal abnormalities. Treatment of mouse, pig and rabbit intra-species cloned embryos with the histone deacetylase inhibitor trichostatin A (TSA) has been reported to improve embryo development to the blastocyst stage (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Zhang et al. 2007 Cloning Stem Cells 9, 357–363; Shi et al. 2008 J. Anim. Sci. 86, 1106–1113). TSA has also increased the number of mouse live offspring after embryo transfer (Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have evaluated the effect of TSA on the ability of domestic cat (DSH, Felis catus) cytoplasts to support nuclear reprogramming and embryo development using black-footed cat (BFC, Felis nigripes) donor nuclei. To observe the effect of TSA on the covalent modification of histone H3, BFC-DSH reconstructed embryos were treated with 0, 5, 50, and 100 nm concentrations of TSA for 2 h before activation and 4 h and 16 h following activation. Levels of trimethylation and acetylation at lysine 9 of histone 3 (H3K9) were assessed by immunofluorescence and confocal microscopy. Average nuclear staining intensity was calculated for each antibody and compared between TSA treatments for each time point. Two to three oocytes were analyzed per group. H3K9 methylation was low or non-detectable for all treatments with or without TSA. H3K9 acetylation levels were not affected at 2 h for each of the TSA concentrations v. control. At 4 h and 16 h post-activation, H3K9 acetylation increased with exposure to TSA in a concentration-dependent manner. Next, BFC-DSH cloned embryos were treated with 50 or 100 nm TSA for 6 h or 24 h to determine the effect of TSA on in vitro development to the blastocyst stage (n = 157) and in vivo development after embryo transfer into domestic cat recipients (n = 255). TSA exposure did not enhance embryo cleavage, and among TSA treated embryos, blastocyst development was observed only in cybrids that were treated with 50 nm TSA for 24 h. The development rate of 2.8% for this group (n = 38) was similar to that of untreated embryos (2.0 to 3.3%, n = 502; P > 0.05). One pregnancy was established after transfer of TSA treated embryos, using a 24 h, 50 nm TSA treatment. The pregnancy rate (50%) and percentage of embryos that implanted following transfer (4.4%, n = 68) corresponded to those of untreated BFC-DSH cloned embryos (57.1% and 4.2%, n = 213; P > 0.05). Similar to untreated controls, all TSA treated embryos that implanted in DSH recipients developed an amorphous fetal mass and started reabsorption by Day 30 of gestation. In summary, administration of TSA did have an effect on the acetylation level of histone H3K9, but did not improve embryo development to the blastocyst stage or in vivo viability of inter-species black-footed cat cloned embryos.