Treatment with a histone deacetylase inhibitor after nuclear transfer improves the preimplantation development of cloned bovine embryos.
We examined the effects of treatment with histone deacetylase inhibitors (HDACi), trichostatin A (TSA) and scriptaid (SCR), on the blastocyst formation rate in bovine somatic cell nuclear transferred (SCNT) embryos derived from fibroblast cells. Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors to produce SCNT embryos (L1, L2 and L3 embryos, respectively). In Experiment 1, we compared the in vitro developmental competence of L1 embryos treated with various concentrations of TSA for different time periods following chemical activation. Embryos treated with 5 nM TSA for 20 h showed a significantly increased blastocyst formation rate compared with untreated controls. In Experiment 2, we examined the effect of TSA (5 nM) treatment of L1, L2 and L3 embryos as well as the effect of treatment of L1, L2 and L3 embryos with various concentrations of SCR on in vitro developmental competence. It was found that 5 nM TSA treatment significantly increased the blastocyst formation rate in L1 and L3 embryos but did not have an influence on the development of L2 embryos. On the other hand, 5 nM SCR treatment significantly increased the blastocyst formation rates of L1 and L2 embryos compared with controls. However, there was no significant increase in the blastocyst formation rate of L3 embryos when they were treated with SCR. In Experiment 3, acetylation of H4K12 was examined in donor cells and pronuclear-stage L1, L2 and L3 embryos treated with 5 nM TSA or 5 nM SCR by immunostaining. The level of H4K12 acetylation was different among donor cells. The staining intensities in the TSA-treated L1 and L3 embryos and SCR-treated L2 embryos were significantly higher than those of untreated embryos. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, the optimal treatment conditions may differ among donor cell lines.
Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science, NARO, Ibaraki, Japan. akagi@affrc.go.jp
Satoshi
S
Akagi
Kazutsugu
K
Matsukawa
Eiji
E
Mizutani
Kazuhiro
K
Fukunari
Masahiro
M
Kaneda
Shinya
S
Watanabe
Seiya
S
Takahashi
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D019976:Cloning, Organism / Q000379:methods*; D004477:Ectogenesis / Q000187:drug effects*; D046149:Embryo Culture Techniques; D005347:Fibroblasts; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D006898:Hydroxylamines / Q000494:pharmacology; D053652:Nuclear Transfer Techniques; D009865:Oocytes; D009994:Osmolar Concentration; D011804:Quinolines / Q000494:pharmacology; D013997:Time Factors
D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D006898:Hydroxylamines; D011804:Quinolines; C410733:scriptaid; C012589:trichostatin A