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Extracorporeal development of porcine somatic cell nuclear transfer (SCNT)-derived embryos is biased by scriptaid-induced epigenetic modulation of adult cutaneous fibroblast cells

The current study was conducted to examine the ex vivo developmental competences of cloned pig embryos reconstructed with oocytes receiving the cell nuclei of adult dermal fibroblast cells that had been epigenetically transformed by treatment with new-generation non-specific inhibitor of histone deacetylases, known as scriptaid (6- (1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide). Cumulus-oocyte complexes (COCs) were matured in vitro for 20 h in Tissue Culture Medium 199 (TCM 199). The maturation medium was enriched with 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP), 10 IU/mL equine chorionic gonadotropin (eCG), 10 IU/mL human chorionic gonadotropin (hCG), 10% foetal bovine serum (FBS), 10% porcine follicular fluid, 10 ng/mL recombinant human epidermal growth factor, 5 ng/mL recombinant human basic fibroblast growth factor and 1.2 mM L-cysteine. Afterwards, the COCs were cultured for an additional 22 to 24 h in the db-cAMP- and eCG+hCG-deprived TC 199 medium. Prior to use for SCNT, the permanent fibroblast cell lines (between passages 1 and 3) that had been established from the primary cultures derived from ear skin biopsies of a prepubertal boar were exposed to 350 nM scriptaid during 24-h contact inhibition. Reconstruction of enucleated in vitro-matured oocytes was accomplished by their electrofusion with epigenetically modulated fibroblast cells. Simultaneous fusion and electrical activation of reconstituted oocytes were triggered using two consecutive DC pulses of 1.2 kV/cm for 60 μs. Immediately after fusion/activation, nuclear-transferred oocytes (clonal cybrids) were incubated in North Carolina State University-23 (NCSU-23) medium supplemented with 5 µg/mL cytochalasin B for 2 h, followed by in vitro culture to morula and blastocyst stages in NCSU-23/BSA/FBS medium for 144 to 168 h. The rates of dividing embryos (187/252; 74.2%A), morulae (145/252; 57.5%A) and blastocysts (76/252; 30.2%C ) that originated from nuclear-transferred oocytes reconstituted with adult cutaneous fibroblast cells undergoing scriptaid treatment were significantly higher than in the scriptaid-unexposed group (133/229; 58.1%B , 101/229; 44.1%B and 42/229; 18.3%D, respectively) [A,B P < 0.001; C,D P < 0.01; χ 2 test]. Altogether, the improvements in not only cleavage activity of porcine cloned embryos, but also their morula/blastocyst yields seem to arise from enhanced abilities for promotion of faithful and complete epigenetic reprogramming of scriptaid-treated adult dermal fibroblast cell nuclei in a cytoplasm of reconstituted oocytes.