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A Comparison of m-Carboxycinnamic Acid Bishydroxamide with Trichostatin A as Histone Deacetylase Inhibitor on the Developmental Competence of Somatic Cell Nuclear Transfer Porcine Embryos

이준희|박상훈|김태석|김진욱|성환우|고영규|윤호백|백상기|이미란|진상진
농업생명과학연구원 (Institute of Agriculture & Life Science, Gyeongsang National University)|2014|농업생명과학연구 (Journal of Agriculture & Life Science)|제48권 제6호|pp. 215-232|DOI http://dx.doi.org/10.14397/jals.2014.48.6.215

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키워드

Porcine SCNTm-Carboxycinnamic acid bishydroxamideTrichstatin A

초록

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer(SCNT) method producing cloned animals in several mammals. In general, faulty epigeneticmodifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainlyresults in low cloning efficiency. The nuclear reprogramming process involves epigeneticmodifications, such as DNA demethylation and histone acetylation, which may be an importantfactor in enhancing the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi),such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increasehistone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmentalcompetence and pluripotency-related gene expression (Oct4, Nanog and Sox2) in porcine clonedblastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treatedwith a 50nM concentration of TSA and 100μM concentration of CBHA during the in vitroearly culture (10h) after cell fusion and then were assessed to cleavage rate, development to theblastocyst stage and pluripotency-related gene expression in NT blastocyst also, level of histoneacetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NTembryos were not different between all groups for cleavage rates, the developmental competenceto the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) comparedto that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, allof pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treatedNT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos andexpression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at thezygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increasedthe developmental competence of porcine NT embryos and pluripotency-related gene expressions(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

Abstract

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer(SCNT) method producing cloned animals in several mammals. In general, faulty epigeneticmodifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainlyresults in low cloning efficiency. The nuclear reprogramming process involves epigeneticmodifications, such as DNA demethylation and histone acetylation, which may be an importantfactor in enhancing the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi),such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increasehistone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmentalcompetence and pluripotency-related gene expression (Oct4, Nanog and Sox2) in porcine clonedblastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treatedwith a 50nM concentration of TSA and 100μM concentration of CBHA during the in vitroearly culture (10h) after cell fusion and then were assessed to cleavage rate, development to theblastocyst stage and pluripotency-related gene expression in NT blastocyst also, level of histoneacetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NTembryos were not different between all groups for cleavage rates, the developmental competenceto the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) comparedto that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, allof pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treatedNT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos andexpression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at thezygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increasedthe developmental competence of porcine NT embryos and pluripotency-related gene expressions(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

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