The developmental potential of mouse somatic cell nuclear-transferred oocytes treated with trichostatin A and 5-aza-2′-deoxycytidine

Abstract

To facilitate nuclear reprogramming, somatic cells or somatic cell nuclear-transferred (SCNT) oocytes have been treated with the histone deacetylase inhibitor trichostatin A (TSA), or the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), to relax epigenetic marks of differentiated somatic cells. TSA-treated SCNT oocytes have increased developmental potential, but the optimal treatment period is unknown. Reduced methylation levels in somatic cells have no positive effect on SCNT oocytes, but the treatment of SCNT embryos with 5-aza-dC has not been investigated. We examined the effect of TSA treatment duration on the developmental potential of mouse SCNT oocytes and the effect of 5-aza-dC treatment on their in vitro and in vivo developmental potential. To determine the effects of TSA treatment duration, nuclear-transferred (NT) oocytes were cultured for 0 to 26 h with 100 nM TSA. SCNT oocytes treated with TSA for 8 to 12 h had the higher rate of development to blastocysts and full-term fetuses were obtained after treatment for 8 to 12 h. When oocytes were treated for 14 h and 26 h, blastocyst rates were significantly decreased and fetuses were not obtained. To examine the effect of 5-aza-dC, 2-cell stage SCNT embryos were cultured with 10 or 100 nM 5-aza-dC for 48 h to the morula stage and transferred. The potential of embryos treated with 5-aza-dC to develop into blastocysts was decreased and no fetuses were obtained after transfer. The findings demonstrated that long-term TSA treatment of SCNT mouse oocytes and treatment with 5-aza-dC inhibit the potential to develop into blastocysts and to fetuses after transfer.

Recent advancements in cloning by somatic cell nuclear transfer

“…This is a necessary part of genetic reprogramming [86]. In fact, several reports clearly showed that HDACi treatment during SCNT cloning improved histone acetylation [87], nascent mRNA production [64] and gene expression [88] in a manner similar to that in normally fertilized embryos. TSA treatment also improved the long-term consistency of genome-wide gene expression regulation: the total number of genes commonly exhibiting up-or downregulation in the TSA-treated clone pups decreased to half of the conventional SCNT pups and the total gene expression profile of the TSA clones came to resemble that of the intracytoplasmic sperm injection (ICSI) pups [89].…”

Role of the donor nuclei in cloning efficiency: can the ooplasm reprogram any nucleus?

“…Reprogramming the meiotic deacetylation process by inhibiting deacetylation with the potent and specific histone deacetylase inhibitor trichostatin A (TSA) has been effective for in vitro and in vivo cloning efficiency (Rybouchkin et al, 2006;Tsuji et al, 2009;Kishigami et al, 2006), but has not induced sufficiently dramatic improvement. Although the low efficiency of SCNT embryos appears to be due to insufficient reprogramming of the donor nucleus in the ooplasm, this has not been clarified and overcoming this insufficient reprogramming presents several challenges.…”

Extended Exposure to Trichostatin A after Activation Alters the Expression of Genes Important for Early Development in Nuclear Transfer Murine Embryos

“…The application of TSA treatment in SCNT has resulted in higher pre-implantation embryonic development in pigs [61], cattle [7] and rabbits [36]. Although the application of TSA has resulted in improvements in the murine SCNT program [18,44], the effect of TSA on SCNT is still an issue in some animal species [26,36,57] and many research groups have suggested that it has various harmful effects on the development of mammalian SCNT embryos [26,44,57]. In our experiment, we investigated the optimal duration of TSA exposure during the early reprogramming event in SCNT embryos.…”