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.

Trichostatin A (TSA) is the most potent histone deacetylase (HDAC) inhibitor known. We previously reported that treatment of mouse somatic cell nuclear-transferred (SCNT) oocytes with TSA significantly increased the blastocyst rate, blastocyst cell number, and full-term development. How TSA enhances the epigenetic remodeling ability of somatic nuclei and the expression of development-related genes, however, is not known. In the present study, we compared the expression patterns of nine genes involved in chromatin structure and DNA methylation, and seven development-related genes in blastocysts developed from SCNT oocytes treated with and without TSA, and in blastocysts developed in vivo and in vitro using real-time reverse transcription-polymerase chain reaction. In vivo-recovered blastocysts and blastocysts developed from TSA-treated SCNT oocytes exhibited similar expression patterns for Hdac1, 2, and 3, CBP, PCAF, and Dnmt3b genes compared with in vitro-developed blastocysts and blastocysts developed from SCNT oocytes without TSA treatment. There were significantly lower expression levels of Hdac1 and Hdac2 transcripts in TSA-treated and in vivo-recovered blastocysts than in TSA-untreated and in vitro-developed blastocysts. The finding that TSA treatment of SCNT oocytes significantly upregulated Sox2 and cMyc transcripts in blastocysts indicated that both transcripts are TSA-responsive genes. Thus, TSA treatment of mouse SCNT oocytes decreased the expression of chromatin structure- and DNA methylation-related genes, and increased the expression of Sox2 and cMyc genes in blastocysts. Such modifications might be a reason for the high developmental potential of mouse SCNT oocytes treated with TSA.

The occurrence of M-D twinning may be avoided by excluding blastocysts that contain decompacting ICMs.

In cases of cleavage failure, an embryonic cell could become tetraploid and may induce abnormal chromosomal configurations. Some cells exposed to cleavage failure may become trophectoderm cells and form placental abnormalities. Even if they develop into trophectoderm cells, the ICM can be susceptible to further cleavage failure and may in turn cause further aneuploidy. For these reasons, it is important to monitor pregnancies and births derived from oocytes that contained sERCs.

Regulatory T cells (Tregs) are a subpopulation of lymphocytes that play a role in suppressing and regulating immune responses. Recently, it was suggested that controlling the functions and activities of Tregs might be applicable to the treatment of human diseases such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease. TNF receptor type 2 (TNFR2) is a target molecule that modulates Treg functions. In this study, we investigated the role of TNFR2 signaling in the differentiation and activation of mouse Tregs. We previously reported the generation of a TNFR2-selective agonist TNF mutant, termed R2agoTNF, by using our unique cytokine modification method based on phage display. R2agoTNF activates cell signaling via mouse TNFR2. In this study, we evaluated the efficacy of R2agoTNF for the proliferation and activation of Tregs in mice. R2agoTNF expanded and activated mouse CD4+CD25+ Tregs ex vivo. The structural optimization of R2agoTNF by internal cross-linking or IgG-Fc fusion selectively and effectively enhanced Treg expansion in vivo. Furthermore, the IgG-Fc fusion protein suppressed skin-contact hypersensitivity reactions in mice. TNFR2 agonists are expected to be new Treg expanders.