Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro.

The beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10-6 to 10-12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10-12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

Slight improvement in full-term development of mouse somatic cell nuclear-transferred embryos by cotransfer of fertilized embryos.

In our previous study (Tsuji et al., 2010 ), administration of hCG to recipients around the timing of implantation significantly increased the in vivo development of mouse embryos after somatic cell nuclear transfer (SCNT) until day 10.5, but did not increase the development to full term. The present study was undertaken to examine whether cotransfer of fertilized embryos or parthenogenetic embryos prevents the embryonic loss of SCNT embryos after day 10.5, allowing them to develop to full term. We found that compared with SCNT embryo transfer alone, full-term development of SCNT embryos slightly, but not significantly, increased by cotransfer of mouse hybrid blastocysts derived from BDF1 (C57BL/6×DBA) female×ICR male into the oviducts of recipients administered hCG (2.0% vs. 5.5%). This was not the case with the cotransfer of blastocysts from an ICR female×ICR male (2.5% vs. 2.2%) or parthenogenetic blastocysts from BDF1 female (3.0% vs. 2.0%). Furthernore, when SCNT blastocysts were transferred into the uteri of recipients, full-term development did not increase even with the cotransfer of hybrid blastocysts. The mechanisms of the effect of cotransfer of fertilized and parthenogenetic embryos on the full-term development of SCNT mouse embryos are discussed.

Administration of cyclosporin A to recipients improves the potential of mouse somatic cell nuclear-transferred oocytes to develop to fetuses.

Somatic cell nuclear-transferred (SCNT) oocytes have a high potential for development in vitro, but a large proportion of embryos that are transferred to recipients is aborted before parturition. The precise mechanism for the high abortion rate is unknown, but abnormal placenta formation is frequently observed in SCNT-cloned pregnancies. The present study examined the effects of treating the recipients with cyclosporin A (CsA), an immunoprotectant, on the proportion of fetuses resulting from SCNT-cloned pregnancies. Cloned embryos developed from enucleated oocytes and receiving cumulus cells from F1 (C57BL/6 × DBA, H-2b/d) females were transferred to outbred ICR (in which the H-2 complex was not fixed) recipient females. Each recipient received an intraperitoneal injection of CsA or vehicle. Compared with vehicle, administration of CsA to recipients on day 4.5 of pregnancy significantly increased the proportion of fetuses observed on day 10.5. The proportion of fetuses at day 18.5 of pregnancy in recipients receiving CsA treatment was slightly higher than that in controls. This study is the first to report that CsA administration increases the proportion of fetuses resulting from SCNT-cloned pregnancies.

Effect of melatonin treatment on the developmental potential of parthenogenetic and somatic cell nuclear-transferred porcine oocytes in vitro.

Melatonin secreted from the mammalian pineal gland is a free-radical scavenger that protects tissues from cell damage. The present study examined the effects of addition of melatonin to the culture medium on the developmental potential of parthenogenetic and somatic cell nuclear-transferred (SCNT) porcine oocytes. Supplementation of the maturation medium with melatonin did not increase the maturation rate, the proportion of oocytes that cleaved and developed into blastocysts after parthenogenetic activation, or the blastocyst cell number compared to controls. When 10-7 M melatonin was added to the culture medium, the proportion of parthenogenetic oocytes that developed to the 2-cell and 4-cell stages was significantly higher than that of controls. The potential of melatonin-treated oocytes to develop into blastocysts was high but not significantly different from that of controls. The addition of 10-7 M melatonin to the culture medium did not increase the preimplantation development of SCNT oocytes. Melatonin treatment significantly reduced the levels of reactive oxygen species in 4-cell parthenogenetic and SCNT embryos, but did not reduce the proportion of apoptotic cells in parthenogenetic and SCNT blastocysts. Although the results indicated that parthenogenetic and SCNT melatonin -treated embryos had significantly lower levels of reactive oxygen species than controls, the potential of melatonin-treated embryos to develop into blastocysts was not significantly higher than that of controls, in contrast to previous reports. The beneficial effects of melatonin on the developmental potential of oocytes might depend on the culture conditions.

Effect of human chorionic gonadotropin and progesterone administration on the developmental potential of mouse somatic cell nuclear-transferred oocytes.

Somatic cell nuclear-transferred (SCNT) oocytes have a relatively high potential to develop into blastocysts in vitro, but a large proportion embryos die at various pre- and postimplantation stages after transfer to recipients. Although the reason for the high mortality of SCNT embryos at the peri- and postimplantation stages is not clear, epigenetic abnormalities of SCNT embryos are considered to be the main cause. Such abnormalities of SCNT embryos may decrease their ability to maintain the corpora lutea, which is necessary for initiating implantation and maintaining fetal development. To examine this hypothesis, human chorionic gonadotropin (hCG) and progesterone were administered at different times to recipients that received SCNT embryos. When hCG was administered daily from day 3.5 to day 6.5 of pregnancy, the implantation and fetal development rates increased significantly compared to those of controls. The potential of SCNT embryos to develop to full term, however, was not greater than that of controls, even if hCG administration was continued to day 11.5 or day 17.5 and progesterone was administered from day 7.5 to day 17.5 after hCG injection. These findings demonstrated that administering hCG to recipients protects the in vivo development of SCNT embryos until day 10.5, but other treatment is necessary to support the progression of the embryos to full-term development.

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

Cloning efficiency has not been dramatically improved after the first success of somatic cell nuclear transfer (SCNT) in sheep in 1997. The reasons for the low efficiency of SCNT embryos must be attributed to the insufficient reprogramming of the donor nucleus in ooplasm. It has been clarified that the methylation and acetylation status are disordered in SCNT embryos and the gene expression pattern is different and widely varied in SCNT embryos, compared with fertilized embryos. In this paper, we focused on the role of the donor nuclei in cloning efficiency, and discuss whether ooplasm can reprogram any nucleus.

The effect of the time interval between injection and parthenogenetic activation on the spindle formation and the in vitro developmental potential of somatic cell nuclear-transferred rat oocytes.

We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in the rat. First, we examined the effect of preincubation time before activation on SCNT rat oocytes produced in the presence of MG132 with regard to spindle formation and the potential to develop into blastocysts. The spindles of SCNT oocytes continued to elongate with an increase in the culture duration and, in approximately half of oocytes, the chromosomes were distributed along the spindles at 120 min after incubation. Such abnormal spindle formation in SCNT oocytes is a possible reason for the low developmental potential of SCNT rat oocytes. To inhibit the formation of abnormal spindle formation, we examined secondly the developmental potential of rat SCNT oocytes that had been preincubated with nocodazole and demecolcine instead of MG132. The developmental rates in SCNT oocytes, however, were decreased. For successful rat somatic cell cloning, two steps might be required: (1) to culture the somatic cell nuclei for a sufficient time in MII oocyte cytoplasm to enhance nuclear reprogramming; and (2) to induce normal spindle formation with normal chromosomal construction.

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

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.

The developmental potential of parthenogenetic and somatic cell nuclear-transferred rat oocytes in vitro.

We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in rat oocytes. First, we compared the effects of two types of inhibitors of spontaneous activation, MG132 and demecolcine, on the developmental potential of parthenogenetic oocytes. The potential of activated oocytes to develop into blastocysts significantly decreased 2 h after oocyte recovery (77% vs. 7%). The developmental potential of oocytes preserved in MG132-supplemented medium for 1 to 4 h was high (62% to 77%), but the potential of those preserved in demecolcine-supplemented medium for 3 and 4 h was low (77% vs. 41% and 37%, respectively). Second, the effect of the duration of parthenogenetic activation on the developmental potential was examined. When oocytes preserved in MG132 for 4 h were treated with 10 mM strontium for 5 or 6 h, the potential of activated oocytes to develop into blastocysts was high (78% and 70%, respectively). Using the optimal conditions for parthenogenetic activation, we examined the potential of rat enucleated oocytes receiving cumulus cells to develop into blastocysts. In contrast to parthenogenotes, the potential of SCNT rat oocytes to develop into blastocysts was low (2%) even if then oocytes were treated with the histone deacetylation inhibitor trichostatin A. The reason for the low developmental potential of rat SCNT oocytes is discussed.

The effects of trichostatin A on mRNA expression of chromatin structure-, DNA methylation-, and development-related genes in cloned mouse blastocysts.

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.

Comparative gene expression analysis of bovine nuclear-transferred embryos with different developmental potential by cDNA microarray and real-time PCR to determine genes that might reflect calf normality.

Placental abnormalities are the main factor in the high incidence of somatic cell clone abnormalities. The expression of several trophoblast cell-specific molecules is enhanced during gestational days 7 to 14. To determine the possible genes whose expression patterns might reflect calf normality, we first compared the gene expression profiles on day 15 between in vitro-fertilized (IVF) embryos and two types of somatic cell nuclear-transferred embryos with either a high (FNT) or low (CNT) incidence of neonatal abnormalities using a cDNA microarray containing 16 of 21 placenta-specific genes developed from tissues collected across gestation. To identify significant genes from the screening of day 15 embryos, genes with a less than two-fold difference in expression between IVF and CNT embryos, and those with a greater than two-fold difference between IVF and FNT and between CNT and FNT were considered to contribute to clone abnormalities. These two comparisons revealed 18 down-regulated and 18 upregulated genes of the 1722 genes examined. We then examined the expression levels of 10 genes with known functions in eight-cell and blastocyst-stage embryos by real-time PCR. The mRNA expression pattern of interferon (IFN)-tau, a trophectoderm-related gene, differed between IVF, CNT, and FNT eight-cell embryos; few or none of the IVF or CNT eight-cell embryos expressed IFN-tau mRNA, but all eight-cell FNT embryos expressed IFN-tau. IFN-tau mRNA expression was significantly higher in IVF blastocysts, however, than in nuclear-transferred blastocysts. Average IFN-tau mRNA expression in FNT blastocysts was not different from that in CNT blastocysts, due to one CNT blastocyst with high expression. The precise relation between early expression of IFN-tau mRNA and inferior developmental potential in cloned embryos should be examined further.

Gene expression in individual bovine somatic cell cloned embryos at the 8-cell and blastocyst stages of preimplantation development.

Aberrant gene expression in somatic cell nuclear-transferred (NT) embryos due to abnormal epigenetic modifications of the donor nucleus likely accounts for much of the observed diminished viability and developmental abnormalities. We compared the expression of 13 developmentally important genes in individual 8-cell and blastocyst stage NT embryos produced from adults female cumulus cells and adult male skin fibroblast cells with low and high incidences of neonatal abnormalities. In vitro-fertilized (IVF) embryos were used as control embryos. Among the genes tested, the relative abundance of Glut-1, IGF-1R, E-cad, and Cx43 transcripts varied significantly between the two types of NT embryos at the 8-cell stage. The relative abundance of manganese super oxide dismutase (MnSOD) and Stat3 transcripts was significantly higher in IVF embryos compared with both types of NT embryos. At the blastocyst stage, there was a significant difference in the relative expression of only one gene, Bcl-2, between the two types of NT embryos. Although the level of Glut-1 expression did not vary between the two types of NT blastocysts, its expression in both types of NT blastocysts was significantly lower than that in IVF blastocysts. The MnSOD expression level tended to be higher in NT blastocysts. The gene expression profile for any single gene, however, was highly variable among individual embryos and was independent of embryo morphology. The present study demonstrated that the expression profiles of the 13 genes examined in Day 9 NT blastocysts produced from two different types of donor cells with different incidences of neonatal abnormalities are largely indistinguishable.

Bovine oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP).

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

Aging of recipient oocytes reduces the development of cloned embryos receiving cumulus cells.

Parthenogenetic activation is an important factor in successful production of cloned mammals. Because it has been reported that aged oocytes are more sensitive to parthenogenetic activation than young oocytes, the present study examined the effects of oocyte aging on the in vitro and in vivo developmental potential of nuclear-transferred (NT) mouse oocytes receiving cumulus cells. The potentials of young NT oocytes (14 h after human chorionic gonadotrophin [hCG] injection) to develop into blastocysts was, however, significantly higher than that of aged oocytes (20 h after hCG injection; 16% vs 6%). When the nuclei of NT oocytes at the 2-cell stage were fused with enucleated fertilized 2-cell embryos, the potentials of the serial NT embryos to develop into blastocysts were no different for both young and aged oocytes (74% vs 74%). Live young, however, were obtained only after transfer of serial NT blastocysts developed from young NT oocytes (2%). In contrast to a report using embryonic nuclei as the nuclear donors, the results of the present study indicate that young oocytes are superior to aged oocytes as a source of recipient cytoplasm for mouse somatic cell cloning.

Effect of the timing of first cleavage on in vitro developmental potential of nuclear-transferred bovine oocytes receiving cumulus and fibroblast cells.

The aim of the present study was to examine whether cumulus and fibroblast cell nuclear-transferred oocytes, which have high and low potential to develop into normal calves, respectively, are different in terms of in their patterns of timing of first cleavage and in their relationships between timing of first cleavage and in vitro developmental potential. The timing of first cleavage was similar in both types of nuclear-transferred and in vitro fertilized oocytes. More than 86% of the oocytes cleaved within 24 h after activation or in vitro fertilization; these oocytes contributed to more than 98% of the total number of blastocysts in all three groups. The potential of oocytes that cleaved at different intervals to develop into blastocysts differed among the groups. The developmental potential of the cumulus cell nuclear-transferred oocytes and in vitro fertilized oocytes decreased with the increase in time required for cleavage. Fibroblast cell nuclear-transferred oocytes that cleaved at 20 h, an intermediate cleaving time, had higher potential to develop into blastocysts. The results of the present study suggest that the type of donor nucleus used for nuclear transfer affects the timing of first cleavage.

Aberrant spindle assembly checkpoint in bovine somatic cell nuclear transfer oocytes.

Nuclear, microtubular dynamics and spindle assembly checkpoint (SAC) in bovine somatic cell nuclear transfer (SCNT) oocytes receiving G1/0 or M phase somatic cell nuclei were studied. SCNT oocytes assembled microtubules, however, the spindles were structurally abnormal, including bi-, tri-polar or elongated spindles with scattered premature chromosome condensation (PCC) in G1/0 phase nuclei, and some miniature spindles with unaligned chromosomes in M phase nuclei. In contrast, demecolcine-treated SCNT oocytes formed chromosome clusters with membrane protrusion and significantly induced maturation-promoting factor (MPF) activity elevation (up to 177%) for 3 hours, indicating that first SAC at second metaphase (MII) is established upon spindle disruption in SCNT oocytes. After parthenogenetic stimuli, unlike MII oocytes which prevent exit from MII arrest with high MPF activity upon spindle disruption by second SAC, demecolcine-treated SCNT oocytes could not prevent exit from MII arrest with inactivation of MPF activities, whereas MG132-treated SCNT oocytes could persist at MII arrest, indicating that SCNT oocytes lack the ability for second SAC establishment, however, two G1/0 phase nuclei in an ooplasm restored second SAC establishment upon spindle disruption. Furthermore, the developmental potential of demecolcine-treated SCNT oocytes receiving G1/0 phase nuclei to blastocyst stage was not significantly different than untreated SCNT oocytes (29% vs 31%). These results indicate that unlike MII oocytes, SCNT oocytes have aberrant spindle morphology and SAC at MII due to insufficient SAC signals from somatic cell nuclei, thus aberrant remodeling has started immediately after somatic cell nuclear transfer and may be responsible for chromosome instability in SCNT embryos as well as the low successful efficiency of cloning.

Comparative studies on the mRNA expression of development-related genes in an individual mouse blastocyst with different developmental potential.

The evaluation of embryo morphology, widely used for selecting mammalian embryos before transfer, is not an adequate standard for selecting nuclear-transferred (NT) embryos. To search for markers useful for predicting the potential of NT embryos to develop into young, we examined the relation between the morphology of embryos with different developmental potential and gene expression of Oct 4, Nanog, Stat3, FGF4, Stella, and Sox2. In the present study, we examined pronuclear-exchanged blastocysts and morula blastomere, embryonic stem (ES) cell, and cumulus cell NT blastocysts, and in vivo-developed and in vitro-developed blastocysts. Based on the small variations in the gene expression levels among the in vivo-developed blastocysts, and the significant differences in gene expression between in vivo-developed (high developmental potential), and ES cell and cumulus cell NT blastocysts (low developmental potential), down-regulation of Sox2 and Oct4 genes is considered to be a candidate marker for the low potential of NT embryos to develop into young.

Analysis of development-related gene expression in cloned bovine blastocysts with different developmental potential.

The high incidence of abnormalities in cloned calves is a most serious problem for bovine somatic cell nuclear transfer (NT) technology. Because there is little information on the differences in mRNA expression in cloned blastocysts with donor cells of different sex and origin, we compared development-related gene expression in two types of cloned bovine blastocysts with different potentials to develop into normal calves, a female adult cumulus cell line (high potential to develop into live calves) and a male fibroblast cell line (low potential to develop into live calves) to examine the correlation between the normality of cloned calves and blastocyst mRNA expression patterns. We analyzed 12 genes involved in apoptosis, growth factor signaling, metabolism, and DNA methylation in blastocysts originating from two types of donor cells and in vitro-fertilized blastocysts using quantitative real-time polymerase chain reaction. Expression of the pro-apoptotic Bax gene and anti-apoptotic Bcl-2 and Glut-1 genes in fibroblast-derived blastocysts was significantly higher than in cumulus cell-derived and in vitro-fertilized blastocysts. The high Bcl-2 and Glut-1 gene expression suggests that some embryonic cells with damaged DNA in fibroblast-derived blastocysts are not removed, and their descendants later manifest abnormal placenta or fetus formation. Transfer of pre-selected cloned blastocysts into recipients is required, however, to determine whether the expression pattern of these apoptosis-related genes reflects differences in the potential to develop into normal calves.

Demecolcine-assisted enucleation for bovine cloning.

The present study demonstrated that demecolcine treatment for at least 30 min produces a membrane protrusion in metaphase II-stage bovine oocytes. The maternal chromosome mass is condensed within the protrusion, which makes it easy to remove the maternal chromosomes for nuclear transfer (NT). Maturation promoting factor activity, but not mitogen-activated protein kinase activity, increased up to 30% in oocytes during demecolcine treatment. One normal healthy calf was obtained after transfer of four NT blastocysts produced following demecolcine treatment. Demecolcine treatment did not increase the potential of NT oocytes to develop into blastocysts. The present study demonstrated that chemically-assisted removal of chromosomes is effective for bovine cloning.

Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer.

Before fertilization, chromatins of both mouse oocytes and spermatozoa contain very few acetylated histones. Soon after fertilization, chromatins of both gametes become highly acetylated. The same deacetylation-reacetylation changes occur with histones of somatic nuclei transferred into enucleated oocytes. The significance of these events in somatic chromatin reprogramming to the totipotent state is not known. To investigate their importance in reprogramming, we injected cumulus cell nuclei into enucleated mouse oocytes and estimated the histone deacetylation dynamics with immunocytochemistry. Other reconstructed oocytes were cultured before and/or after activation in the presence of the highly potent histone deacetylase inhibitor trychostatin A (TSA) for up to 9 h postactivation. The potential of TSA-treated and untreated oocytes to develop to the blastocyst stage and to full term was compared. Global deacetylation of histones in the cumulus nuclei occurred between 1 and 3 h after injection. TSA inhibition of histone deacetylation did not affect the blastocyst rate (37% with and 34% without TSA treatment), whereas extension of the TSA treatment beyond the activation point significantly increased the blastocyst rate (up to 81% versus 40% without TSA treatment) and quality (on average, 59 versus 45 cells in day 4 blastocysts with and without TSA treatment, respectively). TSA treatment also slightly increased full-term development (from 0.8% to 2.8%). Thus, deacetylation of somatic histones is not important for reprogramming, and hyperacetylation might actually improve reprogramming.