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Research Article |
Laboratory of Animal Reproduction, College of Agriculture, Kinki University, Nara 631-8505, Japan
ABSTRACT
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.
assisted reproductive technology, developmental biology, early development, oocyte development
INTRODUCTION
Reprogramming of differentiated somatic nuclei into the totipotent embryonic state by transfer into oocyte cytoplasm is not efficient. Although in some published protocols a considerable number of reconstructed oocytes might reach the blastocyst stage, their development after implantation and to full term is very limited. To make application of nuclear transfer (NT) technology feasible in animal breeding and regenerative medicine, an understanding of the mechanisms underlying the reprogramming process is necessary in order to make it more efficient.
The reprogramming events following transfer of somatic nuclei into oocyte cytoplasm occur at the epigenetic level. One of the many currently-known epigenetic modalities is the global level and local pattern of acetylation of nuclear histones [1]. Whether and to what extent reprogramming after NT depends on reshaping by histone acetylation is not clear. Increased histone acetylation levels on most amino acid residues leads to looser binding of the nucleosome to DNA and/or linker histones, relaxation of the chromatin structure, and formation of a transcriptionally permissive state [2–5]. Moreover, increased histone acetylation is associated with more effective formation of DNA replication complexes and can facilitate cell proliferation [6]. Histone acetylation levels in special chromatin sites are frequently inversely related with the level of methylation of the corresponding DNA, and there are likely multiple and somewhat interdependent relationships between these two epigenetic modalities [7, 8]. Finally, there is some evidence that the pattern of histone acetylation is preserved during mitosis; thus, it likely contributes to the mechanism underlying epigenetic memory of a differentiated state in daughter cells [9, 10]. If this is the case, correct reprogramming of the acetylation pattern following NT would be very important for converting a differentiated somatic nucleus to a totipotent state.
The global level of histone acetylation in the chromatin of mouse oocytes drops dramatically at the meiotic metaphase, and there are also few if any acetylated histones in mouse spermatozoa [11–15]. Acetylation levels, however, become gradually restored in zygotic pronuclei following oocyte activation or fertilization, with a somewhat higher initial acetylation level in the male pronucleus [10, 13]. The importance of these dynamic events is thought to be in erasing the pattern of gene expression (differentiation memory) characteristic of maturing gametes at meiosis, and unhindered establishment of the embryonic pattern following interphase entry at fertilization and oocyte activation, i.e., contribution to epigenetic reprogramming [12, 13, 16]. These interesting and thought-provoking ideas are especially relevant to the mechanisms of reprogramming following somatic cell NT. Indeed, some initial studies demonstrated that after fusion of NIH 3T3 cells with enucleated mouse oocytes, acetylation of many histone amino acid residues decreases markedly, or even totally, within 2 h after fusion [12]. As in normal fertilization, this might indicate a complete or at least partial removal of the somatic, differentiated-style acetylation program from the transferred chromatin and suggests that manipulation of the deacetylation level will influence the efficiency of reprogramming. The actual significance of this meiotic erasing of acetylation for development of NT, as well as normal embryos, however, remains unknown. In the present study, we examined the reprogramming significance of this meiotic deacetylation process by inhibiting deacetylation with the potent and specific histone deacetylase inhibitor trychostatin A (TSA). We have also applied TSA at early postactivation stages to examine the role of the changes in histone acetylation levels in reprogramming or remodeling of somatic nuclei. There were previous attempts to apply TSA and other histone deacetylase inhibitors (sodium butyrate) for NT to study the role of acetylation in nuclear reprogramming; these were performed in cows, and, more importantly, the drugs were applied only to the donor cells before NT per se [17, 18]. Our study extends this line of research to examine the events occurring in somatic nuclei immediately after their placement into the oocyte cytoplasm.
The objectives of this research were as follows: 1) to evaluate the dynamics of deacetylation on some histone residues following the injection of cumulus nuclei into enucleated mouse oocytes; 2) to analyze the role of meiotic deacetylation in the development of NT embryos by its inhibition with TSA; and 3) to investigate the effect of the extension of TSA treatment to the period of nuclear remodeling during oocyte activation on the development and quality of resulting NT embryos.
MATERIALS AND METHODS
All experiments and protocols were performed in strict accordance with the Guiding Principles for the Care and Use of Research Animals adopted by the Kinki University Committee on Animal Research and Bioethics.
Media and Reagents
Salts for preparation of the media and most other reagents were purchased from Sigma Chemical Co. unless stated otherwise. Human chorionic gonadotrophin and eCG were purchased from Sankyo. The mouse oocytes were handled and cultured in flushing holding medium (FHM) and potassium simplex optimized medium (KSOM) media, respectively [19]. Both FHM and KSOM media contained 4 mg/ml BSA and 50 µM EDTA, and had osmolarities of 290 and 270 mOsm, respectively. Water for medium preparation was obtained from Specialty Media and mineral oil to cover culture drops from Nakalai Tesque, Inc. Stock solutions of cytochalasin B and TSA were dissolved in dimethyl sulfoxide (DMSO) at 1 mg/ml and 1mM, respectively, and stored at –20°C. The inhibitory constant of TSA for different known histone deacetylases is between 1.4 and 38 nM; thus, we considered 100 nM to be high enough to have a strong inhibitory effect, and this concentration was used throughout all experiments. Cytochalasin B (5 µg/ml) was used during oocyte enucleation and during the oocyte activation period (6–9 h, depending on experiment set) to give a final concentration of 0.5% DMSO in the incubation medium. TSA was applied before and/or during oocyte activation by dissolving the stock concentration of TSA 10 000 times. An equal volume of the vehicle was added to the control group. Thus, the final concentration of DMSO in the control and TSA treatment groups was 0.01 and 0.51% in the preactivation and activation media, respectively. Anti-acetyl-histone H4 (lysine 12; H4K12) and anti-acetyl-histone H3 (lysine 14; H3K14) rabbit polyclonal antibodies were purchased from Upstate Biotechnology and used at 1:1000 and 1:250 dilution, respectively.
Production of NT Oocytes and Embryos
The method of production of NT oocytes and embryos was essentially the same as the Honolulu technique described elsewhere [20]. In brief, recipient oocytes were recovered from 8- to 10-wk-old B6D2F1 female mice (Clea-Japan, Inc.) following superovulation with 7.5 IU of eCG and 7.5 IU of hCG, 48 h apart. Females were killed 13 to 14 h after injection of hCG and oocyte-cumulus complexes were released into FHM medium with 200 U/ml hyaluronidase (Type I-S). Following a 5-min incubation, cumulus-denuded oocytes were collected and washed in three drops of plain FHM. Groups of oocytes were enucleated in FHM medium with 5 µg/ml of cytochalasin B, washed in plain FHM, and maintained at room temperature before NT. Cumulus cells released by hyaluronidase treatment were collected into 1 ml of FHM in a 1.5-ml Eppendorf tube, washed by centrifugation, and maintained at 4°C before being used as nuclear donors. Immediately before NT, cumulus cells were mixed with 10% clinical grade polyvinylpyrrolinde solution (MediCult).
All micromanipulations were performed at room temperature using a PMM-150FU piezo-actuated micromanipulator unit (PrimeTech) and microneedles shaped from borosilicate glass capillaries.
Following NT and a short 15-min recovery period, oocytes were transferred for culture into KSOM medium with or without TSA and placed in a humidified incubator at 37°C in 5% CO2 in air. The oocytes were activated if necessary in 10 mM SrCl2 and 5 µg/ml cytochalasin B-supplemented calcium-free KSOM with or without TSA for 6 or 9 h. If embryos were cultured to the blastocyst stage (96 or 120 h), they were transferred at 64 h after the start of activation into KSOM supplemented with 1:200 stock solution of essential and nonessential amino acids solutions (Invitrogen) and 3.5 mg/ml glucose.
Analysis of Histone Acetylation Level
All staining procedures were performed at room temperature in 10 mM isotonic PBS supplemented with 1% BSA. Oocytes and embryos in groups of 5 for 3 independent repeats each were fixed for 1 h in freshly-prepared 4% paraformaldehyde in PBS without BSA. They were then washed and permeabilized for 20 min with 0.5% Triton X-100, washed again for 20 min, and incubated with the above-mentioned primary antiacetyl antibodies for 1 h. After incubation and washing for 20 min, the oocytes were incubated with secondary goat anti-rabbit fluorescein isothiocyanate-conjugated antibodies at 2 µg/ml (1:200 of stock; Santa Cruz Biochemistry) for 1 h. They were then washed and mounted for observation into UltraCruz DAPI containing mounting medium. Staining was observed under a Nikon UV eclipse E800m microscope with triple D-F-R and B-2A (excitation 450–480 nm, dichroic mirror 505 nm and barrier filter 520 nm) filter sets. Images were photographed under standard conditions of 1-sec exposure time with an ND4 filter using a Keyence VB6010 CCD camera and VB6000 fluorescence digital microscope camera controller. Fluorescence was measured by analysis of the oocyte photos with Photoshop 8.0 software (Adobe Systems Inc.). Average pixel intensity value for a fixed-pixel-size (5 x 5) area was measured in five different random regions of cytoplasm, and total average was calculated and subtracted from the same total average measured in randomly selected regions of chromosomes (Green channel). This average chromatin intensity staining was measured for a total of 15 oocytes for each antibody and in each time and treatment group.
Full-Term Development Test
Groups of 10 to 13 2-cell-stage NT embryos obtained with and without TSA application were transferred into the same pseudopregnant females (CD1, 0.5 days postcoitus ["c]), but into different oviducts (carriers) to randomize other possible effectors. Expanded NT blastocysts at 96 h of culture were transferred into the uterine horns (carriers) of the same or different 3.5-dpc pseudopregnant females. All females were sacrificed at 19.5 dpc and examined for implantation sites, placentas, and pups. Pups were raised by lactating foster mothers of the CD1 strain, if available.
Differential Staining of Blastocysts for Inner Cell Mass and Trophectoderm Cell Counting
Nuclear transferred embryos reaching the expanded blastocyst stage 96 h after activation were denuded with acid Tyrode solution and incubated in rabbit anti-mouse lymphocyte serum in FHM (1:2) solution for 20 min at 37°C. Afterwards, they were briefly washed in FHM and incubated in 30% guinea pig complement in FHM supplemented with 15 µg/ml propidium iodide (PI) for 20 min. After incubation in complement, blastocysts were briefly washed in BSA-free FHM with 10 µg/ml PI and fixed in ice-cold ethanol for 1 min. After fixation, blastocysts were stained in ethanol with 10 µg/ml Hoechst H33258 for 30 min, transferred to glycerol on a glass slides, and flattened with coverslips. They were immediately observed and photographed for the counting of nuclei under a fluorescent microscope.
Experimental Design and Statistical Analysis
Experiment set 1. Groups of 5 NT oocytes at 20 min and 1, 2, and 3 h after nuclear injection were stained with antibodies to acetylated histones to analyze the timing of deacetylation of transferred chromatin and the inhibitory effect of TSA on this phenomenon. Anti-acetyl-H4K12 and anti-acetyl-H3K14 were used as representative of acetylation level in heterochromatin and euchromatin, respectively [10]. For these residues in particular, the meiotic metaphase deacetylation phenomenon was clearly demonstrated [12], and it is known that TSA in a selected concentration (100 nM) prevents deacetylation of all histones on all 14 known acetylation sites [21]; therefore, we considered proof of the inhibitory effect of TSA on deacetylation of these two residues as being enough to confirm the general efficiency of TSA.
Experiment set 2. To determine the effect of inhibition of deacetylation on the development of NT embryos to the blastocyst stage, each group of concomitantly-injected oocytes was divided into two equal parts and cultured for 3 h with or without TSA in KSOM. Both parts were activated for 6 h in medium without TSA, and their development to blastocysts was measured. The scheme for these experiments was K3K6 (KSOM 3 h before activation and 6 h during activation) or T3K6 (TSA 3 h before activation and KSOM 6 h during activation). All following experimental sets were performed using this scheme. The experiments were repeated three times.
Experimental set 3. The development of NT embryos after continuous TSA treatment (T3T6), and treatment only during the activation period (K3T6) were compared with development of untreated NT embryos (K3K6) to investigate the effect of the enhanced somatic (T3T6) or embryonic (K3T6) pattern of histone acetylation on development. The experiments were repeated nine times.
Experimental set 4. We studied the effect of the duration of activation and treatment with TSA on the development of NT embryos to the blastocyst stage (T2T6 vs. T2T9 vs. K2K6 vs. K2K9). The duration of preactivation incubation was reduced to a maximum of 2 h to activate postovulatory younger oocytes. We also estimated the quality of the resulting blastocysts by counting the number of cells in the inner cell mass (ICM) and trophectoderm (TE). Again, each group of injected oocytes was divided into four parts to randomize other possible influence factors. The experiments were repeated five times.
Experiment set 5. The full-term developmental potential of 2-cell-stage embryos and 96-h blastocysts obtained in K2K6 and T2T6 treatment schemes were compared following transfer to pseudopregnant females.
Data expressed as proportions (percentages) were analyzed by z-test and data on the number of cells were analyzed by t-test, using SigmaStat 3.0 software.
RESULTS
Timing of Histone Deacetylation in Nonactivated NT Oocytes
We have studied the dynamics of deacetylation of lysine 12 of histone H4 and lysine 14 of histone H3 in NT oocytes before activation. In all analyzed NT oocytes, there was only moderately reduced staining for acetylated H4K12 2 h after NT, compared to the intensity of staining at 20 min after NT (Fig. 1, A and B; see also Supplemental Fig. S1 available online at http://www.biolreprod.org). The staining for this modification, however, was barely visible after 3 h of incubation following nuclear injection (Fig. 1C). If the culture medium contained 100 nM TSA, the intensity of staining for both analyzed acetylated residues remained unchanged or even somewhat increased from the initial level at any time after the start of incubation (Fig. 1D). The staining on acetylated H3K14 was no longer visible after 1 h of incubation in plain medium, but was bright even after 3 hrs of culture in TSA-containing medium (data not shown).
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Effect of the Prevention of Meiotic Deacetylation on the Development of NT Embryos to the Blastocyst Stage
After we confirmed that TSA prevented deacetylation of at least some lysine residues in histones, and that this deacetylation indeed occurred in the absence of TSA, we analyzed whether the prevention of deacetylation had any negative effects on the development of NT embryos to the blastocyst stage. TSA-treated and untreated NT oocytes reached blastocyst stage at very similar rates. Moreover, we could observe some slight increase in the percentage of embryos reaching the 2- and 4-cell stages (Table 1).
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Effect of Extension of the TSA Treatment to the Activation Period on the Development of NT Embryos to the Blastocyst Stage
The prevention of meiotic deacetylation of somatic chromatin did not negatively affect development of NT embryos to the blastocyst stage; therefore, we speculated that some forms of reprogramming by deacetylation occur during nuclear remodeling (from the start of activation to the formation of mature, visible pronuclei). To test this assumption, we extended the period of TSA treatment from the moment of somatic nucleus injection to the pronucleus formation in NT embryos (T3T6) to inhibit any possible reprogramming of the somatic acetylation pattern. Additionally, by allowing deacetylation of chromatin in the preactivation period and applying TSA only after activation started (K3T6), we could estimate the effect of the induced somatic chromatin hyperacetylation alone on remodeling and reprogramming of somatic chromatin. There was a significant, increase, almost a doubling, in the proportion of NT embryos developing to the blastocyst stage in both TSA-treated groups (Table 2). The blastocyst rate and especially the hatching rate, however, were significantly higher for the TSA-treated group both before and after beginning activation.
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Analysis of the Timing of TSA Effect and Blastocyst Quality of TSA-Treated Embryos
We further examined whether the beneficial effect of TSA could be further enhanced by extending the duration of the treatment with deacetylase inhibitor from 6 to 9 h. The duration of preincubation of NT oocytes before activation was reduced from 3 to 1–2 h to minimize oocyte aging effects. There was a modest, nonsignificant increase in the blastocyst rate when NT oocytes were treated with TSA for 9 instead of 6 h (Table 3). The duration of activation treatment with strontium had no effect on blastocyst rate; there was no difference in the blastocyst rate between control groups activated for 6 or 9 h (K2K6 versus K2K9). Remarkably, in our experiments, the blastocyst rate of control, NT embryos not treated with TSA varied between 20% and 50% from experiment to experiment, and, in parallel, changes in the blastocyst rate in TSA groups increased or decreased, but were always at least two times higher than in controls.
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We further analyzed the quality of the blastocysts obtained in this series of experiments by counting cell numbers in the ICM and TE. The average cell numbers were very close between both controls (6 and 9 h activation time) and the two TSA-treated groups (Table 3), so these data were pooled together. In total, 94 control and 124 TSA-treated blastocysts were analyzed; there was a highly significant increase in ICM (9 ± 5 versus 14 ± 5, P < 0.01) and TE (36 ± 13 versus 45 ± 15, P < 0.01) cell numbers in TSA-treated groups. Analysis of 23 parthenogenetic blastocysts obtained in these experiments produced cell numbers very close to those of the TSA-treated NT group (16 ± 4 and 43 ± 10 for ICM and TE, respectively).
Full-Term Development of NT Embryos with or Without TSA Treatment
Transfer of the 2-cell stage TSA-treated and untreated NT embryos into oviducts of the same mice demonstrated a significant increase in the implantation rate in the TSA-treated group (Table 4), likely reflecting an increase in the blastocyst development rate found during culture in vitro. There was also some increase in the full-term development rate (3–4 times). It was, however, still too low compared to the number of embryos transferred to reach statistical significance.
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There was approximately the same 3-fold increase in the full-term development rate for the TSA treated embryos, if blastocysts instead of 2-cell embryos were transferred. The difference, however, was not statistically significant. There was also no difference in implantation rate in these blastocyst transfer experiments (Table 5). There was also no difference in the vigor of the delivered pups, and they survived to adulthood normally at an equal rate if feeding mothers were available at the time of delivery. Placental and body weights were also not different between the two groups, and were on average 0.343 and 1.706 g for the TSA-treated group and 0.372 and 1.748 g for nontreated group, respectively.
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DISCUSSION
The present study investigated the importance of reshaping of the global histone acetylation pattern for reprogramming of somatic chromatin in oocyte cytoplasm. Consistent with previous findings [12], transfer of somatic nuclei into nonactivated ooplasm dramatically reduced the global level of histone acetylation. In contrast to the previous report, however, deacetylation of histone H4 on lysine 12 in our study seemed a bit slow, dropping strongly after only 3 h of incubation. This discrepancy might be explained by differences in the type of cells used as nuclear donors. Indeed, the NIH 3T3 embryonic fibroblasts used in the above-mentioned study are fast-proliferating, immortalized cells and might have a chromatin conformation more readily accessible to histone deacetylases. In contrast, the cumulus used in our experiments are nondividing G0 arrested cells and could likely have a more compacted chromatin structure. This is analogous to mature chicken erythrocytes, which have more compacted chromatin, and at the same time a longer turnover time of the acetylated groups in their histones [22]. The other possible explanation is the NT method (electrical fusion in the above-mentioned study and direct nuclear injection in our experiments), because it can influence the timing of accessibility of the transferred chromatin to the cytoplasmic factors. The acetylation of the histone H3 on lysine 14 in transferred cumulus chromatin declined quickly and reached an undetectable level within 1 h after NT.
Consistent with the earlier study [12], incubation of the NT oocytes in medium containing TSA prevents deacetylation in the transferred chromatin. This allowed us to analyze the functional significance of deacetylation by comparing the developmental potential of NT oocytes incubated with and without TSA. Surprisingly, NT oocytes with inhibited deacetylation developed to the blastocyst stage at the same rate as those that were incubated for a long enough time to ensure almost complete histone deacetylation. This finding suggests that reactivation of the genes responsible for blastocyst development in the transferred somatic nuclei is independent from the deacetylation events occurring before oocyte activation. A more detailed analysis of the histone deacetylation events with multiple site-specific or pan-acetylated antihistone antibodies would provide more solid data. The well-studied mode of the inhibitory action of TSA on deacetylation of all histones, however, supports our conclusion. Alternatively or additionally, treatment with TSA could also stimulate DNA demethylation [7, 8], leading, at least theoretically, to improved reprogramming. This is, however, rather unlikely, because of short duration of treatment, absence of DNA replication, and no reported active demethylation events at meiotic metaphase. Overall, our results add further doubts to the earlier published assumptions that meiotic deacetylation has reprogramming significance [12, 13, 16].
It is possible, however, that significant changes in the acetylation pattern by deacetylation of histones at some specific chromatin locations occurred at the time of oocyte activation and/or at the early postactivation period, in spite of or in parallel to the rise in the global level of chromatin acetylation [11, 14, 23]. To examine this possibility, we extended TSA treatment well beyond the start of the activation period. Paradoxically, presumably preventing erasure of the somatic pattern of chromatin acetylation (although possibly modifying it somewhat) by continuing culture in TSA before and as long as 9 h after the start of activation not only did not inhibit the development of NT embryos, but conversely allowed a higher number of embryos to reach blastocyst stage. Moreover, the quality of the obtained blastocysts was better, as judged by the cell number in the ICM and TE. Extension of the TSA treatment from 6 to 9 h after activation had only a minor positive effect on blastocyst rate and no effect on average cell numbers. This suggests that the positive effect of hyperacetylation on nuclear reprogramming (or remodeling) occurs at a very early stage of development. This is further supported by the observation that continuous inhibition of deacetylation before and after activation was more effective for stimulating development of NT embryos than treatment only after the start of activation. Indeed, in the former case, chromatin is already acetylated at the start of activation, and this probably improves its chances for remodeling, whereas later, some time after activation is necessary to reach the basic level of acetylation. Taken together, our results indicate that it is highly unlikely that deacetylation events have a principal role in the reprogramming of the pattern of gene expression from somatic to embryonic type following NT.
The reasons for the higher number of embryos reaching the blastocyst stage following an extended period of TSA treatment warrants further investigation. In general, approximately 30%–50% of cumulus-derived NT embryos reach the blastocyst stage after culture in vitro [24–26]. Moreover, there are reports that NT embryos vary very strongly in the expression level of the imprinted and pluripotency-related genes in contrast to their normal counterparts [27, 28]. It may be speculated that switching on or off the genes affecting blastocyst development occurs in the transferred somatic chromatin by a scenario resembling that occurring during mosaic, random switching off of position effect variegation (PEV) genes [29, 30]. An obvious difference, however, is that with PEV the potentially active genes in some random cells of a given tissue are silenced, whereas cloning-related "reprogramming effect variegation" is likely variegation both in silencing of somatic cell-specific genes and in restoration of the active state of some embryogenesis-important genes. The reasons for the random switching off of the genes at PEV are not well understood, and hypothetically depend on intracellular or intercellular fluctuations of chromatin remodeling factors, but the state of the PEV genes in some models can be shifted towards activity by exposure to chemicals that stimulate histone acetylation [29]. Moreover, in at least some instances, this modified state can be remembered through many cellular generations after withdrawal of hyperacetylation-inducing chemicals [31]. We can therefore speculate that, in contrast to deacetylation, hyperacetylating treatment of the NT embryos with TSA at early stages of nuclear remodeling stimulates more effective remodeling of the somatic chromatin, suppressing reprogramming effect variegation, at least for early genes. Active DNA demethylation occurring early after fertilization in normal embryos, and mostly in hyperacetylated male pronucleus [32, 33], might also be more effective in TSA-hyperacetylated NT embryos [7]. Further comparative study of the molecular events occurring in TSA-treated and untreated NT embryos might provide valuable data on mechanisms of reshaping and fixing of epigenetic memory at reprogramming.
Different culture media can have significant effect on cloned blastocyst rate [34, 35]. Therefore, TSA might somehow adapt cloned embryos to KSOM medium only, and not have an effect in other culture systems. Whether the positive effect of TSA on the blastocyst rate observed in the present study for KSOM medium is culture medium-specific is not clear, and further investigation is required. The increased implantation rate and the tendency towards an increase in the delivery rate after transfer of TSA-NT embryos at the 2-cell stage (therefore exposed to KSOM for only a short period) makes a medium-specificity effect of TSA rather unlikely, although it cannot be excluded.
In addition to the blastocyst rate, the quality of the blastocysts, as measured by cell number, was also improved after TSA treatment. The mean total and TE cell numbers were increased by 25% and cell numbers in the ICM by 50% in TSA-treated embryos as compared to untreated NT embryos. This can be explained by improved reprogramming in the TSA group or otherwise more effective DNA replication, which histone hyperacetylation stimulates in some cells [6, 36].
The significantly improved blastocyst rate and quality obtained with TSA application in our experiments did not significantly increase full-term development of NT embryos compared to untreated embryos. The 3- to 3.5-fold increase in the number of 2-cell- or blastocyst-stage embryos capable of developing to full term was too small, and a very high number of transfers would be required to demonstrate a statistically significant difference. Because there was a difference in full-term development after transfer of blastocysts, however, it is possible that TSA treatment not only has some effect on the proportion of embryos able to achieve preimplantation development, but also has a positive influence on the reprogramming of genes that are important at postimplantation stages. This influence, however, seems minimal. This could, in fact, be expected, because histone acetylation is far from the single, and likely not the main, epigenetic player (others being, e.g., DNA and histone methylation), and control of epigenetic players would be important to improve not only blastocyst rate but also the much more complex process of full-term development. We also cannot exclude that other epigenetic or even genetic defects attributed to mammalian NT embryos [37] may limit the level of the beneficial effect of enhanced histone acetylation on full-term development of the clones. It is likely that controlled reprogramming following nuclear transfer requires fine-tuning of multiple epigenetic players, fine-tuning that oocyte cytoplasm might perform only very randomly. It is foreseeable that TSA will be only one of many tools necessary to fine-tune the reprogramming potential of the oocyte cytoplasm towards somatic chromatin.
The findings of the present study indicate that meiotic deacetylation of somatic histones is not important for further development of NT embryos, whereas inhibition of the removal of acetyl groups during oocyte activation and nuclear remodeling increases the number of embryos reaching the blastocyst stage and the average cell number in the blastocysts. Inhibition of histone deacetylation might also increase the number of NT embryos with nearly perfect reprogramming (able to reach full-term development), but this increase is very modest. Our data will be important for further analysis of the epigenetic mechanisms of reprogramming, and might also help in the development of more effective animal cloning, especially in species in which experimental provisioning of early nuclear remodeling events represent significant technical challenges, e.g., rats [38]. These data might also contribute to the development of technologies that would make human therapeutic cloning clinically feasible [39], by increasing in the blastocyst rate via optimal TSA treatment, because the blastocyst stage is an important source of embryonic stem cells.
FOOTNOTES
1 Supported by grant no. 16200030 from the Ministry of Education, Culture, Sports, Science, and Technology, and a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). 
2 Correspondence. FAX: 81 7 4243 115; tsunoda{at}nara.kindai.ac.jp
Received: 14 September 2005.
First decision: 12 October 2005.
Accepted: 6 February 2006.
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