Mouse Somatic Cell Nuclear Transfer Embryos Research Articles

Reduction of H3K9 methylation by G9a inhibitors improves the development of mouse SCNT embryos

Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear transfer (SCNT). However, this strategy involves the injection of histone demethylase mRNA into embryos, which is limiting because of its invasive and labor-consuming nature. Here, we report that treatment with an inhibitor of G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved the development of mouse SCNT embryos. Intriguingly, G9ai caused an immediate reduction of H3K9me1/2, a secondary loss of H3K9me3 in SCNT embryos, and increased the birth rate of cloned pups about 5-fold (up to 3.9%). G9ai combined with the histone deacetylase inhibitor trichostatin A further improved this rate to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic genome activation. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications.

Aberrant nucleosome organization in mouse SCNT embryos revealed by ULI-MNase-seq.

SummarySomatic cell nuclear transfer (SCNT) can reprogram terminally differentiated somatic cells into totipotent embryos, but with multiple defects. The nucleosome positioning, as an important epigenetic regulator for gene expression, is largely unexplored during SCNT embryonic development. Here, we mapped genome-wide nucleosome profiles in mouse SCNT embryos using ultra-low-input MNase-seq (ULI-MNase-seq). We found that the nucleosome-depleted regions (NDRs) around promoters underwent dramatic reestablishment, which is consistent with the cell cycle. Dynamics of nucleosome position in SCNT embryos were delayed compared to fertilized embryos. Subsequently, we found that the aberrant gene expression levels in inner cell mass (ICM) were positively correlated with promoter NDRs in donor cells, which indicated that the memory of nucleosome occupancy in donor cells was a potential barrier for SCNT-mediated reprogramming. We further confirmed that the histone acetylation level of donor cells was associated with the memory of promoter NDRs. Our study provides insight into nucleosome reconfiguration during SCNT preimplantation embryonic development.

RS-1 enhanced the efficiency of somatic cell reprogramming via activation of RAD51 in somatic cell nuclear transfer

Background & Aim Reprogramming efficiency of somatic cell nuclei is very low in both somatic cell nuclear transfer (SCNT) and iPSCs technologies. Recently, it was reported that the genetic instability and DNA damage arise prior to transcriptional activity during reprogramming. Subsequently, in the absence of mitotic nuclear remodeling, DNA replication is delayed and errors are exacerbated (Egli D et al., 2017). Methods, Results & Conclusion Here, in order to find a regulator of DNA damage during reprogramming events, we observed the effect of RAD51 stimulatory compound (RS-1) supplements in DNA repair process of mouse SCNT embryos in the early embryogenesis and compared with in vitro fertilization (IVF)-derived embryos. First, Rad51 mRNA expression in both embryos of SCNT and IVF groups was observed at 1-cell, 2-cell, and 4-cell stages. In IVF group, relative Rad51 mRNA expression was markedly decreased from 1-cell to 2-cell stages and then restored in 4 cell stage. However, in SCNT group, its expression decreased from 1-cell to 2-cell stages at a similar rate with IVF group, but not recovered at 4-cell stage. Based on these results, we have observed DNA damage detected by rH2AX and DNA repair of double strand breaks by Rad51 expressions. The rH2AX expression was highly localized in the nucleus of IVF embryo when compared with SCNT groups until 10 h after reconstruction of embryos. However Rad51 was not expressed in the nucleus until 10 h after SCNT moreover, at 12h after SCNT, rH2AX showed highly dotted in nuclear in both IVF and SCNT+RS-1 group. A large number of embryos were observed Rad51 expression only in IVF and SCNT+RS-1 groups. In this study, we found different expression of some cell cycle-related genes among IVF, SCNT and SCNT+RS-1 groups by RNA-sequencing. The SCNT+RS-1 group significantly enhanced blastocyst formation rate of the cloned embryos when compared to those of the RS-1-untreated group, and reduced the rate of DNA fragmentation. Furthermore SCNT+RS-1 group improved full term clone mice rates and significantly increased the derivation efficiency of pluripotent stem cells from cloned embryos (SCNT-PSCs). Based on these results, we suggest that DNA damage and Rad51-mediated DNA repair during DNA replication could complement each other and its regulation has plays an important role in somatic cell nuclei reprogramming process.

Chromatin architecture reorganization in murine somatic cell nuclear transfer embryos

The oocyte cytoplasm can reprogram the somatic cell nucleus into a totipotent state, but with low efficiency. The spatiotemporal chromatin organization of somatic cell nuclear transfer (SCNT) embryos remains elusive. Here, we examine higher order chromatin structures of mouse SCNT embryos using a low-input Hi-C method. We find that donor cell chromatin transforms to the metaphase state rapidly after SCNT along with the dissolution of typical 3D chromatin structure. Intriguingly, the genome undergoes a mitotic metaphase-like to meiosis metaphase II-like transition following activation. Subsequently, weak chromatin compartments and topologically associating domains (TADs) emerge following metaphase exit. TADs are further removed until the 2-cell stage before being progressively reestablished. Obvious defects including stronger TAD boundaries, aberrant super-enhancer and promoter interactions are found in SCNT embryos. These defects are partially caused by inherited H3K9me3, and can be rescued by Kdm4d overexpression. These observations provide insight into chromatin architecture reorganization during SCNT embryo development.

Long non-coding RNAs potentially function synergistically in the cellular reprogramming of SCNT embryos

BackgroundLong non-coding RNAs (lncRNAs), a type of epigenetic regulator, are thought to play important roles in embryonic development in mice, and several developmental defects are associated with epigenetic modification disorders. The most dramatic epigenetic reprogramming event occurs during somatic cell nuclear transfer (SCNT) when the expression profile of a differentiated cell is abolished, and a newly embryo-specific expression profile is established. However, the molecular mechanism underlying somatic reprogramming remains unclear, and the dynamics and functions of lncRNAs in this process have not yet been illustrated, resulting in inefficient reprogramming.ResultsIn this study, 63 single-cell RNA-seq libraries were first generated and sequenced. A total of 7009 mouse polyadenylation lncRNAs (including 5204 novel lncRNAs) were obtained, and a comprehensive analysis of in vivo and SCNT mouse pre-implantation embryo lncRNAs was further performed based on our single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs were expressed in a developmental stage-specific manner during mouse early-stage embryonic development, whereas a more temporal and spatially specific expression pattern was identified in mouse SCNT embryos with changes in the state of chromatin during somatic cell reprogramming, leading to incomplete zygotic genome activation, oocyte to embryo transition and 2-cell to 4-cell transition. No obvious differences between other stages and mouse NTC or NTM embryos at the same stage were observed. Gene oncology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and weighted gene co-expression network analysis (WGCNA) of lncRNAs and their association with known protein-coding genes suggested that several lncRNAs and their associated with known protein-coding genes might be involved in mouse embryonic development and cell reprogramming.ConclusionsThis is a novel report on the expression landscapes of lncRNAs of mouse NT embryos by scRNA-seq analysis. This study will provide insight into the molecular mechanism underlying the involvement of lncRNAs in mouse pre-implantation embryonic development and epigenetic reprogramming in mammalian species after SCNT-based cloning.

Effects of Embryo Aggregation and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

To improve the cloning efficiency of somatic cell nuclear transfer (SCNT) and to establish nuclear transfer embryonic stem cells (NT-ESCs) reliably, it is necessary to produce high-quality blastocysts derived from mice SCNT embryos. Therefore, the present study aims to investigate an optimal method for mouse SCNT embryo production and NT-ESCs derivation by comparing the effects of two methods: the treatment of histone deacetylase inhibitor PXD101 after SCNT, embryo aggregation and their combination treatment. The results suggest that embryo aggregation at four-cell stage and 50 nM PXD101 treated for 10 hours during and after activation could improve both mouse SCNT embryos' development (PXD101: 40.0% vs. 18.5%; p < 0.05; aggregation: 40.2% vs. 18.5%; p < 0.05) and also enhance the isolation rate of NT-ESCs (PXD101: 38.2% vs. 12.5%; p < 0.05; aggregation: 39.0% vs. 12.5%; p < 0.05). The combination of their treatments had a higher development rate (43.6%) and significantly higher NT-ESCs isolation rate (54.7%), therefore, we concluded that the combination of these two methods (50 nM PXD101 treated for 10 hours after SCNT and then aggregated at four-cell stage) is considered as the optimal way for the in vitro development of SCNT embryo and subsequent NT-ESCs isolation in mice, providing a new approach for the practical improvement of mouse cloning techniques and opening new opportunities to improve cloning efficiencies in other species.

Effects of Trichostatin A and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with aberrant epigenetic nuclear reprogramming. It has been demonstrated that treatment with histone deacetylase inhibitors (HDACis) enhances developmental potential of SCNT embryos. Previous studies in many species revealed that treatment of SCNT embryos with trichostatin A (TSA)-an HDACi-significantly enhances the in vitro development of SCNT embryos. In this study, we compared two different SCNT protocols with TSA and investigate, for the first time, the effect of another new HDACi, PXD101 (belinostat), on in vitro development of mouse SCNT embryo. Rates of blastocyst development in mouse SCNT embryos treated with either 5 nM TSA during (6 hours) and after (4 hours) activation (39.1%) or with 50 nM PXD101 during (6 hours) and after (4 hours) activation (40.2%) were significantly higher than those of nontreated SCNT embryos (11.5%) and both treatments also significantly improved the subsequent establishment of NT-ESCs in comparison with the nontreated group (38.1% and 40.9% vs. 11.8%). In conclusion, we optimized the TSA concentration and treatment timing and, for the first time, investigated the effect of PXD101 on mouse development of SCNT embryos and establishment of NT-ESCs.

Epigenetic reprogramming, gene expression and in vitro development of porcine SCNT embryos are significantly improved by a histone deacetylase inhibitor--m-carboxycinnamic acid bishydroxamide (CBHA).

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0034-3) contains supplementary material, which is available to authorized users.

Abnormal dynamic changes in β-tubulin in somatic nuclear transfer cloned mouse embryos

The efficiency of somatic cell nuclear transfer (SCNT) cloning remains low, thus limiting the applications of this technique. In this study, we used immunochemistry and confocal microscopy to detect the microtubule component, β-tubulin, in SCNT, parthenogenetic (PA), and intracytoplasmic sperm injection (ICSI) embryos before the first mitotic division. β-Tubulin is the component subunit of microtubule, which plays critical roles in regulating localization of cellular organelles, and the growth, maturation and fertilization of oocytes. Our results demonstrated similar changes of spindle patterns in PA and ICSI embryos. The second meiotic division resumed 1 h post-treatment, and the cytoplasmic asters (CAs) disappeared. After about 4-6 h of treatment, pronuclei formed with the midbodies connecting each other. Meanwhile, the CAs reappeared and a microtubule network developed in the cytoplasm. However, SCNT embryos showed abnormal multipolar spindles, and the pseudopronuclei that contained many nucleoli existed after 6 h of SrCl2 activation. Enucleated oocytes alone did not form spindle-like structures when they were artificially activated for 6 h, indicating that somatic cell chromosomes might be necessary for spindle formation in SCNT embryos. These results demonstrated abnormal changes of β-tubulin in mouse SCNT embryos, compared with PA and ICSI embryos.

27 PSAMMAPLIN A INCREASES DEVELOPMENT AND QUALITY OF MOUSE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Among the many biological and technical factors affecting the success rate of mouse somatic cell nuclear transfer (SCNT), faulty reprogramming of the differentiated donor nucleus to a totipotent embryonic state by the recipient oocyte seems key. Accordingly, treatment of SCNT embryos with epigenetic modifiers such as valproic acid (VPA), a histone deacetylase inhibitor (HDACi), enhances cloning efficiency. Psammaplin A (PsA) is a natural and potent DNA methyltransferase inhibitor and HDACi that has never been used in nuclear reprogramming studies. The purpose of our study was to determine the effect of PsA on the development and quality of mouse SCNT embryos, and to compare it to that of VPA. To this aim, mechanically enucleated oocytes from B6CBAF1 female mice were reconstructed with cumulus cell nuclei, activated, and cultured in the presence of the epigenetic modifier. Embryos that reached the blastocyst stage were differentially stained for counting inner cell mass (ICM) and trophectoderm cells. Alternatively, 2-cell embryos were transferred to CD1 recipient females to assess full-term development. In a first set of experiments, embryos were exposed to different concentrations of PsA (5, 10, and 20 µM) or VPA (2 and 4 mM) for 1 to 2 h after reconstruction and 6 h of activation (total 8–9 h). We found that 10 µM PsA and 2 mM VPA significantly increased blastocyst rates (37.3 and 31 v. 23.3% for the control group), although no differences were found in blastocyst quality (10.4–13.6 ICM cells). In a second set of experiments, we studied the effect of treatment duration by incubating the embryos in 10 µM PsA or 2 mM VPA for 8 to 9, 16, or 24 h after reconstruction. With PsA, all treatments showed equivalent blastocyst rates (35.2–43.3%), which were significantly higher than in the control group (20%), but only treatments for 16 and 24 h yielded blastocysts with significantly higher numbers of ICM cells (16.3 and 18.5 v. 10 for the control group). With VPA, treatments for 8 to 9 h and 16 h were equivalent in terms of blastocyst rates (34.0 and 32.5%) and significantly higher than the control group, but only VPA 16 h yielded blastocysts with a significantly higher number of ICM cells (15.6). In a third set of experiments, we studied the full-term development of embryos treated with 10 µM PsA or 2 mM VPA for 16 h and we found that both treatments, but especially the PsA treatment, resulted in higher birth rates than those obtained in the control group, although the differences were not statistically significant (1.79 and 0.86 v. 0.46%). Finally, when the actin polymerization inhibitor latrunculin A was used instead of cytochalasin B in the SCNT protocol during oocyte micromanipulation and activation, we obtained a 3-fold increase in the birth rate of embryos treated with PsA (5.29%). In conclusion, PsA enhances development and quality of mouse SCNT embryos, to a greater extent than VPA, and when combined with the use of latrunculin A instead of cytochalasin B, it results in an 11.5-fold increase in full-term development. Support from MEC AGL-2011-23784, 2009-SGR-282, and PIF-UAB Fellowships is acknowledged.

70 FIBROBLAST GROWTH FACTOR 4 PROMOTES THE DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN MICE

Somatic cell nuclear transfer (SCNT) embryos can develop during the preimplantation period; however, most of these die after implantation period. A transcription factor, Cdx2, promotes differentiation of extraembryonic tissues and appears to be involved in the segregation of inner cell mass (ICM) and trophectoderm (TE) in preimplantation embryos. So far, we have demonstrated that the expression of Cdx2 in mouse SCNT embryos is delayed and its expression level is significantly lower than that in intracytoplasmic sperm injection (ICSI) embryos. Moreover, the ectopic expression of Oct-3/4 was observed in the TE tissues of SCNT blastocysts, but not in ICSI blastocysts. Fibroblast growth factor (FGF) receptor 2 (FGFR2) is specifically expressed in 8-cell to morula-stage embryos and trophectoderm (TE) and is essential for implantation; however, FGFR2 expression in SCNT embryos significantly decreases compared with IVF embryos. Therefore, it is likely that abnormality of differentiation that is controlled in development of pre-implantation in SCNT embryos leads to a rapid decrease of subsequent developmental ability. Then, we investigated the effects of FGF4 on development of SCNT embryos. Mouse SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998). B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. Data were analyzed by Student’s t-test. First, the timing to start adding FGF4 was decided by FGFR2 expression time about 54 h after cell injection and treated for 3, 6, 12, 24, and 42 h thereafter. In the case of FGF4 concentration at 25 ng mL-1 with treating time of 6 h from the 4- to 8-cell stages, SCNT embryos significantly promoted the development to morula and blastocyst stages (91 and 45%, respectively) compared with IVF embryos (80 and 30%, respectively; P &lt; 0.05). However, longer treatment of 42 h with FGF4 made their morphology considerably worse. Then, concentrations of FGF4 at 5, 25, 50, 250, and 500 ng mL-1 with treating time of 6 h was examined. In case of FGF4 concentration at 25 and 50 ng mL-1, SCNT embryos significantly promoted the development to morula and blastocyst stages (P &lt; 0.05). Immunohistochemical analysis showed segregation of the expression of Oct-3/4 and Cdx2 in ICM and TE, respectively, in FGF4-treated SCNT embryos, unlike in the case of nontreated SCNT embryos, which showed an ectopic expression of Oct-3/4 in TE tissues. Furthermore, after the transplantation of SCNT embryos treated with FGF4 at 50 ng mL-1 and the treating time of 6 h to recipient mice, most of the transferred embryos implanted and cloned mice were successfully produced as well as nontreated SCNT embryos. Therefore, FGF4 facilitates the development of SCNT embryos especially to the morula and blastocyst stages. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

46 EXPRESSION OF TRANSCRIPTION FACTORS SPECIFIC TO THE TROPHOBLAST LINEAGE IN MOUSE SOMATIC NUCLEAR TRANSFER EMBRYOS

Somatic cell nuclear transfer (SCNT) embryos can develop at relatively high rates during the preimplantation period; however, most of these fail after implantation. Development of extraembryonic tissue is indispensable for normal embryonic development. Hence, an abnormality of trophoblast development might be a significant factor in post-implantation lethality of SCNT embryos. A transcription factor, caudal-related homeobox 2 (Cdx2), appears to be involved in the segregation of ICM and trophectoderm (TE) in preimplantation embryos (Niwa et al. 2005 Cell 123, 917–929). Both Cdx2 and Oct3/4 are expressed in all cells at the morula stage, and then Cdx2 expression becomes restricted to the TE and Oct3/4 to the ICM as the blastocyst develops. Mouse embryos deficient in Cdx2 are able to develop to normal blastocysts but die soon after implantation, probably because of defects in the TE lineage. Moreover, dysplasia of the spongiotrophoblast layer might attribute to an abnormality of Tpbpa expression in mouse SCNT embryos (Wakisaka-Saito et al. 2006 Biochem. Biophys. Res. Commun. 349, 106–114). In this study, we examined the expression profiles of transcription factors implicated in trophoblast development in mouse SCNT embryos and intracytoplasmic sperm injection (ICSI) embryos by immunohistochemistry and real-time PCR analysis. SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998 Nature 394, 369–374). In brief, B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. After nuclear transfer, the oocytes were activated and cultured in KSOM to the morula and blastocyst stages. Immunohistochemical analysis demonstrated that in ICSI embryos Cdx2 was only partially expressed at the 8-cell stage but completely in early morulae. In contrast, in SCNT embryos, it was absent at the 8-cell stage and appeared partially at the early morula stage. Thereafter, Cdx2 expression became restricted to the TE cells in both the ICSI and the SCNT blastocysts. However, ectopic expression of Oct3/4 was observed in the TE cells of SCNT, but not in ICSI blastocysts. Real-time PCR analysis showed that at the 8-cell stage, Cdx2 was expressed in ICSI but not in SCNT embryos. In addition, the expression level of Cdx2 in SCNT embryos at the blastocyst stage was only half that in ICSI embryos (P &lt; 0.05). However, there was no significant difference in expression level of Oct3/4 between ICSI and SCNT embryos. Eomesodermin (Eomes) is also implicated in trophoblast development and its expression depends on Cdx2, BMP4, and FGF4. In SCNT embryos, the expression level of Eomes was also only half that in ICSI embryos. These results indicate that the delayed expression of Cdx2 in SCNT embryos may lead to the ectopic expression of Oct3/4 in blastocysts and, along with the limited expression of Cdx2 and Eomes, may contribute to disorders in the function of the trophoblast lineage for normal placental development. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

Tough beginnings: Alterations in the transcriptome of cloned embryos during the first two cell cycles

Cloned embryos produced by somatic cell nuclear transfer (SCNT) display a plethora of phenotypic characteristics that make them different from fertilized embryos, indicating defects in the process of nuclear reprogramming by the recipient ooplasm. To elucidate the extent and timing of nuclear reprogramming, we used microarrays to analyze the transcriptome of mouse SCNT embryos during the first two cell cycles. We identified a large number of genes mis-expressed in SCNT embryos. We found that genes involved in transcription and regulation of transcription are prominent among affected genes, and thus may be particularly difficult to reprogram, and these likely cause a ripple effect that alters the transcriptome of many other functions, including oxidative phosphorylation, transport across membrane, and mRNA transport and processing. Interestingly, we also uncovered widespread alterations in the maternal (i.e., non-transcribed) mRNA population of SCNT embryos. We conclude that gene expression in early SCNT embryos is grossly abnormal, and that this is at least in part the result of incomplete reprogramming of transcription factor genes.

50 DNA METHYLATION PROFILES OF UPSTREAM ELEMENTS OF Oct-3/4 GENE IN IN VITRO FERTILIZATION (IVF) AND SOMATIC CELL NUCLEAR-TRANSFERRED (SCNT) EMBRYOS

Cloning by adult somatic cell nuclear transfer (SCNT) has proven to be successful for the production of clones from many species (Keith 2004 Cytogenet. Genome Res. 105, 285). However, somatic cloning is currently highly inefficient. One of the reasons for this is that SCNT is believed to be associated with epigenetic errors including abnormal DNA methylation of the reconstructed embryo. The Oct-3/4 gene, a member of the POU transcription factor family, is expressed throughout the pre-implantation embryo. Abnormal expression of the Oct-3/4 gene in the nuclear-transferred embryo is either directly or indirectly caused by nuclear transfer and is suggested to be indicative of a general failure to reset the genetic program (Boiani et al. 2002 Genes Dev. 16, 1209). In this study, we investigated the DNA methylation profiles of the Oct-3/4 gene in the genome of SCNT embryos, using bisulfite sequencing analysis. Then, we observed the detailed subcellular localization of Oct-3/4 proteins in SCNT embryos using immunocytochemical (ICC) analysis. Nuclear transfer of cumulus cell nuclei was carried out as previously described (Wakayama et al. 1998 Nature 394, 369). After nuclear transfer, embryos were subsequently cultured in KSOM media to the morula and blastocyst stages. We compared the methylation profiles of 3 transcriptional control elements (distal enhancer, DE; proximal enhancer, PE; and promoter) of the upstream region of the Oct-3/4 gene with the genome of in vitro fertilization (IVF) and SCNT embryos. The methylation rate of CpG sites in the DE and promoter regions of both IVF and SCNT embryos was low at both the morula and the blastocyst stages. What&amp;apos;s interesting is that there was a significant difference in the methylation level on CpG sites in the PE element between IVF and SCNT embryos. At the morula stage, the methylation level on CpG sites in the PE element was very low in the IVF embryo and moderately high in the SCNT embryo (0.9&amp;percnt; and 26.3&amp;percnt;). Conversely, at the blastocyst stage, CpG sites in the PE element showed high methylation in the IVF embryo and low methylation in the SCNT embryo (55.2&amp;percnt; and 10.5&amp;percnt;). CpG sites in the PE element were lightly methylated (3.0&amp;percnt;) in the inner cell mass (ICM) of the IVF embryo. This means that the main portion of methylation in the IVF blastocyst embryo occurred at the trophectoderm (TE). On the other hand, in ICM of the SCNT embryo, the methylation level of each embryonic cell was almost the same in the whole blastocyst embryo (9.8&amp;percnt; and 10.5&amp;percnt;). As a result, it is highly possible that the CpG sites in the PE element of ICM were methylated as in the TE. ICC analysis revealed that some SCNT embryos showed aberrant Oct-3/4 expression in the TE. These results indicate that the methylation of CpG sites in the Oct-3/4 PE element may be related to expression of Oct-3/4 in the mouse IVF and SCNT embryos. These differences in methylation level between IVF and SCNT embryos were reflected as abnormal expressions of Oct-3/4 on SCNT embryos. This study was supported by the 21st COE Program of MEST. M.K. is a JSPS Research Fellow and supported by Grant-in Aid for Scientific Research (No. 1751132) of JSPS.

48 EFFECTS OF TRICHOSTATIN A ON DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Although cloning by somatic cell nuclear transfer (SCNT) has been achieved in various mammalian species, its efficiency has been very low (Han et al. 2003 Theriogenology 59, 33–44). Successful cloning requires conversion from differentiated donor nuclei to embryonic nuclei after transfer of the somatic nuclei into enucleated oocytes. Reprogramming of the transferred somatic nuclei must be completed by the time when normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, both full-term development and pre-implantation development of mouse SCNT embryos were significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The objective of this study was to investigate the effects of TSA on the development of bovine SCNT embryos. Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. The cells were electro-fused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF medium until 168 h post-activation (hpa). The NT embryos were exposed to 0 (control), 5, 50, and 500 nM TSA from the start of activation to 48 hpa. Experiments were repeated 3 times, and the data were analyzed with Fisher's PLSD test following ANOVA. The cleavage rates were the same among the groups (60 to 80&amp;percnt;; P &amp;gt;0.05). However, the blastocyst rate of NT embryos treated with 50 nM TSA was higher than that of control embryos (40&amp;percnt; vs. 19&amp;percnt;, respectively; P &amp;lt; 0.05). On the other hand, the blastocyst rate was lower with 500 nM TSA than with 5 or 50 nM TSA (7&amp;percnt; vs. 33&amp;percnt; or 40&amp;percnt;; P &amp;lt; 0.05). These data suggest that proper TSA treatment after somatic cloning improves the rate of development of bovine cloned embryos to the blastocyst stage. Further research is needed to examine whether NT embryos derived from different cell lines or types have similar susceptibility to TSA.