Development Of Bovine Somatic Cell Nuclear Transfer Embryos Research Articles

TET3 overexpression facilitates DNA reprogramming and early development of bovine SCNT embryos.

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

SiRNA inhibition and not chemical inhibition of Suv39h1/2 enhances pre-implantation embryonic development of bovine somatic cell nuclear transfer embryos.

The efficiency of somatic cell nuclear transfer (SCNT) is low due to the strong resistance of somatic donor cells to epigenetic reprogramming. Many epigenetic drugs targeting DNA methylation and histone acetylation have been used in attempts to improve the in vitro and in vivo development of SCNT embryos. H3K9me3 has been shown to be an important reprogramming barrier for generating induced pluripotent stem cells (iPSCs) and SCNT embryos in mice and humans. In this study, we examined the effects of selective siRNA and chemical inhibition of H3K9me3 in somatic donor cells on the in vitro development of bovine SCNT embryos. Chaetocin, an inhibitor of SUV39H1/H2, was supplemented during the culture of donor cells. In addition, the siRNA knockdown of SUV39H1/H2 was performed in the donor cells. The effects of chaetocin and siSUV39H1/H2 on H3K9me3 and H3K9ac were quantified using flow cytometry. Furthermore, we assessed chaetocin treatment and SUV39H1/H2 knockdown on the blastocyst formation rate. Both chaetocin and siSUV39H1/H2 significantly reduced and elevated the relative intensity level of H3K9me3 and H3K9ac in treated fibroblast cells, respectively. siSUV39H1/H2 transfection, but not chaetocin treatment, improved the in vitro development of SCNT embryos. Moreover, siSUV39H1/H2 altered the expression profile of the selected genes in the derived blastocysts, similar to those derived from in vitro fertilization (IVF). In conclusion, our results demonstrated H3K9me3 as an epigenetic barrier in the reprogramming process mediated by SCNT in bovine species, a finding which supports the role of H3K9me3 as a reprogramming barrier in mammalian species. Our findings provide a promising approach for improving the efficiency of mammalian cloning for agricultural and biomedical purposes.

Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos

Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.

Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development

Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) is a promising technology, but its application is hampered by its low efficiency. Hence, the majority of SCNT embryos fail to develop to term. In this study, the antioxidant melatonin reduced apoptosis and reactive oxygen species (ROS) in bovine SCNT embryos. It also increased cell number, inner cell mass (ICM) cell numbers, and the ratio of ICM to total cells while improving the development of bovine SCNT embryos in vitro and in vivo. Gene expression analysis showed that melatonin suppressed the expression of the pro-apoptotic genes p53 and Bax and stimulated the expression of the antioxidant genes SOD1 and Gpx4, the anti-apoptotic gene BCL2L1, and the pluripotency-related gene SOX2 in SCNT blastocysts. We also analyzed the epigenetic modifications in bovine in vitro fertilization, melatonin-treated, and untreated SCNT embryos. The global H3K9ac levels of melatonin-treated SCNT embryos at the four-cell stage were higher than those of the untreated SCNT embryos. We conclude that exogenous melatonin affects the expression of genes related to apoptosis, antioxidant function, and development. Moreover, melatonin reduced apoptosis and ROS in bovine SCNT embryos and enhanced blastocyst quality, thereby ultimately improving bovine cloning efficiency.

Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, p<0.05), compact morulae formation (60.83 vs. 51.30%, p<0.05), and the blastomere apoptosis index (3.70 ± 1.41 vs. 4.43% ± 1.65, p<0.05) of bovine SCNT embryos. However, vitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, p<0.05) on day 7 and the hatching blastocysts formation rate on day 9 (26.51 vs. 50.65%, p<0.05) compared with that of the untreated group. Vitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

Effects of 3-hydroxyflavone on the cellular and molecular characteristics of bovine embryos produced by somatic-cell nuclear transfer

This study aimed to investigate the effects of 3-hydroxyflavone, a natural antioxidant pigment enriched in vegetables, on the developmental cellular and molecular characteristics of bovine somatic-cell nuclear transfer (SCNT) embryos. There were no significant differences in the cleavage rate at 48 hr of culture or in the inner cell mass (ICM)-to-trophectoderm (TE) ratio between 3-hydroxyflavone addition and untreated (control) groups (P > 0.05). 3-hydroxyflavone (20 µM) did, however, increase the cleavage rate at 24 hr of culture and the blastocyst-formation rate on Days 6 and 7 (P < 0.05); decrease the levels of intracellular reactive oxygen species in two-, four-, and eight-cell stage embryos (P < 0.05); increase H3K9ac levels in two- and four-cell stages (P < 0.05); increase the total cell number; and decrease the apoptosis index in Day-7 blastocysts. Furthermore, the addition of 3-hydroxyflavone resulted in lower expression of the stress-related gene HSP70.1 and pro-apoptotic gene BAX, as well as higher expression of the anti-apoptotic gene BCL-xL and pluripotency-related genes OCT4 and SOX2 in Day-7 blastocysts produced by SCNT (P < 0.05). The addition of 3-hydroxyflavone during in vitro culture thus exerted beneficial effects on preimplantation development of bovine SCNT embryos both at the cellular and molecular levels.

A Cathepsin B Inhibitor, E-64, Improves the Preimplantation Development of Bovine Somatic Cell Nuclear Transfer Embryos

Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.

Valproic acid enhances early development of bovine somatic cell nuclear transfer embryos by alleviating endoplasmic reticulum stress

Despite the positive roles of histone deacetylase inhibitors in somatic cell nuclear transfer (SCNT), few studies have evaluated valproic acid (VPA) and its associated developmental events. Thus, the present study was conducted to elucidate the effect of VPA on the early development of bovine SCNT embryos and the underlying mechanisms of action. The histone acetylation level of SCNT embryos was successfully restored by VPA, with optimal results obtained by treatment with 3mM VPA for 24h. Importantly, the increases in blastocyst formation rate and inner cell mass and trophectoderm cell numbers were not different between the VPA and trichostatin A treatment groups, whereas cell survival was notably improved by VPA, indicating the improvement of developmental competence of SCNT embryos by VPA. Interestingly, VPA markedly reduced the transcript levels of endoplasmic reticulum (ER) stress markers, including sXBP-1 and CHOP. In contrast, the levels of GRP78/BiP, an ER stress-alleviating transcript, were significantly increased by VPA. Furthermore, VPA greatly reduced cell apoptosis in SCNT blastocysts, which was further evidenced by the increased levels of the anti-apoptotic transcript Bcl-xL and decreased level of the pro-apoptotic transcript Bax. Collectively, these results suggest that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage.

Defined media optimization for in vitro culture of bovine somatic cell nuclear transfer (SCNT) embryos

The objective was to establish an efficient defined culture medium for bovine somatic cell nuclear transfer (SCNT) embryos. In this study, modified synthetic oviductal fluid (mSOF) without bovine serum albumin (BSA) was used as the basic culture medium (BCM), whereas the control medium was BCM with BSA. In Experiment 1, adding polyvinyl alcohol (PVA) to BCM supported development of SCNT embryos to blastocyst stage, but blastocyst formation rate and blastocyst cell number were both lower (P < 0.05) compared to the undefined group (6.1 vs. 32.6% and 67.3 ± 3.4 vs. 109.3 ± 4.5, respectively). In Experiment 2, myo-inositol, a combination of insulin, transferrin and selenium (ITS), and epidermal growth factor (EGF) were added separately to PVA-supplemented BCM. The blastocyst formation rate and blastocyst cell number of those three groups were dramatically improved compared with that of PVA-supplemented group in Experiment 1 (18.5, 23.0, 24.1 vs. 6.1% and 82.7 ± 2.0, 84.3 ± 4.2, 95.3 ± 3.8 vs. 67.3 ± 3.4, respectively, P < 0.05), but were still lower compared with that of undefined group (33.7% and 113.8 ± 3.4, P < 0.05). In Experiment 3, when a combination of myo-inositol, ITS and EGF were added to PVA-supplemented BCM, blastocyst formation rate and blastocyst cell number were similar to that of undefined group (30.4 vs. 31.1% and 109.3 ± 4.4 vs. 112.0 ± 3.6, P > 0.05). In Experiment 4, when blastocysts were cryopreserved and subsequently thawed, there were no significant differences between the optimized defined group (Experiment 3) and undefined group in survival rate and 24 and 48 h hatching blastocyst rates. Furthermore, there were no significant differences in expression levels of H19, HSP70 and BAX in blastocysts derived from optimized defined medium and undefined medium, although the relative expression abundance of IGF-2 was significantly decreased in the former. In conclusion, a defined culture medium containing PVA, myo-inositol, ITS, and EGF supported in vitro development of bovine SCNT embryos.

Valproic Acid Improves theIn VitroDevelopment Competence of Bovine Somatic Cell Nuclear Transfer Embryos

The present study was carried out to examine the effect of valproic acid (VPA), an important histone deacetylase inhibitor, on the in vitro development and expression of the epigenetic marker histone H3 lysine 9 (H3K9ac) in bovine somatic cell nuclear transfer (SCNT) embryos. We found that treatment with 4 mM VPA for 24 h could significantly improve the development of bovine SCNT embryos. Compared with the no-treatment group, the cleavage rate was higher (69.79 ± 0.99% vs. 65.11 ± 1.02%, p<0.05), as was the blastocyst rate (39.99 ± 1.29% vs. 34.87 ± 1.74%, p<0.05). Moreover, the rate of apoptosis (1.91 ± 0.48% vs. 5.67 ± 0.40%, p<0.05) in blastocysts was greatly reduced after VPA treatment. Valproic acid treatment also increased the immunofluorescent signal for H3K9ac in SCNT embryos in a pattern similar to that of in vitro fertilized (IVF) embryos. In conclusion, we demonstrated that VPA can significantly improve the in vitro developmental competence and enhance the nuclear reprogramming of bovine SCNT embryos.

High expression of Mfn1 promotes early development of bovine SCNT embryos: Improvement of mitochondrial membrane potential and oxidative metabolism

Mitofusin 1 (Mfn1) is the main mediator of mitochondrial fusion and homeostasis. To determine whether increased Mfn1expression level could promote the fusion of heteroplasmic mitochondria and development of somatic cell nuclear transfer (SCNT) embryos. Embryos were constructed using bovine oocytes as recipient cytoplasm, and Holstein cow fetal fibroblasts with different expression levels of Mfn1 gene as donor nuclei. Mitochondrial membrane potential, ATP and H2O2 generation, as well as the expression level of Mfn1 were detected in different development stages. The results showed that high level of Mfn1 expression significantly improved the embryo development rates by increasing ATP level and Δψm, while reducing H2O2 generation. This study suggests that overexpression of Mfn1 could promote the early development of bovine SCNT embryos via improving oxidative phosphorylation.

Oxamflatin Significantly Improves Nuclear Reprogramming, Blastocyst Quality, and In Vitro Development of Bovine SCNT Embryos

Aberrant epigenetic nuclear reprogramming results in low somatic cloning efficiency. Altering epigenetic status by applying histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos. The present study was carried out to examine the effects of Oxamflatin, a novel HDACi, on the nuclear reprogramming and development of bovine SCNT embryos in vitro. We found that Oxamflatin modified the acetylation status on H3K9 and H3K18, increased total and inner cell mass (ICM) cell numbers and the ratio of ICM∶trophectoderm (TE) cells, reduced the rate of apoptosis in SCNT blastocysts, and significantly enhanced the development of bovine SCNT embryos in vitro. Furthermore, Oxamflatin treatment suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-XL and the pluripotency-related genes OCT4 and SOX2 in SCNT blastocysts. Additionally, the treatment also reduced the DNA methylation level of satellite I in SCNT blastocysts. In conclusion, Oxamflatin modifies epigenetic status and gene expression, increases blastocyst quality, and subsequently enhances the nuclear reprogramming and developmental potential of SCNT embryos.

54 THE EFFECT OF TREATMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS WITH VALPROIC ACID ON THEIR SUBSEQUENT IN VITRO DEVELOPMENT

Recently, trichostatin A (TSA) and some other histone deacetylase inhibitors (HDACi) were reported to enhance the development of mouse somatic cell nuclear transfer (SCNT) embryos. Previously, we have succeeded in improving in vitro development of bovine SCNT embryos significantly with inhibitors of class I, IIa, and IIb HDACs such as TSA or Scriptaid (SCR). In this study, we examined the effect of valproic acid (VPA), a selective inhibitor of class I and IIa HDACi, on in vitro development of bovine SCNT embryos. 3 cell lines (adult male, adult female, and fetal female fibroblast cells) were used as donor cells. SCNT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Reconstructed embryos were chemically activated by treatments with 10 μM calcium ionophore for 5 min and 10 μg mL–1 cycloheximide for 5 h. Then SCNT embryos were cultured in serum-free medium for 7 days. Embryo development data were analyzed by chi-square test. Differences were considered significant at P &lt; 0.05. Each experiment was replicated at least 3 times. At first, to determine the suitable concentration of VPA, bovine SCNT embryos derived from adult female fibroblast were treated with various concentrations of VPA (0, 0.01, 0.1, 1, 10 mM) for 20 h from the start of chemical activation and embryo development was examined. There was no difference in blastocyst formation rates based on the number of cleaved embryos among the groups [46% (166/357), 53% (49/92), 46% (51/112), 55% (56/102), and 44% (44/100), respectively], and the cell numbers of blastocysts were also similar. Next, we examined the effect of duration of VPA treatment on development of SCNT embryos obtained from adult male, female, and fetal female fibroblast cells. Based on the results of previous experiment, bovine SCNT embryos were treated with 1 mM VPA for 20 h or 40 h and those without treatment were used as control. Neither 20 h nor 40 h VPA treatment affected blastocyst formation rates of SCNT embryos from adult male, control: 38% (41/107), 20 h: 45% (47/104), 40 h: 46% (39/85); adult female, control: 47% (166/357), 20 h: 55% (56/102), and 40 h: 36% (12/33); and fetal female fibroblast cells, control: 7% (4/58), 20 h: 13% (7/54), and 40 h: 16% (8/51). In the present study, treatment of bovine SCNT embryos with VPA did not improve in vitro development significantly. Comparing these results with our previous results on TSA or SCR treatment, inhibition of HDAC class IIb may be a key factor to improve development of bovine SCNT embryos.

Trichostatin A Promotes the Development of Bovine Somatic Cell Nuclear Transfer Embryos

We studied the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on the development of bovine somatic cell nuclear transfer (SCNT) embryos by investigating (1) the optimal concentration and treatment time of TSA for development of bovine SCNT embryos, (2) the status of histone acetylation in TSA-treated and control SCNT embryos and (3) the expression of histone acetylation- and deacetylation-related genes in TSA-treated and control SCNT embryos. We observed that 50 nM TSA-treatment for 20 h following fusion resulted in more efficient in vitro development of bovine SCNT embryos to the blastocyst stage. In regard to histone H4K5 acetylation, half of the control SCNT embryos faintly displayed histone H4K5 signals 30 min after electrofusion, while most of the TSA-treated SCNT embryos displayed histone H4K5 signals within 30 min after electrofusion. Furthermore, the expressions of HDAC1 and HDAC2 in the blastocysts were significantly lower (P<0.05) in the TSA-treated SCNT than in the control SCNT. However, the expression of GCN5 and HAT1 did not differ between the TSA-treated and control SCNT. In conclusion, we demonstrated that TSA-treatment after SCNT in bovine embryos can dramatically improve the practical applications of current cloning techniques.

47 EFFECT OF THE TYPE OF CYTOPLASTS, SOURCE OF KARYOPLASTS, AND ACTIVATION PROCESS ON IN VITRO DEVELOPMENT OF BOVINE CLONE EMBRYOS

As the recipient cytoplast plays a key role in nuclear reprogramming after somatic cell nuclear transfer (SCNT), the aim of this study was to compare the type of cytoplast/karyoplast [metaphase II (MII) oocyte, early zygote, somatic cells] and the chemical (CA) or sperm-mediated/spontaneous activation (SA) on in vitro development of bovine SCNT embryos produced by handmade cloning (HMC). After 17 h of in vitro maturation, a group of cumulus–oocyte complexes (COCs, n = 945) was manually bisected following zona removal and segregated as enucleated (MII hemi-Cyt) or non-enucleated (MII hemi-Kar). Another group of COCs was in vitro-fertilized, and, 4 h after the onset of IVF, zona-free zygotes with 2 polar bodies (n = 490) were manually bisected under fluorescent light to obtain IVF hemi-Cyt and IVF hemi-Kar. A somatic cell (SC) culture from an adult cow was used for HMC procedures (SC Kar). In 5 replications, experimental groups were composed of: zona-intact MII oocytes (parthenote control, PG); zona-intact zygotes (IVF control); MII Cyt + MII Cyt + SC Kar (SCNT control); IVF Cyt + MII Cyt + SC Kar (G1); MII Cyt + IVF Kar (G2); IVF Cyt + IVF Kar (G3); IVF Cyt + IVF Cyt + SC Kar (G4); and MII Cyt + MII Kar (G5). Following reconstruction and electrofusion, groups G1 to G5 were further divided into 2 sub-groups each, 1 being chemically activated (ionomycin/6-DMAP) along with the control groups PG and SCNT, whereas the others were cultured to verify sperm-mediated (G1 to G4) or spontaneous (G5) activation. Embryos were in vitro-cultured in the WOW system for 7 days. Cleavage (Day 2) and blastocyst (Day 7) rates were compared by the chi-square and Fisher tests, respectively. Cleavage rates in G1-SA, G2-SA, and G3-SA were lower than in their CA counterparts, which were similar to controls (Table 1). Such decrease in cleavage in G1-SA and G2-SA may be caused by the manipulation process rather than by sperm-mediation, since the observed rates were very similar to the G5-SA group. Cleavage in G3 and G4 were also similar to controls, most likely due to the fusion of 2 sperm-activated IVF hemi-Cyt. Blastocyst rates were generally higher in CA than in SA sub-groups except for G4, for which SA benefited from 2 sperm-activated cytoplasts. The lower blastocyst yield in SA sub-groups may reflect at least 2 possible mechanisms: an increased level of heteroplasmy (G1 and G2), potentially caused by an insufficient sperm-activated IVF hemi-Cyt or by a blocking effect imposed by the M-phase-derived hemi-Cyt, and/or a disruption in karyokinetic events caused by the manipulation in sperm-activated IVF hemi-Kar (G2 and G3). In G4, both mechanisms were probably attenuated by the use of 2 sperm-activated IVF hemi-Cyt and a SC-kar, analogous to conditions in the SCNT and G5 groups. Table 1.Effect of cytoplast type and activation process on in vitro development of bovine SCNT embryos This study was supported by a grant from CAPES/Brazil.

37 EFFECTS OF TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Faulty epigenetic reprogramming is a likely major cause of the low success rate observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been reported that treatment of reconstructed mouse embryos with the potent histone deacetylase inhibitor, trichostatin A (TSA), results in significantly increased developmental capacity of SCNT preimplantation embryos and live offspring (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 240, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089; Kishigami et al. 2006 J. Reprod. Dev. 53, 165–170). Studies investigating similar reprogramming capabilities of TSA in bovine SCNT embryos report conflicting results (Akagi et al. 2007 Reprod. Fertil. Dev. 19, 24 abst; Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 48 abst). In this study, the effects of TSA treatment on in vitro development of bovine SCNT embryos were examined. Bovine fetal fibroblasts were cultured under contact inhibition for 2 to 5 days and used as donor cells for SCNT. Oocytes were aspirated from abattoir-derived ovaries, and matured in vitro for 18 h prior to enucleation. Reconstructed SCNT couplets were electrofused, and then activated 24 h post-maturation using 5 µm ionomycin followed by 2 mm dimethylaminopurine (DMAP) for 4 h. SCNT embryos were subjected to 0 (control; C-NT) or 50 nm TSA for 13 h post-ionomycin (hpi) TSAa-NT) or 13 hpi + 6 h starting from 40 hpi (TSAb-NT). IVF embryos were produced as an additional control. All embryos were cultured in KSOM supplemented with 3 mg mL–1 BSA for 7.5 days, with 5% FBS added on Day 3. Experiments were repeated 3 or 7 times, and data were analyzed a -way ANOVA procedure. Developmental rates to the blastocyst stage and total cell number of blastocysts were determined. Total cell numbers were determined by fixing blastocysts in 4% paraformaldehyde, and staining with bisbenzimide 33342, followed by microslide mounting and visualization using an epifluorescence microscope. No difference was observed in cleavage rates among the four treatment groups, C-NT, TSAa-NT, TSAb-NT, and IVF, with the rates being 66%, 75%, 73.1%, and 82.3%, respectively (P = 0.33); nor was any improvement seen in the rate of blastocyst development of TSAa-NT or TSAb-NT over C-NT embryos: 36%, 40.2%, and 30.2%, respectively (P = 0.22). Furthermore, there was no significant difference in mean total cell number of blastocysts among treatment groups: C-NT, 120.2; TSAa-NT, 124.2; TSAb-NT, 129.3; and IVF, 141.1 (P = 0.29). These results suggest that 50 nm TSA treatment immediately following activation does not affect the development of bovine SCNT preimplantation embryos.

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.