46 EFFECT OF SHORT-TERM TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 123
Author(s):  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
S. G. Hong ◽  
J. T. Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Short Term ◽  
Total Cell Number

Epigenetic reprogramming such as acetylation in somatic cell nuclear transfer (SCNT) has been known as one of problems in cloned embryos. For resolving this acetylation reprogramming, many investigators recently have reported the effect of long-term culture of post-activated SCNT embryos using trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). The objective of this study is to investigate the effect of short-term TSA treatment on in vitro developmental ability and the quality of bovine SCNT embryos. Immature oocytes were aspirated from abattoir-derived ovaries, matured in vitro for 22 h, and enucleated. A bovine fetal fibroblast was placed into the enucleated oocyte and fused by electrical stimulation. The fused couplets were activated by 4-min incubation in 10 μm ionomycin, followed by 4 h of culture in 1.9 mm 6-dimethylaminopurine with or without TSA (0, 50, or 100 nm). The SCNT embryos were subsequently cultured in modified synthetic oviduct fluid medium for 8 days. Developmental competence was assessed by blastocyst formation and total cell number. Total cell numbers were determined by staining with bisbenzimide 33342. As results, developmental competence to blastocysts was higher in 100 nm than control (36.7 v. 27.9%, P < 0.05). In blastocyst hatching rate, TSA 100 nm group (19.5%) at 8 days showed an increased pattern as opposed to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). No significant differences in two cell and morula stage were observed among treatment groups. In terms of development to hatching stage of blastocysts, TSA 100 nm group (19.5%) at 8 days has a significant effect compared to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). Total cell number of blastocysts derived from TSA 100 nm was significantly higher (P < 0.05) than that in TSA 50 nm (116 v. 100), whereas there was not significant difference between control and TSA 100 nm. In conclusion, short-term culture with high concentration of TSA improved the blastocysts formation however total cell number of blastocysts showed contradictory result. The epigenetic modification by TSA treatment on bovine SCNT needs further investigation. This study was financially supported by KOSEF (grant # M10625030005-08N250300510) and the Korean MEST, through the BK21 program for Veterinary Science.

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

2008 ◽  
Vol 20 (1) ◽  
pp. 99 ◽  
Author(s):  
A. E. Iager ◽  
Z. Beyhan ◽  
P. J. Ross ◽  
N. P. Ragina ◽  
K. Cunniff ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Preimplantation Embryos ◽  
Total Cell Number ◽  
Treatment Groups

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.

158 IN VITRO DEVELOPMENT OF WOOD BISON (BISON BISON ATHABASCAE) EMBRYOS BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 178 ◽  
Author(s):  
B. M. Kumar ◽  
E. St. John ◽  
P. M. Mackie ◽  
W. A. King ◽  
G. F. Mastromonaco
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Total Cell ◽  
Bison Bison ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Wood Bison

Wood bison (Bison bison athabascae) are currently classified as threatened in Canada. Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for embryo production in non-domestic species in which access to gametes is limited. Unlike fertilization, SCNT allows preservation of the entire genome, thus avoiding dilution of valuable alleles, an important factor for the preservation of genetic diversity. The present study compared the developmental competence of iSCNT embryos reconstructed from adult female wood bison ear fibroblasts (bison NT) with development of embryos reconstructed from adult female cattle ear fibroblasts (cattle NT). Domestic cattle (Bos taurus) oocytes were used as recipient ooplasm for both donor cell types. In vitro fertilized (IVF) and parthenogenetic (PA) cattle embryos were used as controls. Fibroblast cultures at passages 3 to 5 confluent for 5 days were used for SCNT. Mature oocytes were enucleated, reconstructed by transfer of donor cells, and fused with an electrical stimulus of 1.5 kV cm–1 for 40 μs in 0.28 m mannitol containing 100 μm CaCl2 and MgCl2. Oocytes for parthenogenesis and following reconstruction were activated for 5 min in 5 μm ionomycin followed by 5 h in 10 μg mL–1 cycloheximide. Embryos produced by IVF, PA, and SCNT were cultured in modified synthetic oviductal fluid medium at 38.5°C in 5% CO2, 5% O2, 90% N2. Cleavage, blastocyst development to day 8, apoptosis (TUNEL assay, Roche Diagnostics, IN, USA), and total cell number were evaluated. Statistical analyses were carried out using one-way ANOVA, followed by Tukey post hoc analysis or the equivalent nonparametrical Kruskal-Wallis test. Cleavage rate was significantly (P < 0.05) higher in the IVF group than in all other groups (86.9 ± 2.9% v. 71.6 ± 4.5% to 78.1 ± 5.1%). Blastocyst rates, expressed as a percentage of cleaved embryos, were similar among all treatment groups (33.4 ± 3.3% to 39.8 ± 5.7%) except for bison NT which had significantly (P < 0.05) lower development to blastocyst (19.2 ± 5.5%). The percentages of TUNEL-positive cells among PA embryos (6.6 ± 1.5%) and bison NT embryos (6.7 ± 2.4%) were significantly (P < 0.05) higher than in IVF embryos (4.2 ± 1.0%), but similar to cattle NT embryos (5.4 ± 1.7%), which did not differ from the IVF group. Total cell number was significantly (P < 0.05) higher in the IVF group than in all other groups (133.2 ± 10.2 v. 91.2 ± 7.8 to 100.1 ± 12.9). These results confirm that in vitro-matured domestic cattle oocytes can serve as suitable recipients of wood bison somatic cells and that iSCNT may provide a possible alternative for embryo production and genetic preservation of endangered cattle species. Both the incidence of apoptotic cells and total cell number did not differ between cattle and bison NT embryos; thus other factors must play a role in the significantly decreased blastocyst development observed in bison NT embryos. This work was supported by Endangered Species Reserve Fund, Toronto Zoo, and the Canada Research Chairs program.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer

2015 ◽  
Vol 27 (3) ◽  
pp. 544 ◽  
Author(s):  
H. S. Pedersen ◽  
Y. Liu ◽  
R. Li ◽  
S. Purup ◽  
P. Løvendahl ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Oocyte Growth ◽  
Parthenogenetic Activation ◽  
Selection Of

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer

2003 ◽  
Vol 15 (3) ◽  
pp. 179 ◽  
Author(s):  
Goo Jang ◽  
Byeong Chun Lee ◽  
Sung Keun Kang ◽  
Woo Suk Hwang
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Fertilization ◽  
Chondroitin Sulfate ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
Vitro Fertilization

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.

scholarly journals 68 EFFECT OF XENOPUS EGG EXTRACT TREATMENT OF DONOR CELLS ON PORCINE SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 192
Author(s):  
Y. Liu ◽  
O. Østrup ◽  
J. Li ◽  
G. Vajta ◽  
L. Lin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Permeabilization ◽  
Total Cell Number ◽  
Egg Extract ◽  
Donor Cells

Pretreatment of somatic cells to promote subsequent reprogramming during somatic cell nuclear transfer (SCNT) may significantly improve efficiency of the technique. The aim of this study was to evaluate the effect of Xenopus laevis egg extract pretreatment of porcine fetal fibroblast cells using different permeabilization agents prior to SCNT. Fibroblasts were permeabilized using streptolysin O (SLO; 300 ng mL-1, 30 min, 37°C) or digitonin (7 μg mL-1, 2 min, 4°C), and exposed to egg extract for 1 h or 0.5 h, respectively. Cell membranes were resealed in DMEM supplemented with 2 mM CaCl2 for 2 h. After culture for 1, 3, and 5 days (for SLO) or 3 and 5 days (for digitonin), the SLO extract-treated cells (SETC) and digitonin extract-treated cells (DETC) were used as donor karyoplasts for handmade cloning. Controls were SCNT with nontreated cells. Embryos were evaluated for cleavage rate (Day 2), blastocyst rate (Day 6), and total cell numbers of blastocysts. Statistical differences were analyzed by ANOVA. Results are summarized in Table 1. When SETC were used as donors, blastocyst rates were significantly lower compared with the controls, except when the donor cells were cultured for 3 days after treatment. Blastocysts of the latter group also had higher total cell number. With DETC as donors, blastocyst rates and total cell number of embryos at Day 6 reconstructed with cells cultured for 5 days were higher than those in other groups. Results indicate that extract treatment of the donor cells after SLO-permeabilization can give higher number of cells in cloned blastocysts but not improve overall embryo development. However, digitonin treatment for donor cell permeabilization improved both embryo development and cell number of blastocyst. The latter effect was detected only 5 days after the treatment. In conclusion, qualitative efficiency of porcine SCNT could be improved with a combined donor cell permeabilization and extract treatment. Table 1.Effect of different permeabilization agents prior to SCNT

62 DIFFERENT EFFECT OF TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF PORCINE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETICALLY ACTIVATED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 112 ◽  
Author(s):  
H. X. Wei ◽  
K. Zhang ◽  
Y. F. Ma ◽  
Y. Li ◽  
Q. Y. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Polar Body ◽  
Cell Number ◽  
Cell Numbers ◽  
First Polar Body ◽  
Electrical Fusion

Accumulating evidence suggests that trichostatin A (TSA), a histone deacetylase inhibitor, can increase the success rate of somatic cloning. The objective of this study was to investigate the effect of 50 nm TSA treatment on the development of porcine somatic cell nuclear transfer (SCNT) and parthenogenically activated (PA) embryos. Cumulus-oocyte complexes were matured in vitro. The oocytes with the first polar body (PB1) were chosen for SCNT, and the rest with PB1 or good morphology were selected for PA by a single 100-μs direct current pulse of 1.6 kV cm–1, the same parameter as for electrical fusion. GFP transgenic fetal fibroblast cells were used as nuclear donors. Data were analyzed using SPSS (13.0; SPSS, Inc., Chicago, IL, USA) with one-way ANOVA. In Experiment 1, immediately after electrical fusion and activation, the reconstructed embryos were randomly cultured in porcine zygote medium 3 (PZM3) with 10 μg mL–1 cytochalasin B (CB) and 10 μg mL–1 cycloheximide (CHX), with either 0 nm (control) or 50 nm TSA for the first 4 h, before being cultured for another 20 h in PZM3 without CB and CHX. After being washed, the embryos were cultured in PZM3 medium without TSA until Day 6 at 39.0°C, 5% CO2, 5%O2, 90% N2, and 100% humidity. The same experimental design was used for PA embryos concurrently. The results showed that there were no significant differences in blastocyst rates for SCNT or PA between control and TSA groups (23.0 ± 6.1% v. 27.9 ± 6.3%; 21.0 ± 1.0% v. 17.5 ± 3.2%, respectively). Neither were there differences in the cell numbers of blastocysts (38.3 ± 5.7 v. 32.2 ± 3.4; 42.2 ± 3.5 v. 39.0 ± 1.9, respectively). In Experiment 2, TSA treatment was prolonged to either 36 or 40 h. The blastocyst rates of SCNT were increased (7.3 ± 1.2% (0 h), 13.3 ± 2.6% (36 h), and 20.0 ± 3.3% (40 h)), whereas those of PA were decreased (46.7 ± 5.0% (0 h), 27.7 ± 6.5% (36 h), and 30.8 ± 6.3% (40 h)). The cell numbers of blastocysts from either SCNT or PA were also decreased (SCNT: 47.5 ± 3.8, 37.5 ± 2.0, and 37.1 ± 3.3; PA: 46.1 ± 1.9, 37.5 ± 1.9, and 39.3 ± 2.2; P < 0.05). In Experiment 3, the cell number and the apoptotic index of Day 5, 6, and 7 PA blastocysts treated with 0 or 50 nm TSA were determined by the terminal deoxynucleotide-mediated nick end labeling (TUNEL) assay (Table 1). The results suggested that TSA treatment probably delayed embryo development, which may be one of the reasons for the lower cell numbers in the TSA-treated group. Table 1. Cell apoptosis of PA blastocyst by TUNEL

Production of somatic cell nuclear transfer embryos using in vitro-grown and in vitro-matured oocytes in rabbits

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 494-500 ◽  
Author(s):  
Hironobu Sugimoto ◽  
Yuta Kida ◽  
Noriyoshi Oh ◽  
Kensaku Kitada ◽  
Kazuya Matsumoto ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase Inhibitor ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Deacetylase Inhibitor ◽  
Number Of Cells

SummaryWe examined growing oocytes collected from follicles remaining in superovulated rabbit ovaries, that were grown (in vitro growth, IVG) and matured (in vitro maturation, IVM) in vitro. We produced somatic cell nuclear transfer (SCNT) embryos using the mature oocytes and examined whether these embryos have the ability to develop to the blastocyst stage. In addition, we examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), on the developmental competence of SCNT embryos derived from IVG–IVM oocytes. After growth for 7 days and maturation for 14–16 h in vitro, the growing oocytes reached the metaphase II stage (51.4%). After SCNT, these reconstructed embryos reached the blastocyst stage (20%). Furthermore, the rate of development to the blastocyst stage and the number of cells in the blastocysts in SCNT embryos derived from IVG–IVM oocytes were significantly higher for TSA-treated embryos compared with TSA-untreated embryos (40.6 versus 21.4% and 353.1 ± 59.1 versus 202.5 ± 54.6, P < 0.05). These results indicate that rabbit SCNT embryos using IVG–IVM oocytes have the developmental competence to reach the blastocyst stage.

45 EFFECT OF TRICHOSTATIN A TREATMENT ON THE TERM DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER RABBIT EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 103 ◽  
Author(s):  
Q. Meng ◽  
Z. Polgar ◽  
J. Liu ◽  
A. Dinnyes
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cumulus Cell ◽  
Cell Number ◽  
Pregnant Female ◽  
Rabbit Embryos

The efficiency of somatic cell nuclear transfer (SCNT) is low in the rabbit. So far, there have been few live births reported and most clones died within the first 3 weeks after birth. It has been shown that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, improved cloning efficiency in cattle (Enright et al. 2003 Biol. Reprod. 69, 896–901) and mice (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). Although a recent report indicated that TSA treatment could increase the cell number of rabbit SCNT blastocysts (Xu et al. 2007 Reprod. Fertil. Dev. 19, 165), term development of TSA-treated cloned embryos in this species has not been reported. In this study we investigated the effect of TSA treatment on the term development of somatic cell nuclear transfer (SCNT) rabbit embryos. The oocytes and cumulus cells were collected from superovulated Hycole hybrid rabbits. After staining with Hoechst 33342 and locating following 1–2 s of UV illumination, the nuclei of oocytes were removed by micromanipulation, A cumulus cell was then inserted into the perivitelline space and fused with the cytoplast with three 20 μs 3.2 kV cm–1 DC pulses. Fused embryos were activated using the same electrical parameters 1 h later, treated with 2 mm 6-dimethylaminopurine and 5 μg mL–1 cycloheximide for 1 h, and then subsequently cultured in Earles Balanced Salt Solution (EBSS) with or without 5 nm TSA for 10 h. The embryos were then cultured in EBSS either overnight (before ET) or for 4.5 days. Embryos were transferred at the 2- to 4-cell stages to the recipients 22 h after collection of the oocytes from the donors. Caesarean sections were performed on Day 30 post-ET. In vitro developmental data (Table 1) showed no differences in the cleavage, blastocyst rates, and blastocyst cell numbers between the TSA-treated or untreated cloned embryos. After ET in the TSA group, one pregnant female delivered 7 live and 3 stillborn pups, but all of the live pups died within 1 h to 19 days later. In the untreated group, one pregnant female gave birth to 2 live and 1 stillborn pup. One pup died within 1 h after birth; the other survived (2.5 months old when this abstract was submitted). In conclusion, the results indicate that TSA treatment has a limited effect on in vitro development of SCNT embryos, and both TSA-treated and untreated SCNT clones can develop to term in rabbit. The effects of TSA treatment on the health of clones need further investigation. Table 1. In vitro and in vivo development of SCNT rabbit embryos with or without TSA treatment This study was supported by Wellcome Trust (Grant No. 070246), EU FP6 (MEXT-CT-2003-509582, MRTN-CT-2006-035468), and Chinese-Hungarian Bilateral projects (TET CHN-28/04, CHN-41/05).

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

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