52 EFFECT OF DONOR CELLS WITH VARIOUS DIFFERENTIATED STATUS ON THE DEVELOPMENTAL COMPETENCE OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
J. G. Kim ◽  
E. J. Kang ◽  
M. K. Kim ◽  
S. Y. Choe ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts

Adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 70, 1088–1095). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and have a more flexible potential as donor cells for NT. The aim of this study was to compare the developmental potential of NT embryos using undifferentiated (MSCs) and differentiated cells in the same lineage (osteocyte, adipocyte, and chondrocyte) by assessing the cleavage and blastocyst rates. Fetal fibroblasts were used as NT control. MSCs obtained from the aspirated bone marrow of a neonatal pig were cultured in advanced-DMEM (ADMEM) supplemented with 5% FCS. The differentiation potential was demonstrated by culture of MSCs at passage 3 under the conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143–147). For NT, cells from passages 3–5 were transferred into the perivitelline space of enucleated MII oocytes that had been in vitro-matured after collection from slaughterhouse-derived ovaries. After fusion with a needle-type electrode, eggs were cultured in 7.5 µg mL−1 cytochalasin B for 3 h, and subsequently cultured in PZM-3 medium for 6 days. Statistical significance was tested using ANOVA with Bonferroni and Duncan tests. The results are presented in Table 1. The rates of cleavage and development to blastocyst stage of NT embryos varied among donor cell sources. Most eggs (92.2 ± 2.7%) cloned with MSCs cleaved, and 47.8% of eggs developed to the blastocyst stage. In contrast, NT eggs using differentiated MSCs—osteocytes, adipocytes, chondrocytes, and controls (fetal fibroblasts)—revealed significantly (P < 0.05) lower cleavage (74.5, 63.4, 74.3, and 66.4%, respectively) and blastocyst development (33.7, 30.1, 36.5, and 25.5%, respectively) rates than those using undifferentiated MSCs. The results demonstrate that the genome of donor cells with different differentiated status supports embryonic development to various degrees, and multipotent MSCs might have a greater potential in producing viable cloned porcine embryos. Table 1.Development of NT embryos with undifferentiated and differentiated cells This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

55 INFLUENCE OF DONOR CELL TYPE ON THE DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 136
Author(s):  
K. Lee ◽  
W. L. Fodor ◽  
Z. Machaty
Keyword(s):  
Stem Cells ◽  
Olfactory Bulb ◽  
Progenitor Cells ◽  
Nuclear Transfer ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
Fetal Fibroblasts

Embryonic development after nuclear transfer is very low; the majority of cloned embryos do not survive the pre-implantation stage. Recent reports indicate that the characteristics of nuclear transfer embryos depend on the type of nuclear donor cells. It has been suggested that development after nuclear transfer improves if less differentiated cells are used as nuclear donors. The aim of the present study was to investigate the developmental potential of nuclear transfer embryos reconstructed using differentiated and non-differentiated cells. Two types of non-differentiated cells, skin stem cells and olfactory bulb progenitor cells, were used; fetal fibroblasts were used as differentiated control. Prior to nuclear transfer, the differentiated state of the cells was characterized by Oct-4 immunocytochemistry (Chemicon International, Inc., Temecula, CA, USA); Oct-4 is known to be expressed by pluripotent cells only. During nuclear transfer, the cells were transferred into the perivitelline space of in vitro-matured enucleated oocytes. After fusion, reconstructed oocytes were activated by an electrical pulse followed by incubation in 10 �g/mL cycloheximide and 5 �g/mL cytochalasin B for 5 h. The embryos were subsequently cultured in NCSU-23 medium for 6 days; their developmental data were recorded and compared by ANOVA. Non-differentiated cell types showed strong Oct-4 expression, whereas the marker protein was completely absent in fetal fibroblast cells. A total of 161 embryos were reconstructed using skin stem cells, 171 embryos from olfactory bulb progenitor cells, and 189 embryos from fibroblasts. Of the skin stem cell-derived embryos, 32.9% cleaved, and during subsequent culture, 5.6% developed to the morula/blastocyst stage. In the olfactory bulb progenitor cell group, 19.8% cleaved, and the percentage of embryos that developed to the morula/blastocyst stage was 4.7%. In the control group, 22.7% cleaved; the morula/blastocyst formation was 2.6%. Embryos reconstructed from skin stem cells showed superior cleavage rate compared to embryos from the other cell types (P < 0.05). Also, morula/blastocyst formation from skin stem cells was significantly higher than that from fetal fibroblasts (P < 0.05), and morula/blastocyst formation from olfactory bulb progenitor cell-derived embryos also tended to be higher compared to control embryos (P = 0.08). Furthermore, the formation of morulae/blastocysts per cleaved embryos was the highest in embryos reconstructed with olfactory bulb progenitor cells (23.5% vs. 17.0% using skin stem cells and 11.6% using fibroblasts) implying that embryos from olfactory bulb progenitor cells may have higher developmental potential in later stages of development. The results demonstrate that nuclei of different donor cells support development to various degrees and confirm previous reports that using non-differentiated cells as nuclear donors increases the efficiency of nuclear transfer in the pig.

43 PRODUCTION OF PORCINE EMBRYOS BY NUCLEAR TRANSFER OF BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 130
Author(s):  
H.-F. Jin ◽  
B. Mohana Kumar ◽  
J.-G. Kim ◽  
H.-J. Song ◽  
S. Balasubramanian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Donor Cells ◽  
Fetal Fibroblasts

Recent experimental evidence indicates that adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 71, 1890-1897). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and may have a more flexible potential as donor cells for NT. The aim of this study was to characterize an isolated population of porcine MSCs from bone marrow and to compare the developmental potential of cloned (IVF) embryos with MSCs and porcine fetal fibroblasts (pFFs) by assessing the cleavage and blastocyst rate, total cell numbers, inner cell mass (ICM) ratio and apoptosis. MSCs were obtained from the aspirated bone marrow of 6-8-month-old pigs. Cells were centrifuged, resuspended, and plated in advanced-DMEM (ADMEM) supplemented with 5% fetal bovine serum (FBS). The differentiation potential was demonstrated by culture of MSCs (passage 3) under conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143-147). Oil red O staining revealed that MSCs produced lipid droplets after incubation in adipogenic media. Following osteoinduction, MSCs exhibited robust alkaline phosphatase activity and cells later transformed into mineralized nodules as demonstrated by von Kossa staining. Histological staining of proteoglycan indicated chondrogenic differentiation. Cumulus-oocyte complexes were matured, fertilized, and cultured by the following method (Abeydeera et al. 2000 Theriogenology 54, 787-797). NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Cleavage rate was significantly (P < 0.05) higher in IVF embryos than in NT embryos derived from MSCs and pFFs (84.5% � 4.6 vs. 52.2% � 5.4 and 50.8% � 5.2, respectively). However, blastocyst rates in IVF embryos and NT embryos derived from MSCs (20.6% � 2.5 and 18.5% � 3.0) did not differ but these rates were significantly (P < 0.05) higher than that for NT embryos derived from pFFs (9.5% � 2.1). Total cell numbers and the ratio of ICM to total cells among embryos developed in NT from MSCs (29.4 � 5.2 and 0.38 � 0.08, respectively) were significantly (P < 0.05) higher than for those from pFFs (22.6 � 5.5 and 0.18 � 0.12, respectively). Proportions of TUNEL-positive cells in NT embryos from pFFs (12.8 � 2.5) were significantly (P < 0.05) higher than in those from MSCs (8.6 � 1.8) and in IVF embryos (4.6 � 1.5). The results clearly demonstrate that multipotent bone marrow MSCs can make a suitable alternative to fibroblasts as donor cells and have a greater potential for producing viable cloned porcine embryos. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 137
Author(s):  
A. Lucas-Hahn ◽  
E. Lemme ◽  
K.-G. Hadeler ◽  
H.-G. Sander ◽  
H. Niemann
Keyword(s):  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Ultrasound Guided ◽  
Embryo Production ◽  
Fetal Fibroblasts ◽  
Developmental Rates ◽  
In Vitro Embryo Production

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle

257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 236
Author(s):  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Marrow Mesenchymal Stem Cells

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

32 MicroRNA-29b Improves the Quality and Developmental Potential of Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 155
Author(s):  
W.-J. Zhou ◽  
S. Liang ◽  
X.-S. Cui
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Down Regulation ◽  
Developmental Potential ◽  
Somatic Cell Reprogramming ◽  
Underlying Mechanisms

MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse cellular processes. miR-29b plays a crucial role during somatic cell reprogramming. However, studies of the function of miR-29b in embryogenesis are limited. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared with IVF embryos (P < 0.05). To determine the function of miR-29b in the bovine SCNT embryo, we microinjected a miR-29b mimic and inhibitor into bovine SCNT zygotes. The results showed that miR-29b significantly decreased the expression of Dnmts (Dnmt3a/3b and Dnmt1) in bovine SCNT embryos (P < 0.05). We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency (P > 0.05) but down-regulation inhibits developmental potency (P < 0.05). Although miR-29b overexpression does not improve the developmental potency of bovine SCNT embryos, the quality of bovine SCNT embryos at the blastocyst stage improved significantly (P < 0.05). The expression of pluripotency factors (OCT4 and SOX2) and cellular proliferation rate were significantly higher in blastocysts from the miR-29b overexpression group than the control and down-regulation groups (P < 0.05). In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and down-regulation groups (P < 0.05). Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
B. Mohana Kumar ◽  
H. F. Jin ◽  
J. G. Kim ◽  
S. A. Ock ◽  
H. J. Song ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Dna Hypomethylation ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Donor Cells ◽  
Fetal Fibroblasts

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 �m, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 �m 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P &lt; 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 � 5.2% vs. 65.8 � 5.3% and 69.3 � 4.4%, respectively). Similarly, significantly (P &lt; 0.05) higher blastocyst rates were observed in IVF embryos (27.2 � 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P &lt; 0.05) higher rates of blastocysts (21.3 � 2.2%) than NT embryos from PFF (14.8 � 1.9%). NT embryos from PFF + 5-azaC (33.8 � 4.1) had significantly (P &lt; 0.05) higher total cell numbers than from PFF (24.6 � 3.5), but did not differ in the proportion of apoptotic cells (6.9 � 1.8% and 7.2 � 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 � 3.8, 2.7 � 0.8%, and 53.9 � 3.5, 1.2 � 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

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