44 NUCLEAR TRANSFER IN CATTLE USING SOMATIC CELLS FROM FROZEN TESTICLES WITHOUT CRYOPROTECTANTS

2007 ◽  
Vol 19 (1) ◽  
pp. 140 ◽  
Author(s):  
Y. Hoshino ◽  
N. Kobayashi ◽  
N. Hayashi ◽  
T. Matsuhashi ◽  
K. Saeki ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Somatic Cells ◽  
Frozen Storage ◽  
Cell Number ◽  
Fetal Skin ◽  
Nylon Mesh ◽  
Viable Cells ◽  
Donor Cells ◽  
Bovine Oocytes

Obtaining somatic cells from preserved organs or tissues is useful for the conservation and regeneration of genetic resources by nuclear transfer (NT). Bovine cells for NT have been obtained from cooled carcasses stored at 0�C for several days (Arat et al. 2005 Reprod. Fert. Dev. 17, 164 abst) and from fetal skin tissue cryopreserved with DMSO (Fahrudin et al. 2001 J. Vet. Med. Sci. 63, 1151–1154). However, frozen storage of organs or tissues without cryoprotectants was considered to be quite inappropriate for obtaining viable cells. We report here that viable donor cells for NT were obtained from bovine testicles after frozen storage without cryoprotectants. In the first experiment, we investigated whether viable cells can be recovered from frozen testicles castrated from Japanese Black bulls. The testicles were frozen at -80�C in a freezer for several days; then some were stored in liquid nitrogen for 10 months without cryoprotectants. Before thawing, the testicles were divided into 3 pieces, caput epididymis, cauda epididymis, and testis. Each piece was then put in saline at 42�C for quick thawing. Thawed tissues were minced into 5-mm pieces and incubated at 39�C for 2 h in DMEM containing 0.1% collagenase and 0.2% dispase. After filtration through a 250-�m nylon mesh filter, the filtrates were centrifuged at 250 � 4g for 5 min. Then precipitates were resuspended with MF-start� primary culture medium (TM Cell Research Inc., Fukui, Japan) and incubated at 38.5�C under the atmosphere of 5% CO2 in air with high humidity. After 5 days of incubation, the medium was replaced and nonadherent debris was discarded. Viable cells were obtained from the caput epididymis. These cells actively proliferated and expanded. In the next experiment, to determine whether these cells can be used for NT, the cells were electrically fused with enucleated bovine oocytes. Bovine fibroblasts taken from unfrozen ear tissue were used as controls. The NT embryos were activated by Ca-ionophore treatment, followed by treatment with cycloheximide for 6 h, and then cultured in mSOF for 168 h. NT embryos reconstructed from testicle cells did not significantly differ from NT embryos made with control cells with regard to blastocyst rates (22.1% and 20.2%), cell number of blastocysts [130 � 43 and 121 � 43 (mean � SD)], and ICM ratio (21.1% and 22.6%), respectively (ANOVA). These results suggest that somatic cells derived from bovine frozen testicles can be used for nuclear transfer. Further studies are needed to examine whether viable cells can be obtained from other frozen organs or tissues. This study was partially supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST.

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P < 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P < 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

34 Effect of polysaccharide from Flammulina velutipes on the vitrification of bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 143
Author(s):  
Y. Ihara ◽  
K. Tatakura ◽  
Y. Wada ◽  
H. Kawahara ◽  
K. Yamanaka
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Flammulina Velutipes ◽  
Control Group ◽  
Cleavage Pattern ◽  
Total Cell Number ◽  
Bovine Oocytes

The developmental competence of oocytes after cryopreservation is compromised by the physical injury due to the ice crystallisation. Recent studies have reported that polysaccharide (xylomannan) derived from the mycelium and fruit body of the basidiomycete Flammulina velutipes inhibits the ice recrystallisation in the cryopreserved Chinese hamster ovary cells. In this study, we aimed to clarify the effect of xylomannan from Flammulina velutipes on the developmental competence of bovine vitrified oocytes. Bovine ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) were aspirated from follicles (2-6mm in diameter) using a 19-gauge needle attached to a syringe. The COCs were matured for 22h in tissue culture medium-199 supplemented with 5% fetal bovine serum (FBS), 0.02IUmL−1 FSH, and 10μgmL−1 gentamycin. After maturation, COCs were incubated in base solution (BS: 10% FBS-tissue culture medium-199, control group; n=149) or BS supplemented with 100μgmL−1 xylomannan (xylomannan group; n=175) for 1h before vitrification. All vitrification procedures were performed at room temperature. The COCs were equilibrated in BS with 3% ethylene glycol for 12min and then in vitrification solution (BS with 30% ethylene glycol, 1.0M sucrose) for 1min. The COCs were loaded on a Cryotop (Kitazato) and transferred into liquid nitrogen. The warming procedure was performed on a warm plate (42°C). The COCs were placed into BS supplemented with 0.5, 0.25, 0.125, and 0M sucrose for 5min each. After washing with IVF100 solution (Research Institute for the Functional Peptide), COCs were applied for IVF. The viability of putative zygotes was morphologically evaluated following IVF, and ones that survived were cultured in CR1aa supplemented with 5% FBS. The cleavage pattern was evaluated at 28h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the 1-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The rates of cleavage and blastocyst formation were calculated on 2 and 8 days after culture, respectively. Total cell number and apoptosis of blastocysts were measured by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. All data were obtained from more than four replicates. Viability and invitro development data were analysed using the chi-squared test. Total cell number and apoptosis data were analysed by a Student's t-test. Although no significant differences in viability, cleavage pattern, and cleavage rate (85.8 vs. 80.3%, 17.2 vs. 14.8%, and 35.4 vs. 36.7%, respectively) were observed, the developmental rate to blastocysts in the xylomannan group was significantly higher than that in the control group (68.6 vs. 42.2%; P<0.01). The present results suggest that co-incubation with xylomannan before vitrification is an effective method to improve the vitrification outcome in bovine oocytes.

Cellular reprogramming of adult goat fibroblast: Toward pluripotency

2016 ◽  
Author(s):  
Dharmendra Kumar ◽  
Rakesh Ranjan ◽  
Ajit P. Singh ◽  
Bikash C Sarkhel
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Cellular Reprogramming ◽  
Serum Starvation ◽  
Methyl Transferase ◽  
Donor Cells ◽  
Key Factor ◽  
Gene Transcripts

Cellular reprogramming erases the epigenetic constraints of somatic cells genome and thus considered as key factor for success of somatic cell nuclear transfer technology. To achieve the reprogramming, different strategies are used which are mostly based on arresting the cell cycle at G0 or G1 stage. The present study was based on molecular investigation of reprogrammed cells for expression of pluripotent genes that are crucial for development of cloned embryos. The fibroblast cell lines were treated by four methods to induce cellular reprogramming viz., serum starvation, Roscovitin, aphidicolin and overconfluent. These treated cell lines were used for quantification of pluripotent gene transcripts by using real time PCR machine. The results showed that the relative expression of different pluripotent genes as Oct-4 and Nanog along with DNA methyl transferase gene (Dnmt-1) was observed in four treated cells. In case of normal cells, only Dnmt-1 gene was expressed, but pluripotent genes were not expressed at detection level. The expression of pluripotent genes in the donor cells prior to nuclear transfer have significant impact on cloning as because it facilitates the expression of that gene in the resulting embryo after nuclear transfer. The finding of this study may be extended for stem cell generation as it showed that pluripotent genes could be induced in the somatic cells without any transgenic incorporation.

scholarly journals Production of Blastocysts after Intergeneric Nuclear Transfer of Goral (Naemorhedus goral) Somatic Cells into Bovine Oocytes

2006 ◽  
Vol 68 (11) ◽  
pp. 1167-1171 ◽  
Author(s):  
Byoung-Chol OH ◽  
Jung-Tae KIM ◽  
Nam-Shik SHIN ◽  
Soo-Whan KWON ◽  
Sung-Keun KANG ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Somatic Cells ◽  
Bovine Oocytes

scholarly journals Genome of non-living cells: trash or recycle?

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 497-503 ◽  
Author(s):  
Pasqualino Loi ◽  
Josef Fulka ◽  
Thomas Hildebrand ◽  
Grazyna Ptak
Keyword(s):  
Nuclear Transfer ◽  
Somatic Cells ◽  
Reproductive Technologies ◽  
Biological Assays ◽  
Cell Vitality ◽  
Viable Cells ◽  
Heat Treated ◽  
Freeze Dried ◽  
Temperature Exposure ◽  
Normal Offspring

Reproductive technologies have been often used as a tool in research not strictly connected with developmental biology. In this study, we retrace the experimental routes that have led to the adoption of two reproductive technologies, ICSI and somatic cell nuclear transfer (SCNT), as biological assays to probe the ‘functionality’ of the genome from dead cells. The structural peculiarities of the spermatozoa nucleus, namely its lower water content and its compact chromatin structure, have made it the preferred cell for these experiments. The studies, primarily focused on mice, have demonstrated an unexpected stability of the spermatozoa nuclei, which retained the capacity to form pronuclei once injected into the oocytes even after severe denaturing agents like acid treatment and high-temperature exposure. These findings inspired further research culminating in the production of mice after ICSI of lyophilized spermatozoa. The demonstrated non-equivalence between cell vitality and nuclear vitality in spermatozoa prompted analogous studies on somatic cells. Somatic cells were treated with the same physical stress applied to spermatozoa and were injected into enucleated sheep oocytes. Despite the presumptive fragile nuclear structure, nuclei from non-viable cells (heat treated) directed early and post-implantation embryonic development on nuclear transfer, resulting in normal offspring. Recently, lyophilized somatic cells used for nuclear transfer have developed into normal embryos. In summary, ICSI and SCNT have been useful tools to prove that alternative strategies for storing banks of non-viable cells are realistic. Finally, the potential application of freeze-dried spermatozoa and cells is also discussed.

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

30 OXYGEN DEPRIVATION DOES NOT FURTHER AUGMENT MITOCHONDRIAL MEMBRANE POTENTIAL IN PHARMACOLOGICALLY TREATED FIBROBLASTS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER

2017 ◽  
Vol 29 (1) ◽  
pp. 122
Author(s):  
B. R. Mordhorst ◽  
S. N. Bogue ◽  
K. D. Wells ◽  
J. A. Green ◽  
R. S. Prather
Keyword(s):  
Membrane Potential ◽  
Somatic Cell ◽  
Mitochondrial Membrane Potential ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mitochondrial Membrane ◽  
Somatic Cells ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Donor Cells

Somatic cells commonly used in nuclear transfer primarily utilise the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively, the metabolism of somatic cells contrasts that of cells within early embryos, which predominantly use glycolysis and exhibit Warburg Effect (WE)-like characteristics. We hypothesised that fibroblast cells can become more blastomere-like if driven either pharmacologically or by oxygen constraint and could result in improved in vitro embryonic development after somatic cell nuclear transfer (SCNT). The pharmaceuticals used (PS48 and CPI-613) should decrease mitochondrial use of the tricarboxylic acid (TCA) cycle and promote the PI3K pathway, respectively. Furthermore, we hypothesised that oxygen constraint (1.3%) would hinder TCA cycle activity and promote glycolysis. The goal was to achieve a WE-like effect in donor cells before nuclear transfer (NT) by treating Day 35 porcine fetal fibroblasts with CPI-613 (100 µM), PS48 (10 µM), both drugs combined (MIX), or as controls (CON, 0 µM) for 7 days under stepwise oxygen constraint (OC; 1.3%) or under normal conditions (ON; 5%). Three biological replicates were collected and data were analysed for main effect of treatment via GLM procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA). To determine if our treatments affected mitochondria respiratory capacity (thereby TCA cycle capability) within embryos, we measured mitochondrial membrane potential (Δψm) using JC-10, a biphasic cationic dye. Mitotracker green (MTG) was used to estimate mitochondrial quantity. The percentage of cells with low Δψm was increased (P = 0.02) with any CPI or MIX treatment (treatments ≥ 95%) compared with OC-PS48 and both control (ON and OC) treatments (treatments ≥ 77.4%), whereas ON-PS48 had an intermediate level (90.4%; error = 4.9%). Contrary to our prediction, MTG intensity was lower across all ON treatments compared with OC treatments (NO treatments ≤ 736 AU v. OC treatments ≥ 872 AU; error = 23 AU; P < 0.01). Regardless of oxygen level, controls and PS48 treatments yielded the highest percentages of viable cells (treatments ≥ 94%) and OC-CPI and NO-MIX the lowest (treatments ≤ 86%) with NO-CPI and OC-MIX being intermediate (treatments ≥ 90%; error = 3%; P < 0.01). Oxygen constraint did not promote a reduction in mitochondrial membrane potential in pharmacologically treated fibroblasts. Additionally, intensity of MTG was increased in fibroblasts cultured under oxygen constraint compared with those cultured in 5% oxygen. Our results warrant further investigation of the mitochondrial changes occurring with oxygen deprivation in donor-cells. Experiments are underway to determine if gene expression in cells treated pharmacologically and with oxygen constraint are augmented, and whether these treatments will result in better development after SCNT. This study was funded by Food for the 21 st Century and NIH R01HD080636.

143 DEVELOPMENTAL EFFICIENCY IMPROVEMENTS OF NUCLEAR TRANSFER EMBRYOS BY REPROGRAMMING SOMATIC CELLS USING TESTIS-DERIVED CELL EXTRACTS

2007 ◽  
Vol 19 (1) ◽  
pp. 189
Author(s):  
H.-Y. Choi ◽  
C. Won ◽  
B.-W. Kim ◽  
Y.-J. Kang ◽  
G.-H. Kang ◽  
...  
Keyword(s):  
Germ Cell ◽  
Somatic Cell ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Male Germ Cell ◽  
Kidney Cells ◽  
Cell Extracts ◽  
Donor Cells ◽  
Porcine Testis

Somatic cell cloning has promise for medical treatment using embryonic stem cells derived from cloned embryos. However, porcine cloning by somatic cell nuclear transfer has been inefficient and, even after birth, cloned pigs are found to carry a variety of abnormalities. Moreover, recent molecular analyses of cloned embryos have revealed abnormal epigenetic modifications. Therefore, the prevention of epigenetic errors is expected to lead to the improvement of the success rate in animal cloning. Reports of recent studies indicate that the direct transformation of one differentiated somatic cell type into another is possible and would be advantageous for producing isogenic replacement cells. Therefore, in this study, we modulated the cell fate of somatic donor cells by introducing cell extracts derived from porcine testis. Several porcine somatic cells, including primary and stabilized porcine fibroblasts or epithelial kidney cells, were treated with streptolysin O (SLO; 230 ng mL-1), which reversibly permeablizes plasma membrane, and incubated for 30 min with testis cell-derived cell extracts (4 mg mL-1). To reseal plasma membranes, cells were placed in DMEM containing 30% FBS and 2 mM CaCl2 for 30 min. After resealing the cell membranes, we incubated the cells for 3 weeks and analyzed the expression of testis-specific genes such as protamine 1, protamine 2, SOX 9, mullerian inhibitory substance (MIS), preproacrosine (ACR), phosphoglycerate kinase 2 (PGK-2), protein C, and c-kit ligand. In the reprogrammed primary porcine fibroblasts or epithelial kidney cells, the porcine testis extracts were able to activate the expression of the porcine testis sertoli cell-specific genes. The male germ cell functions were sustained for more than 10 days after the reprogramming process. Then, in vitro-matured oocytes were enucleated and a single cell (either reprogrammed or intact) was injected directly into cytoplasm of the oocytes. The reconstructed embryos were activated electrically and cultured in vitro for 7 days. The rate of blastocyst formation was significantly higher (P &lt; 0.05; chi-square test) in the reprogrammed nuclear donor cells (27/119; 22.7 � 5.0%) than in the control (intact) cells (11/83; 13.3 � 3.2%). Taken together, our results suggest that testis-derived cell extracts can be successfully used to reprogram fibroblasts to express male germ cell function, thus improving the developmental efficiency of the nuclear transfer embryos.

40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 129
Author(s):  
G.-S. Im ◽  
J.-S. Seo ◽  
I.-S. Hwang ◽  
S.-W. Kim ◽  
H.-S. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Electric Pulse ◽  
Chemical Activation ◽  
Cell Mass ◽  
Cell Number ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 �s. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 �g/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5�C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P < 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P < 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P < 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P < 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.

200 REPROGRAMMING OF Oct-4 FOLLOWING EQUINE SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 198
Author(s):  
T. Xiang ◽  
S. Walker ◽  
K. Gregg ◽  
W. Zhou ◽  
V. Farrar ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Donor Cells

Oct-4, a POU domain-containing transcription factor encoded by Pou5f1, is selectively expressed in pre-implantation embryos and pluripotent stem cells, but not in somatic cells. Because of such a unique expression feature, Oct-4 can serve as a useful reprogramming indicator in somatic cell nuclear transfer (SCNT). Compared with data of Oct-4 expression in mouse and bovine cloned embryos, little is known about this gene in equine nuclear transfer. In the present study, we investigated Oct-4 expression in donor cells, oocytes, and SCNT embryos to evaluate reprogramming of equine somatic cells following nuclear transfer. Horse ovaries were obtained from a local slaughterhouse and the oocytes collected from the ovaries were matured in vitro in an M199-based medium (Galli et al. 2003 Nature 424, 635) for 24 h. Donor cells were derived from biopsy tissue samples of adult horses and cultured for 1 to 5 passages. Standard nuclear transfer procedures (Zhou et al. 2008 Mol. Reprod. Dev. 75, 744–758) were performed to produce cloned embryos derived from equine adult somatic cells. Cloned blastocysts were obtained after 7 days of in vitro culture of reconstructed embryos. Total RNA were extracted using Absolutely RNA Miniprep/Nanoprep kits (Stratagen, La Jolla, CA) from oocytes (n = 200), donor cells, and embryos (n = 5). DNase I treatment was included in the procedure to prevent DNA contamination. Semiquantitative RT-PCR was performed with optimized cycling parameters to analyze Oct-4, GDF9, and β-actin in equine donor cells, oocytes, and cloned blastocysts. The RT-PCR products were sequenced to verify identity of the genes tested. The relative expression abundance was calculated by normalizing the band intensity of Oct-4 to that of β-actin in each analysis. No transcript of Oct-4 was detected in equine somatic cells used as donor nuclei, consistent with its expression patterns in other animal species, whereas Oct-4 was abundantly expressed in equine SCNT blastocysts derived from the same donor cell line. Oct-4 transcripts were also detected in equine oocytes and whether any maternally inherited Oct-4 mRNA persisted up to the blastocyst stage was unclear in this study. We selected GDF9 to address this question; GDF9 was abundantly detected in equine oocytes, consistent with its expression pattern in mouse and bovine, but not detected in donor cells and cloned blastocysts, suggesting that the GDF9 mRNA from the oocyte was degraded at least by the blastocyst stage. The results from this study imply occurrence of Oct-4 reprogramming in equine SCNT blastocysts, and future analysis for more developmentally important genes is needed to better understand reprogramming at molecular levels in this species.

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