scholarly journals 62 RELATION OF INTENSITY OF GENE EXPRESSION IN BOVINE RECONSTRUCTED EMBRYOS TO SUBSEQUENT DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 181
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cells ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Critical Event ◽  
Serum Starvation ◽  
Cell Stage ◽  
Sports Science ◽  
Strong Luminescence

During embryo development, embryonic gene activation (EGA) is the first critical event. We previously showed that EGA is also critical for further development in somatic cell-cloned embryos (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). To show this, we reconstructed bovine embryos with bovine somatic cells transfected with chicken β-actin/firefly luciferase fusion gene (β−act/luc+) and showed that only luminescent embryos at 60 hours post-fusion (hpf) developed to the blastocyst stage. In this study, we examined the relation between the intensity of expression of the same reporter gene in embryos reconstructed with bovine β−act/luc+ fibroblasts and their subsequent development to the blastocyst stage. Bovine fibroblasts were transfected with β−act/luc+ as described earlier (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). The stably transfected and cloned cells were cultured for several passages. The cells were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20 h post maturation. Enucleated oocytes were electrofused with the cells, and activated with a calcium ionophore and cycloheximide. The LUC+ signal (luminescence) in the embryos was detected in medium containing 500 μg mL−1 luciferin with an imaging photon counter (ARGUS 50, Hamamatsu, Japan) for 30 consecutive min at 60 hpf. The intensity of luminescence in embryos (4- to 8-cell stage) was graded as being strong (>10 × 104 pixels/embryo), intermediate (5 to 10 × 104 pixels/embryo), weak (<5 × 104 pixels/embryo), or absent. The embryos were cultured separately until 168 hpf, and examined for blastocyst development. Experiments were repeated four times, and the data were analyzed with Fisher's PLSD test following ANOVA by Stat View software (Ver. 5.0; abacus Concepts, Berkeley, CA, USA). Of 125 embryos that were reconstructed, 74 (59%) developed to the 4- to 8-cell stage at 60 hpf. The luminescence was strong in 29 (39%) of the embryos, intermediate in 12 (16%), weak in 19 (26%), and absent in 14 (19%). Blastocysts were obtained from a group of embryos that exhibited strong luminescence (10/29, 34%), but none of the embryos from the other groups developed to blastocysts. These results suggest that active gene expression in embryos reconstructed with somatic cells is important for their subsequent development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

69 RELATION OF SPATIAL GENE EXPRESSION PATTERNS IN BOVINE EMBRYOS RECONSTRUCTED WITH SOMATIC CELLS TO BLASTOCYST DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 142
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
D. Iwamoto ◽  
S. Kameyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Calcium Ionophore ◽  
Gene Expression Patterns ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Reconstructed Embryos ◽  
M Phase ◽  
G1 Cells

Recently, enhanced development to full term was obtained with embryos reconstructed with bovine early G1 cells rather than with G0 cells (Kasinathan et al. 2001 Nat. Biotechnol. 19, 1176-1178; Urakawa et al. 2004 Theriogenology 62, 714-728). However, the reason why donor somatic cells at the early G1 phase are better for embryo reconstruction is unclear. In this study, we investigated the relation of spatial gene expression patterns at the 4- to 8-cell stage to blastocyst development of embryos reconstructed with early G1 cells. Bovine fibroblasts stably transfected with �-act/luc+/IRES/EGFP were used for embryo reconstruction. M phase cells were prepared as described by Urakawa et al. (2004). Early G1 cells were obtained from cultured M phase cells soon after the M phase cells divided. Quiescent cells (cultured in 0.4% serum for 7 days) were used as G0 cells for a control. The cells were electrofused with enucleated bovine oocytes matured in vitro, and activated with a calcium ionophore and cycloheximide. The reconstructed embryos were cultured until 60 hours post fusion (hpf), and zonae pellucidae of 4- to 8-cell embryos were removed by pronase. To determine gene expression, the LUC+ activity (luminescence) in the embryo blastomeres was detected with an imaging photon counter (Hamamatsu Photonics, Hamamatsu City, Shikuoka Prefecture, Japan) for 10 min. The embryos were categorized as being positive, mosaic, or negative depending on whether all, some or no blastomeres were luminescent, respectively. The embryos were cultured in mSOF medium individually until 168 hpf to assess development to the blastocyst stage. Blastocyst development of reconstructed embryos without detection of luminescence was also examined. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. At 60 hpf, 75% (74/99) of embryos reconstructed with early G1 cells and 55% (46/83) of embryos with G0 cells developed to 4- to 8-cell stage embryos. The difference is significant (P < 0.05). The percentages of positive, mosaic, and negative embryos with G1 cells were 49, 35 and 16%, and blastocyst rates were 30, 11, and 0%, respectively. With G0 cells, the percentages were 32, 56, and 12%, and the blastocyst rates were 15, 4, and 0%, respectively. More positive embryos were obtained with early G1 cells than with G0 cells (P < 0.05). Blastocyst rates of the positive embryos with early G1 cells were the same as with G0 cells (P > 0.05). Blastocyst development of positive embryos was higher than that of mosaic and negative embryos in early G1 and G0 groups (P < 0.05). Without detection of luminescence, the blastocyst rates from the reconstructed embryos were 43% (35/81) and 16% (20/125) with early G1 and G0 cells, respectively (P < 0.05). These results suggest that the higher developmental capacity of embryos reconstructed with early G1 cells might be related to the appropriate spatial gene expression at the 4- to 8-cell stage. A part of this study was supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
M. Fahrudin ◽  
K. Kikuchi ◽  
N. W. K. Karja ◽  
M. Ozawa ◽  
T. Somfai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gradient Centrifugation ◽  
Somatic Cells ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.

scholarly journals 72EFFECTS OF GENE EXPRESSION IN BOVINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS TRANSFECTED WITH LUCIFERASE GENE ON THE SUBSEQUENT DEVELOPMENT

2004 ◽  
Vol 16 (2) ◽  
pp. 157
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Luciferase Gene ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Transfected Cells

During embryo development, embryonic gene activation (EGA) is one of the first critical events. Inappropriate EGA results in failure of further development. We have reported that gene expression in bovine embryos reconstructed with fibroblasts begins at 48 hours postfusion (hpf) and reaches a maximum level at 60hpf as detected by their bioluminescence following injection of chicken β-actin/firefly luciferase fusion gene (β-act/luc+) into their nuclei (Saeki et al., 2001 Theriogenology 55, 289). In the present study, effects of gene expression in embryos reconstructed with bovine fibroblasts transfected with luciferase gene on their subsequent development to the blastocyst stage were examined. Cultured bovine fibroblasts taken from an ear of a female calf were transfected with plasmid containing β-act/luc+/IRES/EGFP and neor using GeneJammer (StrataGene, La Jolla, CA, USA). Neomycin-resistant cells were selected by culturing with G418. Then, EGFP-positive colonies were further selected under fluorescence microscopy to obtain stably transfected cells. The transfected cells were cultured for several passages. Growing (50 to 60% confluence, GCs) and serum-starved cells (SCs) were used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20h post maturation. Enucleated oocytes were electrofused with the cells, and activated with calcium ionophore and cycloheximide. Luminescence in the embryos was detected with an imaging photon counter at 0 and 60hpf. Luminescence-positive (P) and -negative (N) embryos were cultured separately at each detection time. Embryos were cultured until 168hpf, and examined for cleavage and blastocyst development. Experiments were repeated 3 times, and totals of 91 and 123 embryos were reconstructed with GCs and SCs, respectively. Data were analyzed with Fisher’s PLSD test following ANOVA by Stat View software (Ver. 5.0). At 0hpf, luminescence was detected in 55 and 4% of embryos reconstructed with GCs and SCs, respectively. At 60hpf, luminescence was detected in 47 and 28% of P and N embryos with GCs, and 17 and 40% of P and N embryos with SCs at 0hpf, respectively. Cleavage rates were not different among groups (P&gt;0.05). Blastocysts were obtained only from the groups of embryos that were N at 0hpf and P at 60hpf (8% with GCs and 17% with SCs). No embryos in the other groups developed to the blastocyst stage. These results suggest that appropriate gene expression in embryos reconstructed with somatic cells is important for their subsequent development and that detecting the reporter gene expression can be used for selection of viable cloned embryos.

scholarly journals 57EMBRYO DEVELOPMENT FOLLOWING INTERSPECIES NUCLEAR TRANSFER OF AFRICAN BUFFALO (SYNCERUS CAFFER), BONTEBOK (DAMALISCUS DORCUS DORCUS) AND ELAND (TAUROTRAGUS ORYX) SOMATIC CELLS INTO BOVINE CYTOPLASTS

2004 ◽  
Vol 16 (2) ◽  
pp. 150 ◽  
Author(s):  
M. Matshikiza ◽  
P. Bartels ◽  
G. Vajta ◽  
F. Olivier ◽  
T. Spies ◽  
...  
Keyword(s):  
South Africa ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Critically Endangered ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Cell Stage ◽  
Wildlife Species ◽  
Interspecies Nuclear Transfer

Wildlife conservation requires traditional as well as innovative conservation strategies in order to preserve gene and species diversity. Interspecies nuclear transfer has the potential to conserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where representative cell lines have been established for the wildlife population while numbers were still abundant. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of somatic cells from the African buffalo (Syncerus caffer), bontebok (Damaliscus dorcus dorcus) and eland (Taurotragus oryx) to enucleated domestic cattle (Bos taurus) oocytes. Skin tissue from the three wildlife species were collected by surgically removing approx. 1.0×1.0cm ear skin notches from animals immobilized with a combination of etorphine hydrochloride (M99; South Africa) and azaperone (Stressnil, South Africa). The biopsies were placed into physiological saline and transported to the laboratory at 4°C within 2h, cleaned with chlorohexidine gluconate and sliced finely in Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Baumgarten and Harley 1995 Comp. Biochem. Physiol. 110B, 37–46) and actively growing fibroblast cultures made available for the nuclear transfer process. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578) using slaughterhouse-derived bovine oocytes. Culture was performed in SOFaaci (Vajta et al., 2003 Biol. Reprod. 68, 571–578) medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). Two identical replicates were made with somatic cells of each species. After successful reconstruction, 57, 42 and 48 nuclear transferred and activated buffalo, bontebok and eland embryos were cultured, respectively. All except for 2 buffalo embryos cleaved; 22 (39%) developed to or over the 8-cell stage, and 2 (3.5%) of them to the blastocyst stage. All but 3 bontebok embryos cleaved, 17 (40%) developed to or over the 8-cell stage, but none of them reached the compacted morula or blastocyst stage. Sixteen (33%) of the eland embryos developed to or over the 8-cell stage with one (2%) reaching the blastocyst stage. In conclusion, buffalo, bontebok and eland embryos developed from reconstruction using their respective somatic cells combined with bovine cytoplasts, however, in vitro developmental ability to the blastocyst stage was limited. Additional basic research that establishes the regulative mechanisms involved with early preimplantation development together with optimising nuclear transfer techniques may have the potential to one day play a role in the conservation of critically endangered wildlife species.

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

2007 ◽  
Vol 19 (1) ◽  
pp. 142 ◽  
Author(s):  
D. Iwamoto ◽  
K. Saeki ◽  
S. Kishigami ◽  
A. Kasamatsu ◽  
A. Tatemizo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Mammalian Species ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Embryonic Genome ◽  
Blastocyst Rate

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&percnt;; P &gt;0.05). However, the blastocyst rate of NT embryos treated with 50 nM TSA was higher than that of control embryos (40&percnt; vs. 19&percnt;, respectively; P &lt; 0.05). On the other hand, the blastocyst rate was lower with 500 nM TSA than with 5 or 50 nM TSA (7&percnt; vs. 33&percnt; or 40&percnt;; P &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.

178 RELATIVE ABUNDANCE OF Oct-4, Nanog, AND Sox-2 TRANSCRIPTS IN PORCINE OOCYTES AND CLEAVAGE-STAGE EMBRYOS PRODUCED VIA FERTILIZATION IN VITRO OR PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 168
Author(s):  
L. Magnani ◽  
R. Cabot
Keyword(s):  
Gene Expression ◽  
Similar Pattern ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Developmental Expression ◽  
Fertilization In Vitro ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Parthenogenetic Embryos

Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.

scholarly journals Efficiency of Dimethyl Sulphoxide and Ethylene Glycol on Subsequent Development of Vitrified Awassi Sheep Embryos

2020 ◽  
Vol 25 (2) ◽  
pp. 60
Author(s):  
Omar R Mardenli ◽  
MS Mohammad ◽  
HA Hassooni
Keyword(s):  
Ethylene Glycol ◽  
Large Range ◽  
Survival Rates ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Dimethyl Sulphoxide ◽  
Three Solutions ◽  
Cell Stage ◽  
Awassi Sheep

The use of cryoprotectants in vitrification would reduce the critical damages to the embryos, thus increase the survival rates. This research was conducted in the laboratory of reproductive biotechnology at the faculty of Agriculture of Aleppo University.  The study aimed to evaluate the viability and survivability of early Syrian Awassi embryos under the influence of dimethyl sulphoxide (DMSO) and ethylene glycol (EG) following vitrification. Embryos were vitrified in three solutions of cryoprotectants (A: DMSO (3 ml), B: EG (3 ml), and C which was composed of a combination of DMSO (1.5 ml) and EG (1.5 ml)). After thawing, embryos that had been vitrified in C solution achieved the highest rates of cleavage (P&lt; 0.01) comparing with A and B solutions for 2-16 cell stage (50.00% Vs 30.77% and 36.36%), morula (9.00% Vs 44.44% and 40.00%) and blastocyst stage embryos (92.86% Vs 58.33% and 50.00%) respectively. Down to the hatching blastocyst stage, 2-16 cell stage vitrified embryos in C solution achieved an encouraging rate comparing with A and B solutions (39.20% Vs23.08% and 22.73% respectively). The rates of arrested embryos decreased significantly (P&lt; 0.05) after thawing across the three solutions especially the morula and blastocyst stage (0.00 and 3.70% respectively) (C solution). No significant differences were observed in the three types of embryos across all stages and solutions despite the large range among these rates. Given the apparent benefit of the participatory effect of cytoprotectants, it is advised to use a mixture of DMSO and EG (1:1) in vitrification of ovine embryos.

51 AGGREGATION REDUCES THE VARIATION OF GENE EXPRESSION LEVELS IN BOVINE CLONES

2006 ◽  
Vol 18 (2) ◽  
pp. 134
Author(s):  
S. Kurosaka ◽  
N. A. Leu ◽  
K. J. McLaughlin
Keyword(s):  
Gene Expression ◽  
Coefficient Of Variation ◽  
Blastocyst Stage ◽  
Average Level ◽  
Cdna Libraries ◽  
Cell Stage ◽  
Glucose Transporter 1 ◽  
Expression Levels ◽  
Gene Expression Levels

Mammalian somatic cell clones frequently exhibit abnormal gene expression that presumably results from errors in reprogramming of the transplanted genome. In the mouse, aggregation of 4-cell stage clones with each other improves reprogramming with respect to Oct-4 expression in blastocysts and an increase in term development (Boiani et al. 2003 EMBO J. 22, 5304-5312). To determine if clone-clone aggregation has a similar beneficial effect in the bovine, we aggregated 8-16 cell bovine clones with each other and profiled gene expression levels in bovine clones and clone-clone aggregates at the blastocyst stage. Clone embryos were produced from fibroblasts and cultured in vitro in SOF supplemented with fetal bovine serum at 39�C in an atmosphere of 5% CO2, 5% O2, and 90% N2. For aggregation of embryos, we first removed the zonae pepellucidae by treatment with 0.5% pronase at the 8-16 cell stage and then placed two zona-free embryos per well into deep microwells produced on the bottom of a culture dish by pressing a heated darning needle onto the surface. Seven to 10 microwells in close proximity were covered by a culture 50-�L drop of culture medium, and embryos were cultured until Day 7. Real-time RT-PCR analysis for Oct-4, DNA methyltransferase 1 (Dnmt1), Dnmt3, glucose transporter 1 (Glut1), Glut3, and Poly(A) polymerase (PolyA) was performed on reusable Dynabead Oligo (dT)25-cDNA libraries synthesized from individual blastocysts at Day 7. In vitro-fertilized embryos were used as controls. To compare the variation of gene expression in each embryo within the group, the coefficient of variation (COV; standard deviation/mean) was calculated. Although spatial distribution of Oct-4 transcript is normal in bovine blastocyst stage clones (Kurosaka et al. 2004 Reprod. Fertil. Dev. 16, 147), we detected disturbances in the level of Oct-4 expression in clones: 44.4% (8 of 18) of clones expressed Oct-4 within a range of 0.5- and 1.5-fold of the average level of expression in IVF embryos, compared to 81.8% (9 of 11) of IVF embryos. Only 22.2% (4 of 18) of clones expressed all genes examined within a range of 0.5- and 2.0-fold of the average level of IVF embryos, versus 45.5% (5 of 11) of IVF embryos. Clone-clone aggregation did not increase the proportion of clones with normal expression levels but did reduce the coefficient of variation of gene expression levels between individual clones for the genes Oct-4, Dnmt1, Dnmt3a and PolyA, but not for Glut1 and Glut3. Interestingly, bovine clone-clone aggregates (n = 25) had less variation between individual embryos compared to IVF aggregates (n = 11) for all genes except Glut1 and Glut3, although variation of single clones was larger than that of single IVF embryos. Analysis of Oct-4 and �-Actin transcripts in mouse clone blastocysts indicated a similar decrease in gene expression variation subsequent to aggregation of mouse clones. These results demonstrate that bovine pre-implantation stage clones exhibit a high degree of variation in gene expression levels and suggest that aggregation of clones is beneficial in reducing the variation in expression of some genes.

58 PRODUCTION OF CLONED BOVINE EMBRYOS DERIVED FROM AMNIOTIC CELLS OF PREGNANT COWS

2008 ◽  
Vol 20 (1) ◽  
pp. 110
Author(s):  
S. Taniguchi ◽  
N. Hayashi ◽  
Y. Abe ◽  
D. Iwamoto ◽  
S. Kishigami ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Lamp Method ◽  
Developmental Capacity ◽  
Grand Island ◽  
Cloned Bovine ◽  
Amniotic Cells

Progeny tests are widely used for selection of sires for beef and dairy cattle. A less costly method might be to clone the sire candidates at their earliest developmental stage possible. To produce cloned bulls, we obtained amniotic cells as donors for nuclear transfer by transvaginal aspiration of pregnant cows. However, the collected cells may include some maternal cells. In this study, we examined collection methods to obtain only fetal cells from the collected fluid. We also examined the developmental capacity of the embryos cloned from these cells. Amniotic fluids were aspirated from pregnant cows by ultrasound-guided aspiration. We collected amniotic fluids from 27 pregnant Japanese black beef cattle (between 58 and 132 days of gestation). In Method 1, cells were recovered from the whole amniotic fluid (approximately 15 mL). In Method 2, the initial 5 mL of aspirated fluid was discarded and then the next 10 mL sample was collected. Cells were recovered from the collected fluids. The cells in the fluids were washed twice by centrifugation and then cultured in AmnioMAX™-II medium (GIBCO, Grand Island, NY, USA). After 3–4 passages, the sex of the cell lines was determined by the loop-mediated isothermal amplification (LAMP) method (Eiken Chemical Co., Ltd., Tokyo, Japan). For the cell lines that were determined as 'male' by the LAMP method we further analyzed the sex of individual cells (137–620 cells of each cell line) by fluorescent in situ hybridization (FISH) using a bovineY chromosome-specific probe (Kobayashi et al. 1998 Mol. Reprod. Dev. 51, 390–394). The percentage of male cells obtained from Methods 1 and 2 were 0–0.4% (from 4 animals) and 93.7–99.5% (from 6 animals), respectively. Then, we used confluent amniotic cells from 3 cell lines obtained by Method 2 as donor cells for nuclear transfer and examined the developmental capacity of the cloned embryos. Bovine fibroblasts cultured under serum starvation were used as a control. The cells were electrically fused (2.7 kV cm–1, 11 µs, 2 times) with enucleated bovine oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF until 168 h post-activation. The data were analyzed with Fisher's protected least-squares difference (PLSD) test following ANOVA. The rates of fusion, cleavage, and development to the blastocyst stage of the cloned embryos were the same as those of the control embryos (78% v. 81%, 75% v. 75%, and 22% v. 27%, respectively; P > 0.05). Furthermore, the rate of male blastocysts derived from the cloned embryos with the three cell lines was 95% (19/20). These results indicate that the amniotic fluids collected from pregnant cows by Method 2 contained fewer maternal cells, and that the embryos cloned from the cells developed in a manner similar to that of embryos cloned from the fibroblasts. This work was supported byWakayama Prefecture CREATE, JST.

142 DYNAMICS OF Tet FAMILY DURING PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 183 ◽  
Author(s):  
J. Teson ◽  
K. Lee ◽  
L. Spate ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Profile ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Dna Demethylation ◽  
Dimensional Structure ◽  
Cell Stage ◽  
Donor Cells

One of the key regulators of gene expression in mammals is DNA methylation. The Tet family (Tet1–3) is suggested to be involved in regulating the level of methylation by hydroxylating a methyl group from 5-methylcytosine to form 5-hydroxymethylcystosine. This hydroxylation alters the 3-dimensional structure of the DNA and results in altered gene expression. Previous studies conducted in the mouse have shown that Tet1 is important for inner cell mass specification by regulating the apparent level of methylation on a specific promoter region in blastocysts and Tet3 is related to the apparent paternal DNA demethylation after fertilization by hydroxylating the paternal genome. The objective of this study was to investigate the expression profile of the Tet family in porcine oocytes and pre-implantation-stage embryos derived from IVF and somatic cell nuclear transfer (SCNT). The RNA was isolated from donor cells, germinal vesicle (GV), MII and 2-cell and blastocyst stage embryos (20 oocytes or embryos per group). Levels of mRNA for each Tet gene were measured by quantitative real-time RT-PCR. The levels of each mRNA transcript were compared to YWHAG, a housekeeping gene that shows a constant level of expression throughout pre-implantation embryo development and normalized to the GV stage. The analysis was repeated with 3 biological replications and 2 experimental replications. Differences in gene expression were compared by ANOVA and P < 0.05 was considered significant. No difference was found in the levels of the Tet family members between GV and MII stage oocytes. Compared with GV stage oocytes, up-regulation of Tet3 at the 2-cell stage was detected in both IVF and SCNT embryos, 4.7 and 6.2 fold, respectively. A dramatic increase in Tet1 was also observed at the blastocyst stage in IVF and SCNT embryos when compared with the GV stage, 65.7 and 79.7 fold increases, respectively. Interestingly, the level of Tet3 was down-regulated in blastocyst embryos at a 25 or more fold decrease compared with GV. The level of Tet2 remained constant throughout embryo development. Embryos (2-cell and blastocyst) compared from IVF and SCNT showed no difference in Tet expression levels. Donor cells had significantly lower levels of Tet2 and Tet3 when compared with GV. Our results indicate that the Tet family shows a dynamic expression profile during porcine pre-implantation embryo development. High expression of Tet3 in 2-cell stage embryos suggests its importance during the post-activation demethylation process. The increase of Tet1 transcript in blastocysts suggests that Tet1 is involved in regulating the type of methylation at the blastocyst stage. These results are consistent with results from previous mouse studies. There was no misregulated expression of the Tet family in SCNT embryos compared with IVF embryos, thus indicating successful reprogramming of the Tet family after SCNT. Lower levels of Tet2 and Tet3 would indicate that Tet1 is important for maintaining type of methylation in donor cells. This is the first report on the profile of the Tet family during porcine pre-implantation embryo development and further studies are needed to clarify their role during this stage.

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