80 EFFECT OF MEDIA, SYNCHRONIZATION OF FIBROBLAST CELLS, CULTURE TIME, OXYGEN CONCENTRATION, AND ACTIVATION ON NUCLEAR-TRANSFERRED PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 157
Author(s):  
J. H. Quan ◽  
H. B. Seok ◽  
S. K. Kim
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Atmospheric Condition ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Donor Cells ◽  
The Impact ◽  
Cells Cultured

The purpose of this study was to investigate the impact of culture medium, culture duration, and atmospheric condition on the fusion and in vitro development rates of nuclear transfer porcine embryos constructed by the microinjection of fetal fibroblast cells into in vitro-matured oocytes. Single fetal donor cells were deposited into the perivitelline space of enucleated oocytes, followed by electrical fusion and activation. Activated embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at 38.5�C for 6 to 8 days in 5% CO2 and air. In Experiment 1, the fusion rates of nuclear transfer embryos did not differ from those of fetal fibroblast cells incubated in 5% FBS + NCSU-23 or 5% FBS + TL-HEPES medium, nor did fusion rates of donor cells differ among 1–8-h incubation durations. Fusion rates for the 4 treatment subclasses ranged from 72.1% to 78.0%. In Experiment 2, pre-synchronization in medium containing 0.1 �g mL-1 Hoechst(H) 33342 increased during the period from 0 and 8 h of culture up to 15 h, the end of the synchronization period, at which time there was a significantly increased percentage of porcine fibroblast cells at the G2/M stage (12.4%, 17.5%, and 47.6%; P < 0.01). Neither an increase in the concentration of H 33342 (0.2–1.6 �g mL-1) nor a longer exposure time (12 h, 18 h, and 24 h) increased the proportion of porcine G2/M fibroblasts. In Experiment 3, fusion rates did not differ significantly between nuclear transfer embryos constructed using donor cells cultured in 5% FBS + NCSU-23 medium for 1–2, 6–8, or 12–14 days (60.0%, 73.3%, and 62.5%, respectively). The cleavage rate for nuclear transplant embryos using fetal fibroblast cells cultured for 1–2 days was 44.0%, which was significantly less than the 56.7% and 50.0% for 6–8 or 12–14 days of culture, respectively (P < 0.05). In Experiment 4, the proportions of nuclear transfer embryos that developed to the e2 cell and to the blastocyst stage were not affected significantly by culture medium (5% FBS + NCSU-23 or 5% FBS + TL-HEPES) or by O2 concentration during culture (5% vs. 10%). The developmental rates to the e2 cell stage ranged from 65.9% to 70.1%, and those to the blastocyst stage ranged from 9.8% to 12.5%, for the 4 treatment subclasses. Blastocyst rate was highest for embryos cultured in 5% FBS + NCSU-23 under a gas atmosphere of 5% O2 in air.

scholarly journals 65 PRODUCTION OF PORCINE NUCLEAR TRANSFER EMBRYOS USING FETAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

2005 ◽  
Vol 17 (2) ◽  
pp. 182 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
State Committee ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Functional Quality ◽  
Donor Cells ◽  
Morphological Transformations

One of the most important factors that determine the developmental potential of mammalian cloned embryos is the structuro-functional quality of nuclear donor cells. Biochemical changes that are some of the earliest symptoms of apoptosis signal transduction are not reflected in the morphological features of somatic cells. Therefore, an appropriate system of cell selection would enable the sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cloning. The aim of our study was to examine the in vitro developmental competencies of porcine nuclear transfer (NT) embryos reconstructed with fetal fibroblast cells that had been analyzed for apoptosis by live-fluorescent labelling. Frozen/thawed fetal fibroblast cells, which had been in vitro-cultured to a confluent state, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, a single cell suspension of nuclear donor cells was subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The recipient cells were in vitro-matured oocytes. Maternal chromosomes were removed by a chemically assisted microsurgical technique. Then, single nuclear donor cells were inserted into the perivitelline space of enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with two consecutive DC pulses of 1.2 kV/cm for 60 μs. Reconstructed embryos were in vitro cultured in 50-μL drops of NCSU-23 medium supplemented with 0.4% BSA-V for 6 to 7 days at 38.5°C in a humidified atmosphere of 5% CO2 and 95% air. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of approximately 50 different random samples collected from the population of fetal fibroblast cells, that had been labelled with YO-PRO-1 dye, it was found that a relatively high proportion of donor cells revealed ultrastructural apoptotic changes. The percentage of late apoptotic cells with advanced morphological transformations was about 40% of the total pool of the fibroblast cells. A total of 262/270 (97.0%) enucleated oocytes were subjected to reconstruction and 141/262 (53.8%) were successfully fused with non-apoptotic nuclear donor cells. Following the simultaneous fusion/activation protocol, reconstituted oocytes were selected for in vitro culture. Out of 262, 133 (50.8%) cultured NT embryos cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/133 (36.0%) and 10/133 (7.5%), respectively. In conclusion, morphology is a sufficient selection factor for detection of apoptosis in the cultured (confluent) fetal fibroblast cells to be used for cloning. Moreover, it was found that YO-PRO-1 fluorochrome may be not able to detect the early phases of apoptosis, because only the morphologically abnormal cells emitted the YO-PRO-1-derived fluorescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-KBN-084/P06/2002/4.2 from years 2003 to 2005.

185 NONSURGICAL EMBRYO TRANSFER FOR PRODUCTION OF PIGS BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 251
Author(s):  
J.-G. Yoo ◽  
M.-R. Park ◽  
H.-N. Kim ◽  
Y.-G. Ko ◽  
J.-Y. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Transfer ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Vitro Fertilization ◽  
Miniature Pigs ◽  
Donor Cells

Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.

Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 81-87 ◽  
Author(s):  
P.N. Moreira ◽  
R. Fernández-Gonzalez ◽  
M.A. Ramirez ◽  
M. Pérez-Crespo ◽  
D. Rizos ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Differential Effects ◽  
Glut 1 ◽  
Mouse Somatic Cell

It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMα, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMα, as well as on in vivo cultured and MEMα cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMα cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

185 EMBRYONIC LOSS IN PIGS ASSOCIATED WITH OVIDUCT TRANSPLANTATION OF EARLY-STAGE EMBRYOS WITH DAMAGES IN THE ZONA PELLUCIDA

2006 ◽  
Vol 18 (2) ◽  
pp. 200
Author(s):  
S. Ueno ◽  
M. Kurome ◽  
R. Tomii ◽  
K. Hiruma ◽  
N. Maeda ◽  
...  
Keyword(s):  
Immune Response ◽  
Zona Pellucida ◽  
Nuclear Transfer ◽  
Early Stage ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Embryo Recovery ◽  
Embryonic Loss ◽  
The Impact

It is assumed that if porcine early-stage embryos with damages in their zonae pellucidae are transplanted to the recipient's oviduct, they may suffer from mechanical and immunological stresses by oviduct contraction and the recipient's immune response. This study aimed to examine the impact of zona pellucida damages, which might arise during nuclear transfer and intra cytoplasmic sperm injection (ICSI), on the development and survival of transplanted embryos. Cumulus-oocyte complexes were collected from ovaries obtained at a local slaughterhouse and matured in vitro in NCSU23 to prepare MII-stage oocytes. The zonae pellucidae of these oocytes were either penetrated with 8- to 10-�m square-ended microinjection pipettes or incised with 35- to 40-�m beveled enucleation pipettes. Intact oocytes were used as controls. The oocytes were electroactivated to induce parthenogenesis and transplanted to the oviducts of estrus-synchronized recipient gilts (estrus-synchronized with 1000 IU eCG and 1500 IU hCG). After 5 to 7 days, the recipient uteri were flushed with PBS supplemented with 1% fetal bovine serum (FBS) to collect embryos, and their development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-penetrated, 129 zona-incised, and 57 intact embryos were transplanted to four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (59.0 to 81.8%). However, the zona-penetrated and zona-incised embryos showed inconsistent development and survival compared with controls; the development and survival rate were 92.6% (25/27) to 96.7% (29/30) and 77.8% (21/27) to 96.7% (29/30) in control embryos, respectively, whereas those of zona-penetrated embryos were 57.1% (28/49) to 95.7% (22/23) and 8.2% (4/49) to 78.3% (18/30), and those of zona-incised embryos were 47.6% (30/63) to 92.3% (36/39) and 23.8% (15/63) to 92.3% (22/23), respectively. Large foci of cells that appeared to be macrophage giant cells were observed at the surface or inside of the degenerated zona-damaged embryos. These results indicate that the recipient's immune response may impair development after transplantation of the embryo to the oviduct, when there is damage in the zona pellucida. This may be one of the factors attributable to the reduced efficiency of live progeny production by ICSI and nuclear transfer. This work was supported by PROBRAIN.

42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A

2008 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
J. Li ◽  
Y. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
K. Villemoes ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Embryonic Genome ◽  
Donor Cells ◽  
Deacetylase Inhibitor ◽  
Vitro Development

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells

70 FETAL FIBROBLAST CELLS PERMEABILIZED WITH STREPTOLYSIN O IMPROVE THE RATES OF FUSION AND IN VITRO DEVELOPMENT OF PORCINE RECONSTRUCTED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 152
Author(s):  
K. Naruse ◽  
Y. M. Shin ◽  
Y. S. Quan ◽  
C. S. Park ◽  
D. I. Jin
Keyword(s):  
Cell Number ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Streptolysin O ◽  
Donor Cells ◽  
Developmental Rates ◽  
Vitro Development

Streptolysin O (SLO) is known to bacterial proteins that form very large pores in the plasma membrane of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effect of permeabilization of donor cells using SLO on in vitro development of porcine reconstructed embryos. Porcine fetal fibroblast cells were treated with Ca2+-free DMEM medium containing 200 ng mL−1 of SLO for 50 min before or after trypsinization. Those SLO-treated donor cells were injected into enucleated oocytes, fused with 2 DC pulses (1.2 kV cm−1, 30 µs) and cultured in procine zygote medium-3 (PZM-3) for 6 days. In vitro development of the reconstructed embryos was examined. SLO treatment after trypsinzation significantly increased (P &lt; 0.05) the percentage of fusion rates and blastocyst developmental rates compared with that before trypsinization or in the nontreated group. Additionally there were no significant differences in fusion rates, cleavage rates, blastocyst developmental rates, and total cell number of blastocysts between the SLO-treated group before trypsinzation and the nontreated group. Next, after the trypsinzation treatment, fetal fibroblast cells were incubated in Ca2+-free DMEM containing 200 ng mL−1 of SLO for 0, 30, 50, and 70 min and SLO-treated donor cells were also tested for fusion rate and developmental capability following reconstruction. The 50-min group of SLO-treated cells significantly increased (P &lt; 0.05) the percentage of fusion rates (90.6 vs. 77.6, 85.4, and 78.5%) and blastocyst developmental rates (24.7 vs. 13.5, 11.2, and 13.5%) compared with the other groups (Table 1). However, there was no significant difference in the total cell number of blastocysts among SLO-treated groups. Although cleavage rates the in SLO-treated groups were not significantly different from those of the nontreated group, there the cleavage rates were slightly in SLO-treated groups. In conclusion, permeabilization of porcine fetal fibroblast cells with SLO improves the fusion rates and in vitro development of porcine reconstructed embryos. Table 1.Effects of SLO treatment of fetal fibroblasts by different exposure times on in vitro development of porcine reconstructed embryos

scholarly journals 43 DNA SYNTHESIS, PREIMPLANTATION DEVELOPMENT AND Oct-4 EXPRESSION OF BOVINE CLONES RECONSTRUCTED WITH OOCYTES PREACTIVATED OR ENUCLEATED AFTER SPINDLE DISASSEMBLY

2005 ◽  
Vol 17 (2) ◽  
pp. 171
Author(s):  
S. Kurosaka ◽  
K.J. McLaughlin
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Immunofluorescence Assay ◽  
Donor Cells ◽  
Spindle Disassembly ◽  
Student’S T

Enucleation procedures applied in mammalian cloning remove not only the oocyte's chromosomes but presumably also the spindle-associated factors. If these factors are beneficial for reprogramming, alternative protocols that limit enucleation of factors in addition to the chromosomes may improve cloning efficiency. In this study, we evaluated the enucleation in combination with various activation protocols on clone development and gene expression. Clones produced by nuclear transfer into pre-activated bovine oocytes rather than non-activated oocytes can develop in vitro (Kurosaka et al. 2002 Biol. Reprod. 67, 643–647; Tani et al. 2003 Biol. Reprod. 69, 1890–1894). We produced bovine clones using four different nuclear transfer protocols and, in clones of all groups, examined timing of DNA synthesis in the first cell cycle, pre-implantation development, and gene expression at the blastocyst stage. Protocols applied were: (A) donor cells were fused with non-activated oocytes; (B) donor cells were fused with oocytes at 2 hours after activation with ethanol (7%, 7 min); (C) oocytes were enucleated after spindle disassembly with nocodazole treatment (0.3 μg/mL, 30 min) and donor cells were fused with non-activated oocytes; and (D) oocytes were enucleated after spindle disassembly and donor cells were fused with oocytes at 2 h after activation. Fused couplets in all treatment groups were treated with 10 μg/mL cycloheximide for 6 h, 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. The onset of DNA synthesis was determined by an immunofluorescence assay of 5-bromo-deoxyuridine incorporation at 6 and 9 h post-fusion (hpf). Oct-4 mRNA distribution in clone blastocysts was examined by whole mount in situ hybridization using a bovine Oct-4-specific antisense riboprobe. Data were statistically analyzed with Student's t-test. In the majority of clones DNA synthesis had not commenced 6 hpf but had initiated 9 hpf. Although the cell cycle of activated oocytes (protocols B and D) was 2 hours advanced compared to non-activated oocytes (protocols A and C), clones produced by all protocols had a similar onset of DNA synthesis at 6 to 9 h post-fusion. Developmental rates to the blastocyst stage of clones were not significantly different between the four protocols (48.5%–57.7%, P < 0.05). Oct-4 distribution in clones produced by all four protocols was not different from that of IVF embryos used as a control in that Oct-4 mRNA signal was typically restricted to the ICM (87.0%–100.0%, P < 0.05). We conclude that in bovine clones produced in this study, nocodazole-treated enucleation and activation status of recipient oocyte did not influence the pre-implantation development and spatial pattern of Oct-4 expression. This work was supported by the Lalor Foundation.

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.

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 134
Author(s):  
P. Q. Cong ◽  
E. S. Song ◽  
E. S. Kim ◽  
Z. H. Li ◽  
Y. J. Yi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pulse Number ◽  
Donor Cells ◽  
First Polar Body ◽  
Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

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