28 GENERATION OF REACTIVE OXYGEN SPECIES IN BOVINE CULTURED SOMATIC CELLS AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DURING MICROMANIPULATION PROCEDURES AND EARLY IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 120 ◽  
Author(s):  
H. K. Bae ◽  
J. Y. Kim ◽  
I. S. Hwang ◽  
C. K. Park ◽  
B. K. Yang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Serum Starvation ◽  
Ros Generation ◽  
Oh Radical ◽  
Oxygen Species ◽  
Donor Cells

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in the donor cells, recipient oocytes, and somatic cell nuclear transfer (SCNT) embryos during nuclear transfer procedures. Bovine ear skin cells were classified by serum starvation, confluence, and cycling cells. Bovine metaphase II (MII) oocytes matured in vitro for 22 h and denuded by vortexing were enucleated and electrofused with serum-starved donor cells, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture for 4 h. In vitro fertilization (IVF) was performed for controls. SCNT and IVF embryos were cultured in CR1aa supplemented with 3 mg mL–1 BSA for ∼36 h. Donor cells, recipient oocytes, and SCNT embryos were stained in 10 μM dichlorohydrofluorescein diacetate (DCHFDA) or 10 μM HPF dye each for 30 min at 39°C to measure the H2O2 or ·OH radical levels after various micromanipulation steps. SCNT and IVF embryos were also stained at the 1-, 2-, and 4-cell stages after 8, 24, and 42 h of fusion or insemination, respectively. The fluorescent emissions from the samples were recorded as JPEG file using a digital camera (F5.0, 4 s) attached to a fluorescent microscope with filters at 450 to 480 nm for excitation and at 515 nm for emission. The images were analysed using ImageJ software 1.37 (NIH) by the intensity of fluorescence (pixels) in each cell (total 70 to 75 cells in each group), oocyte and embryo (total 50 to 60 eggs or embryos in each group). 4 to 7 replicates were performed for each experiment, and data were analysed by Duncan′s multiple-range tests. H2O2 and ·OH radical levels of cultured somatic cells were high in confluence group and significantly low in serum starvation group (P < 0.05). During micromanipulation, H2O2 levels in recipient oocytes and SCNT embryos were increased by enucleation (37.2 pixels), electrofusion (49.7 pixels), and activation (40.6 pixels) treatments (P < 0.05) compared to that in MII oocytes (33.1 pixels), and the level of H2O2 was extremely increased immediately after electrofusion. ·OH radical levels were significantly higher during manipulation procedures (51.6 to 55.7 pixels; P < 0.05) compared to MII oocytes. During in vitro culture, the H2O2 and ·OH radical levels of SCNT embryos were significantly higher (P < 0.05) compared to IVF embryos at 1- (32.4 v. 17.3 and 52.0 v. 29.6 pixels, respectively), 2- (27.2 v. 22.0 and 33.4 v. 26.0 pixels, respectively), and 4-cell (25.1 v. 16.5 and 26.9 v. 20.7 pixels, respectively) stages. These results suggest that the culture type of donor cells can affect the ROS generation level and the cellular stress during micromanipulation procedures also can generate the ROS in bovine SCNT embryos, which may lead the cellular damages in bovine SCNT embryos. This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008–313-F00067).

scholarly journals 44 IMPROVED DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS BY THE TREATMENT OF NUCLEAR DONOR CELLS WITH APOPTOSIS INHIBITORS DURING SERUM STARVATION

2005 ◽  
Vol 17 (2) ◽  
pp. 171
Author(s):  
C.-K. Lee ◽  
Bon-sik Koo ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
D.-H. Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Tunel Assay ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Embryonic Fibroblasts ◽  
Crown Rump Length ◽  
Donor Cells

In somatic cell nuclear transfer, serum starvation is a widely used method to synchronize donor cells at the quiescent stage (Go) of the cell cycle. However, it has been shown that serum starvation during culture of mammalian cells could induce cell death via apoptosis by removing growth factors and increasing intracellular stress. Therefore, apoptosis caused by serum starvation in somatic cells could induce damages to nuclear DNA contributing to a lower efficiency of nuclear transfer. This study was performed to characterize apoptosis during serum starvation of bovine embryonic fibroblasts (BEFs) and to determine the effects of BEFs treated with apoptosis inhibitors on the development of bovine embryos after nuclear transfer. BEFs, collected from a fetus with a 3–4-cm crown-rump length, were cultured for 7 days in starvation medium consisting of Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum to induce quiescence. Cells were also placed in starvation medium containing the apoptosis inhibitors, β2-macroglobulin (broad-range protease inhibitor: MAC; 1.4 pM) and glutathione (GSH: reactive oxygen species scavenger; 2.0 mM). Apoptosis of serum starved BEFs with or without apoptosis inhibitors were analyzed morphologically with light and electron microscope, and biochemically using a TUNEL assay. Somatic cell nuclear transfer was performed by our standard procedure as follows. Bovine oocytes were matured in vitro and enucleated after 22 h. Nuclear donor cells were collected randomly before injection. The reconstructed embryos were placed into the fusion chamber in a solution containing 0.28 M mannitol and aligned manually. A double pulse of 1.8 kV/cm for 15 μs was used to fuse the cells and activate the embryos simultaneously. The fused embryos were cultured for 4 min in 5 μ­M ionomycin and 4 h in 2 mM 6-DMAP. Then, embryos were moved to culture media and cultured in 5% CO2 and 39°C in 100% humidity. Development of NT embryos was recorded at 120 h post NT (morulae) and 168 h (blastocysts) with experiments being repeated three times. Serum starved BEFs showed typical morphology of apoptotic cells such as chromatin condensation and membrane blebbing. Also, when stained for DNA fragmentation by TUNEL assay, 22.6% ofBEFs showed apoptosis, in contrast to 0.1% in actively growing cells. However, when BEFs were cultured with MAC and GSH, the proportions of apoptotic BEFs were greatly reduced, 6.0% and 2.1%, respectively. After nuclear transfer with BEFs, embryos reconstructed with BEF treated with apoptosis inhibitors showed significant improvement in in vitro development compared to the controls (Table 1). In conclusion, while there are a number of factors affecting the nuclear transfer procedure, damage to the donor nuclei by serum starvation is likely to reduce the efficiency of the procedure; the addition of apoptosis inhibitors could reduce this unnecessary damage to donor nuclei and result in improvement in the development of nuclear transferred embryos. Further experiments are needed to assess the effect of apoptosis inhibitors on improvement of overall nuclear transfer efficiency. Table 1. Development of bovine embryos nuclear transferred with embryonic fibroblasts treated with or without apoptosis inhibitors

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 &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 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.

73 RE-CLONING BY SOMATIC CELL NUCLEAR TRANSFER FROM A CLONED KOREAN NATIVE GOAT

2007 ◽  
Vol 19 (1) ◽  
pp. 154
Author(s):  
J. K. Park ◽  
S. Y. Jung ◽  
S. H. Park ◽  
A. N. Ha ◽  
J. I. Jin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Pregnancy Rate ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser System ◽  
Serum Starvation ◽  
Factors Affecting ◽  
Estrous Synchronization ◽  
The Difference

Nuclear transfer (NT) is one of the most advanced technologies to increase animal efficiency in livestock production. Re-cloning can be utilized to investigate more effective methods for agricultural, biological, and medical research. However, few studies have been undertaken on re-cloning from cloned animals. The present study was conducted to examine some factors affecting in vitro development of re-cloned embryos and pregnancy by somatic cell nuclear transfer (SCNT). Ear fibroblast cells as karyoplast donors were isolated from a cloned Korean goat, Jinsoonny, 3 weeks after birth and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. Recipient oocytes were surgically collected by flushing the oviducts at 35 h after hCG injection from FSH-stimulated goats. The zonae pellucidae of the oocytes were partially drilled using a laser system and each somatic cell was individually transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at 39�C in an atmosphere of 5% CO2, 5% O2, 90% N2 for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients. Estrous synchronization was induced by 10 days of treatment with a CIDR and a single injection of PGF2�. Pregnancy was diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion and cleavage rates of re-cloned oocytes were 87.5% (182/208) and 56.0% (102/182), respectively. A total of 175 re-cloned embryos were transferred into 28 recipients. Eleven (39.3%) and 4 recipients (14.3%) were confirmed pregnant on Days 21 and 60, respectively. In comparison of pregnany rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were confirmed pregnant on Days 21 and 63, while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. There were no differences in pregnancy rate when the recipients were in estrus within 12 h of the donors (40 to 60%). However, the pregnancy rate was significantly decreased when the difference was greater than 24 h (0 to 35%; P &lt;&lt; 0.05). Re-cloning can be used for many purposes, and synchronization between donors and recipients may be an important factor for further success of nuclear transfer.

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.

24 DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF EMBRYONIC CULTURED CELLS ISOLATED FROM SOMATIC CELL NUCLEAR TRANSFER BLASTOCYSTS

2008 ◽  
Vol 20 (1) ◽  
pp. 92
Author(s):  
X. J. Bai ◽  
J. L. Yu ◽  
M. Murakami ◽  
Y. Zhang ◽  
Y. J. Dong
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cultured Cells ◽  
Es Cells ◽  
Donor Cells ◽  
The Mean ◽  
Development In Vitro ◽  
Chi Square Analysis

Embryonic stem (ES) cells derived from somatic cell nuclear transfer (NT) bovine embryos would increase the utility of the cow as a large animal model for human cell therapy. They would also be useful for studies of cell differentiation. Such cells exhibit full pluripotency, and cloned offspring were obtained from them following a second NT in mice, indicating that the reprogramming that produced pluripotent ES cells could be reversed (Wakayama et al. 2001 Science 292, 740–743). The objective of this study was to examine if there would be any beneficial effects of using somatic cell NT-derived embryonic cultured cells as donors for cloning in cattle. Cloned embryos were produced from a single cell line of bovine fetal fibroblasts (FF) and adult ear-tip cells (AEC) (passages 1 to 5) by NT, as described previously (Dong et al. 2004 Asian–Aust. J. Anim. Sci. 17, 168–173). NT embryos that reached the blastocyst stage were cultured separately to isolate embryonic cultured cells derived from FF (NT-FF) and AEC (NT-AEC) according to previous methods (Dong et al. 2003 Acta Genet. Sin. 30, 114–118). More than 80% of the generated embryonic cultured cells stained positive for alkaline phosphatase. Embryonic cells cultured for 7 to 35 days were used as the donor cells for NT in the NT-FF and NT-AEC groups. Cloned embryos were produced using individual cell lines of FF, AEC, NT-FF, and NT-AEC (passages 1 to 5, putative cell cycle stage of G0 or G1) as donor cells, and their development in vitro is summarized in Table 1. The FF and AEC groups include data from the initial round of NT. The rates of fusion and embryo development were compared by chi-square analysis. Duncan's multiple range test was used to compare the mean cell numbers of blastocysts. The percentage of embryos that developed into blastocysts was significantly higher (P < 0.05) in the FF group than in the AEC group. Interestingly, we observed that the developmental potential in vitro and the mean cell number of blastocysts tended to be higher in the NT-FF and NT-AEC groups than in the FF and AEC groups. A total of 15 and 6 good quality Day 7 embryos in the NT-FF and NT-AEC groups were nonsurgically transferred to 5 and 3 synchronized recipients (2 to 3 embryos/female), respectively. On Day 30 of gestation, 3 (60%) and 1 (33%) females in the NT-FF and NT-AEC groups, respectively, were diagnosed as pregnant via ultrasonography. One (20%) recipient cow in the NT-FF group remained pregnant at Day 60 of gestation, but lost the pregnancy by Day 90. These results suggest that cloning of bovine embryonic cultured cells generated from fetal and adult somatic cells by NT can produce transferable embryos and initiate pregnancies, although none of the pregnancies has developed beyond the first trimester at this time. Table 1. Development in vitro of bovine NT embryos produced from different donor cell types

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.

35 USE OF METAPHASE DONOR CELLS AND ACTIVATION WITH ROSCOVITINE FOR SOMATIC CELL NUCLEAR TRANSFER IN BOVINE

2017 ◽  
Vol 29 (1) ◽  
pp. 125
Author(s):  
G. V. Landschoot ◽  
V. Savy ◽  
N. Canel ◽  
S. Ferraris ◽  
D. Salamone
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
M Cells ◽  
In Vitro Development ◽  
Donor Cells ◽  
G1 Cells ◽  
Vitro Development

Cloning of domestic species by somatic cell nuclear transfer (SCNT) continues to be inefficient, probably due to an incomplete reprogramming of the reconstituted embryo. The ability of the embryonic cytoplasm to support reprogramming fluctuates within the cell cycle (Egli et al. 2007 Nature 447, 679–85). In this context, we compared the development capability and second polar body (2PB) extrusion of embryos produced by metaphase (M) cells, in comparison with G0/G1 cells, which are commonly used as nuclear donors. Because M cells have 2 sets of chromosomes (in contrast with G0/G1 cells, which have only 1 set), an activation protocol that impedes 2PB extrusion is required to produce reconstituted embryos with the correct ploidy. Therefore, we performed SCNT with M or G0/G1 cells, followed by different activation protocols, and evaluated in vitro development and 2PB extrusion of the reconstituted embryos. Cow oocytes were in vitro matured and enucleated as described by Gambini et al. (2014 PLoS One 14, 9). A group of cells at 70 to 80% confluence was synchronized in M stage using 0.05 μg mL−1 demecolcine for 3 to 4 h and used as nuclear donors for SCNT (M group). Another group of cells was induced into quiescence by serum starvation for 3 to 4 days before SCNT (G0/G1 group). For activation, reconstituted embryos were treated with 5 µM ionomycin (Io) for 4 min followed by 5-h incubation in 50 μM roscovitine for M group, or in 50 μM roscovitine and 5 μg mL−1 cytochalasin B for G0/G1 group. Parthenogenetic controls were activated with Io followed by 50 μM roscovitine alone (ROSCO) or with 5 μg mL−1 cytochalasin B (ROSCO/CB). Hoescht 33342 staining was performed 16 h post-Io to evaluate 2PB extrusion. Other activated oocytes were cultured in SOFaa medium and rates of cleavage, morulas, and blastocysts were evaluated at Days 2, 5 and 7 of in vitro development, respectively. Data were analysed by Fisher’s exact test (P < 0.05). Rates of 2PB extrusion were 72.72 (n = 33), 65.63 (n = 32), 80 (n = 15), and 42.86 (n = 14) for M, G0/G1, ROSCO, and ROSCO/CB, respectively. Results of in vitro development are shown in Table 1. In conclusion, somatic M cells can be used as donors to produce cloned embryos. The M and G0/G1 groups were able to induce cloned blastocysts, even though rates did not differed statistically from controls groups (ROSCO and ROSCO/CB). The M group was as effective as G0/G1. Although further analysis is required to establish the quality of the embryos, our results are encouraging for use in SCNT. Table 1.In vitro development of NT embryos produced with M and G0/G1 donor cells

29 BIRTH OF A FOAL CLONED BY ADULT SOMATIC CELL NUCLEAR TRANSFER WITH ROSCOVITINE-TREATED DONOR CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
Y. H. Choi ◽  
Y. G. Chung ◽  
D. D. Varner ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Direct Injection ◽  
Donor Cell ◽  
Mixed Gas ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Significant Difference

Only one horse foal produced from adult somatic cell nuclear transfer has been reported in the scientific literature (Galli et al. 2003 Nature 425, 680); a second foal from the same laboratory was reported in the popular press in 2005. In these reports, the blastocyst rates were 3 and 17%, and efficiency to birth of a live foal from total reconstructed oocytes was 0.1 and 0.5%, respectively. In cattle, roscovitine treatment of donor cells has been associated with a decrease in blastocyst development, but an increase in live births (Gibbons et al. 2002 Biol. Reprod. 66, 895-900). The present study was performed to determine the effect of roscovitine treatment of donor cells on blastocyst production after equine nuclear transfer and to evaluate the viability of pregnancies established via this treatment. In Experiment 1, fibroblasts were either grown to confluence or treated with 15 �g/mL roscovitine, for 24 h. Enucleated in vitro-matured oocytes were reconstructed by direct injection of fibroblasts using a piezo drill. Recombined oocytes were activated by injection of stallion sperm extract, followed by culture in the presence of 2 mM 6-DMAP for 4 h. They were then placed in culture in DMEM/F-12 with 10% fetal bovine serum (FBS) under mixed gas for 8 days and evaluated for blastocyst development. In Experiment 2, oocytes recombined with either confluent or roscovitine-treated donor cells were activated as above either alone or with the addition of 10 �g/mL cycloheximide at the time of 6-DMAP treatment. Resulting blastocysts from Experiment 2 were transferred transcervically to the uteri of recipient mares. One embryo was transferred per mare. In Experiment 1, there was no difference in rates of cleavage (73-19%) or blastocyst development between confluence and roscovitine treatments (2/55, 3.6% vs. 2/56, 3.6%, respectively). In Experiment 2, there was no significant difference in rates of cleavage (78-18%) or blastocyst development (0-1%; 4/105, 0/104, 0/106, 2/108) among donor cell or activation treatments. Six blastocysts were transferred to mares: two from confluent donor cells and four from roscovitine-treated donor cells. One mare, which received an embryo from the roscovitine donor/6-DMAP treatment, established pregnancy after transfer. The pregnancy continued normally and the mare delivered a colt with minimal assistance on Day 389. Typing for 13 equine microsatellites confirmed that the colt was of the same DNA type as the donor fibroblasts. The colt has grown and developed normally. Results of these studies show that roscovitine treatment of equine donor cells does not negatively affect the proportion of recombined oocytes that progress to the blastocyst stage. A viable colt resulted from an embryo produced with roscovitine-treated donor cells. More work is needed on methods to increase blastocyst rates after nuclear transfer in this species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

Analysis of Heterogeneous Mitochondria Distribution in Somatic Cell Nuclear Transfer Porcine Embryos

2008 ◽  
Vol 14 (5) ◽  
pp. 418-432 ◽  
Author(s):  
Zhisheng Zhong ◽  
Yanhong Hao ◽  
Rongfeng Li ◽  
Lee Spate ◽  
David Wax ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Mouse Fibroblast ◽  
Mitochondrial Morphology ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Donor Cells

AbstractWe previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro–fertilized (IVF) embryos (Katayama, M., Zhong, Z.-S., Lai, L., Sutovsky, P., Prather, R.S. & Schatten, H. (2006). Dev Biol299, 206–220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Sign in / Sign up

Export Citation Format

Share Document