Timing Factors Affecting Blastocyst Development in Equine Somatic Cell Nuclear Transfer

2015 ◽  
Vol 17 (2) ◽  
pp. 124-130 ◽  
Author(s):  
Young-Ho Choi ◽  
Isabel C. Velez ◽  
Beatriz Macías-García ◽  
Katrin Hinrichs
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Development ◽  
Factors Affecting

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 139 ◽  
Author(s):  
S. Mitalipov ◽  
Q. Zhou ◽  
J. Byrne ◽  
W.-Z. Ji ◽  
D. Wolf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Lamin A ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Morula Stage

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

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.

scholarly journals Somatic cell nuclear transfer: failures, successes and the challenges ahead

2019 ◽  
Vol 63 (3-4-5) ◽  
pp. 123-130 ◽  
Author(s):  
Marta Czernik ◽  
Debora A. Anzalone ◽  
Luca Palazzese ◽  
Mami Oikawa ◽  
Pasqualino Loi
Keyword(s):  
Endangered Species ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Transgenic Animals ◽  
Factors Affecting ◽  
Potential Applications ◽  
Mitochondrial Defects ◽  
Species Production

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before.

32 CRYOPRESERVATION OF DONOR CELLS FOR NUCLEAR TRANSFER: EFFECT OF CELL FREEZING METHOD ON THE EFFICIENCY OF SOMATIC CELL NUCLEAR TRANSFER IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
J. Estrada ◽  
E. Lee ◽  
J. Piedrahita
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Development ◽  
Freezing Method ◽  
Donor Cells ◽  
First Polar Body ◽  
Cell Freezing

Donor cell quality is one of the most important factors affecting somatic cell nuclear transfer (SCNT) in mammals. Many studies have been carried out to improve the donor cell characteristics in nuclear transfer, including studies on cell type, cell cycle stage, cell passage, and handling of donor cells before the SCNT. Even though most SCNT work is done with donor cells that have been previously frozen and thawed, no studies have been conducted to evaluate the effect of the cell freezing rate on the SCNT efficiency. The objective of this experiment was to evaluate the effect of the cell freezing method on development of pig SCNT embryos in vitro. Fibroblasts were collected from a 29-day-old female fetus, suspended in DMEM-F12 + 40% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO), and placed in 1.6-mL cryovials for freezing. Vials were randomly assigned to two treatments: In treatment 1, cells were frozen at a controlled rate of 1�C/min in a programmable machine (P) until -40�C, and then plunged into liquid nitrogen (LN2; -196�C). In treatment 2, the traditional system (T), vials were placed in a styrofoam box and left overnight in a freezer at -80�C. The next day samples were plunged into LN2 (196�C). For each treatment, cells were thawed and cultured until confluence before being used for SCNT. Cells were used at passages 2 and 6. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 39 h in TCM 199 supplemented with 10% porcine follicular fluid (pFF), 5 �g/mL insulin, 10 ng/mL epidermal growth factor (EGF), 0.6 mM cysteine, 0.2 mM pyruvate, 25 �g/mL gentamycin and 5 �g/mL each of equine and human chorionic gonadotropin (eCG and hCG). Oocytes were stained with bisbenzimide and enucleated in manipulation media with 7.5 �g/mL cytochalasin B by removing the first polar body and metaphase plate by means of a 16-�m beveled glass pipette. Cells from each treatment were injected into the perivitelline space of recipient enucleated oocytes and fused by two DC pulses of 140 V for 50 �s in fusion media. The fusion rate was evaluated 1 h later, and reconstructed oocytes were activated by two DC pulses of 120 V for 60 �s. After activation, oocytes were placed in bicarbonate-buffered NCSU-13 with 0.4% BSA and cultured at 38.5�C, 5% CO2 in a humidified atmosphere. Embryos were observed for cell cleavage at Day 2, and blastocyst development rate and cell number counting were done at Day 7 of culture. Every experiment was repeated three times. The temperature descending rate for P was slower and more linear (1�C/min vs. 2�C/min) than for the T method. Fusion rate was not significantly affected (P < 0.05) by the freezing method when they were evaluated either individually at each passage or accumulated regardless the passage (78.9 � 3.6% vs. 79.4 � 6.3%) for P and T, respectively. The same trends were observed for cleavage (61.2 � 5.2% vs. 64.3 � 5.2%), blastocyst development (4.2 � 1.8% vs. 5.0 � 2.8%), and number of cells at the blastocyst stage (19.4 � 3.1 vs. 19.8 � 6.2) for P and T, respectively. The present findings indicate that blastocyst development after SCNT does not differ when fetal fibroblasts donor cells are frozen by the two methods tested.

scholarly journals Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e38309 ◽  
Author(s):  
Séverine A. Degrelle ◽  
Florence Jaffrezic ◽  
Evelyne Campion ◽  
Kim-Anh Lê Cao ◽  
Daniel Le Bourhis ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Development ◽  
Embryonic Tissues

91 PRODUCTION OF A CLONED BOER GOAT (CAPRA HIRCUS) BY SOMATIC CELL NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 163
Author(s):  
Y. Tao ◽  
W. Han ◽  
M. Zhang ◽  
J. Ding ◽  
X. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Capra Hircus ◽  
Blastocyst Development ◽  
Tissue Slices ◽  
Boer Goat ◽  
Reconstructed Embryos ◽  
Significant Difference

We reported the birth of a goat clone produced by somatic cell nuclear transfer. The fusion and activation protocols of reconstructed oocytes and embryo transfer procedure were optimized. The donors of somatic cells were fibroblasts derived from ear skin of a Boer goat while the recipient ooplasm was in vitro-matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were activated by ionomycin, 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB) singly or simultaneously (termed as Ionomycin, Ionomycin+6-DMAP, and Ionomycin+6-DMAP+CB). The result showed that the cleavage rate in single ionomycin was significantly lower than that in Ionomycin+6-DMAP and 6-DMAP+CB (34.38% vs. 69.85% and 72.02%; P &lt; 0.05). However, the cleavage rates and blastocyst rates had no significant difference after in vitro culture (P &gt; 0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development rate increased. The reconstructed embryos were equilibrated 1–3 h, 3–6 h, and 6–9 h after fusion, and then activation was undertaken by ionomycin+6-DMAP. We found that the cleavage rates had no significant difference during 1–3 h and 3–6 h (72.58% vs. 72.97%; P &gt; 0.05), but both were significantly higher than during 6–9 h (64.40%) (P &lt; 0.05). A total of 491 reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural estrus. One of those who were treated with hCG after transfer was pregnant and gave birth to a live kid on 153 days. The lamb died accidentally 8 h after birth. The cloned offspring was then dissected and proved well in all organs. Staining of paraffin tissue slices of the viscera suggested that the organs developed well. Microsatellite analysis indicated that the lamb was derived from the somatic cell donor doe genetically.

scholarly journals Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 559-567 ◽  
Author(s):  
Irina Lagutina ◽  
Giovanna Lazzari ◽  
Roberto Duchi ◽  
Silvia Colleoni ◽  
Nunzia Ponderato ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Reconstruction Method ◽  
Blastocyst Development ◽  
Adult Fibroblasts

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

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