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 << 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.

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

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

MicroRNA-145 Inhibitor Significantly Improves the Development of Bovine Somatic Cell Nuclear Transfer Embryos In Vitro

2016 ◽  
Vol 18 (4) ◽  
pp. 230-236 ◽  
Author(s):  
Wenzhe Li ◽  
Yongjie Xiong ◽  
Fengyu Wang ◽  
Xin Liu ◽  
Yang Gao ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

scholarly journals Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts

2007 ◽  
Vol 75 (2) ◽  
pp. 250-264 ◽  
Author(s):  
Aaron J. Bonk ◽  
Rongfeng Li ◽  
Liangxue Lai ◽  
Yanhong Hao ◽  
Zhonghua Liu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Aberrant Dna Methylation
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