• 한국수정란이식학회지
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• 제22권4호
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• pp.235-243
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• 2007

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional $10{\sim}14$ h. Control oocytes were denuded after $40{\sim}44$ h of IVM. The size of the perivitelline space was larger at 40 h of IVM ($11.7{\sim}11.8{\mu}m$) than at 30 h ($8.9{\mu}m;$ p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ ($19.4{\mu}m$) and D- oocytes ($18.9{\mu}m$) than in control oocytes ($25.5{\mu}m;$ p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.

• 한국발생생물학회지:발생과생식
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• 제22권1호
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• pp.73-83
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• 2018

This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.223-228
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• 2009

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum-starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or $150\;{\mu}M$ roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine-treated groups (27.6%) than that of the roscovitine-treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine-treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine-treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine-treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine-treatment creates a more suitable condition for nuclear reprogramming after SCNT.

• 한국수정란이식학회지
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• 제25권2호
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• pp.117-125
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• 2010

Several cloned animals have been produced using somatic cell nuclear transfer (SCNT) and have interested in producing the transgenic cloned animals to date. But still its efficiency was low due to a number of reasons, such as sub-optimal culture condition, aberrant gene expression and nuclear reprogramming. The purpose of this study was to analyze gene expression pattern in in vitro fertilized (IVF) or SCNT pre-implantation embryos. IVF- or SCNT-embryos were cultured in media supplemented with different proteins (FBS and BSA) or energy sources (glucose or fructose). Blastocysts from IVF or SCNT were analyzed using semi-quantitative RT-PCR in terms of developmentor metabolic-related genes. Culture medium supplemented different proteins or energy sources had affected on the expression of developmental or metabolic genes in the SCNT blastocysts.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.229-234
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• 2005

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ for 4 h (immediate activation after injection; IAI), or cultured in vitro for $2\~3$ h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation $2\~3$ h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of $36.6\%\;(15/41),\;39.5\%\;(17/43)\;and\;46.3\%$ (25/54), respectively. However, in the ABI group, only one embryo ($1.8\%$, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage $(4.9\%\cdot2/41)$. However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제30권4호
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• pp.585-592
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• 2017

Objective: The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods: Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results: The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture ($143.8{\pm}10.5$ to $159.2{\pm}14.8$) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups ($31.4{\pm}8.3$ to $33.4{\pm}11.1$). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05) between sexes. Conclusion: The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.127-131
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• 2007

Embryonic germ (EG) cells are undifferentiated stern cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of differentiation both in vitro and in vivo have been established. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide inexhaustible source of karyoplasts in nuclear transfer (NT). In this study the efficiencies of NT using porcine EG and fetal fibroblast cells were compared. Two different techniques were used to perform NT. With conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes, the rates of development to the blastocyst stage in EG and somatic cell NT were 16.8% (59/351) and 14.5% (98/677), respectively. In piezo-driven microinjection (Honolulu method) of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 9.4% (9/96), respectively. Regardless of NT methods used in this study, EG cell NT gave rise to comparable rate of blastocyst development to somatic cell NT. Overall, EG cells can be used as karyoplast donor in NT procedure, and embryos can be produced by EG cell NT that may be used as an alternative to conventional somatic cell NT.

• 한국가축번식학회지
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• 제26권1호
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• pp.33-39
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• 2002

소 체세포 핵이식 시 융합액 내 $Ca^{2+}$ 농도에 따른 융합율, 핵형의 변화, 배 발달율을 검토한 결과는 다음과 같다. 1. 융합액 내 $Ca^{2+}$ 농도를 0.05, 0.1, 0.5 및 1.0 mM로 각기 다르게 처리한 결과, 0.5와 1.0 mM의 CaC$_2$에서 융합율이 80.5 와 84.3%로 나타나 0.05 mM CaCL$_2$에서의 융합율 56.6%에 비하여 유의적(P＜0.01)으로 높았다. 2. 융합액 내 $Ca^{2+}$ 농도에 따른 재구축배의 핵형을 검토한 결과, CaCL$_2$농도 0.05와 0.1 mM 에서는 88.4와 84.0%의 난자가 PCC이후 염색질괴를 형성한 반면, 0.5와 1.0 mM 에서는 54.5와 59.3%가 PCC 형태를 거치지 않고 직접 전핵을 형성하여, $Ca^{2+}$ 농도가 증가함에 따라 PCC 형태를 거치지 않고 직접 전핵을 형성하는 난자의 비율이 증가하였다. 3. 핵이식란의 체외 발육율을 검토한 결과, 1.0 mM CaCl$_2$에서는 배반포 발육율이 30.6%로 나타난 반면, 0.1 mM CaCl$_2$에서 는 20.0%로 나타나 유의적(P＜0.05)인 차이를 나타냈다. 본 연구의 결과는 융합액 내 $Ca^{2+}$농도의 증가가 소 체세포 핵이식란의 융합율 및 배반포 발육율을 향상시킬 수 있음을 시사한다.

• Reproductive and Developmental Biology
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• 제31권4호
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• pp.235-240
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• 2007

This study was conducted to examine the viability of Korean native striped cattle (Bos namadicus Falconer, Chikso) clone embryos after embryo transfer. Chikso somatic cell nuclear transfer (SCNT) embryos were produced by fusion of ear skin cells derived from a female Chikso with enucleated oocytes matured in vitro for 18-24 hr. After in vitro culture of SCNT embryos for 7 to 8 days, fresh or vitrified blastocysts derived from SCNT were transferred into a uterine horn of recipient cows. Fifteen of total 43 recipients were pregnant at Day 50 and 4 recipients were maintained to term. Three IVF-derived calves and 1 clone Chikso calf were born. Pregnancy rate was higher when fresh embryos were transferred to recipients compared to vitrified embryos, but development to term was not different between both groups. The clone Chikso calf died at 5 days after birth due to the fullness of amniotic fluid in rumen and the infection of umbilical cord. The result of the present study shows that clone Chikso calf can produced from the embryo transfer of SCNT embryos, however, solution of abortion problem is necessary to improve the cloning efficiency.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.73-73
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• 2002

Nuclear transfer (NT) techniques have advanced in the last years, and cloned animals have been produced by using somatic cells in several species including pig. However, it is difficult that the nuclear transfer porcine embryos development to blastocyst stage overcoming the cell block in vitro. Abnormal segregation of chromosomes in nuclear transferred embryos on genome activation stage bring about embryo degeneration, abnormal blastocyst, delayed and low embryo development. Thus, we are evaluated that the correlations of the frequency of embryo developmental rates and chromosome aberration in NT and In viかo fertilization (IVF) derived embryo. We are used for ear-skin-fibroblast cell in NT. If only karyotyping of embryonic cells are chromosomally abnormal, they may difficultly remain undetected. Then, we evaluate the chromosome aberrations, fluorescent in situ hybridization (FISH) with porcine chromosome 1 submetacentric specific DNA probe were excuted. In normal diploid cell nucleus, two hybridization signal was detected. In contrast, abnormal cell figured one or three over signals. The developmental rates of NT and IVF embryos were 55% vs 63%, 32% vs 33% and 13% vs 17% in 2 cell, 8 cell and blastocyst, respectively. When looking at the types of chromosome aberration, the detection of aneuploidy at Day 3 on the embryo culture. The percentage of chromosome aneuploidy of NT and IVF at 4-cell stage 40.0%, 31.3%, respectively. This result indicate that chromosomal abnormalities are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT associated with lower implantation rate, increase abortion rate and production of abnormal fetuses.