• Asian-Australasian Journal of Animal Sciences
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• 제19권11호
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• pp.1574-1579
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• 2006

This study was performed to determine the developmental potentials of nuclear transfer (NT) embryos using life-span extended cells transfected with a foreign gene as donor cells. A life-span extended bovine embryonic fibroblast cell line was transfected with an expression vector in which the human type II collagen (BOMAR) and ear fibroblasts were used as a donor cell. Cytogenetic analysis was performed to analyze the chromosomal abnormality of donor cells. The fusion rate of 1.8 kV/cm for $15{\mu}sec$ given twice was significantly higher than that of other groups (p<0.05) and the embryos lysed were significantly higher after 1.8 kV/cm for $20{\mu}sec$ given once compared to other groups (p<0.01). The blastocyst development in the ear cell group was statistically significant compared to both BOMAR groups (p<0.01). Both BOMAR groups cultured more than 40 passages (>40 passages) had a lower number of chromosomes; however, fresh granulosa cell (GC) and BOMAR groups cultured less than 20 passages had normal chromosome numbers. Both >40 passages BOMAR groups had numerous obscure debris in metaphase spreads. The transfected foreign gene was expressed in all BOMAR groups, but not in the GC group. Based on these results, the lower developmental potential of NT embryos using life-span extended donor cells transfected with a foreign gene might be a cause of chromosomal abnormality in donor cells.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.69-69
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• 2003

Although effect of TGF$\beta$$_1$ on preimplantation embryo development was reported at mice, little information relevant to this subject is known in bovine. The objectives of this study were to investigate TGF$\beta$$_1$, and TGF$\beta$$_1$ receptors type I and II expression, known as important factors in the embryo development, at unfertilized oocytes and fertilized embryos that will be used as basic data to be compared to NT embryos. We postulated that TGF$\beta$$_1$ may have a beneficial effect on the preimplantation embryo and show different expression patterns as embryo stages change. We have used immunocytochemistry to investigate the presence in unfertilized oocytes and preimplantation embryos of TGF$\beta$$_1$ and the essential components of the TGF$\beta$$_1$ signalling pathway, TGF$\beta$$_1$ receptors type I and II. We found that both receptors, as well as TGF$\beta$$_1$, were present in the unfertilized oocytes. This indicates that TGF$\beta$$_1$, is a maternally expressed protein. At the morulae and blastocyst stages the TGF$\beta$$_1$ receptor type II was not present, but the TGF$\beta$$_1$ receptor type I was present at both stages and we can confirm the TGF$\beta$$_1$ expression of high level at 8-cell stage. These findings support our hypothesis that the TGF$\beta$$_1$, and TGF$\beta$$_1$ receptors may interact with the oocyte and preimplantation embryo, and that TGF$\beta$$_1$ signalling may be important for the development of the oocyte and the preimplahtation embryo.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.175-180
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• 2005

본 연구는 demecolcine 처리에 의한 탈핵과 수핵란 세포질의 세포 주기가 소 체세포 핵이식란의 발육에 미치는 영향을 검토하였다. 체외에서 $16\~20$시간 성숙배양된 난자를 극체 방출 유무 및 MI, MII기 난자로 구분하여 $0.4\;\muL/mL$ demecolcine으로 40분간 처리 후 염색체 부위가 돌출된 난자는 탈핵 후 핵이식에 공시하였다. 소의 귀 피부 세포를 탈핵란에 이식하여 전기융합과 활성화 처리(Ca-ionophore+DMAP)를 거쳐 체외 배양하였다. Demecolcine처리 후 $86.2\%$의 난자가 염색체 부위의 돌출을 보여 이 중 $98.8\%$가 탈핵에 성공하였다. Demecolcine은 핵이식란의 발육에 영향을 주지 않았다. 제1극체 방출란 유래 핵이식란의 배반포 발육율은 극체 미방출란 유래 핵이식란에 비하여 유의적으로 높았다($18.2\%\;vs.\;4.6\%\cdot$, P<0.05). 한편, MI 난자 유래 핵이식란의 분할율 및 배반포 발육율은($69.4\%$와 $5.9\%$) MII 난자 유래 핵이식란에 비하여 유의적으로 낮았다($96.7\%$와 $23.9\%$, P<0.05). 본 연구의 결과는 demecolcine 처리가 소 난자의 탈핵에 매우 효과적이며 MII기 난자가 MI기 난자에 비하여 수핵란 세포질로 더 적절하나 극체 미방출란 및 MI기 난자도 비록 제한적이기는 하지만 핵이식란의 배반포 발육을 지원할 수 있음을 보여준다.

• 한국수정란이식학회지
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• 제27권2호
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• pp.113-119
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• 2012

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.228-228
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• 2004

Interferon-tau (IFN-τ) is the primary agent responsible for maternal recognition of pregnancy in cattle. Bovine embryos begine to express IFN-τ as the blastocyst forms. Pregnancy recognition in ruminants occurs when IFN-τ from the trophoblast prevents the increase of oxytocin receptors, disrupting luteolytic pulses of prostaglandin (PG) F2a by oxytocin. The expression of IFN-τ is strongly associated with the degree of methylation of the CpG islands in promoter region. (omitted)

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.138-138
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• 2005

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.138-138
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• 2005

• 한국가축번식학회지
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• 제26권3호
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• pp.235-243
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• 2002

본 연구는 몇 몇 포유류로부터 얻은 공여세포핵의 proliferation을 돕기 위한 수핵난자로 소 난자의 세포질을 사용하였으며, 공통 세포질로써의 능력을 시험하였다. 소 난자와 소, 사람, 돼지, 생쥐의 체세포와의 결합에서 유래한 각 종별 핵이식란의 융합율, 분할율, 배발달률 그리고 염색체 수를 조사하였다. 1. 소, 돼지, 생쥐, 사람 각각의 체세포를 이용한 핵이식란의 융합율은 70.2% 70.2% 72.4%와 63.0 %로 유의차를 보이지 않았다. 2. 소, 돼지, 생쥐, 사람 각각의 체세포를 이용한 핵이식란의 체외분할 ($\geq$2cell cleavage)율 또한 60.6%, 63.7%, 54.1%와 62.7%로 유의차가 없었다. 3. 각 종별로 체외분할이 일어난 시기를 조사한 결과 종에 관계없이 대체로 활성화 처리 후 24시간째에 대부분 일어나는 것을 볼 수 있었다. 그러나 점차적으로 분할이 계속 이루어지면서 발달시기가 종 특이적인 특성을 나타내는 것을 볼 수 있었다. 4. 이종간 핵이식의 후기 배발달 (상실배와 배반포) 률을 살펴보면 소와 사람의 체세포를 사용했을 경우 17.5%, 4.3%의 결과를 나타내었으며, 돼지와 생쥐의 체세포를 사용한 경우 16세 포기 이후 단계에 발달중지 현상을 볼 수 있었다. 5. 4~8 세포기 정도의 각 종별 핵이식란 할구를 분석에 사용하였을 때, 공여핵으로 사용된 종의 염색체 수와 일치하는 결과를 볼 수 있었다. 이상의 결과를 종합해 볼 때 성숙한 소 난자의 세포질은 소뿐만 아니라 돼지, 생쥐, 사람과 같은 다양한 포유류의 핵을 사용하여 핵이식을 하였을 때에도 발달이 가능하다는 것을 보여주고 있다.

• 한국수정란이식학회지
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• 제20권2호
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• pp.105-112
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• 2005

본 연구는 재래산양에서 복제 수정란의 생산효율을 향상시키기 위한 기초 자료를 제시하고자 체세포 핵이식을 실시하여 공핵세포의 종류, 핵이식란의 활성화 처리 방법 및 수핵난자의 조건이 체외발달율에 미치는 영향을 조사, 검토하여 핵이식란 생산을 위한 최적의 조건을 규명하고자 실시하였다. 공핵세포의 종류에 따른 핵이식란의 체외발달율은 융합이 이루어진 핵이식란의 활성화 처리 후 분할율은 귀 유래 섬유아세포를 공핵세포로 사용하였을 때가 $40.5\%$로서 태아 유래 섬유아세포를 공핵세포로 사용하였을 때의 $55.5\%$와 유의적인 차이가 없었다. 또한 상실배 또는 배반포기로의 발달율도 각각 $6.7\%$ 및 $16.0\%$로서 유의적인 차이가 없었다. 핵이식란의 활성화 방법에 따른 체외발달율은 ionomycin+6-DMAP 처리를 하였을 때 분할율은 $79.0\%$로서 전기자극을 주었을 때의 $9.5\%$보다는 유의적(P<0.05)으로 높았다. 상실배 또는 배반 포기로의 발달율도 ionomycin+6-DMAP 처리를 하였을 때는 $15.6\%$가 발달하였으나, 전기 자극을 주었을 때는 4-세포기 이후로의 발달이 전혀 이루어지지 않았다. 체세포 핵이식란은 단위발생란에 비하여 분할율$(66.1\%\;vs\;59.18\%)$ 및 상실배 또는 배반포배로의 발달율$(19.0\%\;vs\;0.0\%)$이 유의적 (P<0.05)으로 낮았다. 단위발생란의 분할율은 체내 성숙난자에서 $86.8\%$로서 난포란의 $69.0\%$보다는 유의적(P<0.05)으로 높았다 단위발생란의 상실배 또는 배반포기로의 발달율에 있어서도 체내 성숙난자$(50.0\%)$가 난포란$(23.6\%)$보다 유의적 (p.<0.05)으로 발달율이 높았다. 이상의 결과로 볼 때 재래산양의 체세포를 이용한 복제수정란의 생산효율을 향상시키기 위해서는 다수의 난자 확보를 위한 과배란처리 방법의 개선, 난포란의 이용효율 개선 및 활성화 처리방법 등이 확립되어야 하며, 후기배로의 발달율 향상을 위해서는 최적의 체외 배양조건 확립이 시급한 것으로 생각된다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.