• 한국발생생물학회지:발생과생식
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• 제25권1호
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• pp.43-53
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• 2021

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 µM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.181-184
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• 2010

Osmolarity of culture media is one of the most important factors affecting in vitro development. This study was conducted to investigate the DNA methylation status of Pre-1 and satellite sequence in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaG or 0.05M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. Previous our studies have reported that pNT embryos cultured in both hypertonic media showed significantly higher blastocyst formation rate than that of control. The DNA methylation status of the satellite sequences in blastocyst was characterized using bisulfite-sequencing technology. The satellite region had a similar methylation pattern of in vivo blastocyst among two culture groups excepting the control group. Each level of methylation is that the satellite DNA moderately methylated (43.10% of PZM-3; 56.12% of NaCl; 55.06% of sucrose; 60.00% of in vivo embryos). As a result of the sequence of PRE-1, CpG methylation pattern was similar to three groups, including in vivo group. In case of the satellite DNA region, the osmolarity conditions were affected CpG DNA methylation status while PRE-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. These results indicate that the modification of osmolarity in a culture media may influence to spatially change of DNA methylation of repetitive sequence for pNT embryo development.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.269-279
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• 2013

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2'deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

• 한국가축번식학회지
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• 제22권4호
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• pp.419-424
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• 1998

토끼 수정란의 전핵에 MT-hGH 유전자를 주입하고 핵이식 기법으로 형질전환 복제수정란의 생산효율과 PCR 검색으로 복제수정란에서 유전자존재 여부를 조사한 바 다음과 같은 결론을 얻었다. 1. MT-hGH 유전자를 주입하여 8- 및 16- 세포기로 자란 수정란을 공핵란으로 사용하여 핵이식을 실시하였던 바, 세포융합률은 각각 60.0%, 62.8% 로 비슷하였으나 정상수정란을 공급핵으로 사용한 80.4% 보다 유의적으로 낮은 융합률을 보였다. 그러나 이들 복제수정란의 체외발달률은 처리군간에 유의적인 차이는 인정되지 않았다. 2. 유전자 주입 후 8- 및 16- 세포기로 자란 수정란의 할구를 이용하여 핵이식으로 복제하고 체외에서 배반포까지 자란 수정란을 PCR -screening으로 유전자를 검출한 결과, 각각 23% 와 33% 의 유전자 양성 수정란을 감별하였다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.15-22
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• 2003

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at $38^{\circ}C$ and 5% $CO_2$ in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 ${\mu}m$). Significant differences were observed in cleavage rates ($\geq$3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates ($\geq$3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p<0.05). On the contrary, significantly higher cleavage (43 vs 6%, p<0.05) and developmental rates (14 vs 0%, p<0.05) were observed in the large blastomere nuclear donor group of the 32-cell stage embryos. When the cell cycle stages were controlled by a microtubule polymerization inhibitor (Demicolcine, DEM) or the combined treatment of DEM and Aphidicolin (APH), a DNA polymerase inhibitor, fusion rates were 88-96% for the 16-cell donor group (without DEM treatment), which were greater than the 32-cell donor group (54-58%). Cleavage rates were also greater in the transplants derived from G1 nuclear donor group (93-95%) than those from the DEM and APH combined treatment (73%) for the 16-cell donor group (p<0.05). No significant difference was detected in the morula/blastocyst rates in either donor cell stage (p>0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at $G_1$ phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.

• Asian-Australasian Journal of Animal Sciences
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• 제22권2호
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• pp.168-173
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• 2009

Human follicle-stimulating hormone (hFSH) is a pituitary glycoprotein that regulates follicular development and ovulation. Clinically, hFSH has been used to induce follicular growth in infertile women. The hormone is composed of heterodimers, including a common ${\alpha}$ subunit among the gonadotropin family and a hormone-specific ${\beta}$ subunit. Since assembly of the heterodimer is a rate-limiting step in the production of functional hFSH, transgenic clone cows carrying a single-chain hFSH transgene may efficiently produce functional hormone. Genes encoding the ${\alpha}$ and ${\beta}$ subunits of hFSH were linked using the C-terminal peptide sequence from the ${\beta}$ subunit of human chorionic gonadotropin. Bovine fetal fibroblasts were transfected with the gene construct, including the goat ${\beta}$-casein promoter and a single-chain hFSH coding sequence. Transfected fibroblasts were transferred into enucleated oocytes, and individual nuclear transfer (NT) embryos developed to the blastocyst stage were analyzed for the transgene by polymerase chain reaction. Seventy eight blastocysts (30.8%) were developed from 259 reconstructed embryos. Among these blastocysts, the hFSH gene was detected in 70.8% (34/48) of the embryos. Subsequent transfer of hFSH-transgenic clone embryos to 31 recipients results in 11 (35.5%) early pregnancies. However, all fetuses were lost before reaching day 180 of gestation. The results from this study demonstrated that bovine NT embryos carrying single-chain hFSH could be produced, and further extensive studies in which NT embryos are transferred to more recipients may give rise to single chain hFSH-transgenic cows for biomedical applications.

• 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.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.74-74
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• 2002

Production of u 1-antitrypsin ($\alpha$AT) in transgenic cows has a great value in the field of medicine. The present study was conducted to determine the effect of chemically defined KSOM media on in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human $\alpha$AT was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human $\alpha$AT target gene into a pcDNA3 plasmid. Cumulus cells as donor nuclei in NT were collected from a Holstein cow and transfected by lipid-mediated method using FuGene6 (Roche Molecular Biochemicals, USA) as reagent. GFP expressed cumulus cells were introduced into recipient oocytes under DIC microscopy equipped with FITC filter set. After electrical fusion and chemical activation, reconstructed embryos were cultured in 1) SOF + 0.8% BSA, 2) KSOM + 0.8% BSA, 3) KSOM + 10% FBS and 4) KSOM +0.01% PVA for 192 h at 39$^{\circ}C$ with 5% $CO_2$, 5% $O_2$ and 90% $N_2$in humidified condition. The development of the embryos was recorded and the GFP expression in blastocyst was determined under FITC filter. The average fusion rate was 73.8% (251/340; n=8). The development rates to 2-4 cells, morula, blastocysts and expression rates in blastocysts varied from 70.3 to 76.5%, 30.2 to 33.8%, 25.4 to 33.8% and 11.8 to 15.6%, respectively. The difference in development and expression rates of embryos among 4 culture groups was not significant (P>0.05). This study indicates that chemically defined KSOM medium is also able to support development of bovine transgenic NT embryos at similar rate of SOF or KSOM supplemented with BSA or serum.

• Reproductive and Developmental Biology
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• 제31권1호
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• pp.29-33
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• 2007

본 연구는 소 체외수정란과 체세포 복제란의 초자화 동결 및 응해 후 생존능을 검토하였다. 배반포로 발육된 체외수정란 및 체세포 복제란을 초자화 동결법에 의해 동결하였다가 융해하여 생존율 및 배양 후 부화율을 검사하였다. 체외수정란 배반포를 초자화 동결 응해한 결과, 확장배반포가 배반포기 난자에 비하여 생존율(82.1%, 96/117)과 부화율(64.1%, 75/117)에서 모두 유의적으로 높았다(p<0.05). 핵이식 배반포 복제란을 초자화 동결 응해한 경우도 체외수정란과 비슷한 경향을 보여 확장 및 부화배의 생존율과 부화율이 각각 81.1%(30/37)와 78.3%(29/37)로, 배반포(각각 71.8 및 53.8%)에 비하여 다소 높게 나타났다. 본 연구의 결과는 초자화 동결 방법에 의해서 소 체외수정란과 체세포 복제란을 성공적으로 동결할 수 있으며, 특히 후기 배반포기 단계에서 초자화 동결 시 높은 생존율과 부화배 형성율을 얻을 수 있음을 보여준다.