• 한국수정란이식학회지
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• 제22권2호
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• pp.121-126
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• 2007

본 연구에서 돼지 난포란에서 채취된 난모 세포들을 체외성숙 후 형태적으로 선별하거나 극체 방출란을 선별하여 활성화 처리 후 48시간째에 분할란을 선별할 때 배발달율이 어느정도 향상되는지를 검토하였다. 난모 세포를 48시간 성숙 배양 후 형태적 선별과 극체의 방출 유무를 검사하고, 선별된 난모 세포들을 $16{\sim}18$시간 추가 배양한 후 7% ethanol로 활성화시키고 $5{\mu}g/ml$ cytochalasin B에 5시간 노출 후 PZM-5 배 양액으로 7일간 배양하였으며, 배양 중 4일째 5% FBS를 추가하였다. 48시간 성숙 후 형태적으로 선별하였을 때, 21.9%가 제거되고 78.1%가 선별되었으며, 극체 방출란을 선별하였을 때, 32.1%가 제거되고, 67.9%가 선별되었다. 형태적으로 선별한 난자를 활성화 처리하여 48시간째에 분할율을 검사하였을 때, 15.8%가 분할하지 않았으며, 52.6%가 정상 분할하였고, 31.6%가 과분할하였으며, 극체 방출란을 선별하여 활성화 처리 후 분할율을 검사하였을 때 7.1%가 분할하지 않았으며, 73.1%가 정상 분할하였고, 19.8%가 과분할하였다. 체외 성숙된 난모세포를 형태적으로 선별하고 활성화 처리 후 분할란을 선별하지 않았을 때, 16.7%가 배반포기로 발달하였고, 형태적으로 선별하고 분할란을 추가로 선별해 배양했을 때 31.7%가 배반포기로 발달하였으며, 극체 방출란만을 선별하여 활성화 처리 후 분할란을 선별하지 않았을 때 39.0%가 배반포기로 발달하였고, 극체 선별과 분할란 선별을 하였을 때 배반포기 발달율이 49.0%에 이르렀다. 48시간째 미분할 난자와 정상 분할 난자, 과분할 난자를 배양하였을 때 48시간째 미분할 난자는 배반포기로 발달하지 못했으며, 정상 분할 난자는 42.5%, 과분할 난자는 4.5%가 배반포기로 발달하였다. 분할하는 시기를 활성화처리 후 12시간 간격으로 조사하였을 때 $0{\sim}12$시간 사이에 4.1%가 분할하였고, $12{\sim}24$시간 사이에 68.6%, $24{\sim}36$ 시간 사이에 19.1%, $36{\sim}48$시간 사이에 2.3%, 48시간까지 미분할 난자가 5.9%였으며, $0{\sim}12$시간 사이에 분할한 난자나 $36{\sim}48$시간 사이에 분할한 난자에서 배반포기로 발달한 난자는 없었으며, $12{\sim}24$시간 사이에 분할한 난자의 39.1%, $24{\sim}36$시간 사이에 분할한 난자의 9.5%가 배반포기로 발달하였다. 이상의 결과로 극체 방출란만을 선별하여 $12{\sim}36$시간 사이에 분할하는 난자들만을 선별하여 배양한다면 배발생능을 가진 난자들의 비율을 높일 수 있을 것으로 사료된다.

• 생명과학회지
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• 제18권7호
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• pp.952-957
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• 2008

본 실험에서는 THP-1 세포의 PMA에 의하여 유도되는 초기 세포부착에 관한 메카니즘을 이해하기 위하여 다양한 요인(혈청, 신규 단백질의 합성, 세포 골격 저해제, 단백질 인신화 저해제)들의 효과를 조사하였다. 또한 본 실험에서는 이들 세포부착의 정도를 일반적으로 세포증식 분석에 사용되고 있는 SRB염색법을 도입하여 세포부착 분석에 간편한 방법의 조건을 확립하였다. PMA에 의한 초기 세포부착에는 배양액중의 혈청의 유무는 영향이 없었으나, 신규 단백질의 합성이 요구되는 것을 확인하였다. 또한 이들 초기 세포부착에 PMA처리에 의한 PKC의 활성화는 필수적이나, 그 하류 활성화 인자로 잘 알려진 MAP-kinase (erk1/2)의 인산화는 필요치 않음을 알 수 있었다. 흥미롭게도 액틴 중합 저해제인 cytochalasin D의 PMA와 공 처리는 오히려 세포부착을 PMA 단독 처리시 보다 증가시켰다. 또한 본 실험에서 사용된 SRB 염색법을 통한 세포부착 분석법은 최근 암 등 다양한 질환의 신약 표적 분자로 주목을 받고 있는 PKC 저해제의 초기 세포 기반 분석에 매우 유용하리라고 생각된다.

• 한국연초학회지
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• 제26권2호
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• pp.152-158
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• 2004

Among short-term in vitro genotoxicity assays, micronucleus assays are rapid, inexpensive, and less labor-intensive system. We have undertaken a comparative study of sensitivity of cigarette smoke condensate(CSC) by general micronucleus(MN) assay and cytokinesis-block micronucleus(CBMN) assay. In this study, V79 Chinese hamster cells were employed to evaluate and compare the genotoxicity of CSC of Kentucky Reference Cigarette 2R4F by 2 kinds of in vitro MN assay methods. To determine the optimum concentration of cytochalasin B(CYB) to obtain the maximal number of binucleated cells for CBMN assay, triplicate cultures of growing cells were treated with CYB for 15 h. CYB treatments caused a concentration-dependent increase in cytotoxicity($1\~4{\mu}g/mL$) and proportion($0.25\~1\;{\mu}g/mL$) of binucleated cells. These data suggested that 1 ug/mL of CYB is as an optimum dose for CBMN assay in binucleated V79 cells. Short treatment(4 h) of CSC induced a micronucleated cells with a concentration-dependent response in the presence or absence of CYB, but CSC-induced MNs were weakened when S9 was present. Long treatments(19 h) of CSC also induced a significant increase MN formation with a concentration-dependent response. At a concentration of 75 ${mu}g/mL$, the MN cell frequencies of general MN assay and CBMN assay were $6.5\%\;and\;11.7\%$, respectively. Linear regression analysis revealed a good correlation in CBMN assay between a concentration of CSC and MN cell frequency. All these data indicated that CBMN assay is more sensitive to the induction CSC-induced MN than general MN assay.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.313-317
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• 2011

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM $SrCl_2$ in Ca-free CZB medium in the presence of 5 II ${\mu}$g/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6~7 week-old mice(53.3%) than in oocytes from 8~9(80.9%) and 10~11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium(51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6~7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.

• 한국수정란이식학회지
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• 제28권2호
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• pp.133-139
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• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.22-22
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the different fusion and activation conditions as assessments of subsequent development and chromosome in the embryos. New Zealand White rabbits were used throughout the study. Fetal fibroblasts collected from 22-d of fetuses were cultured in DMEM ＋ 10% FBS in 5% $CO_2$ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept In frozen. From rabbits treated with FSH in 30% PVP solution and hCG, oocytes were surgically collected from oviducts at 14 h post-hCG injection and stripped off their cumulus cells by re-pipetting in a 300 IU hyaluronidase solution. Oocytes with an extruded first polar body and dense cytoplasm were enucleated by micromanipulation in Ham's F-10 medium＋7.5 g/$m\ell$ cytochalasin B. Euncleation was confirmed under a fluorescence microscope after staining with 5 g/$m\ell$ bisbenzimide for 2 min. Each enucleated oocyte was injected with a fetal fibroblast into a perivitelline space. Reconstructed eggs were compared fusion rates either at 2.0 ㎸/cm or 1.6 ㎸/cm(60 sec, double pulses). After fusion, all eggs were activated with the combination of 5 M ionomycin (5 min) and 10 g/$m\ell$ cycloheximide (CHX, 3h), and cultured in CRlaa medium and transferred into TCM199＋10% FBS on day 3. Although there was not significantly differ in fusion rate between treatments (60%, 2.0 ㎸/cm vs. 79.4%, 1.6 ㎸/cm), none of them in the eggs fused with 2.0 ㎸/cm developed to blastocyst. In comparison of development and chromosome status between different activation treatments (Group 1; 5 M ionomycin/10 g/$m\ell$ CHX, Group 2; 5 M ionomycin/5 g/$m\ell$ CHX ＋ 2 mM DMAP after fusion with 1.6 ㎸/cm), there were not differ in cleavage and development rates (67.3% and 28.9% in Group 1; 67% and 33% in Group 2). All out of 8 embryos evaluated in Group 1 appeared a normal diploid chromosome sets and mean number of cells (Mean SEM) on day 4.5 of culture was 141.5 23.15 (n=8). It can be concluded that the use of cycloheximide has not happened in chromosome abnormalities, and fetal fibroblasts can be used for cloning in rabbit.

• 한국수정란이식학회지
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• 제18권1호
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• pp.15-25
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• 2003

발생학에서 키메라(chimera)는 2개 이상의 다른 유전자형의 세포, 또는 다른 종의 세포로부터 만들어진 1개의 생물개체를 뜻하는 말로, 이는 초기 수정란의 발달과 포유류의 분화를 연구하는데 이용되고 있다. 키메라를 만드는 방법에는 할구와 내세포괴를 응집시키는 방법과 배반포 내에 여러 종류의 세포를 주입하는 방법이 있다 본 실험에서는 서로 다른 두 가지 방법의 활성화 처리법, 즉, ionomycin 처리 후 Cycloheximide (CHX) 또는 6-Dimetylaminopurine (6-DMAP)에 따른 단위 발생란의 분할과 단계적인 발달율을 살펴 보고자 하였으며, 서로 다른 방법에 의해 생산된 단위발생란 유래의 할구와 체외수정란 유래 할구를 응집하여 키메라 수정란(chimeric embryo)를 만든 후 체외수정란과 발달율을 비교해 보았다. 도축장 유래의 난소에서 난자를 채취하여 체외에서 22~24시간 성숙시킨 후 난구세포를 제거하고 metaphase II 단계의 난자를 5 $\mu$M ionomycin에 4분간 처리한 후, 10 $\mu$g/ml CHX/5 $\mu$g/ml cytochalasin B (CCB)에 5시간 또는 1.9 mM 6-DMAP에 4시간 처리하여 분할율과 배반포기 발달율을 비교 조사하였다. 난자 분할율에서는 체외수정란과 6-DMAP처리 단위 발생란에서 각각가 83.7 와 85.5%로 CHX/CCB 처리 단위발생란의 57.9%보다 유의적으로 높게(P<0.05) 나타났으며, 배반포기 발달율에 있어서는 체외수정란의 발달율이 27.8%로 6-DMAP처리 활성란 12.3%와 CHX/CCB 처리 활성란 5.3%보다 유의적으로 높게 (P<0.05) 나타났다. 키메라 수정란(chimeric embryo)은 서로 다른 두 가지 처리에 의해 생산된 단위발생란의 할구 2개와 체외수정란 유래의 할구 2개를 빈 투명대 내에서 응집시켜 제조하였다 빈 투명대 내에 키메라 수정란(chimeric embryos)의 8 세포기까지의 발달율은, 체외 수정란 할구 2개와 CHX/CCB 처리에 의한 할구 2개를 응집한 그룹은 46.1%, 체외 수정란 할구와 6-DMAP 유래 할구 2개를 응집한 그룹은 52.8% 였으며, handled control은 54.7%로 체외 수정란 77.7%에 비해 유의적으로 낮게(P<0.05) 나타났다. 배반포기까지의 발달율은 체외 수정란과 CHX/CCB에 의해 생산된 키메라 수정란(chimeric embryo)은 12.8%, 체외 수정란과 6-DMAP에 의한 키메라 수정란(chimeric embryo)은 18.8%로 handled control의 21.4%에 비해 유의적으로 낮았으며(P<0.05), 이들 키메라 수정란(chimeric embryos)은 체외 수정란의 34.9%에 비해 유의적으로 낮게(P<0.05)나타났다. 6-DMAP 처리 단위발생이 유기된 수정란 할구 2개와 체외수정란의 할구 2개의 응집에 의한 키메라 수정란(chimeric embryos)의 발달율이 CHX/CCB와 체외수정란의 응집에 의한 키메라 수정란(chimeric embryos)에 비해 다소 높게 나타났으나, 유의적인 차이는 없었다. 본 실험의 결과 서로 다른 방법에 의한 단위 발생란 유래의 할구와 체외 수정란 유래의 할구가 응집에 의한 재조합이 가능하였고 이들을 체외에서 배양하여 배반포기의 수정란까지 발달시켰다.

• 한국수정란이식학회지
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• 제32권3호
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• pp.95-104
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• 2017

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB ($7.5{\mu}g/mL$), CB ($7.5{\mu}g/mL$) + CHX ($10{\mu}g/mL$), CB ($7.5{\mu}g/mL$) +DC ($0.4{\mu}g/mL$), and CB ($7.5{\mu}g/mL$) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

• 한국가축번식학회지
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• 제20권3호
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• pp.239-250
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• 1996

The objective of the present experiments were to determine whether micromanipulative and electro-stimulation conditions for blastomere survival overlapped those for oocyte activation in porcine. Eggs selected for in vitro development potential of blastomeres isolated from 4-cell embryos and oocyte activation by electrostimulation were equilibrated for 5~10 min, in 0.3M sucrose solution containing 7.5$\mu\textrm{g}$/ml cytochalasin B, and then electrostimulated for 30$\mu$sec using one pulse of 100, 120, 150 or 180 volts DC with electrodes 0.2mm apart. Single blastomeres were inserted into empty zona pellucida prior to electrostimulaticn. Then they were cultured in 20${mu}ell$ drops of fresh BECM to observe their developmental ability in vitro in a humidified incubat or at 38.5$^{\circ}C$. The results obtained from these experiments are as follows : 1. When one pulse of 100, 120, 150 or 180 volts DC for 30$\mu$sec were applied to porcine oocytes having the slit formed on zona pellucida for activation, activation rates were 65.1, 66.7, 70.7 and 91.7%, respectively. Higher activation rate was observed in 180V. 2. Infact oocytes incubated for 30 min, in 0.3M sucrose solution after electrostimulation were significantally different from control group with increasing of voltages(p<0.05). When voltages used for electrostimulation were increased, activation rates of oocytes were improved in all treatment groups. 3. When zona punctured-oocytes were only electrostimulated, or incubated in 0.3M sucrose solution for 30 min. after electrostimulation at 180 volt DC, activation rates were 90.5 and 95.5%, respectively. And activation rates of zona punctured-oocytes were significantly different from the groups for which zona pellucida was not punctured(P<0.05). 4. When single blastomeres form 4-cell transferred into empty zona pellucida were incubated for 0, 15 and 30 min. in 0.3M sucrose solution after electrostimulation using one pulse of 180 volt DC for 30 $\mu$sec, developmental rates of electrostimulated-single blastomeres to blastocyst were 72.5, 59.0 and 51.2%, respectively, and the ratio of control group developed to blastocyst were 80.0%. 5. The average cell number in electrostimulated-blastomeres developed to blastocyst were 7.9~10.8, and reduced than the cell number in diploid control ; Also cell number decreased with increasing of voltages. The results of these experiments indicate that the optimal condition for achieving in vitro developmental ability of single 4-cell blastomeres and oocyte activatin is 1 pulse, duration 30 $\mu$sec. in 180 volt, and incubation of blastomeres and oocytes in 0.3M sucrose solution after electrostimulation was not significantally different from another treatment groups. The results also show that this condition is suitable for nuclear transplantation using porcine eggs.

• Reproductive and Developmental Biology
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• 제28권1호
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• pp.13-20
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 $\mu\textrm{g}$/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at $39^{\circ}C, 5% CO_2$ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).