• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.1-7
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• 2004

연구 목적: 본 연구의 목적은 마우스에서 배양액의 용량이 배반포 배 형성과 세포수에 미치는 영향을 조사하기 위하여 실시하였다. 연구 재료 및 방법: $3{\sim}4$주령 ICR 암마우스에게 48시간 간격으로 5 IU PMSG와 hCG 주사 후 (hCG 주사 후 수컷과 동숙) $46{\sim}50$시간에 난관으로부터 총 138개의 2-세포기 배를 회수하여 2 ml (group I) 또는 $50{\mu}l$ (group II)의 배양액 (Dulbecco's Modified Eagle Medium + 20% human follicular fluid)에서 72시간 동안 배반포기까지 배양하였다. 배반포 배는 zona-intact (ZiB)와 zona-escape (ZeB)로 등급을 구분하고 나서, propidium iodide와 bisbenzimide를 이용한 differential staining 방법으로 염색하여 평균 세포수, 내세포괴(ICM) 세포수, 영양배엽(TE) 세포수, 총 세포수에 대한 ICM의 비율 (%ICM) 및 ICM:TE 비율을 조사하였다. 결과에 대한 유의성 검정은 $X^2$ test와 t-test를 이용하였으며, p<0.05일 때 통계적인 차이가 있는 것으로 하였다. 결 과: Group I과 II에서, 총 배반포 ($62.3{\pm}20.7%$ vs. $63.8{\pm}22.9%$), ZiB ($31.9{\pm}24.0%$ vs. $30.4{\pm}18.2%$)와 ZeB 형성율 ($30.4{\pm}20.8%$ vs. $33.3{\pm}22.3%$)은 차이가 없었다. 87개의 배반포 배를 염색 시도하였는데, 명확하게 differential staining된 41개의 배반포 배만을 대상으로 세포수를 조사하였다. 평균 세포수 ($61.6{\pm}19.5$ vs. $63.7{\pm}26.8$), ICM 세포수 ($13.0{\pm}10.6$ vs. $12.8{\pm}10.5$), TE 세포수 ($49.0{\pm}19.0$ vs. $47.8{\pm}18.7$), %ICM ($21.0{\pm}12.6%$ vs. $21.1{\pm}13.2%$) 및 ICM:TE 비율 (1:$3.77{\pm}4.9$ vs. 1:$3.72{\pm}4.8$)에서도 group I과 II에서 차이가 없었다. 결 론: 마우스에서 배 발생 능력의 척도로 쓰이는 배반포 배 형성율 배반포 배의 등급 세포수 및 %ICM 등이 20% 난포액을 첨가한 MEM 배양액의 용량에 따라 영향을 받지 않았다.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.203-209
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• 2011

Objective: This study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined. Methods: Eight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes ($Rbm3$, $Birc5$, $Sod1$, $Sod2$, $Cirbp$, $Caspase3$, $Trp53$, $Hsp70.1$) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos. Results: There were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers ($107.0{\pm}21.0$ vs. $115.0{\pm}19.7$), or ICM cell numbers of blastocysts ($11.3{\pm}5.2$ vs. $11.1{\pm}3.7$). Cell numbers of blastocysts were significantly ($p$ <0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of $CirbP$ and $Hsp70.1$ were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences. Conclusion: These results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

• Asian-Australasian Journal of Animal Sciences
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• 제20권3호
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• pp.334-339
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• 2007

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of $CaCl_2$ and 0.1 mM of $MgCl_2$ for $30{\mu}s$ using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of $H_2O_2$ in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and $100{\mu}M$ L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and $100{\mu}M$ L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and $200{\mu}M$ L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the $H_2O_2$ level in developing embryos in a dose-dependant manner. The $H_2O_2$ level (pixels/ embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and $200{\mu}M$ L-ascorbic acid, respectively. So, we recommend to supplement 50 or $100{\mu}M$ L-ascorbic acid in porcine in vitro culture medium.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.213-217
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• 1999

In our previous study, we observed that hydrosalpingeal fluid (HSF) adversely effect mouswe embryo development and hatching. The aim of this study was to evaluate the effect of HSF as assessed by the blastocyst development rate (BDR) and by cell counting in vitro. HSF was collected from ninie patients undergoing salpingoneostomy to correct hydrosalpinx. Two-cell embryos were obtained from superovulated ICR mice. T6 medium and $T6{\pm}0.4%$ bovine serum albumin were used as control media. T6 medium containing 10% or 50% HSF and 100% HSF from each patient were used as test media. Nine to 15 embryos were cultured in micro drops prepared from each of these media. To assess the total cell number within each blastocyst, the blastocysts were fixed and stained with Hoechst 33342 to facilitate cell counting. The mean BDR in two control media were 88.89% and 85.40%. The mean BDR in media containing 10%, 50%, 100% HSF were 85.87%, 89.58% and $75.57%^*$, respectively ($^*$: p<0.05). The overall mean cell count $({\pm}SEM)$ in control media were $87.6{\pm}9.65\;and\;90.12{\pm}11.38$. The BDR was affected adversely only by 100% HSF and not in media containing 10% or 50% HSF. Mean cell counts were decreased significantly only in blastocysts cultured 100% HSF ($63.8{\pm}13.66$; p<0.01) but not in blastocysts cultured in 10% or 50% HSF ($91.3{\pm}12.44\;and\;82.9{\pm}18.27$, respectively). Thus, it is concluded that HSF has no embyotoxic effect but has a mildly negatively effect on embryonic growth and development.

• 한국수정란이식학회지
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• 제30권3호
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• pp.219-224
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• 2015

The purpose of this study is to develop transgenic cell line expressing targeted human granulocyte colony stimulating factor (hGCSF) and green fluorescence protein (GFP) genes as well as production of Somatic Cell Nuclear Transfer (SCNT) embryos derived from co-expressed transgenic donor cells. Constructed pPiggy-mWAP-hGCSF-EF1-GFP vector was chemically transfected into bovine fetus cells and then, only GFP expressed cells were selected as donor cells for SCNT. Cleavage and blastocyst rates of parthenogenetic, SCNT embryos using non-TG cell and hGCSF-GFP dual expressed SCNT embryos were examined (cleavage rate: $78.0{\pm}2.8$ vs. $73.1{\pm}3.2$ vs. $70.4{\pm}4.3%$, developmental rate: $27.2{\pm}3.2$ vs. $21.9{\pm}3.1$ vs. $17.0{\pm}2.9%$). Result indicated that cleavage and blastocyst rates of TG embryos were significantly lower (P<0.05) than those of parthenogenetic and non-TG embryos, respectively. In this study, we successfully produced hGCSF-GFP dual expressed SCNT embryos and cryopreserved to produce transgenic cattle for bioreactor system purpose. Further process of our research will transfer of transgenic embryos to recipients and production of hGCSF secreting cattle.

• 한국수정란이식학회지
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• 제29권3호
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• pp.235-240
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• 2014

생쥐 2세포기, 4세포기, 8세포기를 각 발생 단계에서 채취하여 동결 보호제를 첨가한 서로 다른 배양액에서 배양하고, 배아의 동결 보존 및 융해 시 급속 처리와 저속 처리 단계로 비교하여 이들 조건이 배아의 생존과 발현에 미치는 영향을 조사하여 다음의 결과를 얻었다. 동결 보호제로 처리하여 배양액을 달리한 경우, 급속 단계에서는 모든 배양액에서 비슷한 발생율을 보였고, 저속단계의 4세포기와 8세포기는 D-PBS에서 높은 발생율을 보였다(P<0.05, P<0.01). 배아의 발생 시기에 따른 동결 보존 후, 발생율은 2, 4, 8세포기로 넘어갈수록 발생율의 증가를 보여 8세포기에서 발생율이 가장 높았다(P<0.01). 동결 보호제의 처리단계에 따른 발생율은 2세포기의 급속 단계에서는 유사하였으나, 4세포기와 8세포기는 저속단계에서 높은 발생율을 보였으며(P<0.05), 특히 8세포기에서 가장 높았다(P<0.01).

• 한국수정란이식학회지
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• 제31권1호
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• pp.1-7
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• 2016

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of $IFN-{\gamma}$. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제44권2호
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• pp.90-95
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• 2017

Objective: We investigated whether the insemination method (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]) affected morphokinetic events and abnormal cleavage events in embryonic development. Methods: A total of 1,830 normal fertilized embryos were obtained from 272 IVF and ICSI cycles that underwent ovum retrieval culture using a time-lapse system (Embryoscope) from June 2013 to March 2015. All embryos were investigated by a detailed time-lapse analysis that measured the developmental events in the hours after IVF or ICSI insemination. Results: No significant differences were observed between the two groups regarding clinical outcomes (p>0.05). ICSI-derived embryos showed significantly faster morphokinetics than those derived from conventional IVF, from the time to pronuclear fading to the time to 6 cells (p<0.05). However, no significant differences were found from the time to 7 cells to the time to expanded blastocyst (p>0.05). There were no differences in abnormal cleavage events between the two groups (p>0.05); they showed the same rates of direct cleavage from 1 to 3 cells, 2 multinucleated cells, 2 uneven cells, and reverse cleavage. Conclusion: The morphokinetics of embryo development was found to vary between IVF- and ICSI-fertilized oocytes, at least until the 6-cell stage. However, these differences did not affect the clinical outcomes of the embryo. Additionally, no significant differences in abnormal cleavage events were found according to the fertilization method.