• Clinical and Experimental Reproductive Medicine
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• 제34권2호
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• pp.117-124
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• 2007

목 적: 본 연구는 생쥐 전핵시기 배아를 완만동결법과 유리화동결법으로 동결-융해 후 배아의 생존율과 성장률을 비교하고자 시행하였다. 연구방법: 과배란을 유도한 생쥐로부터 전핵시기 배아를 획득하여 10% SSS가 첨가된 HTF 배양액으로 약 1시간 동안 배양한 후 두 개의 전핵이 관찰되는 정상적인 형태의 배아만 선별하여 동결하였다. 동결방법으로는 1.5 M PROH에 0.1 M sucrose가 함유된 완만동결법과 40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 혼합된 EFS40 용액과 EM grid를 이용하여 이용한 유리화동결법을 실시하였다. 동결-융해 후 전핵시기 배아의 회수율, 생존을 및 부화 포배기로의 성장률과 부화율을 비교하였다. 결 과: 각각의 방법으로 동결-융해 후 24시간 동안 배양하였을 때 2-세포기까지의 성장률은 완만동결군이 59.1%이었고 유리화동결군이 77.0%로 두 군간에 유의한 차이를 보였고 (p<0.003), 48시간 동안의 배양에서도 완만동결군이 53.3%이고 유리동결군이 72.6%로 유의하게 유리화동결군에서 높은 수세포기까지의 성장률을 보였으며 (p<0.003), 72시간 배양하였을 때의 상실배로의 성장률 역시 완만동결군이 46.7%이고 유리화동결군이 67.3%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.001). 융해 후 144시간 동안 배양하였을 때의 부화포배기로의 성장률은 완만동결군이 26.3%이고 유리화동결군이 43.4%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.005). 결 론: 생쥐 전핵시기 배아의 동결보존에서 유리화동결법은 완만동결법 보다 시간이 단축되고 비싼 장비가 필요없어 경제적이고 간단했을 뿐 아니라 동결-응해 후 전반적으로 높은 생존율과 성장률을 나타내었다.

• 한국수정란이식학회지
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• 제29권3호
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• pp.273-281
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• 2014

This study was carried out to establish the system of OPU derived embryo production, management of recipients as well as offspring production. OPU derived embryo production system was carried out of aspiration of immature oocytes 2 times per week, total 24 times for 3 months by an ultrasonographic guided follicular aspiration system and then produced in vitro-produced blastocysts by in vitro maturation, fertilization and culture system. This work was collected total 13,866 oocytes, average $8.2{\pm}4.5$ oocytes per session and 8,170 G1 + G2 grade oocytes, average 4.8 oocytes per session by 1,692 times session of total 71 donors for 4 years from 2010 to 2013. The rate of cleavage and blastocyst developmental competence were obtained 11,825 (85.3%) and 5,032 (36.3%) that was $7.0{\pm}3.8$ cleaved embryos and $3.0{\pm}2.5$ blastocysts per session. OPU derived embryo transfer were taken place in 2, 4, 6 and 7 local governments at 2010, 2011, 2012 and 2013 for 4 years and pregnancy rate were obtained 41.2, 43.9, 46.5 and 49.7% in each years. It means that pregnancy rate was continuously improved according of every year for 4 years. Pregnancy rate was significantly different according to individual local government in which was 62.7% in B, but 24.2% in F at 2012. Paternity identification was carried out total 26 offspring in C local government of 2012 and then confirmed 100% agreement of its analysis. In conclusion, the results obtained the possibility of mass production of elite cow embryos as well as offspring by OPU derived embryo production system, of which could be decreased the required time of genetic improvement.

• 한국임상수의학회지
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• 제24권3호
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• pp.295-299
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• 2007

체세포 핵이식을 통한 형질전환 소를 생산 시 초기 수정란 발육 시 발생하는 주요 유전자의 이상 발현을 규명을 목적으로 본 연구를 수행하였다. 형질전환 복제수정란의 낮은 발육능의 원인을 규명하기 위해 하기 위하여 단위 발생란, 체외수정란 및 형질전환 복제수정란에서 초기 배 발육에 중요한 유전자인 IFN-t, Oct4 및 Fgf4유전자의 발현량을 비교 분석하였다. RNA는 각각 10개 수정란에서 추출한 후 reverse-transcription하여 first cDNA를 합성하고 이를 가지고 실시간 중합효수 연쇄반응을 실시하였다. IFN-t유전자의 발현은 형질전환 복제란에서 유의적으로 높게 나왔다 (P<0.05).그러나 Oct4 및 Fgf4 유전자의 경우 형질전환 복제란에서 체외수정란에 비해 유의적으로 낮게 나옴을 확인하였다. 이상의 결과로, 형질전환 복제수정란의 낮은 발육능이 원인이 발육에 중요한 유전자의 이상 발현에 기인된다고 사료된다.

• 한국동물생명공학회지
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• 제34권1호
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• pp.10-19
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• 2019

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: $79.9{\pm}3.8%$ vs G2: $57.5{\pm}4.6%$) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO ($0.1{\mu}M$, MT) treatment (G2: $68.4{\pm}3.2%$ vs G2 + MT: $73.9{\pm}1.4%$). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

• 한국수정란이식학회지
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• 제33권4호
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• pp.211-220
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• 2018

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to $0.1{\mu}M$ SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and $10{\mu}M$ during the first 22 h of IVM to determine a suitable concentration, $0.1{\mu}M$ SNAP (54.2%) exhibited a higher blastocyst formation than 0 and $10{\mu}M$ SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

• Animal Bioscience
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• 제34권4호
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• 한국산학기술학회논문지
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• 제22권3호
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• pp.339-351
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• 2021

본 연구에서는 초, 중, 고 학생의 부모-자녀 간 대화 빈도에 대한 인식 차이에 따른 차별적인 특징을 갖는 유형을 탐색하고, 탐색된 유형에 따라 초, 중, 고 학생의 진로개발역량과 학업적 특성에 차이가 있는지 검증하였다. 이를 위해 초·중등 진로 교육 현황조사(2018) 자료를 활용하였으며, 잠재프로파일분석 방법을 사용하였다. 분석 결과, 초, 중, 고 학생의 부모-자녀 간 대화 빈도에 대한 인식 차이는 초등학생 6개 잠재계층(자녀 학교생활 대화 상, 자녀 상-부모 학교 생활 대화 상, 자녀 하-부모 하, 자녀 상-부모 상, 자녀-부모 학교생활 대화 상, 자녀 하-부모 중), 중학생 6개 잠재계층(자녀 하-부모 하, 자녀 중-부모 중, 자녀 상-부모 하, 자녀 상-부모 중상, 자녀 하-부모 상, 자녀 최상-부모 최상), 고등학생 7개 잠재계층(자녀-부모 학교생활 대화 상, 자녀 상-부모 하, 자녀 중-부모 중, 자녀 중-부모 상, 자녀 하-부모 하, 자녀 상-부모 중상, 자녀 최상-부모 최상)이 존재하는 것으로 확인되었다. 그리고 초, 중, 고 모두 잠재계층에 따라 진로개발역량(자기이해와 사회성, 직업이해, 진로탐색, 진로설계와준비도)과 학업적 특성(자율적 학습 동기, 자기주도학습)에 차이가 있는 것으로 나타났으며, 대체로 부모-자녀 모두 대화 빈도가 높다고 인식한 집단과 부모보다 자녀가 인식한 대화 빈도 수준이 높은 집단의 진로개발역량과 학업적 특성 점수가 높은 것으로 확인되었다. 본 연구는 초, 중, 고 학생은 부모와의 대화 빈도를 부모와 다르게 인식할 수 있으며, 이와 같은 인식의 차이가 진로개발역량과 학업적 특성에 영향을 미칠 수 있음을 시사한다.

• 한국가축번식학회지
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• 제24권4호
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• pp.375-383
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• 2000

본 연구는 공여난소의 황체 형태가 소 미성숙난자의 체외 성숙과 발생에 미치는 영향을 감수분열과 수정란 생산을 비교하여 조사하였다. 공여난소를 황체의 형태에 따라 다음과 같이 4 group 으로 분류하였다. Group 1 ; 최근에 배란한 흔적으로 보이는 혈포가 있는 난소. Group 2 ; 황체는 적색 또는 갈색이며, 혈관이 황체의 가장자리에 국한되어 분포하고 있는 난소. Group 3 ; 황체는 오렌지 또는 황갈색이며, 혈관이 황체의 정상부 까지 분포한 난소. Group 4 ; 황체는 잃은 황색 또는 백색이며, 황체 정상부의 혈관분포가 사라진 난소. 미성숙난자의 체외성숙과 배발생을 위하여 각각 TCM 199과 TLP-PVA을 기본배지로 사용하였다. 각 group에서 채취한 미성숙 난자를 4, 14, 24시간 체외성숙시켰을 때 metaphase I과 metaphase II에 도달한 난자의 비율은 각 group 사이에 차이가 없었으며, 각 group 에서 metaphaseII난자는 체외성숙 후 14시간에 나타나기 시작하여 24시간에 성숙이 종료하였다. 체외 배발생 후 2세포기와 8세포기까지의 발생에는 공여난소 황체의 형태가 영향을 미치지 않았으나, 상실배와 배반포로 발생한 수정란의 비율은 group 1과 3에서 채취한 난자에서 group 2와 4에서 채란한 난자에 비하여 유의적으로 증가하였다 (p＜0.05). 이러한 결과는 공여난소의 번식상태가 체외 수정란생산에 유의적인 영향을 미치는 것으로 암시하고 있다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.

• Animal Bioscience
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• 제36권8호
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• pp.1180-1189
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• 2023

Objective: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. Methods: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. Results: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. Conclusion: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.