• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.62-62
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• 2001

Development of effective activation protocols is of great importance for improving the success of cloning and subsequent transgenic. Three methods for oocyte activation, including 5μM ionomycin (5 min) alone, ionomycin＋1.9 mM 6-dimetylaminopurine (DMAP, 3 hrs) and ionomycin＋10㎍/㎖ cycloheximide(CHX, 3 hrs) were compared for their effects of pronuclei(PN) formation, development, developmental velocity and ploidy of parthenotes to IVF control in bovine. In group of ionomycin＋DMAP, the oocytes having more 3 PN were significantly(P〈0.05) higher than in groups of ionomycin alone and of ionomycin＋CHX (45.5% vs. 0 and 0%, respectively). Activation with the ionomycin alone, ionomycin＋DMAP and ionomycin＋CHX resulted in cleavage rates of 30, 85.5 and 57.9%, respectively. The blastocysts rate of parthenotes activated by ionomycin＋DMAP treatment was significantly higher (12.3%, P〈0.05) than those of other treated groups. Chromosome analysis shows that ionomycin＋DMAP treatment greatly increases the incidence of chromosomal abnormality of the parthenotes. When compared the developmental velocity at 24 hrs after insemination and activation, 27% eggs in IVF control and 55% in DMAP treatment out of total cleaved eggs developed to 2-cell stage, respectively. Developmental velocity of parthenotes activated by ionomycin ＋DMAP treatment was significantly (P〈0.05) faster than others. From the results, we may conclude that DMAP treatment to the oocytes accelerates developmental velocity resulting in both the higher incidence of chromosome abnormality and of PN formation suggesting that CHX combined with ionomycin is suitable DMAP for the purpose of successful nuclear transplantation.

• 한국가축번식학회지
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• 제16권1호
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• pp.15-20
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• 1992

These experiments were undertaken to investigate the rate of in vitro fertilization of bovine follicular oocytes treated with glycosaminoglycans(GAGs). Bovine follicular oocytes were obtained from the ovary of slaughtered animal and matured in media containing the various concentrations of hydluronic acid, chondroitin sulfate or heparin for 26 hours. Epididymal spermatozoa were capacitated and insemination was made by introducing about 10~15 matured oocytes into the suspension of spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then examined the embryo development. After in vitro insemination, percentages of ova fertilized were 61.3 or 48.3%, respectively, for the cumulus intact or removed in the percentages of GAGs. However, in case of cumulus-free oocytes treated with GAGs, the fertilization rates were 58.8, 62.1, 58.8, and 61.8%, respectively, showing significant effect compared to 48.3% in cumulus-free oocytes. Our findings suggest that cnondroitin sulfate and heparin are superior to hyaluronic acid in the fertilizatin and pronuclear formation of bovine oocytes.

• 한국수정란이식학회지
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• 제22권4호
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• pp.265-269
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• 2007

This study was conducted to investigate the efficacy of vitrification procedure for the cryopreservation of porcine oocytes and the utilization of vitrified oocytes as recipient cytoplasts for somatic cell nuclear transfer (NT), and observed that porcine oocytes are evaluated by pronuclear formation, and parthenogenetic development. Single fetal donor cells were deposited into the perivitelline space of vitrified enucleation oocytes, followed by electrical fusion and activation. NT embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at $38.5^{\circ}C$ in 5% $CO_2$ and air. 1. When the developmental rates of the oocytes after being culture for $0{\sim}10$ hours vitrified with EDS and ETS were 42.0%, 38.0%, respectively. This results were lower than the control group(62.2%). 2. When the developmental rates of the oocytes after being culture for $0{\sim}10$ hours vitrified-thawed with sucrose and glucose, 5% PVP, NCSU-23 supplemented with 10% FBS were 33.3%, 25.9%, respectively. This results were lower than the control group(55.6%). 3. The fusion and development to the blastocyst stage between the NT embryos constructed with the vitrified and non-vitrified oocytes were significant differences. Developmental rate of oocytes and NT embryos constructed with the vitrified or non-vitrified oocytes were $13.0{\pm}2.4%\;and\;23.2{\pm}2.4%$, respectively.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.301-309
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• 1997

본 연구에서는 체외수정, 난자내 정자 직접주입, 난자내 정자 두부 미두 주입 후의 핵과 미세소관의 변화를 관찰하였다. 핵과 미세소관의 움직임은 형광염색을 실시한 후 공초점주사현미경을 이용하여 관찰하였다. 체외수정에서 관찰된 바와 동일하게 정자를 난자에 직접주입 한 직후 정자 중편부에서 성상체가 형성되었고, 이 성상체에 의해 자성 웅성 전핵이 융합되는 것으로 관찰되었다. 그러나 난자내 정자를 직접주입하였을 경우 웅성전핵으로 발달하는 비율이 낮았다. 이는 주입된 정자가 원형질막과 perinuclear theca에 싸인 체 난자내로 들어가 난자내의 sperm nucleus decondensing factor와 정자 핵과의 반응이 억제되기 때문으로 생각된다. 정자 두부 만을 주입하였을 경우 성상체가 형성되지 않았지만 자성 웅성 전핵 사이 또는 그 주위에서 두터운 미세소관층이 관찰이 되었다. 따라서 소에 있어서는 정자의 중편부에 위치하여 microtubule organizing center (MTOC)의 역할을 하는 중심립 또는 중심체 없이도 모계에서 유래된 미세소관이 형성되어 이것이 전핵의 융합과 세포분열에 관여하는 것으로 생각된다. 정자의 미부 만을 주입하였을 경우 성상체가 형성이 되지 않았으며, 자성핵 사이에 형성된 미세소관과 떨어져서 관찰되었다. 따라서 주입된 정자의 꼬리는 미세소관형성과 관련이 없는 것으로 생각된다. 이러한 결과는 소에 있어서, 수정 시 정자로부터 유래되는 중심립 또는 중심체가 없이도 미세소관을 형성하여 미세소관에 의해 이후의 배발달이 정상적으로 일어남을 보여주고 있다.

• 한국발생생물학회지:발생과생식
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• 제16권3호
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• pp.195-204
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• 2012

Recently, we reported growth arrest-specific gene 6 (Gas6) as a new maternal effect gene (MEG), that expressed in the oocytes but functioned principally during embryogenesis. Gas6 RNAi-treated oocytes developed to metaphase II (MII) stage but they have affected M-phase promoting factor (MPF) activity and incurred abnormal pronuclear (PN) formation during fertilization. Gas6 is a ligand of TAM family members (Tyro3, Axl and Mertk) of receptor tyrosine kinase (RTK). Purpose of the present study was to evaluate the expression of Tyro3, Axl and Mertk transcripts in oocytes and early embryos. Expression of Gas6 and Mertk mRNA was detectable in oocytes and follicular cells, while Tyro3 and Axl mRNA was expressed only in follicular cells. Expression of Mertk mRNA was relatively constant during oocytes maturation and embryogenesis, but the other receptors, Tyro3 and Axl, were not expressed in oocytes and PN stage of embryos at all. Knockdown of Mertk mRNA and protein by using sequence-specific Mertk double strand RNA (dsRNA) did not affect oocytes maturation. In this case, however, contrary to the ligand Gas6 RNA interference (RNAi), MPF activity had not been changed by Mertk RNAi. Therefore, we concluded that the Gas6-Mertk signaling is not directly related to the oocyte maturation. It is still required to study further regarding the function of Mertk as the receptor of Gas6 during preimplantational early embryogenesis.

• 한국발생생물학회지:발생과생식
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• 제4권1호
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• pp.7-12
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• 2000

수정에 의한 배 발생은 정자가 난자 내로 침입하여 정자와 난자의 반수체 핵질이 융합되고 이어 유사분열로 이어지는 과정에서 시작된다. 하지만 수정 및 초기 배 발생 동안 자웅 핵질과 난 세포질 구성 요소 상호간의 작용기전에 관해서는 명확히 알려져 있지 않은 부분이 많다. 수정보조기법인 세포질 내 정자 직접 주입법의 개발은 남성불임치료에 혁신적인 기술로 자리잡고 있을 뿐만 아니라 포유동물의 수정과정을 이해하는데 많은 도움을 주고 있다. 핵치환에 의한 복제동물 생산기법도 분화된 핵이 난 세포질 내에서 재 분화 (reprogramming)하여 발생하는 유일한 과정으로 세포질 구성요소들의 상호작용과 발생 조절 기능을 이해하는데 도움을 준다. 최근 몇 년간 돼지 난자 세포질에 정자 및 원형정자 직접주입, 세포질 이식, 세포질 융합 및 핵치환 한 후 난자의 발생과정을 간접 면역형광 분석법과 주사 전자현미경으로 조사하였다. 이러한 연구를 통해 체외수정, 세포질 이식 및 정자직접 주입법 등과 같은 임상치료기술 과 핵치환에 의한 복제동물생산 기법의 개선에 필요한 기초자료를 얻을 수 있었고, 포유동물 난자의 후생적 발생과정 (epigenesis)에 관해 공부할 수 있었다.

• Journal of Animal Science and Technology
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• 제47권3호
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• pp.363-370
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• 2005

Experiments were conducted to determine the effects of beta-mercaptoethanol ($\beta$-ME) supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and intracellular glutathione (GSH) concentration. Porcine cumulus-intact oocytes were matured in TCM-I99 medium containing porcine follicular fluid, sodium pyruvate, D-glucose, FBS, hormonal supplements, and $\beta$-ME (0, 25, 50 and 100 ${\mu}$M) for 36 to 46h. After culture, cumulus-free matured oocytes were co-incubated with epididymal spermatozoa for 18h. There were no significant differences in the maturation rate among treatment groups. However, increases (P < 0.05) in intracellular GSH concentration before and after. fertilization were observed in 50 ${\mu}$M $\beta$-ME supplements to the IVM medium. Also, increases (P < 0.05) in male pronuclear formation after IVF were observed in same treatment group. In conclusion, supplementing $\beta$-ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.

• 한국수정란이식학회지
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• 제17권3호
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• pp.239-249
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• 2002

Embryos derived from pig oocytes matured in mSOF are able to develop to blastocysts after IVF. Experiment 1 evaluated the effects of two maturation media (TCM-199 vs mSOF) on maturation rate, fertilization parameters, including penetration, polyspermy, male pronuclear formation, and the mean number of sperm penetrated per oocyte. Experiment 2 and Experiments 3 examined the effects of two maturation media on zona pellucida solubility and cortical granule distribution by transmissible electron microscopy, respectively. Experiment 4 assessed the effects of two maturation media on the in vitro embryo cleavage rate and development to blastocyst. Lastly, experiment 5 examined the cell number of blastocyst. An effect of media (P<0.05) was detected for mSOF on the mean number of sperm per oocyte. In TCM group, zona digestion time (196.5$\pm$15.5 vs 131.6$\pm$20.1 before IVF, 397.5$\pm$30.3s vs 185.3$\pm$16.4s after IVF, p<0.05) was higher in TCM-199 group. No significant effects of media was observed on cortical granule distribution between two groups by TEM. An effect (P<0.05) was observed on embryo development to blastocyst (16% vs 8%) but not on cleavage rates. No significant effects of media was observed on total cell number of blastocyst. We found that the high mean number of sperm penetrated per oocyte and the weaker zona pellucida on the basis of the digestion time was shown in pig oocytes matured in mSOF, however, porcine oocyte maturation with supplemented synthetic oviduct fluid medium (mSOF) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.718-724
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• 2007

Considerable attention has been focused on the cloning of mammalian embryos, as a consequence of poor development, in order to enhance the application of genetic engineering. Experiments were conducted to compare the developmental competence of parthenotes and reconstructed (NT) rabbit eggs with fetal fibroblasts (FFs) following various activation regimens. Oocytes and NT eggs were exposed to: electric stimulation (EST, Group 1) and EST followed by 6-dimethylaminopurine (DMAP, Group 2), cycloheximide (CHX, Group 3) or DMAP/CHX (Group 4). Pronuclear (PN) status, cleavage, blastocyst development and the ploidy were assessed. In parthenote groups 1, 2, 3 and 4, the PN formation differed significantly. And, the cleavage and blastocyst rates were 41.7 and 5%, 75.6 and 53.7%, 68 and 36%, 82.1 and 52.6%, respectively, among treatments. Polyploidy was observed in 17.2% of EST plus DMAP and 44.9% of EST plus DMAP/CHX groups. In SCNT groups (Group 1, 2, 3 and 4), the cleavage and blastocyst rates were 28.6 and 7.1%, 58.3 and 29.2%, 56.8 and 24.1%, 64.5 and 27.8%, respectively. The chromosomal composition differed significantly (p<0.05) among treatments. In Group 2 and 3, 53.8% and 81.8% of embryos revealed diploid chromosomal sets, respectively. However, in Group 4, 53.3% of embryos showed abnormal ploidy (mixoploid). Although DMAP or combination with DMAP/CHX resulted in higher in vitro development of rabbit SCNT embryos, higher incidence of chromosomal abnormality may induce problems related to fetal loss of at late stage of development.

• 한국가축번식학회지
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• 제22권4호
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• pp.395-403
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• 1998

본 연구에서는 돼지난자내 원형정자 주입후 수정율과 체외 배 발달을 조사하였다. 원형정자 주입 2시간 전에 인위적 전기자극을 주고 원형정자를 주입했을 때 난자들의 수정율이 주입 직후에 전기자극을 준 것들과 전기 자극을 주지 않은 것들보다 수정율이 높았다. 원형정자와 원형 정자핵을 각각 주입 한 후 전핵 형성율과 전핵 이동율을 조사하였으나 유의차를 발견할 수 없었다. 전핵의 형성과 이동중 미세소관의 움직임을 간접형광면역법 및 공춧점 현미경을 사용하여 조사해본 결과, 난활성 직후 난자의 표층에서 미세소관이 발생해서 이것에 의해 웅성 및 자성전핵이 난자 중심부로 이전됨을 볼 수 있었다. 원형정자와 원형정자핵 주입후 배양 6일째에 각각 25% 와 27%의 배반포로 형성되었고, 8 일째 형성된 배반포를 염색하여 관찰한 철과 세포수가 각각 평균 87에서 99개가 형성되었음을 알 수 있었다. 이러한 결과는 체외 성숙된 돼지 난자 내에 원형정자 혹은 원형정자 핵을 미세 주입하는 방법에 의해 정상척인 배발달을 하는 돼지 수정란 생산이 가능한 것을 보여주는 것이다.