핵이식을 이용한 복제송아지 생산에 관한 연구 II. 효율적인 복제수정란 생산을 위한 난자의 활성화, 공여핵의 세포주기조절 및 적정 배양조건

황우석,노상호,이병천,Hwang, Woo-suk,Roh, Sang-ho,Lee, Byeong-chun 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.3

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplanted embryos. The oocytes collected from slaughterhouse ovaries were matured 24h in TCM199+10% FBS and exposed to $39^{\circ}C$ or room temperature to allow cytoplasmic maturation and gain activation competence. Donor embryos were treated for 12h with $10{\mu}g/ml$ nocodazole or $0.05{\mu}g/ml$ demicolcine to synchronize the cell cycle stage at 26h after the onset of culture. The blastomeres and recipient oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. In the treatment of oocyte activation and cell cycle regulation of donor nuclei, the room temperature exposure and nocodazole treatment group had significant effect on the developmental rates to morula/blastocyst(21.7% vs 12.1~16.7%), but had no significant effect on the fusion rates between donor blastomeres and recipient oocytes. The developmental rates of bovine nuclear transplanted embryos appeared to be higher significantly in mTALP medium under 5% $O_2$ condition and in TCM199 with bovine oviduct epithelial cell under 20% $O_2$ condition(22.2%) than other groups. In embryo transfer of nuclear transplanted embryos, there were no significant differences in calving rates between the use of excellent and good grade donor embryos.

Studies on the In vitro Culture of Early Bovine Embryos

황우석,권오경,조충호,Hwang Woo-Suk,Kweon Oh-Kyeong,Jo Chung-Ho The Korean Society of Veterinary Clinics 1990 한국임상수의학회지 Vol.7 No.2

소 난소로부터 난자를 채취하여 체외성숙과 체외수정을 실시하였다. 난의 성숙배양을 위한 배양액은 소 태아혈청을 첨가한 TCM 199으로 $CO_2$배양기에서 24시간 배양하였다. 체외수정에는 동결융해정액을 사용하였으며 Caffeine과 Ca Ionophore 처리후 매정에 공하였다. 배양난자중 40%가 Metaphase II까지 성숙하였다. 성숙율에 대한 소 태아혈청의 농도변화와 HEPES 첨가여부의 효과는 없었다. 체외수정율과 분할율의 성적은 각각 23.1%와 14.4%였다. In vitro maturation and fertilization of oocytes collected from slaughtered bovine ovaries were investigated. Immature bovine extrafollicular oocytes were cultured for 24 hrs. in TCM 199 supplemented with fetal calf serum in a humidified CO$_2$ incubator. Fertilization in vitro was performed using frozen-awed bull semen which was treated by Ca Ionophore A23187. Fourty percentage of oocytes cultured had matured to the metaphase II ; There were-no effects of the concentration of fetal calf serum and of the addition of HEPES on the maturation rate. The mean proportions of in vitro fertilized eggs and of cleaved eggs were 23.1% and 14.4%, respectively.

Studies on the Production of Twins in Cattle

황우석,Hwang Woo-Suk The Korean Society of Veterinary Clinics 1990 한국임상수의학회지 Vol.7 No.2

소에 있어서 효율적인 쌍자생산 방법을 검토하기 위하여 이분수정란의 이식, 저단위 FSH에 의한 다배란 유도후의 인공수정, 인공수정후의 수정란이식, 수정란 2개의 양측 및 단측 자각에의 이식 등의 방범을 적용하여 본 실험을 수행하였다. 각 실험군의 60일 수태율은 각각 34.8%, 65.0%(평균), 66.7%, 62.5%, 57.1%였으며 쌍태임신율은 8.7%, 15.0%(평균), 16.7%, 18.7%, 21.4%였다. 위의 결과에서 볼 때 저단위 FSH 투여법(Group B1)과 수정란 2개의 단측 자궁각 이식방법에 있어 쌍태율이 높게 나타나는 경향이 있었으나 실용적 측면에서는 낮은 수준이었다. Induction of twinning was attempted by transfer of two whole- or demi-embryos in Holstein cows and heifers. Cows were superovulated with follicle stimulating hormone(FSH) administered twice daily in intra-muscular injection. On day of 6.5 to 7 post-estrus, embryos were collected nonsurgically, Normal morulae and early balstocysts were obtained from superovulated cows. The embryos were hisected with a micro-blade made from a razor. Twenty seven pairs of half embryos were successfully produced and 23 pairs of half embryos were transferred to recipients( Group A). Twenty cows were treated with low unit of FSH (3mg${\times}$2,2mg${\times}$2 ; Group B1, 2mg${\times}$2, 1mg${\times}$2, 1mg${\times}$2 ; Group 32) to induce double or triple ovulations. The cows of Group B1 and B2 were Inseminated artificially(A1) at following estrus. Twenty four heifers were bred by A1 and received an additional embryo into the uterine horn contralateral to the corpus luteum(CL) 6 days later(Group C). One embryo was transferred into each uterine hem of 16 heifers 6 days after estrus(Group D). Fourteen heifers were received two embryos into the uterine horn ipsilateral to the CL at day 6 of estrous cycle(Group I). Pregnancy rates at 60 days in Group A, B1, B2, C, D and E were 34.8%, 70.0%, 60.0%, 66.7%, 62.5% and 57.1%, respectively. Twinning rates were 8.7%, 20.0%, 10.0%, 16.7%, 18.7% and 21.4%, respectively. The present experiments demonstrate that FSH treatment( Group 31) and ipsilateral transfer( Group I ) of two whole-embryos are more useful methods to produce twins than the others.

절단마우스 이분배의 동결보존실험 1. 마우스 절단이분배의 체외 발육능에 대하여

황우석,Hwang Woo-Suk 한국임상수의학회 1985 한국임상수의학회지 Vol.2 No.1

Mouse embryos of 8-cell stage and compacted morulae(approximately 16 cells) containing different number of blastomeres were bisected and cultured in vitro to determine the developmental potentials of the divided embryos compared with those of unmanipulated control embryos. The results were as follows. 1. Micromanipulation was performed successfully by means of a simple manipulator which holds a fine glass, needle, without the use of any micro-instruments for support. 2. The percentage of bisected morulae with 7-9 blastomeres that developed to eu-blastocyst was 94.1% while only 64.8% of the bisected 8-cell embryos with 4 blastomeres developed to eublastocysts (p<0.05). 3. The percentage of eu-blastocysts decreased, while that of pseudoblastocysts and trophectodermal vesicle increased as the number of blastomeres decreased in the bisected embryos of the two stages. 4. The time of the blastocoele re-formation of the bisected and control embryos was not significantly different in morulae stage embryos, but it was significantly delayed in the 8-cell stage embryos (Eu-B, Pseudo-B) compared with control embryos (P<0.01, P<0.05 respectively).

한국인 천식환자의 Monocyte chemoattractant protein 1(MCP-1) 유전자 다형성에 대한 분석

황우석,정승연,김진주,정희재,정승기,Hwang, Woo-Suk,Jeong, Seung-Yeon,Kim, Jin-Ju,Jung, Hee-Jae,Jung, Sung-Ki 대한한방내과학회 2008 大韓韓方內科學會誌 Vol.29 No.1

Background : Monocyte chemoattractant protein-1(MCP-1), one of the CC chemokines, appears to play a significant role in asthma pathogenesis. It was reported that polymorphism in the MCP-1(-2518 A/G promoter) was associated with asthma in Caucasians, but the association of this polymorphism and asthma patients in the Korean population has not yet been clarified. Objective : We investigated the possible association between 2 polymorphisms (-2518 A/G promoter and Cys35Cys) and asthma patients in a Korean population. Materials and Methods : DNA samples were obtained from 86 Korean asthma patients and 270 healthy controls. MCP-1 genomic variants (-2518 A/G promoter and Cys35Cys polymorphism) were detected by PCR-RFLP. Level of MCP-1 was measured by ELISA for each genotype (n=8) (AA, AG, GG) and allele types of -2518 A/G promoter polymorphism for control subjects. Results : The Cys35Cys polymorphism was associated with asthma patients in Korean population [genotype distribution ($X^{2}=16.011$, P<0.001)]. Comparison of the two groups revealed no detectable differences in genotype and allele frequencies of the -2518 A/G polymorphism. Haplotype frequencies analysis revealed significant difference $(X^{2}=51.70$, P<0.001). MCP-1 serum level of subjects with G genotype of -2518 A/G promoter polymorphism was statistically higher than that with AA genotype (P<0.05). Conclusion : Our data indicate that no association exists between the MCP-1 -2518 A/G polymorphism and asthma susceptibility in the Korean population. However, it is noteworthy that the high prevalence of the -2518 G allele in the Korean population suggests a potentially important ethnic variation in the regulation of MCP-1 production. This variation must be considered in gene-association studies in different ethnic populations.

체외수정 유래 소 핵이식란의 배양에 관한 연구

황우석,조충호,Hwang, Woo-suk,Jo, Choong-ho 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.1

The fusion rates of nuclear transplant embryos with various DC voltage were 55.6-79.2%. The significantly higher fusion rates of nuclear transplant embryos were achieved at the electric field strenght of DC 1.0-2.0kV/cm(72.0-79.2%) than DC 0.75kV/cm(55.6%, P<0.05). No significant differences in the percentage of embryos that cleaved(48.1, 55.4 and 42.6% respectively) and the percentage of cleaved embryos that developed to morulae/blastocyst(1.9, 5.3 and 1.9% respectively) could be found among the three types of in vitro culture system (Granulosa cell, BOEC co-culture and SOF, P>0.01). The age of the recipient cytoplast(30 vs 40hr) had no effect on the fusion rates and the rates of cleavage development(36.9 vs 44.1%, P>0.01).

핵이식 기법을 이용한 한우 쌍태생산에 관한 연구 I. Ovum pick-up(OPU), 전기적 세포융합 및 체외배양 기법을 이용한 복제수정란 생산

황우석,신태영,노상호,이병천,Hwang, Woo-suk,Shin, Tae-young,Roh, Sang-ho,Lee, Byeong-chun 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.1

The purpose of present study is to improve the efficiency of fusion and the developmental rates of nuclear transplanted embryos to produce genetically identical twins from Korean native cattle. The diameter of aspirated follicles had no significant effect on the recovery rates of oocytes collected by ovum pick-up technique. The fusion rates of nuclear transplanted embryos were significantly higher in 50 and $100{\mu}s$ DC duration groups(73.3 and 72.0% ; respectively) than that in $30{\mu}s$ group(55.6% ; p<0.05). The cleavage rates of nuclear transplanted embryos appeared to be significantly higher in donor nuclei derived from in vivo (65.0%) than in those from in vitro (50.5% ; p<0.01), but the developmental rates to morulae and blastocysts were not significantly different between them(13.7 vs 10.9%, respectively).

황체 호르몬 방출기구(PRID)가 젖소의 번식효율에 미치는 영향 1. 임상적 관찰

황우석,Hwang Woo-Suk 한국임상수의학회 1984 한국임상수의학회지 Vol.1 No.1

The effects of progesterone releasing intravaginal device (PRID) on the fertility levels in dairy cows were studied in 2 experiments. In experiment I, 70 lactating cows at 45 days postpartum were allotted to 3 groups and the treatments imposed were either: 1: Untreated control, 2: PRID with a capsule containing long of oestradiol benzoate (ODB) attached, inserted for 12 days, 3: PRID inserted for 12 days with long of prostaglandin F$\_$2${\alpha}$/ administration 24 h before PRID removal. Treated cows were inseminated 56 h after PRID removal and at an observed oestrus during the subsequent 48-day period. The control group was inseminated at an observed oestrus during this 60-day period. In experiment II, 60 ovarian disorder cows were divided into 5 groups and PRID+ODB inserted for 12 days. 1: atrophied ovary, 2: smooth ovary, 3: persistent corpus luteum, 4: follicular cyst, 5: luteal cyst. Treated cows were inseminated 56 h after PRID removal and at an observed oestrus over a period from the first insemination to 46 days. The results obtained were as follows: 1. The device produced a vaginal discharge in some animals. In experimenet I : 2. For treatments 2 and 3, respectively, conception rates to the fixed time insemination were 45% and 52%. 3. The conception rates of cows inseminated to the fixed time insemination and at an observed oestrus during a 60-day period were 65%, 86% and 91% for control, treatment 2 and 3, respectively. 4. Mean interval from calving to conception and inseminations per conception were 133, 91 and 86 days and 2.4, 2.1 and 1.9 for control, treatments 2 and 3, respectively. In experiment II; 5. The conception rates to the fixed time insemination for each group 20, 50, 70, 20 and 50%, respectively. 6. The total conception rates for the 48 days period of each group were 60, 70, 100, 60 and 90%, respectively. 7. The inseminations per conception of each group were 2.8, 2.1, 1.4, 2.7 and 1.9, respectively.

핵이식 수정란의 동결, 융해 및 이식에 의한 클론동물의 생산 II

황우석,조충호,이창우,이병천,Hwang, Woo-suk,Jo, Choong-ho,Lee, Chang-woo,Lee, Byeong-chun 대한수의학회 1993 大韓獸醫學會誌 Vol.33 No.3

This study was carried out to investigate the best condition for in vitro and in vivo culture after freezing and thawing of nuclear transplant 2 cell embryos. When nuclear transplant embryos were submitted to electrofusion, the significantly higher fusion rates of 2 cell donor nuclei were achieved at the electric field strength of DC 1.5 kV/cm for 100 and $150{\mu}sec$, DC 2.0 kV/cm for 100 and $150{\mu}sec$ than DC 1.0 kV/cm for 100 and $150{\mu}sec$(p<0.01). The significantly higher fusion rates of 4 cell donor nuclei were achievecl at DC 2.0 kV/cm for 100 and $150{\mu}sec$ than DC 1.0 kV/cm for 100 and $150{\mu}sec$(p<0.01). The fusion rates in 8 cell donor nuclei were 94.2~99.3%. The developmental potency to blastocyst in 2 cell donor nuclei was significantly higher in DC 2.0 kV/cm for $150{\mu}sec$ treated group(p<0.01). The significantly higher developmental potency to blastocyst in 4 cell donor nuclei were achieved at the electric field strength of DC 2.0 kV/cm for $150{\mu}sec$ than DC 1.5 kV/cm for 100 and $150{\mu}sec$, DC 2.0 kV/cm for $100{\mu}sec$ treated group(p<0.01). The develop mental potency to blastocyst in 8 cell donor nuclei was significantly higher in DC 2.0 kV/cm for $100{\mu}sec$ treated group(p<0.01). The developmental potency to blastocyst after nuclear transplantation was significantly higher in 2 cell donor nuclei than in 8 cell donor nuclei(p<0.01). When the recovered embryos in normal morphology were cultured in vitro, there were no significant differences in the developmental potency to blastocyst between the freezing methods and the concentrations of cryoprotectant(p<0.01). The production rates of offspring after transfer of nuclear transplant embryos to recipient mouse were no significant difference in 2, 4 and 8 cell donor nuclei.

Gonadotropin-Releasing Hormone의 투여(投與)가 산욕기(産褥期)의 유우(乳牛)와 난소낭종유우(卵巢囊腫乳牛)의 번식효율(繁殖效率)에 미치는 영향(影響)

황우석,Hwang, Woo Suk 대한수의학회 1981 大韓獸醫學會誌 Vol.21 No.1

Holstein-Friesian cows(n=284) were given $100{\mu}g$ of gonadotropin-releasing hormone(GnRH) or saline solution by intramuscular injection at 10 to 22 days after parturition, and were investigated their reproductive performance and frequency of ovarian cysts. Among them 28 cystic cows were injected with $150{\mu}g$ of GnRH intramuscularly and examined the recovery rate. The results obtained in this study were summarized as follows: 1. The interval from calving to 1st ovulation was reduced from 28.2 days in controls to 16.5 days for cows given GnRH (p<0.01). 2. The intervals from calving to 1st estrus and from calving to conception were extended significantly in control group (p<0.05). 3. Inseminations per conception and conception rate at 1st insemination did not reveal difference between two groups. 4. Frequency of ovarian cysts was reduced from 14.0% in control to 4.20% for cows given GnRH (p<0.05). 5. Of the 28 cystic cows receiving $150{\mu}g$ of GnRH, 23(82.1%) responded to 1st treatment and returned to estrus $24.2{\pm}4.3$ days after treatment. 6. These data provide evidence for reduction in infertility and reproductive disorders in early postpartum dairy cows given GnRH as a prophylactic.