생쥐 상실배의 초급속동결

백청순,서병희,이재현,이경광,Baik, C.S.,Suh, M.D.,Lee, J.H.,Lee, K.K. 대한생식의학회 1990 Clinical and Experimental Reproductive Medicine Vol.17 No.1

We cryopreserved mouse morulae by a simple ultra-rapid method of freezing embryos directly in $LN_2$ after holding 2min in a $LN_2$ vapor, and thawed them in $37^{\circ}C$ water bath. The time requirements for permeation and dehydration by 2.0 M glycerol and 0.2 M sucrose before freezing were studied. When the embryos were equilibrated for 10 min, the optimun post-thaw survival was obtained. Embryos those developed normally to blastocyst after in vitro culture for over 24hrs were regarded as survival ones. Two experiments to assess post-thaw survival following predehydration in various mixtures of glycerol and sucrose were also accomplished. When sucrose was held constant (0.2 M) and glycerol concentration varied (1.5-3.5 M), post-thaw survival was best (78.0%) in 3.0 M glycerol. When glycerol was held constant (3.0M) and sucrose concentration varied (0.0-1.0M), optimun post-thaw survival (78.0%) was found in 0.2 M sucrose.

Study on the Analysis of Chromosome Abnormality by Flow Cytometric and Cytogenetic Methods

백청순,김묘경,이상민,김진희,백용균,이훈택,정길생,Baik, C.S.,Kim, M.K.,Lee, S.M.,Kim, J.H.,Baik, Y.K.,Lee, H.T.,Chung, K.S. The Korean Society for Reproductive Medicine 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.1

골수나 유산물질에 대한 세포유전학적 검사에 있어 통상적인 염색체검사는 검사에 적합한 중기핵상을 얻기 어려워 실패하는 경우가 많다. 이러한 경우에 진단이나 치료에 도움을 줄 수 있는 방법으로 유식세포분리기를 사용하여 단일 세포내 DNA량에 따른 aneuploidy를 추적할 수 있는 가를 확인하기 위해 본 실험을 실시하였다. 79 (혈액 30, 골수 37, 유산물 12)예에서 염색체 검사와 유식세포 분리검사를 동시에 실시하여 각각의 결과를 비교한 결과 79.7% (63/79)의 일치율을 얻었다. 그러나 염색체의 손실이 없는 전좌와 역위의 경우는 물론 작은 조각의 염색체 부분이 늘어나거나 줄어든 경우에 있어서는 유식세포분리방법에 의해서 추적되지 못하였지만, 염색체 검사의 결과를 얻는데 실패한 경우에는 유식세포분리방법이 DNA량의 변화에 대한 정보를 얻을 수 있다는 것을 확인할 수 있었다. 따라서 본 연구결과는 세포유전학적 검사에서 유식세포분리방법이 염색체 검사보다 신속하며 염색체검사가 불가능한 시료에서도 DNA양에 따른 aneuploidy의 추적이 가능하다는 것을 시사한다.

염색체 분석에 의한 생쥐 분할란의 성감별

백청순,한용만,이경광,정길생,김종배,고대환 ( C . S . Baik,Y . M . Han,K . K . Lee,K . K . Chung,J . B . Kim,D . W . Ko ) 한국축산학회 1986 한국축산학회지 Vol.28 No.11

These experiments were carried out to obtain basic information necessary to produce the offsprings which were sexed by chromosomal analysis prior to transfer in the mouse. Morula stage embryos were obtained at 3 clays post-coitum. Embryos were mechanically bisected by a glass microneedle and then the pairs of demi-embryos were cultured in BMOC-3 medium under the humidified atmosphere of 5% CO₂ at 37℃ for 22-26 hours. To increase the appearance of metaphase, bisected embryos normally developed to the blastocyst were cultured in MOC-3 medium containing colcemid (0.04㎍/㎖) under the same condition for 3 hours. One of both demi-embryos was sexed by chromosomal analysis and the other was transferred to the uterine horn of a foster mother. The results obtained in these experiments were summarized as follows; 1. Of 241 pairs of demi-embryos cultured for 22-26 hours, 162 pairs (67.2%) were normally developed to the blastocysts. In the control groups, the percentage of intact and zona-free embryos normally developed to the blastocysts was 96.5% and 93.9%, respectively. 2. The mean Mitotic Index of 121 demi-embryos subjected to chromosomal analysis was 11.3±13.2%r and 18 demi-embryos (14.9%) were sexed. 3. Of 18 demi-embryos transferred to recipients after sexing, 2 offsprings were born and their sex was corresponded to the sex determined by chromosomal analysis.

생쥐 2세포기배의 동결보존

백청순,서병희,이재현,이경광,Baik, C.S.,Suh, B.H.,Lee, J.H.,Lee, K.K. 대한생식의학회 1989 Clinical and Experimental Reproductive Medicine Vol.16 No.1

For the cryopreservation of human embryos this study was accomplished as a preliminary experiment. The purpose of this study is to obtain optimal cryoprotectant, addition and dilution method of cryoprotectant and cooling rate for raising survival of frozon and thawed 2-cell mouse embryos. Seeding was done at $-7^{\circ}C$ and the straw contained embryos was plunged at $-30^{\circ}C$ when the slow cooling was ended. Embryos those developed normally to blastocyst after in vitro culture for over 96 hours were regarded as survival ones. The survival was the rate of number of survival embryos against the recovered embryos. The results are followed : 1. The survivals were 6.3, 71.2 and 67.4% respectively, when Glycerol, DMSO and 1,2-Propanediol were used as cryoprotectant. 2. When sucrose was added in freezing solution, the survival was 69.0%. That was higher than the survival of embryos frozen without sucrose in freezing solution. The difference was not significant. 3. Addition and dilution of cryoprotectant by 4 stepwise raised the survival than by direct, but that was not significant. 4. When embryos were frozen by -0.3, -0.5 and $-1^{\circ}C/min$ before plunged into $LN_2$, the survivals were 67.9, 78.0 and 37.0% respectively. The differnce was significant.

Sister Chromatid Exchange (SCE) Frequency and In Vitro Development of Mouse Zygote Cryopreserved by Vitrification

김묘경,백청순,엄상준,김은영,윤산현,박세필,정길생,임진호,Kim, M.K.,Baik, C.S.,Uhm, S.J.,Kim, E.Y.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.3

본 연구는 생쥐 1-세포기 수정란의 초자화 동결과 동결액 노출에 따른 수정란의 배발달율과 SCE 빈도를 조사하고자 실시하였다. 체외수정된 생쥐 1-세포기 수정란의 동결은 EFS40 (40% ethylene glycol, 30% Ficoll, 0.3 M sucrose)의 초자화동결법을 이용하였으며, $25^{\circ}C$에서 30 초동안 EFS40에 노출시킨 다음, 곧바로 액체질소에 침투시키거나, 동결액의 독성 검사를 위해 동결없이 배양하여 다음과 같은 결과를 보였다. 융해후, 2-세포기 까지 생존율은 EFS40에 노출 혹은 초자화동결시 각각 95.2, 98.5% 로서 대조군의 100%와 비교했을때 큰 차이가 없었다. 그러나 배반포와 탈출배반포까지의 배발달율에 있어서 초자화 동결된 군 66.7, 50.0%는 동결액에 노출만된 군 94.0 78.8%와 대조군 93.9, 81.8%에 비해 낮았다 (p<0.05). 동결액에 노출 혹은 초자화동결된 생쥐 1-세포기의 체외 발달에 따른 SCE 빈도를 조사한 결과, 동결액 노출 ($20.2{\pm}2.1$) 혹은 초자화동결시 ($21.4{\pm}3.2$) SCE 빈도가 대조군 ($16.8{\pm}1.5$)에 비해 증가하였다. 이러한 결과를 종합하여 고찰할 때, 초자화동결된 1-세포기 수정란의 배반포 또는 탈출 배반포까지의 낮은 배발달율은 동결액의 영향을 받지 않았으나, SCE 빈도는 동결액에 노출 혹은 초자화 동결시 증가 된다는 것을 알수 있다. This study was undertaken to investigate the sister chromatid exchange (SCE) frequency and embryonic development after exposure to cryoprotectants and vitrification of mouse zygotes. Mouse IVF zygotes were cryopreserved by vitrification using vitrification solution, EFS40 (40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). After mouse zygotes were exposed to EFS40 at $25^{\circ}C$ for 30 sec., they were immediately plunged into $LN_2$ or cultured for cryoprotectant toxicity test without freezing. The results obtained in these experiments were summarized as follows: After thawing, survival rates to the 2-cell stage of zygotes exposed to or vitrified in EFS 40 (98.5%, 95.2%) were not significantly difference compared with that of control (100%). However, the developmental rates upto blastocyst and hatching blastocyst in vitrified groups (66.7, 50.0%) were lower than those of control (93.9, 81.8%) or exposed group (94.0, 78.8%) (p<0.05). When the influence of vitrification and exposure to cryoprotectant on the in vitro development of mouse zygotes was assessed by the SCE frequency, the SCE frequency in exposed ($20.2{\pm}2.1$) to or vitrified embryos ($21.4{\pm}3.2$) was higher than that in control embryos ($16.8{\pm}1.5$). These results suggest that the frequency of SCE was increased after cryoprotectant exposure or Vitrification although developmental rates of zygotes upto blastocysts and /or hatching blastocysts were not afected by cryoprotectant.

생쥐에 있어서 Ethanol 처리에 의한 단위발생의 유기

한용만(Y . M . Han),백청순(C . S . Baik),이경아(K . A . Lee),이경광(K . K . Lee),정길생(K . S . Chung) 한국축산학회 1987 한국축산학회지 Vol.29 No.9

These experiments were carried out to obtain the basic informations and to establish the best conditions needed for the activation of mouse eggs by ethanol treatment. The influence of ethanol concentration and the age of eggs, time from hCG injection to recovery, on activation frequency of eggs was investigated, To activate the mouse eggs parthenogenetically, cumulus masses including eggs were released into a fresh solution of ethanol for 5min and then rinsed out 5-6 times with ethanol-free medium. After incubation of cumulus masses in the drops of medium for 5 hrs at 37℃ in the humidified atmosphere of 5% CO₂ in air, the adherent cumulus cells were removed with hyaluronidase (100unit/㎖). The types of parthenogenetic eggs were morphologically classified into haploid (H), immediate cleavage (IC) and diploid (D) under a inverted microscope. These parthenogenetic eggs were then incubated into the drops of mKRB medium for 96 hrs under the same conditions as above. The results obtained in these experiments were summarized as follows : 1. ICR female mice (1) When the eggs were treated with a variety of ethanol concentrations, 0 to 11%, the highest parthenogenetic activation rate (94.4%) was observed at 7% ethanol concentration. (2) Activation frequency was comparatively high when the eggs were recovered from the oviducts in more than 18 hrs after hCG injection. 2. (C₃H × C57BL) F₁ hybrid female mice (1) Of 341 F₁ eggs treated with 7% ethanol for 5 min at 18 hrs after hCG injection, 317 eggs (93.0%) were activated and a majority of parthenogenetic eggs were haploids (71.9%). (2) After 96 hrs of in vitro culture, the developmental rate of haploid, immediate cleavage and diploid eggs to morphologically normal morula or blastocyst stage was 16.7, 66.7 and 72.3%, respectively. (3) Of 18 haploid eggs recovered from the uteri on the 4th day of pregnancy after transfer to the oviducts of recipients, 12 eggs (60.0%) were normally developed to morula or blastocyst stage.