인간 난관 상피세포와의 공동배양이 생쥐와 인간수정란의 체외발달에 미치는 영향에 관한 연구

고정재,정미경,도병록,엄기붕,윤태기,차광열,Ko, J.J.,Chung, M.K.,Do, B.R.,Oum, K.B.,Yoon, T.K.,Cha, K.Y. 대한생식의학회 1992 Clinical and Experimental Reproductive Medicine Vol.19 No.2

We examined effects of co-culture with human oviduct epithelial cells (HOEC) on the development of mouse and human embryos from early embryonic· stage to late morula or blastocyst stage (LM or B). In human, embryos were transferred and pregnancy rate was investigated. The HOEC, collected from surgically removed fallopian tube, were cultured in medium-199 supplemented with 20 % fetal cord serum (FCS). The HOEC were characterized by using immunocytochemical staining with anticytokeratin antibody and then used for cultures of mouse and human embryos. Results obtained from co-culture system were as follows. Development rate of mouse embryos was improved by co-culture system at late developmental stage (p<0.025). Human supernumerary embryos remained after transfer, unsuitable for freezing because of their poor quality, were co-cultured for 72hrs. Co-culture (78.79%) or conditioned medium (78.26%) system improved the developmemt rate, significantly, in comparision with control (11.11%)(p<0.00l). Co-cultured (85.71%) human zygotes for 24hrs showed the better development rate in comparision with control (50.00%) (p<0.01). When we transferred embryos cultured with the HOEC to patients, we obtained one pregnancy. Co-cultured human zygotes for 24hrs showed the better quality and viability for the replacement in comparision with control (p<0.01). In addition, improved pregnancy rate was obtained. Our results suggest that the co-culture system can rescue early degenerating embryos by improving early development and yield a resonable number of blastocyst for the appropriate replacement. The effect provided by cultured HOEC is not species specific for the development of embryos and it can be used to overcome in vitro blocks for the development. And also the co-culture system offers the possibility to freeze embryos at blastocyst stage which is more sucessful stage for the freezing. The HOEC monolayer may provide some stimulus via specific factor, which is unknown, to the development of embryos. Our results showed that the co-culture system with HOEC can be an alternative to conventional culture system.

해양환경에 노출시킨 콘크리트의 내염성능 평가

고정재(Ko, Jeong-Jae),김영웅(Kim, Young-Ung),김동철(Kim, Dong-Chul),신도철(Shin, Do-Chul),김상용(Kim, Sang-Yong),변대봉(Byun, Dae-Bong) 한국콘크리트학회 2004 한국콘크리트학회 학술대회 논문집 Vol.2004 No.5

접합자 난관내 이식 환자에 있어서 수정 실패와 항정자 항체와의 관계 및 난자와 정자의 재처리에 관한 연구

정미경,고정재,도병록,구정진,한세열,차광열,Chung, M.K.,Ko, J.J.,Do, B.R.,Koo, J.J.,Han, S.Y.,Cha, K.Y. 대한생식의학회 1992 Clinical and Experimental Reproductive Medicine Vol.19 No.2

Previous studies have indicated that immunological factor is responsible for the infertility. We have detected sperm antibodies in ZIFT patients which grouped as fertilization failure (A; n=18) and low fertilization rate (${\leq}50%$)(B; n=20). Patients, however, had normal oocytes and sperms. We collected serum from wives and semen from husbands and donors (fertile sperm), if it was needed. We examined class, binding patterns and amounts of antisperm antibodies(ASA) by direct and indirect immunobead binding assay. In group A, 11 husbands were ASA positive showing 62.2% and 61.1% binding with IgA and IgG, respectively, and two wives were ASA positive showing 70.0% and 71.0% binding with IgA and IgG, respectively. Binding sites were mainly at the head of sperms (84%). In group B, 8 husbands were ASA positive showing 37.5% and 40.0% binding with IgA and IgG, respectively, and two wives were ASA positive showing 41.3% and 42.0% binding with IgA and IgG, respectively. Binding sites were also mainly at the head of sperms (78%). For the treatment of ZIFT patients who had fertilization failure at the first trial, we used albumin fractionation method and dilution method with 30% fetal cord serum (FCS) to reduce the titer of ASA. We used partial zona dissection (P.Z.D.) method for wives who have antisperm antibodies in their serum. According to represented method, we could inhance the fertilization rate to 60.0% by albumin fractionation and 20.0% by P.Z.D., respectively. We concluded that the use of micromanipulation like P.Z.D. or the other sperm processing methods is required to increase a chance of fertilization. This result suggested that it should be a prerequisite to test antisperm antibodies prior to entering assisted reproductive technologies (ART) programs.

Laser Captured Microdissection을 이용한 유전자 발현에 대한 연구 (I): RT-PCR을 위한 난자의 RNA 추출 및 증폭을 위한 최소한도의 확립

박창은,고정재,차광렬,이경아,Park, Chang-Eun,Ko, Jung-Jae,Cha, Kwang-Yul,Lee, Kyung-Ah 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.3

Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

생쥐수정란에 대한 H - Y 항체처리가 산자의 성비에 미치는 영향

심호섭,고정재,김종배,박홍양,정길생 ( H . S . Shim,J . J . Ko,J . B . Kim,H . Y . Park,K . S . Chung ) 한국축산학회 1986 한국축산학회지 Vol.28 No.12

These experiments were carried out to control the sex of offsprings in mice by sexing embryos using immunological means prior to transfer to pseudopregnant recipients. H-Y antisera were prepared in inbred SD female rats by repeated immunization of testis supernatant and spleen cells from males of same strain. ELISA and indirect immunofluorescence test were used to detect H-Y antibody in antisera. Eight- to 16-cell mouse embryos were cultured in medium with H-Y antibody and complement (treated embryos) and in medium with BSA (control embryos). After 24-48 hr of culture, embryos were observed their morphological characteristics under the phase contrast microscope. Embryos developed to normal blastocyst were transferred to pseudopregnant recipients and sex of resultant offspring was investigated. The results obtained in these experiments were summarized as follows: 1. Production of H-Y antibodies in antisera obtained from immunized rats was confimed by ELISA and indirect immunofluorescence test. 2. Of 270 embryos treated with H-Y antibody and complement, 126 embryos (46.7%) were developed to normal blastocysts. 3. Following transfer of 126 blastocysts, 16 embryos (12.6%) were survived to term and 13 females (81.3%), significantly high ratio of female offspring, were produced.

Chimera 생쥐의 생산

안치용,노승찬,고정재,정길생,이경광 ( C . Y . Ahn,S . C . Rho,J . J . Ko,K . S . Chung,K . K . Lee ) 한국축산학회 1986 한국축산학회지 Vol.28 No.8

These experiments were carried out to develop new technique essential for the production of chimeric animals. Embryos obtained from inbred ICR, C3H and C57BL mouse were subjected to in vitro aggregation and aggregated chimeric embryos were developed to blastocysts and then transferred to pseudopregnant recipients. The results obtained were summarized as follows; 1. Production rate of live young following transfer of intact mouse morula to the uterus of recipient mouse on day 2,3 and 4 of pseudopregnancy wre 24.7, 61.9 and 21.8%, respectively, and those of intact morula transferred on 3,4 and 5 days of pseudopregnancy were 58.8, 63.4 and 17.3%, respectively. 2. Chimeric mouse embryos produced by in vitro aggregation of two embryos of the same stage and developed to morula or blastocyst by in vitro culture were transferred to pseudopregnat recipients and 12.2 and 9.2% of chimeric morulas and blastocysts transferred were developed to live chimeric young. 3. Among transferred multiple chimeric mouse embryos produced by in vitro aggregation of 3 to 7 embryos, 9.6% were developed to live chimeric young. 4. Of total 236 chimeric embryos prouced by the aggregation of two embryos of different stage, 78.8% were developed to normal chimeric blastocyst by in vitro culture.

여성의 난소 피질조직의 초자화 냉동보존

이경아,이숙현,하상덕,윤세진,고정재,이우식,윤태기,차광열,Lee, K.A.,Lee, S.H.,Ha, S.D.,Yoon, S.J.,Ko, J.J.,Lee, W.S.,Yoon, T.K.,Cha, K.Y. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.2

The present study was conducted to evaluate whether vitrification could be used for ovarian tissue preservation. The important issue here is that the vitrification is very simple, easy, and economical compared to the conventional cryopreserving method that using automatic freezing instrument. Human ovarian cortical tissues were cryopreserved by vitrification with 5.5 M ethylene glycol and 1.0 M sucrose as cryoprotectant. Three points of temperature ($4^{\circ}C$, room temperature, and $37^{\circ}C$) and two points of duration (5 or 10 minutes) for cryoprotectant treatment were examined to determine the best condition for vitrification of the human ovarian cortical tissues. After thawing, viability of the isolated primordial follicles was examined by dye-exclusion method. Histological appearance of tissues before and after the cryopreservation was evaluated. There was no toxic effect of the 5.5 M ethylene glycol on the primordial follicles. However, when the tissues were treated with cryoprotectant at $37^{\circ}C$ for 10 minutes and exposed to liquid nitrogen, it seems likely that there is certain deleterious effects on the viability of the primordial follicles. The highest viability of the primordial follicles was obtained with the treatment of cryoprotectant at room temperature for 10 minutes. Follicles and oocytes survived after freezing and thawing had the similar normal shapes as was seen in the specimens before cryopreservation. It would be useful to apply vitrification in establishing ovarian tissue banking for clinical purposes.

사람 다수정난자의 체외배양시 Fragmented Embryo와 Non-fragmented Embryo에서의 Methionine 유입량 및 미토콘드리아 분포양상의 비교

도병록,정미경,장미경,이경아,고정재,윤태기,차광열,Do, B.R.,Chung, M.K.,Chang, M.K.,Lee, K.A.,Ko, J.J.,Yoon, T.K.,Cha, K.Y. 대한생식의학회 1995 Clinical and Experimental Reproductive Medicine Vol.22 No.3

Despite the frequent incidence of embryo fragmentation in early human embryos, the reason of the embryo fragmentation has not been known yet. This study was conducted to investigate the histological difference(s) between fragmented (FR) and non-fragmented (NFR) human embryos focusing on comparison of mitochondrial distribution and protein synthesis. Multi-pronuclei zygotes (MPZ) such as three or more pronuclei containing in human in vitro fertilization and embryo transfer (IVF-ET) program were used for this study. MPZ were cultured in TCM-199 supplemented with 10% of human fetal cord serum (hFCS) in 5% $CO_2$ incubator at $37^{\circ}C$ for 24 hours. The cleaved embryos to 2-4 cells after 24 hours were grouped by their grade of fragmentation. Embryos were stained with Rhodamine123 (Rh123) and fluorescence was evaluated under the fluorescence microscope through PB 450-490 filter (Leitz). Regarding to protein synthesis during early human embryogenesis, there is no significant difference in the amount of synthetic proteins between FR and NFR embryos. Distribution of cytoplasmic organelles in embryos was evaluated by transmission electron microscope (TEM). The cytoplasmic distribution of mitochondria was different between FR and NFR embryos. The mitochondrial distribution was even in NFR, whereas severely aggregated in FR. It is not able to clarify in the present study whether this uneven mitochondrial distribution in FR embryo is the reason for embryo fragmentation or is the result from fragmentation. Physiological disparity related to the mitochondrial distribution may be one of the reasons for embryo fragmentation. Further studies should be addressed to investigate the physiological differences between FR and NFR embryos.

불임환자에 있어서 Partial Zona Dissection(PZD) 의한 임상적인 결과

박성은,최동희,노환철,고정재,박종영,차광열,Park, Sung-Eun,Choi, Dong-Hee,Rho, Hwan-Cheol,Ko, Jung-Jae,Park, Jong-Young,Cha, Kwang-Yul 대한생식의학회 1993 Clinical and Experimental Reproductive Medicine Vol.20 No.1

Micromanipulation procedures have been used to improve fertilization rates in patients with male factor or with unexplained infertility. Partial zona disseetion(PZD), a method using mechanical force to open the zona pellucida increase the chances of fertilization. The purpose of this study is to increase rates of fertilization and pregnacy in the ART program by using PZD. The influence of PZD on the fertilization rate was investigated in 57 couples with semen defects, antisperm antibodies(ASA), or unknown factors. PZD directly performed in 35 couples with a history of fertilization failure in previous cycle (Group 1), and PZD applied in 22 couples with the failure of initial fertilization in the same cycle (Group 2). The fertilization rates of the male facor, ASA positive factor and unknown factor in Group 1 were 37.6%, 20.0% and 59.2%, respectively. The rates of fertilization of male factor, ASA positive factor and unknown factor in Group 2 were 34.8%, 20.0% and 26.5%, respectively. The incidences of polyspermy in Group 1 and Group 2 were 5.9% and 9.0%, respectively. Among 35 patients of Group 1, one patient was pregnant and successfully delivered, whereas 1 of 22 patients of Group 2 became pregnant, but aborted at 7 weeks.

시험관아기 시술시 미세조작에 의한 임신율의 증진에 관한 연구

노환철,김은경,구정진,고정재,윤태기,차광열,Rho, Hwan-Cheol,Kim, Eun-Kyung,Koo, Jung-Jin,Ko, Jung-Jae,Yoon, Tae-Ki,Cha, Kwang-Yul 대한생식의학회 1993 Clinical and Experimental Reproductive Medicine Vol.20 No.2

This study was carried out to improve pregnancy rate in IVF-ET program through Assisted Hatching (AH) by the use of micromanipulation technique. Among 72 IVF patient, randomized 29 IVF patients were performed for AH by Partial Zona Dissection(PZD). Two to eight cell embryos were micromanipulated just before uterine transfer. The results were as follows: 1. The implantation rates of embryos between PZD group and control group were 10.0%, 4.9%, respectively. 2. The clincal pregnancy rates of both groups were 34.5%,20.9%, respectively. 3. Among 131 PZD embrys, only 2 embryos were damaged mechanically. Although there were no statistical difference in the rates of implantation and pregnancy between PZD group and control group due to small sample size, the PZD group had increasing trend in the rates of implantation and pregnancy. In conclusion, it would be thought that PZD could be adequately used to improve implantation rate and pregnancy rate in IVF-ET program as an assisted technique if much more studies were done. Also the risks resulting from this study can be reduced because of technical stability, which showed the low rate of damaged embryos.