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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.
Formation and Differentiation of Human Fetal Ovarian Follicles
도병록,이창주,송강원,윤현수,노성일,윤용달 한국발생생물학회 2000 발생과 생식 Vol.4 No.2
포유류 원시난포의 분화에는 뇌하수체에서 분비되는 gonadotropins 외에도 다양한 성장인자들 뿐 아니라 스테로이드호르몬 등이 관여하며, 난황낭에서 기원된 생식세포들과 중신에서부터 유입되는 기질세포들의 복잡한 상호작용에 의해 이루어진다. 특히 사람의 경우 태아기에 분화가 시작된 원시난포들은 성장을 개시한 후 배란이 되거나 혹은 폐쇄되어 난소에서 제거되는데, 일부 원시난포는 성장이 개시되기까지 50년 이상 원시난포의 상태로 유지된다. 그러나 원시난포의 The regulatory mechanisms of the initiation and the formation of ovarian follicles during fetal stage of mammals are largely unknown. In addition to the gonadotropins secreted from pituitary, various growth factors, and steroid hormones are believed to be involved in the differentiation and initiation of growth of primordial follicles consisting of primordial germ cells migrated from yolk sac and streamed cells from mesonephric somatic cells. In human, primordial follicles that have already initiated differentiation at fetal stage undergo either folliculogenesis to ovulate or atresia after growth. Some of primordial follicles remain without growth for 50 years or longer. The objective of this paper is to review the mechanism of the formation, growth arrest, and initiation of primordial follicles in human fetal and neonatal ovaries.
황체호르몬 수용체의 발현이 저반응 환자군의 임신에 미치는 영향
이정복,도병록,김은수,김명희,천은경,정현정,노성일,김문규,윤현수,Lee, Jung-Bok,Do, Byung-Rok,Kim, Eun-Soo,Kim, Myung-Hee,Chun, Eun-Kyung,Jeong, Hyeon-Jeong,Roh, Sung-Il,Kim, Moon-Kyoo,Yoon, Hyun-Soo 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.2
Objectives: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. Materials and Methods: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)$\leq$3ea), Group II (n=80) is normal responder (retrieved oocytes>3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by $\beta$-actin. Statistical analysis was performed by using $X^2$ test, Student's t-test and Pearson correlation. Results: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). Conclusion: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.
인간 조혈모 줄기세포의 냉동보존에 미치는 항산화제의 영향
김응배,홍순갑,도병록,김경숙,이준영 한국발생생물학회 2008 발생과 생식 Vol.12 No.1
Oxidative damage resulted from reactive oxygen species(ROS) is one of the main causes for the decrease of the viability during in vitro culture and cryopreservation process. This experiment was performed to determine the effects of antioxidants on the human hematopoietic stem cell(HSC) during cryopreservation procedure. HSCs cultured in vitro with or without antioxidants were frozen and then examined for stem cell potential after thawing. The cell viability of thawed HSC was increased in αtocopherol and ascorbic acid treatment group compared to control group(62.7±8.0%) and it was higher in 150uM αtocopherol treatment group(70.5±7.0%). No significant difference was observed in the membrane integrity in all groups. In auto-differentiation rate, no significant difference was appeared in all groups, but was lower in 150uM αtocopherol(7.3±2.6%) compared to control group(10.1±1.6%).These results demonstrate that treatment of antoxidants improves the efficiency of cryopreservation for HSC and αtocopherol may be considered effective antioxidant for the protective effect on HSC. Reactive oxygen species(ROS)에 의한 산화적 손상은 냉동보존 과정과 체외 배양과정 중 세포 생존률 감소의 주된 요인 중 하나이며, 특히 줄기세포의 경우 냉동보존 후 쉽게 분화하거나 사멸하는 경향이 있음이 잘 알려져 있다. 따라서 본 연구는 체외 배양된 인간 조혈모 줄기세포의 냉동보존 시 선별된 항산화제를 처리하여 항산화제가 줄기세포의 생존 및 자동분화에 미치는 영향을 조사하고자 하였다. 해동 후 세포의 생존률은 α‐tocopherol과 ascorbic acid처리군이 대조군(62.7±8.0%)에 비해 높은 생존률을 보였고 그 중 150uM α‐tocopherol처리군(70.5±7.0%)이 가장 높은 생존률을 보였다. 세포막 손상은 대조군 및 실험군 모두에서 나타나지 않았다. 자동분화율에 있어서는 모든 실험군에서 대조군(10.1±1.6%)과 유의한 차이를 보이지 않았으나, 150uM α‐tocopherol(7.3±2.6%)처리군에서 가장 낮은 자동분화율을 나타내었다. 본 실험의 결과 항산화제는 인간 조혈모 줄기세포 냉동보존 시 생존율을 향상 시키며 특히 α‐tocopherol은 인간 조혈모 줄기세포의 냉동보존 과정 동안 효과적인 항산화제로 작용할 것이라 생각된다.
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.