성선자극호르몬이 흰쥐 난소의 GnRH와 GnRH mRNA의 발현에 미치는 영향

백원영,정파진,박신근,김완영,이종학,김종화,김명옥,최완성,Paik, Won-Young,Chung, Pa-Jin,Park, Shin-Keun,Kim, Wan-Young,Lee, Jong-Hak,Kim, Jong-Hwa,Kim, Myeong-Ok,Choi, Wan-Sung 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.1

Expression of gonadotropin releasing hormone(GnRH) has been described in the rat ovary. It remains, however, unkown whether GnRH is synthesized as a prohormone. Therefore, this study was performed to verify the expression of pro-GnRH by in situ hybridization and further to investigate the effect of gonadotropin on GnRH or GnRH mRNA in rat ovary by immunohistochemical and in situ hybridization techniques. Adult female Sprague-Dawely rats were used and the estrous cycle was synchronized by intraperitoneal injection of pregnant mare's serum gonadotropin(PMSG). Ovaries were fixed with 4% paraformaldehyde and embedded with G.C.T. compound and cut by cryostat. For immunohistochemistry, avidin-biotin peroxidase complex(ABS) method was employed and for in situ hybridization, $^{35}S$-end labeled oligonucleotide was used and followed by autoradiography. By in situ hybridization using GnRH oligomer and GAP(GnRH associated protein) oligomer, GnRH mRNA and GAP mRNA were co-localized in the fullicular cells, luteal cells, interstitial cells and theca cells. GnRH or GnRH mRNA signals in the ovary increased by human chorionic gonadotropin(hCG) injection. At the 3 and 6 hrs after hCG injection, the number of GnRH and GnRH mRNA containing cells increased rapidly and the density of GnRH and GnRH mRHA culminated at 9 hrs after heG injection. With the follicular development, the high expression of GnRH and GnRH mRNA was also observed within the follicles. After ovulation, the density of GnRH or GnRH mRNA decreased in the follicles but increased in the corpus lutea.

생쥐 난자의 초자화동결 시 Cytochalasin B가 Cytoskeleton에 미치는 영향

백원영,최원준,김세나,이종학,Paik, Wong-Young,Choi, Won-Jun,Kim, Se-Na,Lee, Jong-Hak 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Objective : The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. Methods : Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at $37^{circ}C$ for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at $4^{circ}C$ overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. Results: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05). Conclusion: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.

단세포군 항체를 이용한 태아의 OXYTOCIN 및 VASOPRESSIN 분비세포의 출현에 관한 연구

백원영(PAIK Won Young),김종화(KIM Jong Hwa),이종학(LEE Jong Hak),이봉희(LEE Bong Hee),최완성(CHOI Wan Sung),백상호(BAIK Sang Ho) 대한체질인류학회 1988 대한체질인류학회지 Vol.1 No.1

전자간증 산모의 태반에서 Death Domain Receptor-3, Caspase-10, Insulin-Like Growth Factor Binding Protein-3의 발현

백원영 ( Won Young Paik ),최원준 ( Won Jun Choi ),이순애 ( Soon Ae Lee ),이종학 ( Jong Hak Lee ),최완성 ( Wan Sung Choi ) 대한주산의학회 2002 大韓周産醫學會雜誌 Vol.13 No.4

연구목적:전자간증 산모의 태반에서 정상 산모의 태반과 비교하여 DDR-3, caspase-10, IGFBP-3 유전자 발현 양상을 조사하고자 하였다. 방법:경상대학교 병원 산부인과에 입원하여 전자간증으로 진단 받은 산모로부터 적출된 태반10예와 정상 산모의 태반 15예를 사용하였으며, DDR-3, caspase-10, IGFBP-3의 mRNA에 대하여 RT-PCR을 한 후 전기영동을 시행하였다. 결과분석을 위해 NIH image program을 이용하여 이들 유전자의 발현을 비교하였다. 결과:정량적으로 평가한 결과에서 전자간증 산모의 태반에서 정상 산모의 태반에 비하여 DDR-3와 caspase-10의 발현은 유의하게 증가되었으며, IGFBP-3의 발현은 유의하게 감소되었다. 결론:전자간증 산모의 태반에서 DDR-3와 caspase-10 유전자의 발현 증가 및 IGFBP-3 유전자의 발현 감소는 전자간증 산모 태반의 apoptosis 조절기전에 관여할 것으로 생각된다. Objective: To determine that preeclampsia is associated with differential expression of death domain receptor-3 (DDR-3), caspase-10, andinsulin-like growth factor inding protein-3 (IGFBP-3). Methods: Placenta were collected from 10 preeclampsias and from 15 normal pregnancies and the samples were immediately frozen and stored at -70℃. Total mRNA was extracted from placental tissue and then analysis of the expressions of DDR-3, caspase-10, and IGFBP-3 were performed by quantitative RT-PCR. Results: Quantitative RT-PCR analysis demonstrated significantly increased DDR-3 and caspase-10 expression levels and significantly decreased IGFBP-3 expression level in the placenta of preeclampsia compared with normal pregnancy Conclusion: Placental apoptosis is associated with DDR-3, aspase-10, and IGFBP-3. These results may be one of possible mechanisms in the placental apoptosis of preeclampsia.

GnRH - receptor mRNA의 임신 시기별 발현 양상

백원영(Won Young Paik),신반철(Ban Cheol Shin),신정규(Jeong Gyu Shin),최원준(Won Jun Choi),이순애(Soon Ae Lee),이종학(Jong Hak Lee),최완성(Wan Sung Choi),김명옥(Myeong Ok Kim) 대한산부인과학회 1998 Obstetrics & Gynecology Science Vol.41 No.12

N/A Objective: Our previous study demonstrated that the placental GnRH and GnRH mRNA did not parallel the time course of hCG secretion, though it is thought to be one of the potential paracrine regulators of hCG secretion from the trophoblasts. The present study was designed to examine the potential variation in GnRH-receptor mRNA expression in the placenta, which may account for the GnRH-mediated action of hCG secretion during pregnancy. Methods: Human placentas in firt, second, and third trimester of normal pregnancy were obtained. These placentas were fixed with 4% paraformaldehyde and embedded in OCT compound, and sectioned by cryostat. For in situ hybridization, S labeled RNA probes were used and followed by autoradiography. Results: The GnRH-receptor mRNA signals were present in both cytotrophoblast and syncytiotrophoblast cell layers. Signal intensities varied with gestational ages and were abundant at 6-7 weeks, peaked at 9-12weeks, declined at 14 and 24 weeks, and were barely detectable at term. The present study demonstrates that GnRH-receptor mRNA exhibits changes paralleling the time course of hCG secretion during pregnancy Conclusion: These data provide mechanistic understanding that the paracrine/autocrine regulation of hCG secretion by placental GnRH is mediated through an increase followed by a decline in GnRH-receptor mRNA expression from the first trimester to term placenta.

Cytochalasin B가 생쥐 난자의 초자화동결 시 생존률과 체외발달률에 미치는 영향

최원준 ( Won Jun Choi ),이순애 ( Soon Ae Lee ),이종학 ( Jong Hak Lee ),백원영 ( Won Young Paik ) 대한산부인과학회 2003 Obstetrics & Gynecology Science Vol.46 No.2

목적 : Microfilament inhibitor로서 세포질막을 보다 신축성 있게 만들며 난자의 미세구조를 안정화시킨다고 알려진 Cytochalasin B (CCB)를 사용하여 초자화동결 시 생쥐 난자의 생존률 및 체외발달률을 조사하고자 하였다. 연구 방법 : PMSG와 hCG를 사용하여 과배란을 유도하여 얻은 생쥐 난자를 EM grid를 사용하여 초자화동결과 융해를 하였다. 현미경하에서 초자화동결 및 융해를 거친 난자의 생존률과 체외발달률을 조사하였 Objective : The purpose of this study was to evaluate the effect of CCB treatment on the survivability and in vitro development of mouse oocyte frozen by vitrification. Methods : Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated b

cDNA expression array를 이용한 전자간증 태반의 Apoptosis 및 매개인자에 대한 연구

최원준 ( Won Jun Choi ),최완성 ( Wan Sung Choi ),이종학 ( Jong Hak Lee ),백원영 ( Won Young Paik ) 대한산부인과학회 2003 Obstetrics & Gynecology Science Vol.46 No.2

목적 : 본 연구는 apoptosis가 전자간증 산모의 태반에서 어떠한 양상으로 나타나는지를 조사하고 어떤 종류의 매개인자가 관여하는지를 밝히고자 하였다. 연구 방법 : 전자간증 산모의 태반에서 apoptotic body를 확인하고자 TUNEL법을 사용하였고, cDNA expression array를 이용하여 전자간증 산모의 태반에서 apoptosis에 관여하는 미지의 유전자를 밝히고자 하였다. 결과 : 전자간증 산모의 태반에서 정상 산모의 태반에 비해 Objective : The present study was designed to examine whether preeclampsia is associated with an increase in placental apoptosis and to detect differential expression of mediators involved in apoptosis. Methods : Placental tissues from 10 cases of preecla

초자화동결된 생쥐 Preantral Follicle의 체외성장과 배란

박지권,백원영,Park, Ji-Kwon,Paik, Won Young 대한생식의학회 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.2

Objective: To define an appropriate vitrification condition of preantral follicle that yields high survival and to evaluate growth and ovulation rate of mouse follicles during in vitro culture after vitrification. Methods: Preantral follicles were isolated mechanically from mouse ovaries that were surgically recovered from mice aged 14 days. Retrieved preantral follicles were placed in EG (Ethylene Glycol) for 2, 5, 10 minutes and transferred to EFS-40 (40% EG, 18% Ficoll-70, 0.5 M sucrose) for 0.5, 1, 2 minutes. And then, preantral follicles were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing was carried out at room temperature. After defining the most appropriate vitrification condition that yields high survival, in vitro growth and ovulation rate of follicles were evaluated. Results: Appropriate vitrification condition that yield high survival rate ($83.2{\pm}2.1%$) of preantral follicle was EG for 5 minutes and EFS-40 for 0.5 minutes. In vitro survival rate of the vitrified preantral follicles were $85.5{\pm}0.5%$, $67.9{\pm}0.8%$ and $40.2{\pm}0.5%$ on day 2, 6 and 10. And in vitro growth of the vitrified preantral follicles were $107.1{\pm}16.1{\mu}m$, $117.1{\pm}18.4{\mu}m$, $178.4{\pm}45.6{\mu}m$ and $325.4{\pm}54.4{\mu}m$ on day 0, 2, 6 and 10. Although in vitro survival rate and growth of vitrified preantral follicles were lower than that of non-vitrified preantral follicles, the patterns of survival and growth were similar in vitrified and non-vitrified preantral follicles. The ovulation rate of antral follicles that was grown from vitrified preantral follicles was $32.6{\pm}1.2%$. Conclusion: Vitrified preantral follicles could be grown to antral sizes, and mature oocytes that can be used for IVF-ET programs were produced successfully. These data suggest that cryopreservation of preantral follicle by vitrification can be used for the preservation of the fertility.

전자간증 산모의 태반에서 Apoptosis에 관한 연구

최원준 ( Won Jun Choi ),신정규 ( Jeong Gyu Shin ),최완성 ( Wan Sung Choi ),이종학 ( Jong Hak Lee ),백원영 ( Won Young Paik ) 대한주산의학회 2002 Perinatology Vol.13 No.3

목적:전자간증 산모의 태반에서 정상 산모와 비교하여 apoptosis의 차이를 확인하고, apoptosis를 조절하는 매개인자인 bcl-2, bax, caspase-3의 발현양상을 조사하여 전자간증과 apoptosis와의 관련성을 알아보고자 하였다. 방법:전자간증 산모 10예와 정상 산모 15예의 태반을 연구에 사용하였으며, TUNEL 염색법을 이용하여 apoptotic index를 구하여 두 군을 비교하였고, 면역조직화학염색법으로 bcl-2, bax, caspase-3의 발현의 차이를 조사하였다. 결과:전자간증 산모의 태반에서 정상 산모에 비해 apoptotoc index가 주로 영양막에서 유의하게 증가 되었다. 합포영양막에서 주로 발현된 bcl-2는 전자간증 산모의 태반에서 감소하였으나 유의한 차이는 없었고, 세포영양막에서 주로 발현된 bax는 전자간증 산모의 태반에서 증가하였지만 유의한 차이는 없었다. Caspase-3의 발현 부위는 주로 세포영양막이었으며 전자간증 산모의 태반에서 정상 산모에 비해 유의하게 증가되었다. 결론:전자간증 산모의 태반에서 apoptosis는 증가되었고, 적어도 caspase-3의 발현 증가는 전자간증의 병리기전에 중요하게 작용할 것으로 생각되며 여기에는 추후 더 많은 연구가 필요할 것으로 사료된다. Objective:To investigate the change of placental apoptosis and the expression of their mediator in preeclampsia women. Methods:Placental tissues from 10 cases of preeclampsia and 15 cases of normal pregnancy were analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling(TUNEL) staining. Expressions of bcl-2, bax, caspase-3 was also assessed using immunohistochemistry. Results:In TUNEL staining, the number of apoptotic nuclei were significantly increased in the trophoblast of preeclampsia than normal pregnancy. Bcl-2 was mainly expressed in syncytiotrophoblast and bax was mainly expressed in cytotrophoblast. Bcl-2 expression was decreased and bax expression was increased in the preeclampsia than normal, but the difference was not significant. Caspase-3 was mainly expressed in the cytotrophoblast and expression was significantly increased in the preeclampsia than normal pregnancy(p<0.05). Conclusion:Placental apoptosis, especially accompanied with increased expression of caspase-3 in cytotrophoblast, might be related with in the pathogenesis of preeclampsia.

양막 세포 공배양이 생쥐배아의 발달 및 착상에 미치는 영향

최원준(Won Jun Choi),신정규(Jeong Gyu Shin),이순애(Soon Ae Lee),이종학(Jong Hak Lee),백원영(Won Young Paik) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.2

목적 : 사람의 양막 세포와 생쥐의 배아를 공배양함으로써 양막 세포의 공배양의 효과를 알아 보고자 한다. 연구 방법 : 제왕절개술을 시행 받은 산모에서 얻은 양막세포를 2일간 배양하여 준비한 후, 생쥐에서 얻은 2세포기, 8세포기 배아와 공배양하여 포배기 배아의 형성과 부화 및 착상을 관찰하고, 공배양하지 않은 대조군과 비교 분석하였다. 결과 : 2세포, 8세포의 모든 경우에서 공배양의 유무와 관계없이 모두 팽창된 배아(expanded blastocyst)로 진행하는 빈도는 차이가 없었지만, 배아가 부화(hatching) 하는 빈도에서는 공배양을 한 경우에서 공배양을 하지 않은 경우 보다 의미 있게 증가된 소견 (2세포: 71.6% vs 47.0% / 8세포: 74.0% vs 56.1%)을 보였다. 그리고 2세포를 공배양을 한 경우에서는 공배양을 하지 않은 경우 보다 착상률에서도 의미 있게 증가된 소견(31.6% vs 13.7%)을 보였다. 결론 : 체외수정 시술에서 사람의 양막 세포의 공배양은 배아의 포배기로의 발생률을 증가시키고, 착상률을 증가시키므로 체외수정 시술에서 배아의 질적 향상에 도움이 되리라 사료된다. Objective : This study was performed to investigate the influence of coculture with human amnion cell on growth, development, implantation in mouse embryos. Methods : Two-cell and Eight-cell stage mouse embryos were cocultured for 96 hrs with amnion cell. Embryos not treated with amnion were served control. The percentages of embryos which developed to the expanded, hatched blastocyst stage and in vitro implantation at 24hrs, 48hrs, 72hrs were determined. Results : The percentages of fully expanded murine blastocysts in coculture with amnion cell were not significantly different from control. The percentages of hatched blastocysts were significantly higher in coculture group at two-cell stage (71.6%) compared to control (47.0%; p<0.05). The percentages of hatched blastocysts were significantly higher in coculture group at eight-cell stage (74.0%) compared to control (56.1%; p<0.05). In two-cell stage embryos, the percentages of implanted blastocyst in vitro were significantly higher following coculture with amnion cell (31.6%) compared to control (13.7%). Conclusion : Amnion cell coculture may have a stimulatory role in embryonic development, implantation compared to control. The coculture condition which induces the best effect on in vitro growth and development might be the mimics of the physiologic natural condition, just like the coculture with the tubal epithelium.