Analysis of Genes Expressed in Mouse Ovaries of Early Developmental Stages

전은현,윤세진,차광렬,김남형,이경아 한국발생생물학회 2003 발생과 생식 Vol.7 No.2

본 연구에서는 이러한 초기 난포 발달 과정 중 원시난포-1차 난포 변화과정 시기에 발현하는 유전자를 알아보고 자 수행하였다. 원시난포로만 이루어져 있는 생쥐의 생후 1일자 난소와 원시난포 및 1차 난포로만 이루어져 있는 5일자 난소의 RNA와 총 80개의 annealing control primer(ACP) primer를 사용하여 PCR을 수행하여 서로 다르게 발현하는 유전자 (differentially expressed genes; DEG) 41개를 The present study was conducted to investigate gene expression profile of mouse ovaries during the primordial-primary follicle transition. We isolated total RNA from mouse ovaries at day1(contains only primordial follicles) and day5(contains both primordial and primary follicles) and synthesized cDNA using annealing control primers(ACP, Seegene, Inc., Seoul, Korea). Using 80 different ACPs for PCR, we cloned, sequenced, and analyzed identities of 41 differentially expressed genes(DEGs). According to BLAST analysis, sequences of 33 clones significantly matched database entries, 4 clones were novel, and 4 clones were ESTs. We selected 8 DEGs with interesting functions, Anx11 and Pepp2-Pending highly expressed in day1 ovary, while Apg3/Autlp-like, BPOZ, Ches1, Kcmf1, NHE3, Nid2, Ninj1, SENP3, Suil-rsl, and TIAP/m-survivin highly expressed in days ovary, and confirmed their different expression between day1 ovaries and days ovaries using semi-quantitative RT-PCR. There was no false positive result. Using in situ hybridization, we found that almost all of genes studied were expressed in the oocyte from primordial follicle stage but expression decreased from primary follicle stage. Meanwhile their expression was increased in cuboidal granulosa cells. Different expression of BPOZ and TIAP/m-survivin between primordial and primary follicles was confirmed by using laser capture microdissection followed by real-time PCR BPOZ and TIAP/m-survivin expressed 4.5 and 3.4 fold higher in primary than primordial follicles, respectively. List of genes obtained from the present study will provide insights for the study of mechanism regulating primordial-primary follicle transition.

Gonadotropin Bioactivity and Steroids in Ovarian Follicle Matured by Hyperstimulation

윤용달,전은현,김문규,권혁방,Yoon, Yong-Dal,Chun, Eun-Hyun,Kim, Moon-Kyoo,Kwon, Hyuk-Bang The Korean Society for Reproductive Medicine 1989 Clinical and Experimental Reproductive Medicine Vol.16 No.2

본 연구는 생식주기중 폐쇄여포액내에서 생물학적, 면역학적 특성을 나타내는 GTH 의 변화를 조사하고 steroid hormone과의 상관관계를 조사하며 국부조절인자로서 의 GTH의 역 활을 조사하고자 하였다. 가임기간중 215개의 여포와 IVF과정에서 185개의 여포를 얻어 여포액내 GTH의 생물학적 또는 면역학적 활성을 측정하였다. Bioactive LH(bLH)는 생쥐의 Leydig cell-testosterone production assay, bFSH는 흰쥐의 Sertoli cell aromatase assay로 측정 하였 다. Immunological GTH(iLH , iFSH) 는 MaiaClone RIA , Delfia kits를 사용하였다. 여포액내 iLH, iFSH , ihCG 는 hyperstimulation에 의해 형성된 여포의 크기와는 무관하였다. 또 hMG, huFSH 의 처리와도 상관성이 없었다. T의 농도가 높은 여포액내의 iFSH는 현저히 낮았으며 E, P 가 고농도인 여포의 ihCG 양은 현저히 낮았다. 과배란이 유도된 난소의 여포액내 iLH는 LH specific RIA로 측정시 3mIU/ml 이하이었다. 생식주기중 여포액내 bLH, bFSH는 배란기에 현저히 증가 하였다. 혈청내 GTH B/I ratio는 엘정한 반면 여포액내 LH,FSH의 생물학적, 면역학적 활성은 미수정란을 가지거나 폐쇄된 여포내의 활성보다 현저하게 높았다. 위의 결과로 보아 여포액내 생식소자극호르온은 면역학적활성보다 높은 생물학적 활성을 가지며, 생리적 현상의 지표가 된다고 추론된다. 또한 steroid, bGTH는 여포의 선택, 폐쇄를 구분하는 지표로 사용가능하며, 여포가 폐쇄될때 여포액내 B/I ratio가 현저히 낮아지는 것으로 보아 GTH의 활성이 감소되는 것으로 판단된다.

Ultrasensitive Enzymeimmunoassay for Testosterone in Human Saliva

윤용달,전은현,이창주,도병록,이준영 한국발생생물학회 2000 발생과 생식 Vol.4 No.1

남성 및 여성의 타액내 testosterone의 농도를 측정하기 위하여 초감도 효소면역측정법 (ultrasensitive enzymeim-munoassay; EIA)을 정립하고, 이 EIA를 이용하여 한국인 정상 여성 및 남성의 타액에서 testosterone의 정상농도를 얻고자 하였다. 정상 월경주기를 가진 여성 18명에서 아침 6~9시 사이에 타액을 채취하였고, 남성의 타액도 오전 (9~10시)중에 채취하였다. EIA의 표지물질로는 horseradi A few enzymeimmunoassay (EfA) for testosterone (T) have been reported but was not suitable for all biological samples. The present study was designed to develop a rapid, ultrasensitive EIA and to apply this technique for study the physiological changes of T in biological samples. Saliva samples were collected at 06:00~09:00 hour during one menstrual cycle from 18 normally menstruating women and on 09:00~10:00 hour from 20 normal men. The present study shows an established EIA for testosterone, using horseradish peroxidase (HRP), which was covalently bonded to testosterone-3-carboxymethyloxime (T-3-CMO). One batch of T-antisera was also covalently linked to microcrystalline cellulose particles by a mixed anhydride method in order to facilitate separation of bound and free steroids. The established EIA was validated in terms of sensitivity, accuracy, specificity, precisions etc., comparing with conventional radioimmunoassay. The sensitivity of the established EIA was less than 25 pg/tube. The correlation coefficients between the expected T-values and observed T-values measured by EIA or RIA were r=0.985 and r=0.941 respectively. The cross reactivity of antiserum in EIA was a little higher than that of RIA, especially by 5 -DHT. The intra- and inter-assay precisions of the present EIA were similar to those of RIA. The present study also demonstrates that the normal T-values in saliva of Korean male & female samples are 265.6515.80 pmol/l and 109.74 12.01 pmol/l, respectively. The present EIA seems to be established and suitable for use in the endocrinological studies. The advantages of this EIA system also might make the present T-EIA an ideal procedure for use in a routine assay of ordinary laboratory with a conventional spectrophotometer.

正常 및 自發性 糖尿病動物(KK Mouse)의 血糖量에 미치는 Nicotine 및 Ethanol의 相互作用

노원소,전은현,김기순 한양대학교 의과대학 1985 한양의대 학술지 Vol.5 No.2

Several studies using interviews or questionnaries have demonstrated that a high correlation exists between heavy drinking and heavy smoking and that the similar correlation is observable even among alcoholics. It has been reported that intravenous administration of nicotine influenced human smoking behavior, and that the action of nicotine in central nervous system had small but clearly demonstrable role as a primary reinforcer of the smoking habit. Recently it was suggested, from study on effect of ethanol on the disposition of nicotine in rats, that the ethanol-related decreases in nicotine plasma level may reinforce the need for increased smoking during ethanol consumption. There are numerous reports in the literature indication that cigarette smoking or administration of nicotine causes an increase in blood sugar level in man and animals. On the other hand investigations concerning the effect of ethanol on concentration of sugar in blood of men and experimental animals have resulted in conflicting data. Now it appears to be generally recognized that ethanol-induced hypoglycemia results from inhibited gluconeogenesis by ethanol and exhaustion of glycogen stores of liver in fasted men and animals. The present study was designed to investigate effect of ethanol and nicotine on blood sugar levels in normal and diabetic (KK) mice. Experimental animals were divided into the control, nicotine, ethanol, ethanol-after nicotine, nicotine-after ethanol group administration of either nicotine (2mg/Kg) or ethanol (3g/Kg) preceded that of another by 60 minutes. The results obtained from the present study were as follows; 1. In both normal and diabetic mice intraperitoneally administered nicotine invariably caused hyperglycemia showing the peak responses (approximately 70mg% and 100mg%) in 60 minutes. 2. Following ethanol administration in the ethanol-after nicotine group of normal and KK mice, blood sugar concentration further increased (228.4±22.0mg% and 320.7± 12.9mg%) from the initial hyperglycemic level produced by nicotine administration (179.6±12.7mg% and 290.0±12.8mg%). 3. After aministration of ethanol blood sugar level increased invariably in normal mice as well as the diabetic, but the hyperglycemic responses tended to be exaggerated in KK mice. The blood sugar concentartion of normal mice elevated to maximum level (80mg%) in 30 minutes and decreased to its original level in 150 minutes while blood sugar concentration of KK mice rose to maximum level in 60 minutes and did not return to previous level even in 180 minutes. 4. Following nicotine administration in the nicotine-after ethanol group of both normal and diabetic mice, no further hyperglycemic response was observed. It is strongly suggested that hyperglycemic effect of nicotine4 is masked in the ethanol pretreated mice.

Laser Capture Microdissection을 이용한 유전자 발현 연구 (III) -생쥐 착상 부위 자궁 내강상피 조직에서 배아 병치 기간 동안 일어나는 유전자 발현에 관한 Microarray 분석-

윤세진,전은현,박창은,고정재,최동희,차광열,김세년,이경아,Yoon, Se-Jin,Jeon, Eun-Hyun,Park, Chang-Eun,Ko, Jung-Jae,Choi, Dong-Hee,Cha, Kwang-Yul,Kim, Se-Nyun,Lee, Kyung-Ah 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Object: The present study was accomplished to obtain a gene expression profile of the luminal epithelium during embryo apposition in comparison of implantation (1M) and interimplantation (INTER) sites. Material and Method: The mouse uterine luminal epithelium from IM and INTER sites were sampled on day 4.5 (Day of vaginal plug = day 0.5) by Laser Captured Microdissection (LCM). RNA was extracted from LCM captured epithelium, amplified, labeled and hybridized to microarrays. Results from microarray hybridization were analyzed by Significance Analysis of Microarrays (SAM) method. Differential expression of some genes was confirmed by LCM followed by RT-PCR. Results: Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were EST/unknown function, and the remain 53 were categorized to the structural, cell cycle, gene/protein expression, immune reaction, invasion, metabolism, oxidative stress, and signal transduction. Of the 24 structural genes, 14 were related especially to extracellular matrix and tissue remodeling. Meanwhile, among 13 genes up-regulated at INTER, 8 genes were EST/unknown function, and the rest 5 were related to metabolism, signal transduction, and gene/protein expression. Among these 58 (53+5) genes with known functions, 13 genes (22.4%) were related with $Ca^{2+}$ for their function. Conclusions: Results of the present study suggest that 1) active tissue remodeling is occurring at the IM sites during embryo apposition, 2) the INTER sites are relatively quiescent than IM sites, and 3) the $Ca^{2+}$ may be a crucial for apposition. Search for human homologue of those genes expressed in the mouse luminal epithelium during apposition will help to understand the implantation process and/or implantation failure in humans.

Effect of 19-Norandrostenedione on the Spermatogenesis in Rat Testis

윤용달,전은현,양현원,김종민,Yoon, Yong-Dal,Chun, Eun-Hyun,Yang, Hyun-Won,Kim, Jong-Min The Korean Society for Reproductive Medicine 1990 Clinical and Experimental Reproductive Medicine Vol.17 No.1

19-nortestosterone(NORA)와 19-norandrostenedione(NT)는 정소내 aromatization과정중 중간 대사물로 검출된다. 본 연구는 이들을 장기간 투여하여 정소 및 부속기관의 무게, 혈청내 testosterone(T)의 농도, 정자형성에 미치는 영향을 조사하였다. NORA, NT 및 TE를 $300/50{\mu}l$농도로 주당 3회씩 12주간 정소에 직접 주사(intratesticular injection, i.t.)하였다. 또한 GnRH antagonist(RS68439)를 처리하여 혈청내 생식소자극호르몬 (GTH)을 감소시킨후 위 호르몬들을 동일방법으로 처리하여 이들의 보상작용을 조사하였다. NORA는 정소무게를 감소시키지 않았으나 GnRH antagonist를 처리하여 감소된 정소무게를 현저하게 보상하였다(P<0.05). NORA, NT, TE는 모두 부속기관의 무게를 증가시켰으며, RS68439가 감소시킨 부속기관의 무게를 현저히 증가시켰다. 그러나 이들은 부정소(epididymis)에는 영향을 주지않았으며 RS68439처리군에서는 보상작용을 나타내었다. 이들의 처리로 혈청내 LH농도는 완전히 감소하였으나 FSH의 농도에는 영향을 나타내지 않았다. 그리고 혈청내 T의 농도를 증가시켰다. NORA는 정자형성과정중 7단계의 spermatid의 수를 현저히 감소시켰다. 위 결과로 보아 NORA는 GnRH antagonist로 FSH의 분비가 억제된 쥐의 FSH분비를 촉진하며, T의 농도를 증가시켜 정자형성과정을 억제하는 것으로 추론된다.

Mobile transposon-like element, clone MTi7:RNA interference를 이용한 역할 규명

박창은,신미라,전은현,조성원,이숙환,김경진,김남형,이경아,Park, Chang-Eun,Shin, Mi-Ra,Jeon, Eun-Hyun,Cho, Sung-Won,Lee, Sook-Hwan,Kim, Kyung-Jin,Kim, Nam-Hyung,Lee, Kyung-Ah 대한생식의학회 2003 Clinical and Experimental Reproductive Medicine Vol.30 No.4

Objectives: The present study was conducted to evaluate the mobile transposon-like element, clone MTi7 (MTi7) expression in the mouse ovary and to determine its role(s) in the mouse oocytes by RNA interference (RNAi). Methods: MTi7 mRNA expression was localized by in situ hybridization in day5 and adult ovaries. Double stranded RNA (dsRNA) was prepared for c-mos, a gene with known function as control, and the MTi7. Each dsRNA was microinjected into the germinal vesicle (GV) stage oocytes then oocyte maturation and intracellular changes were evaluated. Results: In situ hybridization analysis revealed that MTi7 mRNA localized to the oocyte cytoplasm from primordial to preovulatory follicles. After dsRNA injection, we found 43-54% GV arrest of microinjected GV oocytes with 68%-90% decrease in targeted c-mos or MTi7 mRNA. Conclusions: This is the first report of the oocyte-specific expression of the MTi7 mRNA. From results of RNAi for MTi7, we concluded that the MTi7 is involved in the germinal vesicle breakdown in GV oocytes, and MTi7 may be implicated with c-mos for its function. We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions.

Concentrations of Bioavailable Testosterone and Dihydrotestosterone Determined by Luminescence Immunoassay in Serum

윤용달,이창주,전은현,이준영,Yoon, Yong-Dal,Lee, Chang-Joo,Chun, Eun-Hyun,Lee, Joon-Yeong The Korean Society for Reproductive Medicine 1988 Clinical and Experimental Reproductive Medicine Vol.15 No.2

혈액에서 생물학적 활성을 나타내는 (bioavailable) steroid hormone은 주로 비결합형(free form)과 알부민 결합형(albumin-bound form)으로 구성된다. 특히 Testosterone (T)과 5 alpha-Dihydrotestosterone (DHT)의 활성적 분획이 전체의 T, DHT 양에 비해 생리적 현상과 보다 잘 일치하는 것으로 알려지고 있다. 본 연구는 섬광면역측정법(Luminescence immunoassay, LIA)으로 혈청내 활성적 T 및 DHT의 농도의 측정에 이용하고져 하였다. 항체는 T- 또는 DHT-3-CMO-BSA를 항원으로 토끼에 면역주사하여 얻었다. 추적자는 T-3-CMO, DHT-3-CMO에 aminobutylethylisoluminol(ABEI)를 부착시켜 사용하였다. 항체중 IgG분획을 Protein-A-Sepharose CL-4B로 분리한 후 Immunobead(Bio-Rad)에 부착시켜 Solid-phase LIA를 실시하였다. 본 연구에서 LIA는 정확도(accuracy), 정밀도(precision), 감도(sensitivity), 교차반응도(specificity)등을 조사하고, 기존의 방사면역측정법(RIA)과 비교하여 만족할만한 결과를 얻었다. 혈청내 T및 DHT의 활성적 분획의 농도를 측정한 결과는 다음과 같았다. T의 경우는 남성에서 T의 전체량의 33% 이상으로 $7.1{\pm}1.5nmol/l$, 여성에서는 26% 이상으로 $0.28{\pm}0.05nmol/l$이었다. DHT의 활성적 분획은 남성의 경우 $601.7{\pm}85.8pmol/l$, 여성의경우 $52.4{\pm}19.9\;pmol/l$이었다. 이상의 결과를 보아 본 연구에서 이용된 LIA는 혈청내 활성적 농도를 측정하기에 충분하다고 사료된다. 또한 이 방법을 이용하여 여성의 Androgenicity 및 남성 정소기능등의 제어방법에 응용될 수 있을 것으로 판단된다.

Change of Steroid Receptor Number of and Bioactivity of Gonadotropins in the Follicular Fluid of Porcine Ovarian Atretic Follicles(II):Testosterone Receptor

윤용달,이창주,전은현,김문규,이준영,Yoon, Yong-Dal,Lee, Chang-Joo,Chun, Eun-Hyun,Kim, Moon-Kyoo,Lee, Joon-Yeong The Korean Society for Reproductive Medicine 1989 Clinical and Experimental Reproductive Medicine Vol.16 No.1

Estradiol (E)은 난소내 과립세포 (granulosa cell, GC)를 증가시키고 생식소 자극호르몬과 협동으로 배란을 유도한다. Androgen은 E의 작용과 반대의 작용을 나타내며 여포의 폐쇄요인으로 알려지고 있다. Testosterone(T)이 폐쇄여포의 여포액내 다량 존재하는 것이 알려짐에 따라 난소내에도 자가조절분비(autocrine)또는 paracrine regulation에 의해 작용을 나타낼 것으로 가정되어 난소내 여포가 폐쇄됨에 따라 그 수용체의 변화하는 양상을 조사하고져 하였다. 흰쥐의 과립세포의 세포질에는 $51.3{\pm}6.1$fmol/mg protein의 Estradiol 수용체(ER) ; $153.1{\pm}25.3$의 Testosterone수용체(TR) ; 또한 $35.1{\pm}8.1$의 Progesterone수용체(PR)가 존재하였다. 과립세포내 ER용 세포질내 E를 제거한 후에 정량이 가능하였고 또한 과립세포내에도 TR이 사람에서는 $23.4{\pm}7.2$ fmol/mg protein, 돼지는 $98.5{\pm}23.1$로 상당량 존재함을 관찰하였다. Dihydrotestosterone Enanthate(DHTE)를 100ug/흰쥐의 농도로 처리한 결과 난소내 TR의 농도는 변화가 없이 ER의 농도만 현저히 저하되고 쥐의 난소무게 역시 감소하는 것을 발견하였다. 위의 결과로 보아 난소내에도 스테로이드 호르몬은 autocrine(자가조절)방법으로 작용하며 An-drogen이 난소의 무게를 감소시키는 것은 ER의 수를 감소시켜 E의 작용이 억제되고 여포들이 폐쇄를 일으켜 그 증식이 저하된 때문으로 사료된다.

타액 내 testosterone 측정을 위한 효소면 역 측정 법 과 응용

장철수 ( C S Chang ),전은현 ( E H Jun ) 대한임상검사과학회 1992 대한임상검사과학회지(KJCLS) Vol.24 No.1

A few enzymeimmunoassay(EIA) for steroid hormones especially testosterone(T) have been reported but was not suitable for all biological samples. The present study was designed to develop a rapid, sensitive EIA and to apply this technique for study the physiological changes of T in biological samples. Saliva samples were collected on 06 : 00-09 : 00 hour during one menstrual cycle from 18 normally menstruating women and on 10 : 00-12 : 00 hour from 20 normal men. The present study shows an established T-EIA, using horse radish peroxidase(HRP), which was covalently bonded to testosterone-3-carboxy methyl (T-3-CMO). One batch of T-antisera was also covalently linked to microcrystalline cellulose particles by an mixed anhydride method in order to facilitate separation of bound and free steroid. The established EIA was validated in terms of sensitivity, accuracy, specificity, precisions etc., comparing with conventional radioimmunoassay, which has also been established in the present study. The sensitivity of the established EIA was less than 25 pg/tube. The correlation coefficients between the expected T -values and observed T-values measured by EIA or RIA were r=0.985 and r=0.941 respectively. The cross reactivity of antiserum in EIA was a little higher than that of RIA, especilly by 5 alpha-DHT. The intra-and inter-assay precision of the present EIA were extremely similar to those of RIA. The present study also demonstrates that the normal Tvalues in saliva of Korean male and femal samples are 265.65±15.80 pmol/1 and 109.74±12.01 pmol/1 respectively. The present EIA seems to be established and suitable for use in the endocrinological studies. The advantages of this EIA system also might make the present T-ElA an ideal procedure for use in a routine steroid laboratory especially in underdeveloping poor country.