It was conducted the experiment, divided into three groups as normal, poor and polycystic ovary syndrome, to detect the change of protein patterns in follicular fluid on ovarian response following controlled ovarian hyperstimulation for human IVF outcome. In the normal group, it was confirmed reproducible 57 spots in the detected total 81 spots. Then 1 spot was not found in the other groups. In the poor responder group, it was found reproducible 53 spots in the detected total 98 spots. 6 spots were down-regulation and 7 spots were up-regulation comparable with normal group. There were not 5 spots in poor responder group comparable with other groups. In the polycystic ovary syndrome group, it was expressed reproducible 53 spots in the detected total 80 spots and 3 spots were just expressed in this group. However, 4 spots were not found in polycystic ovary syndrome. 9 spots were up-regulation comparable with normal group. Significant up and down-regulation spots among the each groups were identified. The results were a cytosolic carboxypeptidase, a signal-induced proliferation-associated protein 1, a ceruloplasmin, a keratin(type Ⅱ cytoskeletal 1), a polypeptide N-acetylgalactosaminyltransferase 2, a serine/threonine-protein phosphatase 4 regulatory subunit 4. It was identified that 8 spots, 6 kinds of protein are corresponded with NCBInr database research, but 10 spots were failed in the identification. In conclusion, it has been confirmed change and expression of protein on the ovarian response following COH of human.

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro condition is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. This study analyzed change of proteins in follicular fluids during the porcine follicular development. Follicular fluids were collected from follicles of diameter 1～2 mm, 2～6 mm and 6～10 mm in ovary of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. We confirmed totally 27 same spots, 1 spot from follicle fluid of 2～6 mm follicle and 5 spots from follicle fluid of 6～10 mm in diameter were analyzed by MALDI mass spectrometry and searched on NCBInr. In results, spot No. 28 from 2～6 mm follicle was Ig lambda chain C region, and spot No.32 and 33 from 6～10 mm was Apolipoprotein A-(APOA4). Spot No.29 and 31 were failed to analyze. These results indicate that the porcine oocyte during in vitro maturation depend on specific different expressed proteins may play an important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

본 연구는 돼지 미성숙 난포란의 유리화 동결액에 노출시 독성과 삼투압 스트레스가 가장 적은 유리화 동결액을 조사하기 위하여 실시하였으며, 그 결과를 요약하면 다음과 같다. 미성숙 난포란을 EFS(40% ethylene glycol +18% ficoll +0.3 M sucrose) 용액, ES(5.5 M ethylene glycol +1.0 M sucrose) 용액, GE(10% glycerol +20% ethylene glycol) 용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙하여 관찰한 체외성숙율은 ES 용액과 대조구 에서 EFS 용액과 GE 용액보다 유의적으로 높았으며(P<0.05), 유리화 동결액의 독성에 의한 난포란의 퇴화율은 대조구, ES 용액, EFS 용액, GE 용액 순으로 유의적으로 낮은 퇴화율을 나타내었다(P<0.05). 미성숙 난포란을 EFS 용액, ES용액, GE용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙, 체외수정하여 관찰한 정상 수정율은 대조구가 세 종류의 유리화 동결액보다 유의적으로 높았고(P<0.05), 유리화 동결액 간에는 유의적인 차이가 없었다. 다정자 침입율과 난자당 침입한 평균 정자수는 모든 처리구간에 유의적인 차이가 없었다. 미성숙 난포란을 EFS 용액, ES 용액, GE 용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙, 체외수정, 체외 배양하여 관찰한 수정 3 일째의 분할율은 ES 용액과 대조구가 EFS 용액과 GE 용액보다 유의적으로 높았으며(P<0.05), 수정 7일째의 배반포 형성율은 모든 처리구간에 유의적인 차이가 없었다. 배양기간 동안 나타난 퇴화율은 대조구가 세 가지 유리화 동결액에 비해 유의적으로 낮았고(P<0.05), ES 용액은 EFS 용액과 GE 용액보다 유의적으로 낮은 퇴화율을 나타내었다(P<0.05).

The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37/with hFF, Group II: 32/withGroup III: 37/without, Group IV:32 /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.91.9%. After 24 hours culture, sperm motility was 23.52.1% (Group I), 8.11.1% (Group II), 10.4 1.4% (Group III) and 4.00.8% (Group IV), respectively. After 48 hours, the motility had been changed as 322.3% (Group I), 14.31.7% (Group II), 5.3 1.4% (Group III) and 4.30.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37 was higher than those of group II at 32. But, sperm viability of group I cultured at 37 was lower than those of group II at 32 (54.44.1% vs. 59.43.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.

본 연구는 사람의 시험관아기 프로그램에서 체외 수정란의 질을 향상시키는 한 방법으로 난포액으로 처리된 정자를 체외 수정에 사용하여 생산된 체외 수정란의 전핵 등급과 발달 능력을 조사하였다. 정상적인 시험관 아기 시술을 시행한 실례 환자를 대상으로 과배란을 유기하여 배란 직전의 난자를 채취하여, 난포액으로 처리된 정자와 체외 수정시킨 후 사람 체외 수정란의 전핵 등급과 체외발달율을 조사하였다. 체외수정을 위한 정자의 처리 방법으로 synthetic ser

포유동물의 암컷 생식기관에 존재하는 다양한 종류의 matrix metallo-proteinase (MMP)는 난소와 자궁의 구성성분의 주기적인 변화를 조절하며 이중 난소의 MMP는 난포의 성장과 배란 그리고 퇴화 동안 조직재구성에 매우 중요한 역할을 한다고 알려져 있다. 본 실험에서는 근래에 새로 발견된 사람의 난포액에 존재하는 분자량 약 110kDa인 MMP-2 isoform GA110을 동정하고 자 하였다. 난포액으로부터 GA110 단백질을 분리하기 위하여 난포액에 5mM ethylenediaminetetraacetic acid(EDTA)를 처리한 후 DEAE Sepharose Fast Flow를 이용한 chromatography를 시행하였다. 그 결과, 난포액 단백질들은 0.2M NaCl 의 분획에서 GA110 활성을 나타내었고 anti-human MMP-2 antibody에 대한 면역반응도 뚜렷이 나타났다. DEAE Sepharose Fast Flow에서 얻은 분획 중 GA110의 활성과 면역반응을 모두 나타내는 분획만을 모아 Gelatin Sepharose 4B affinity chromatography로 다시 분리하였다 분리한 결과 resin에 흡착된 단백질 (eluate) 분획들에서 매우 뚜렷한 GA110 gelatinase 활성을 나타내었으며 면역반응 또한 관찰되었다. 이 분획들의 단백질을 농축한 후 zymography를 시행하여 나타난 GA110 단백질 band를 잘라 내었으며 이로부터 단백질을 electroelution하여 농축한 후 reducing agent인 2-mercaptoethanol를 처리하였다. 이를 전기영동 후 MMP-2 (propeptide region) antibody를 사용하여 immunoblotting 한 결과 70-72kDa의 단백질만이 면역반응을 나타내었다. 마지막으로 위와 같이 준비된 70-72kDa 단백질의 아미노산 서열을 Edman degradation 방법으로 분석하였다. 그 결과 이 단백질의 N 말단의 10개의 아미노산 배열 순서가 알려진 사람의 proMMP-2의 전체 배열순서 중 propetide domain의 N 말단에서부터 다섯 번째에서 시작하여 10개의 아미노산의 서열과 정확하게 일치하였다. 위 결과들로 미루어 사람의 난포액에 존재하는 MMP-2의 새로운 isoform인 GA110은 70-72kDa의 ProMMP-2가 disulfide bond를 통해 homodimer 구조를 이루고 있는 것으로 여겨진다.

난포액내 함유되어 있는 단백질성분 중에서 sucrose 층으로부터 정자의 swim-up 이동을 자 극하는 성분을 분리하기 위하여 paper chromatography (PC) 및 reverse phase column (RPC) 과 superose column (SC)를 이용한 액체 chromatography의 분리효과를 조사하였던 바 결과는 다음과 같다. 1. Chromatography용 paper로 분리한 각 band 의 성분은 첨가농도가 증가할수록

정자의 무분별한 이동을 충분히 억제하면서 운동성을 저해하지 않는 sucrose 층으로부터의 swim-up 분리체계를 구축하기 위하여, 정자의 이동에 장애가 될 수 있는 sucrose 층에 첨가되는 sucrose수준과 정자의 이동과 운동을 극대화시킬 수 있는 배양시간 및 sucrose 이중층의 형태를 조사하였던 바, 얻어진 결과는 다음과 같다. 1. 10mM의 sucrose 층은 정자의 swim-up 이동을 억제하였다. 2. Sucrose 층을 이용하지

Follicular fluid influxed into the oviduct during ovulation may affect movement of sperm for fertilization Thus, in this study, the effect of follicular fluid, obtained from follicles of l0mm in diameter, on number and quality of sperm recovered by swim-up separation was investigated and sperm-movement stimulating components extracted from follicular fluid with methanol and isooctane were separated by gel filtration with Sepadex G-1O, G-25 and G-1OO gels, and were isolated by electrophoresis with SDS-PAGE mini gel. The results obtained were as follows; 1. Diluted follicular fluid stimulated sperm movement. 2. Sperm-movement stimulating factors were in methanol extract. 3. Sperm-movement stimulating effect of methanol extract appeared in fraction I among fractions recovered after gel filtration. And the fraction I contained proteins indicating 4 major bands as about 47, 43, 25 and 14 kilodaldons and 5 minor bands as about 67, 58, 23, 22 and 21 kilodaldons. 4. The fraction I recovered from G-100 gel showed significantly low percentage of motile sperm and had no protein indicating the band of 67 kilodaldons among the minor bands.

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

The studies on the carried out to investigate the effects of co-culture with uterine fluids and uterine epithelial cells on the in-vitro fertilization and developmental rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% ECS for 46~48 hrs in a incubator with 5% in air at 38.5 and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The results obtained in these experiments were summarized as follows ; 1.The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with uterine fluids in TCM-199 medium were 68.0% arid 55.7%, the rates were higher than of control, 56.5% arid 38.7%. 2. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the fertilization rate was 60.3%, the rates were higher than that of control, 35.7%. 3. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the development rate to be blastocyst was 12.4%, the rates were higher than that of control, 9.2%(p<0.05).