• Clinical and Experimental Reproductive Medicine
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• 제20권2호
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• pp.107-115
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• 1993

Procedures to separate motile. normal & motile and acrosome-reacted sperm with high efficiency have clinical application in Assisted Reproductive Technology in terms of increasing the probability of fertilization by a normal sperm and subsequent normal embryonic development. This study evaluated the effects of 10 sperm preparation techniques [Swim-up from a washed pellet (SU). Swim-up from semen (SO). Continuous Percoll Gradients I (PIC). Discontinuous Percoll Gradients I (PID). Continuous Percoll Gradients II(P II C). Discontinuous Percoll Gradients II(P II D), SpermPrep (SFC). Wang's tube (WT). Albumin Gradients (AG), Low temperature capacitation (LTC)] on motility (%), normal morphology (%), motile sperm recovery rate(%). morphologically normal & motile sperm recovery rate (%), true acrosome reaction (%) and fertilizing ability. A P II D proved to be an effective means of separating morphologically normal & motile sperm. Our results indicated the P II D has advantages as compared with other methods in terms of recovery rate. enhancement of motility and normal morphology. And a LTC seems to be an effective means of enhancing the true acrosome reaction and fertilizing ability. These results suggest that the combined method of LTC and P II D for separation of morphologically normal & motile sperm and acrosome reacted sperm may be a useful procedure for intrauterine insemination and in vitro fertilization in the management of male factor infertility as well as for isolation of subpopulation of sperm for basic research.

• Parasites, Hosts and Diseases
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• 제26권3호
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• pp.155-162
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• 1988

Toxoplasmn gondii이 강독주인 RH주와 조직내 cyst 형성주인 Fukaya주의 세포막 단배 성분을 SDS 존재하에 서 전기영동하여 분석하였다. 먼저 RH tachyzoite와 Fukaya의 cyst를 각각 마우스의 복강액과 뇌조직으로부터 분리하였는데, 불연속 Percoll density-gradient서 원심분리하여 tachygoite는 50 U와 605 Percoll용액 경계면에서, cyst는 40%와 50%의 경계면 및 50%와 60 % 경계면에서 얻었으며, cyst는 저장액으로 처리하여 bradyzoite를 얻었다. Lactoperoxidase를 촉매로 세포막에 방사성 요오드를 표지시킨 후 자가방사표지그림을 얻었을 때, bradyzoite 는 15 KDa와 14 KDa의 분자량을 가진 단백질이 주요 단백질로 나타났으며, tachyzoite에서는 30 KDa 단백질이 주요 단백질로 나타났다. 또, 당단백질의 존재를 파악하기 위해서 lectin blotting을 시행하였는데, concanavalin A는 bradyzoite에서 200K∼50KDa의 여러 단백질을, .그리고 tachyzoite에서는 52KDa 단백질을 주로 하는 33K∼20 KDa단백질을 검출하였으며, phytohemagglutinin은 두·유형에서 아무런 단백질도 검출하지 못하였다. 한편, 이들을 효소면역이적법으로 항 Fukfya항체와 항 RH항체로 반응시켰을 때, 많은 교차 반응을 보였으나, bradyzoite에서는 15 KDa 단백질이, 그리고 tachyzoite에서는 52 KDa, 30 KDa 및 25 KDa 단백 짙이 각각 유형 특이 항원 단백으로 나타났다. 위의 결과들로, bradyzoite에서는 15 KDa 단백질이 당단백질은 아니지만 특이 항원성을 갖는 주요 백으로 나타났으며, tachyzoite에서는 지금까지 주요 세포막 단백으로. 알려진 P3O외에 당단백질이며 성을 갖는 세포막 단백으로 SaKDa 단백 (gps2)을 확인할 수 있었다.

• 한국수정란이식학회지
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• 제15권1호
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2004년도 제47차 추계학술대회
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• pp.84-84
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• 2004

• 한국임상수의학회:학술대회논문집
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• 한국임상수의학회 2008년도 춘계학술대회
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• pp.105-105
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• 2008

• 한국임상수의학회:학술대회논문집
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• 한국임상수의학회 2009년도 춘계학술대회
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• pp.304-304
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• 2009

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2003년도 제45차 추계학술대회
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• pp.133-133
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• 2003

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.494-498
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• 1989

AIDS 환자의 치명적인 2차 감염을 유발하는 Cryptosporidium parvum 의 Infective stage 인 sporozoites의 단일군 항체를 분리하였다. Oocysts를 효소처리하여 sporozoites를 excystation시킨 후 Isopycnic percoll gradients를 이용하여 sporozoites를 순수분리한 후 단일군 항체 생산을 위한 항원으로 사용하였다. 두 달된 BALB/c 쥐를 immunize한 후 splenocytes와 P3-X63-Ag8 myeloma cells를 융합시킨 후 hybridoma 기술을 이용해 Kor1(IgGl), Ea2(Ig2a) 두 clones을 분리하였으며 정제된 sporozoites를 SDS-PAGE로 분리한 후 Western blot을 이용하여 단일군 항체 Kor1과 Ea2는 20,000 daltons 크기의 항원을 인식하였다. Immunofluorescent assay에서 단일군 항체가 sporozoites 표면에 반응하는 것으로 보아 20-kDa 단백질 항원은 sporozoites 표면에 위치하는 항원으로 밝혀졌으며 C. parvum에 감염되었을 때 항체생성에 관여하는 중요 항원 중 하나일 것으로 추정되었다.

• 한국가축번식학회지
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• 제17권2호
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• pp.103-111
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• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles (3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated (39$^{\circ}C$, 5% CO2 in air) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1993년도 제2회 신약개발 연구발표회 초록집
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• pp.150-150
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• 1993

목적 :크롬친화세포는 카테콜아민 (CA)을 분비하는 새포로서 무신 수질내에 주로 존재한다. 따라서 부신피질의 영향을 배제하여 여러가지 자율신경계 작용 약물의 약리작용을 연구하는데 중요한 조직이다. 그러므로, 부신으로부터 크롬친화세포를 분리하여 배양하는 방법을 습득하여 자율신경계 작용약물연구에 이용코자 히스타민을 이용하여 CA 분비작용을 연구하였다. 방법: 도살장에서 소를 즉사시킨 후 좌우양측 부신을 분리하여 collagenase digestion으로 부신수질로부터 분리하고 Percoll gradient에 의해서 chromaffine cell을 정제하였다. 이렇게 정제한 크롬친화세포는 Dulbecco's modified Eagle medium에 10% fetal calf serum과 함께 culture dish에다 넣어 5% $CO_2$, incubator에서 유지시켜 주면서 실험을 시행하였다. 배양세포는 분리후 일주일 이내에 사용하였다. Catecholamine측정은 electrochemical detector를 연결하여 HPLC로 측정하였다.