Search
Search

연구성과물 검색 타이틀 이미지

HOME ICON HOME > Search by Achievements Type > Reports View

Reports Detailed Information

https://www.krm.or.kr/krmts/link.html?dbGubun=SD&m201_id=10006991&local_id=10019669
돼지 난자의 체외성숙과 체외수정시 plasminogen activator 활성의 변화
Reports NRF is supported by Research Projects( 돼지 난자의 체외성숙과 체외수정시 plasminogen activator 활성의 변화 | 2004 Year | 사수진(강원대학교) ) data is submitted to the NRF Project Results
Researcher who has been awarded a research grant by Humanities and Social Studies Support Program of NRF has to submit an end product within 6 months(* depend on the form of business)
사업별 신청요강보기
  • Researchers have entered the information directly to the NRF of Korea research support system
Project Number F00003
Year(selected) 2004 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2007년 10월 30일
Year type 결과보고
Year(final report) 2007년
Research Summary
  • Korean
  • 본 연구는 plasminogen activators (PAs) 활성이 돼지 난자의 체외성숙 및 수정에 미치는 영향을 검토하기 위해서 수행되었다. 우선, 돼지의 정자와 난자에서 PAs의 존재여부와 체외수정시 수정배지내에 PA의 특이적 기질인 plasminogen을 첨가가 체외수정에 미치는 영향을 검토하였다. 돼지정자를 체외수정용 배양액 (mTBM)에서 0, 2, 4 및 6시간동안 배양하였을 경우, tPA-PAI, tPA 및 uPA의 활성이 정자배양액(sperm incubation medium)과 정자(sperm)에서 관찰되었으며, 정자배양액에서의 PA 활성은 배양시간이 연장됨에 따라 증가하는 경향을 보였다. 난자-난구세포 복합체(COCs)를 체외에서 0, 22 및 44 시간동안 성숙배양한 결과, 채란직후의 난구세포 제거난자에서는 아무런 PA 활성이 관찰되지 않았다. 그렇지만, 성숙배양액에서 22시간동안 배양 후 PA 활성을 검토한 결과, tPA-PAI와 tPA 활성이 검출되었으며, 난자에서의 PA 활성 역시 배양시간이 연장됨에 따라 점차 증가하였다. 한편, PAs의 특이적 기질인 plasminogen이 체외수정에 미치는 영향을 검토한 결과, 수정배지에 대한 plasminogen의 첨가는 난자의 투명대(ZP)에 대한 정자의 부착을 증가시키고, 웅성전핵형성을 증가시켰다. 또한, plasminogen은 활성화된 난자에서 pronase에 의한 투명대의 용해시간을 증가시켰으며, 난자에 대한 정자침입율과 다정자침입은 억제시켰다.
    ROS 생성조건인 xanthine-xanthine oxidase system하에서 돼지정자를 전배양한 후, 정자의 생존성, 첨체반응, lipid peroxidation 및 PA 활성을 검토하였다. 그 결과, 이 실험에서 이용된 낮은 농도의 X-XO system은 정자의 생존성과 lipid peroxidase에는 영향을 미치지 않았다. 그렇지만, X-XO system에 의해 생성된 ROS는 첨체반응이 일어난 정자의 비율과 정자배양액(sperm incubation medium)에서의 PA활성을 증가시켰다.
    또 다른 연구에서, X-XO system하에서 배양된 돼지난자의 체외성숙과 PA활성에 대한 난구세포의 영향을 검토하기 위하여, 난구세포가 제거 또는 부착된 난자를 X-XO system하에서 성숙배양을 실시한 결과, 난구세포가 제거된 난자가 X-XO system에 의해 생성된 ROS에 노출되었을 경우 난자의 퇴행, DNA cleavage 및 capase-3 활성이 증가하였으며, 이 경우 높은 tPA의 활성이 관찰되었다. 그렇지만, 난구세포 부착난자의 경우에는 ROS에 의한 DNA cleavage나 capase-3 활성이 다소 증가하였으나, 유의적인 차이는 나타나지 않았으며, tPA 활성 뿐만 아니라 tPA-PAI 활성이 검출되었다.
    이와 같은 연구결과로부터, PA들은 정자의 첨체반응과 난자의 성숙시에 방출 또는 생산되며, 수정배지에 대한 plasminogen의 첨가와 그에 따른 plasmin 생성의 증가는 돼지의 체외수정능력 향상에 유리한 영향을 미치는 것으로 사료된다. 또한, ROS에 의해 증가된 tPA 활성은 돼지의 체외성숙시 산화스트레스에 의해 야기되는 apoptosis와 관련이 있는 것으로 추측된다.
  • English
  • These studies were performed to understand the changes of plasminogen activator (PA) activity on in vitro maturation and fertilization in porcine oocytes. In chapter 1, the effects of plasminogen on sperm-oocyte interaction during in vitro fertilization in the pig was examined. It was found that when boar spermatozoa were preincubated in fertilization medium (mTBM) for 0, 2, 4 or 6 h, the activities of tPA-PAI, tPA and uPA were observed in sperm incubation medium and spermatozoa. These PA activities in sperm incubation medium increased with duration of culture prolonged. When oocytes with cumulus cells were cultured for in vitro maturation, no PA activities were observed in cumulus free-oocytes just after aspiration from follicles. However, the activity of tPA-PAI and tPA was observed at 22 h of culture and increased with duration of culture prolonged. The addition of plasminogen, the major substrate for PAs, to fertilization medium increased the sperm binding to zona pellucida (ZP), ZP dissolution time (sec) by pronase and male pronucleus formation, but not penetration and polyspermy.
    Chapter 2 was performed to examine the effects of reactive oxygen species (ROS) on sperm function and PA activity in porcine spermatozoa. The low concentration of the xanthine-xanthine oxidase (X-XO) system used in this study was not significant influence in either sperm viability or lipid peroxidation. However, ROS generated by the X-XO system increased the percentage of acrosome-reacted spermatozoa and activities of PA in the sperm incubation medium.
    Chapter 3 was conducted to examine the effects of cumulus cells on plasminogen activator activity in matured porcine oocytes under X-XO system. When porcine COCs were cultured in maturation medium (NCSU-23) for 44 h, the activities of tPA-PAI, tPA and uPA were observed. However, tPA-PAI and tPA were detected in oocytes that were separated from porcine COCs at 44 h of maturation culture, and only tPA was produced in oocytes that were denuded before the onset of maturation culture. The proportion of oocytes remaining GV stage, oocytes degenerated, DNA cleavage and caspase-3 activity were increased in cumulus free-oocytes exposed to ROS generated by the X-XO system. The higher activity of tPA were observed in porcine DOs underwent apoptotic changes by oxidative stress. In COCs, however, tPA-PAI as well as tPA was detected and apoptotic changes such as DNA cleavage or caspase-3 activation were not observed.
    In conclusion, porcine gametes produce PAs during culture in vitro. The supplement of plasminogen to fertilization medium during IVF, and thereby increased generation of plasmin, may participate in improving of fertilization ability in vitro in the pig. The increase of acrosome reaction by ROS resulted in increase of PAs activity in the sperm incubation medium. The increased activity of tPA may be relevant to apoptotic changes by oxidative stress during in vitro maturation in the pigs. These results suggest that PAs activity may influence to not only apoptosis but also in vitro maturation and fertilization in porcine oocytes.
Research result report
  • Abstract
  • These studies were performed to understand the changes of plasminogen activator (PA) activity on in vitro maturation and fertilization in porcine oocytes. In chapter 1, the effects of plasminogen on sperm-oocyte interaction during in vitro fertilization in the pig was examined. It was found that when boar spermatozoa were preincubated in fertilization medium (mTBM) for 0, 2, 4 or 6 h, the activities of tPA-PAI, tPA and uPA were observed in sperm incubation medium and spermatozoa. These PA activities in sperm incubation medium increased with duration of culture prolonged. When oocytes with cumulus cells were cultured for in vitro maturation, no PA activities were observed in cumulus free-oocytes just after aspiration from follicles. However, the activity of tPA-PAI and tPA was observed at 22 h of culture and increased with duration of culture prolonged. The addition of plasminogen, the major substrate for PAs, to fertilization medium increased the sperm binding to zona pellucida (ZP), ZP dissolution time (sec) by pronase and male pronucleus formation, but not penetration and polyspermy.
    Chapter 2 was performed to examine the effects of reactive oxygen species (ROS) on sperm function and PA activity in porcine spermatozoa. The low concentration of the xanthine-xanthine oxidase (X-XO) system used in this study was not significant influence in either sperm viability or lipid peroxidation. However, ROS generated by the X-XO system increased the percentage of acrosome-reacted spermatozoa and activities of PA in the sperm incubation medium.
    Chapter 3 was conducted to examine the effects of cumulus cells on plasminogen activator activity in matured porcine oocytes under X-XO system. When porcine COCs were cultured in maturation medium (NCSU-23) for 44 h, the activities of tPA-PAI, tPA and uPA were observed. However, tPA-PAI and tPA were detected in oocytes that were separated from porcine COCs at 44 h of maturation culture, and only tPA was produced in oocytes that were denuded before the onset of maturation culture. The proportion of oocytes remaining GV stage, oocytes degenerated, DNA cleavage and caspase-3 activity were increased in cumulus free-oocytes exposed to ROS generated by the X-XO system. The higher activity of tPA were observed in porcine DOs underwent apoptotic changes by oxidative stress. In COCs, however, tPA-PAI as well as tPA was detected and apoptotic changes such as DNA cleavage or caspase-3 activation were not observed.
    In conclusion, porcine gametes produce PAs during culture in vitro. The supplement of plasminogen to fertilization medium during IVF, and thereby increased generation of plasmin, may participate in improving of fertilization ability in vitro in the pig. The increase of acrosome reaction by ROS resulted in increase of PAs activity in the sperm incubation medium. The increased activity of tPA may be relevant to apoptotic changes by oxidative stress during in vitro maturation in the pigs. These results suggest that PAs activity may influence to not only apoptosis but also in vitro maturation and fertilization in porcine oocytes.
  • Research result and Utilization method
  • 돼지정자에서 tPA-PAI, tPA 및 uPA의 활성이 관찰되었으며, 난자-난구세포 복합체의 체외성숙시 tPA-PAI와 tPA 활성이 증가하였다. PAs의 특이적 기질인 plasminogen은 난자의 투명대에 대한 정자의 부착, 웅성전핵형성 및 활성화된 난자에서 pronase에 의한 투명대의 용해시간을 증가시킨 반면 난자에 대한 다정자침입은 억제시켰다.
    ROS는 첨체반응이 일어난 정자의 비율과 정자배양액에서의 PA활성을 증가시켰다. 한편, 난구세포가 제거 또는 부착된 난자를 X-XO system하에서 성숙배양을 실시한 결과, 난구세포가 제거된 난자가 X-XO system에 의해 생성된 ROS에 노출되었을 경우 난자의 퇴행, DNA cleavage 및 capase-3 활성이 증가하였으며, 이 경우 높은 tPA의 활성이 관찰되었다. 그렇지만, 난구세포 부착난자의 경우에는 ROS에 의한 DNA cleavage나 capase-3 활성의 증가가 크지 않았으며, tPA뿐만 아니라 tPA-PAI 활성이 동시에 검출되었다.
    이와 같은 연구결과로부터, PA들은 정자의 첨체반응과 난자의 성숙시에 방출 또는 생산되며, 수정배지에 대한 plasminogen의 첨가와 그에 따른 plasmin 생성의 증가는 돼지의 체외수정능력 향상에 유리한 영향을 미치는 것으로 사료된다. 또한, ROS에 의해 증가된 tPA 활성은 돼지의 체외성숙시 산화스트레스에 의해 야기되는 apoptosis와 관련이 있는 것으로 추측된다.
    최근 발생공학연구의 진전에 따른 transgenic 동물을 작출해 장기이식용 donor 및 병리질환 모델로서 돼지를 이용하려는 연구가 활발히 진행되고 있다. 그러나 가축에서는 유전자 발현율이 1% 이하로 transgenic 동물의 작출효과가 매우 낮기 때문에 이들 재료로 이용되는 수정란을 대량으로 생산하는 것이 상기의 목적을 달성하기 위해 무엇보다 중요하다. 본 연구의 결과는 돼지의 체외수정계에서 정자의 수정능획득, 첨체반응 및 정자-난자의 결합을 조절하는 메카니즘을 밝혀 정상수정란의 생산효율을 낮추는 가장 큰 요인인 체외수정후 높은 비율로 발생하는 다정자침입 및 낮은 전핵형성율을 향상시키고, 체외수정 이후 초기배 발육중에 빈번하게 발생하는 발육억제현상을 극복하기 위한 기초연구자료로 활용될 수 있을 것으로 기대된다.
  • Index terms
  • Plasminogen activators, Maturation, Fertilization, Reactive oxygen species (ROS), Oocyte, Sperm, Pig
  • List of digital content of this reports
데이터를 로딩중 입니다.
  • This document, it is necessary to display the original author and you do not have permission
    to use copyrighted material for-profit
  • In addition , it does not allow the change or secondary writings of work
데이터 이용 만족도
자료이용후 의견
입력
트위터 페이스북
NRF Daejeon
(34113) 201, Gajeong-ro, Yuseong-gu, Daejeon, Korea
Tel: 82-42-869-6114 / Fax: 82-42-869-6777
NRF Seoul
(06792) 25, Heonreung-ro, Seocho-gu, Seoul, Korea
Tel: 82-2-3460-5500 / Fax: 82-2-3460-5759
KRM Help Center
Tel : 042-869-6086 Fax : 042-869-6580
E-mail : krmcenter@nrf.re.kr