[논문]돼지 단위발생 배아의 발달과정에서 세포사멸에 관한 연구

이재달

doi:10.5762/kais.2013.14.8.3843

초록
AI-Helper

이배체 단위발생 돼지 난자를 체외 배양시 배반포 형성 단계에서 FBS (우태아 혈청), BSA (우혈청 알부민), EGF(상피세포 성장인자)를 배양액에 첨가하였을 때 이배체 단위발생 에서 총세포수, 세포사멸 및 세포사멸에 관여하는 유전자의 발현 효과를 조사하고자 본 연구를 수행하였다. 0.4% BSA를 배양액에 첨가 하였을 때 2 세포기 단계 단위발생의 발달은 배반포 까지는 강화 되었다 (p<0.01). FBS 처리 시는 배반포의 세포 수는 감소시켰으나 세포 사멸률은 증가하였다(p<0.01). 하지만 EGF가 존재할 때 BSA 처리는 총 세포수를 증가 시켰다. RT-PCR의 결과에 의하면 EGF는 0.4% BSA가 존재하는 배양액에서는 Bcl-xL mRNA 발현을 증가시키고 BSA와 EGF 가 단독으로 존재 할 때는 효과가 없었다. 하지만 FBS 처리시 Bcl-xL 유전자 발현은 감소하고 Bak 유전자의 발현은 증가시킨다. 이러한 결과 세포사멸에 관여하는 유전자의 발현은 배양액의 첨가물에 따라 유의적으로 영향을 받으며, 돼지 배아의 체외 배양시 세포사멸과 초기발달에 관여함을 시사한다.

Abstract ▼ AI-Helper

This study was conducted to determine the effects of fetal bovine serum (FBS), bovine serum albumin (BSA) and epidermal growth factor (EGF) on blastocoel formation, total cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. The addition of ...

This study was conducted to determine the effects of fetal bovine serum (FBS), bovine serum albumin (BSA) and epidermal growth factor (EGF) on blastocoel formation, total cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell stage parthenotes to the blastocysts stage (p<0.01). Treatment of FBS reduced cell numbers of blastocysts (p<0.01) and increased the percentage of apoptosis in blastocysts (p<0.001). However, BSA increased cell numbers, only in the presence of EGF reverse-transcriptase polymerase chain reaction revealed that EGF enhanced the mRNA expression of Bcl-xL in the presence of 0.4% BSA but BSA and EGF alone had no effect. However, Treatment of FBS reduced Bcl-xL mRNA expression (p<0.05) and enhanced Bak expression. This result suggests that apoptosis related gene expression is significantly affected by supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

주제어

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

가설 설정

Exposure of early cattle embryos (2 to 8 cell) to FBS significantly reduced the formation of blastocoel, blastocysts cell numbers[13]. Supporting the idea that EGF reduces apoptosis and FBS enhances apoptosis are our observations on the expression of two apoptosis-related genes during blastocyst development in vitro. Supporting the idea that EGF reduces apoptosis induced by BSA factors are our observations on the expression of two apoptosis-related genes during blastocyst development in vitro.

제안 방법

Experiment 2 was conducted to determine the effect of protein supplements on the apoptosis and total cell numbers in the blastocysts. In vitro cultured embryos obtained as described in experiment 1 were harvested at the blastocyst stage on day 7 and then used in the TUNEL assay of apoptosis.
Experiment 3 was conducted to evaluate the effect of protein supplements on Bcl-xL and Bak gene expression in the porcine parthenotes and in vivo produced embryos. Presumptive diploid parthenotes were obtained as described in Experiment 1 were harvested at the blastocyst stage on day 7, washed in PBS and stored at –70℃ before RT-PCR.
In the present study we demonstrated the effects of exogenous protein supplements on porcine blastocyst production. The experiments were performed with embryos of homogeneous quality by selecting good quality porcine diploid parthenotes at the 2-cell stage. We found that porcine presumptive diploid parthenotes at 30h after activation developed to the blastocyst stage at a relatively high rate in the presence of BSA as compared to those in the presence of either PVA or none-supplement control.
EGF or BSA by itself had no effect on apoptosis. These observations are in line with the effect of BSA and EGF on cell numbers, as blastocyst cell numbers are the end result of a combination of cell proliferation and cell death processes. In the present study FBS decreased cell numbers in blastocysts cultured from 24h.
To investigate whether different supplements can modulate mRNA expression of apoptotic-related genes in diploid porcine parthenotes developing in vitro, mRNA was prepared from single blastocysts cultured in the NCSU 23 media containing different supplements. Gene expression of Bcl-xL and Bak were examined by RT-PCR analyssis (Table 4).

데이터처리

The scores in stage of blastocyst, mean cell number of blastomeres, percentage of apoptosis and relative abundance of gene expression were subjected to analysis of variance using the general linear model (PROC-GLM) in SAS program (Anon, 1992). When the significance of the main effects was detected in each experimental parameter, the treatment effects were compared by the least square method.

이론/모형

Apoptotic cells in individual embryos were measured by the TUNEL assay. There was no difference in apoptosis (fragmented cell number/total cell number) of blastocysts in the control and PVA, PVA+EGF or BSA-derived embryos.
The scores in stage of blastocyst, mean cell number of blastomeres, percentage of apoptosis and relative abundance of gene expression were subjected to analysis of variance using the general linear model (PROC-GLM) in SAS program (Anon, 1992). When the significance of the main effects was detected in each experimental parameter, the treatment effects were compared by the least square method. Differences of p<0.

성능/효과

Diploid parthenotes were recovered after 24 h of culture in NCSU 23 medium containing 0.4% BSA, Presumptive 2-cell stage (30 h) embryos were collected and washed three times in NCSU 23 medium without (control) or with 0.1% PVA (w/v, P-1763, Sigma), 10% FBS (v/v, 16140-071, Gibco), 0.4% BSA (w/v, A-8806, Sigma), 0.1% PVA added 10 μg/ml murine EGF (Sigma, St. Louis, USA) or 0.4% BSA added 10 ng/ml murine EGF and then randomly cultured in the same medium containing none (control), 0.1% PVA, 10% FBS, 0.4% BSA, 0.1% PVA + 10 ng/ml EGF or 0.4% BSA + 10μg/mll EGF.
In conclusion, our data indicate that conditions of supplementing PVA, FBS, BSA or EGF in culture medium, affect on developing, cell numbers and apoptosis. BSA enhanced in vitro development of porcine diploid parthenotes, while FBS reduce embryo viability.
In the present study FBS decreased cell numbers in blastocysts cultured from 24h. The ratio of TUNEL-labeled DNA to total DNA area of FBS-derived blastocysts was greater than in its absence medium-derived counterparts (7% vs. 4%). This result is consistent with previous results in cattle.

참고문헌 (14)

Maurer, H.R. Towards serum-free, chemically defined media for mammalian cell culture. In: Freshney RI , Animal Cell Culture:A Practical Approach. Oxford: Oxford University:15-.1992
Wei, Z., Park, K.W., Day, B.N. and Prather, R.S. Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.Mol Reprod Dev. 60:457-462, 2001. DOI: http://dx.doi.org/10.1002/mrd.1110

상세보기
Chia, C.M., Winston,R.M.L. and Handyside, A.H.. EGF, TGFa, and EGFR expression in human preimplantation embryos. Development. 121:299-307,1995.
Lee, E.S. and Fukui, Y. Effect of various growth factors in a defined culture medium on vitro development of bovine embryos matured and fertilized in vitro. Theriogenology. 44:71-83,1995. DOI: http://dx.doi.org/10.1016/0093-691X(95)00149-3

상세보기
Byrne, A.T., Southgate, J, Brison, D.R. and Leese, H.J. Analysis of poptosis in the pre-implantation bovine embryo using TUNEL. J. Reprod. Fertil. 117:97- 105,1999. DOI: http://dx.doi.org/10.1530/jrf.0.1170097

상세보기
Cory, S. and Adams, J.M. Matters of life and death: programmed cell death at cold spring harbor. Biochim. Biophys. Acta. 1377:R25-44, 1998.
Jurisicova, A. Antenos, M. Varmuza, S. and Tilly, J.L. Casper RF. Expression of poptosis-related genes during human preimplantation embryo development: potential roles for the harakiri gene product and caspase-3 in blastomere fragmentation. Molecular. Human. Reproduction. 9:133-141, 2003. DOI: http://dx.doi.org/10.1093/molehr/gag016

상세보기
Warner, C.M., Exley, G.E., Mcelhinny, A.S. and Tang, C. Genetic regulation of preimplantation mouse embryo survival. The Journal of Experimental Zoology. 282:272-9 , 1998. DOI: http://dx.doi.org/10.1002/(SICI)1097-010X(199809/10) 282:1/2 3.0.CO;2-U

상세보기
Van, T.N., Harayama, H. and Miyake, M. Characteristics of preimplantational lopment of porcine parthenogenetic diploids relative to the existence of amino acids in vitro. Biol. Reprod. 67:1688-1698, 2002. DOI: http://dx.doi.org/10.1095/biolreprod.102.004812

상세보기
Cui, XS., Xu, Y.N., Shen X.H., Zhang, L.Q,. Zhang, J.B,. Kim, N.H.. Trichostatin a modulates apoptotic gene expression and improves embryo viability in cloneed embryos. cell reprogram 13 (2) 179-189, 2011.

상세보기
Cui, X.S,. Shen, X.H,. Kim, N. H.. High mobility group box 1 (HMGB1) is implicated in preimplantation embryo development in the mouse. Mol Reprod dev 75(8)1290-1299, 2008. DOI: http://dx.doi.org/10.1002/mrd.20694

상세보기
Fisher, D.A.and Lakshmanan, Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocr. Rev. 11:418-442, 1990. DOI: http://dx.doi.org/10.1210/edrv-11-3-418

상세보기
Hagemann, L.J., Weilert, .LL., Beaumont, S.E. and Tervit, H.R. Development of bovine embryos in single in vitro production (sIVP) systems. Mol. Reprod. Dev. 51:143-147, 1998. DOI: http://dx.doi.org/10.1002/(SICI)1098-2795(199810) 51:2 3.0.CO;2-Q

상세보기
Jurisicova, A., Latham, K.E., Casper, R.F. and Varmuza, S.L. 1998. Expression and regulation of genes associated with cell death during murine pre-implantation embryo development. Mol. Reprod. Dev. 51:243-253, 1998. DOI: http://dx.doi.org/10.1002/(SICI)1098-2795(199811) 51:3 3.0.CO;2-P

상세보기

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증

돼지 단위발생 배아의 발달과정에서 세포사멸에 관한 연구
Effect of Apoptosis on Porcine Parthenotes Development in vitro 원문보기

초록
AI-Helper

Abstract ▼ AI-Helper

주제어

AI 본문요약
AI-Helper

가설 설정

제안 방법

데이터처리

이론/모형

성능/효과

참고문헌 (14)

이 논문을 인용한 문헌

저자의 다른 논문 :

관련 콘텐츠

원문 보기

원문 URL 링크

오픈액세스(OA) 유형

이 논문과 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

연합인증

돼지 단위발생 배아의 발달과정에서 세포사멸에 관한 연구 Effect of Apoptosis on Porcine Parthenotes Development in vitro 원문보기

초록 용어보기논문에서 용어와 풀이말을 자동 추출한 결과로, 시범 서비스 중입니다. AI-Helper

Abstract ▼ AI-Helper

주제어

AI 본문요약 엑셀 다운로드 AI-Helper

가설 설정

제안 방법

데이터처리

이론/모형

성능/효과

참고문헌 (14)

이 논문을 인용한 문헌

저자의 다른 논문 :

이재달 (9)

관련 콘텐츠

원문 보기

원문 URL 링크

오픈액세스(OA) 유형

이 논문과 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

돼지 단위발생 배아의 발달과정에서 세포사멸에 관한 연구
Effect of Apoptosis on Porcine Parthenotes Development in vitro 원문보기

초록
AI-Helper

AI 본문요약
AI-Helper