이차원 전기영동(2-DE)을 이용하여 체외성숙동안 돼지 난자의 단백질 패턴

소개

포유동물 난소의 난포 내에서 난자의 성숙과 분화는 체세포성 난포세포와 발달중인 생식세포 사이의 긴밀한 관계에 달려있다. 완전히 성장한 포유동물 난자의 체외성숙은 초기 난핵포 붕괴(GVBD; germinal vesicle break down)와 제 1차 감수분열 시에 미세소관의 재정렬, 제 1극체 방출과 2차 감수분열 중기의 차단에 의해 특징되어진다. 오로지 완전히 성숙된 난자만이 수정이 일어날 수 있고 접합자 단계에 진입할 수 있게 된다. 이러한 관찰은 대부분 형태학적 평가에 기원을 두고 있지만, 이러한 과정이 일어나는 분자적 사건은 명확히 알려져 있지 않다. 이러한 난자의 성숙과 분화 과정은 특정한 단백질의 합성에 의해 수반되어 일어난다. 특이하게 난자에서는 mRNA level과 다량의 단백질 사이에는 명백히 나타나는 상호작용이 드러나 있지 않다. 게다가, 단백질의 활성은 세포질 성장, 분화, 세포 주기와 감수분열과정과 같은 세포내 과정을 조절하는 특이적 kinase와 phosphatase에 의해 조절되어 진다. 다시 말해 난자의 감수분열 성숙 동안 단백질 분해효소의 활성, MPF (maturation promoting factor)와 MAPK(mitogen-activated protein kinase)가 뛰어난 역할을 담당한다.
본 연구에서는 분자적 조절과정의 이해를 도모하기 위하여 이차원 전기영동과 단백질을 동정할 수 있는 질량 분석기를 이용하여 체외 성숙 레벨에서 난자의 성숙과정 동안 다르게 발현되어지는 것을 확인하고자 하였다.

목차

Ⅰ. RESEARCH BACKGROUND

Ⅱ. INTRODUCTION

Ⅲ. MATERIAL & METHODS
1. Culture Media
2. Collection of Porcine Oocytes and In Vitro Maturation
3. Two Dimension Electrophoresis (2-DE)
4. Staining 2-DE Gels
5. Proteomic Analysis
6. Identification of proteins by MALDI-TOF/MS

Ⅳ. RESULTS
1. In Vitro Maturation of Porcine Oocytes
2. Separation of Protein from Porcine Oocytes using a 2-DE
3. Identification of Oocyte Proteins by MALDI-TOF MS

Ⅴ. DISCUSSION

Ⅵ. REFERENCES

SUMMARY IN KOREAN

본문내용

In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. Cellular maturation and differentiation processes are accompanied by the expression of specific proteins. Especially in oocytes, there is no reliable strict linear correlation between mRNA levels and the abundance of proteins. Furthermore, the activity of proteins is modulated by specific kinases and phosphatases which control cellular processes like cellular growth, differentiation, cell cycle and meiosis. During the meiotic maturation of oocytes, the activation of protein kinases, namely of the MPF and MAPK play a predominant role.
In the present study, to better understand the molecular regulation processes, It was utilized two-dimensional electrophoresis (2-DE) and mass spectrometry to identify proteins that are differentially expressed during oocyte maturation in vitro maturation (IVM) the level. To investigate protein patterns during porcine oocyte maturation in vitro, first of all, GV as control and M II phase was analyzed. The GV stage porcine oocytes (GV) were put in culture with TCM-199 from time zero to 44hr for M II stage oocytes IVM period. Total proteins were extracted from 1200 GV and M II stage arrested oocytes, separated on 2-DE gel and stained with silver. In general, the overall protein-staining pattern between the two gels was remarkably similar with most proteins spots. Analysis of the gels indicated that was seemingly up and down-regulated between the GV and M II stage gel.

참고자료

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