Proteomic profiling of murine oocyte maturation

被引:67
作者
Vitale, Alejandra M. [1 ]
Kennedy Calvert, Meredith E.
Mallavarapu, Mallika
Yurttas, Piraye
Perlin, Julie
Herr, John
Coonrod, Scott
机构
[1] Cornell Univ, Weill Med Coll, New York, NY 10021 USA
[2] Univ Virginia, Dept Cell Biol, Charlottesville, VA 22903 USA
关键词
proteomics; nucleoplasmin; oocyte maturation;
D O I
10.1002/mrd.20648
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD61P), stress-inducible phosphoprotein (STI1), importin alpha 2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD61P, importin alpha 2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RTPCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrestedoocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage.
引用
收藏
页码:608 / 616
页数:9
相关论文
共 29 条
[1]   Variations in the response of mouse isozymes of adenylosuccinate synthetase to inhibitors of physiological relevance [J].
Borza, T ;
Iancu, CV ;
Pike, E ;
Honzatko, RB ;
Fromm, HJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (09) :6673-6679
[2]   Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos [J].
Burns, KH ;
Viveiros, MM ;
Ren, YS ;
Wang, P ;
DeMayo, FJ ;
Frail, DE ;
Eppig, JJ ;
Matzuk, MM .
SCIENCE, 2003, 300 (5619) :633-636
[3]   Identification and cloning of Xp95, a putative signal transduction protein in Xenopus oocytes [J].
Che, SL ;
El-Hodiri, HM ;
Wu, CF ;
Nelman-Gonzalez, M ;
Weil, MM ;
Etkin, LD ;
Clark, RB ;
Kuang, J .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (09) :5522-5531
[4]   Oolemmal proteomics [J].
Coonrod, SA ;
Wright, PW ;
Herr, JC .
JOURNAL OF REPRODUCTIVE IMMUNOLOGY, 2002, 53 (1-2) :55-65
[5]   MASSIVE PHOSPHORYLATION DISTINGUISHES XENOPUS-LAEVIS NUCLEOPLASMIN ISOLATED FROM OOCYTES OR UNFERTILIZED EGGS [J].
COTTEN, M ;
SEALY, L ;
CHALKLEY, R .
BIOCHEMISTRY, 1986, 25 (18) :5063-5069
[6]  
de Moor CH, 2001, INT REV CYTOL, V203, P567
[7]  
Ferrell JE, 1999, BIOESSAYS, V21, P833, DOI 10.1002/(SICI)1521-1878(199910)21:10<833::AID-BIES5>3.0.CO
[8]  
2-P
[9]  
Gachet Y, 1999, J CELL SCI, V112, P1257
[10]   Synthesis and function of Mos: The control switch of vertebrate oocyte meiosis [J].
Gebauer, F ;
Richter, JD .
BIOESSAYS, 1997, 19 (01) :23-28