Protein Expression Profile of the Mouse ... - ACS Publications

Sep 20, 2008 - A proteome reference database for the mouse oocyte was ... This database is the most detailed mouse oocyte proteomic database to date...
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Protein Expression Profile of the Mouse Metaphase-II Oocyte Minyue Ma,†,‡,§,# Xuejiang Guo,†,# Fuqiang Wang,† Chun Zhao,† Zichuan Liu,‡ Zhonghua Shi,† Yufeng Wang,† Ping Zhang,† Kemei Zhang,† Ningling Wang,† Min Lin,† Zuomin Zhou,† Jiayin Liu,† Qingzhang Li,§ Liu Wang,‡ Ran Huo,*,† Jiahao Sha,† and Qi Zhou*,‡ Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China, State Key Laboratory of Reproductive Biology, Institute of Zoology, The Chinese Academy of Science, Beijing 100080, China, and College of Life Sciences, Northeast Agricultural University, Harbin 150030, China Received May 29, 2008

The mature oocyte contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins have yet to be characterized. In this study, two-dimensional electrophoresis (2-DE) of mouse metaphaseII (MII) oocyte proteins, stained with silver staining or Pro-Q Diamond dye, was performed to describe the proteome and phosphoproteome of the mouse oocyte derived from ICR mice. A total of 869 selected protein spots, corresponding to 380 unique proteins, were identified successfully by mass spectrometry, in which 90 protein spots representing 53 unique proteins have been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All identified proteins were bioinformatically annotated in detail and compared with the embryonic stem cell (ESC) proteome. A proteome reference database for the mouse oocyte was established from the protein data generated in this study, which can be accessed over the Internet (http://reprod.njmu.edu.cn/2d). This database is the most detailed mouse oocyte proteomic database to date. It should be valuable in expanding our knowledge of the regulation of signaling in oogenesis, fertilization, and embryo development, while revealing potential mechanisms for epigenetic reprogramming. Keywords: Bioinformatics • Mouse oocyte • Phosphoproteome • Proteome • Reprogramming factors

Introduction Mammalian oocytes are highly specialized cells with the ability to reconstruct sperm DNA and initiate zygotic development. Maternal proteins and mRNAs play an important role during fertilization, oocyte activation, zygotic gene activation (ZGA), and early embryogenesis. The oocyte can also confer totipotency or pluripotency on somatic cells by somatic cell nuclear transfer (SCNT) or cell fusion.1-7 But the mechanisms involved in the ability of oocytes to reverse the epigenetic changes that direct terminal differentiation and permanent exit from the cell cycle remain largely unknown. Recently, the establishment of induced pluripotent stem (iPS) cells demonstrated that, by introducing a combination of defined factors, mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state.8-12 This is a breakthrough in the stem cell field and it opens an avenue for generating * To whom correspondence should be addressed: Prof. Dr. Qi Zhou, State Key Laboratory of Reproductive Biology, Institute of Zoology, The Chinese Academy of Science, Beijing 100080, China. E-mail, [email protected]; tel, 86-10-62650042; fax, 86-10-62529248. Additional corresponding author: Dr. Ran Huo, Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China. E-mail, [email protected]; tel, 86-25-86862038; fax, 86-25-86862908. † Nanjing Medical University. ‡ State Key Laboratory of Reproductive Biology. § Northeast Agricultural University. # These authors contributed equally to the work. 10.1021/pr800392s CCC: $40.75

 2008 American Chemical Society

patient- and disease-specific stem cells. However, its low efficiency and potential risks, such as oncogenicity and intersional mutagenesis, prompted us to look for other factors that are also required to induce complete reprogramming and totipotency in oocytes. An understanding of oocyte components would be useful in searching for such factors and illustrating the molecular mechanism of reprogramming. Gene expression analyses of oocytes have been performed by several research groups.13-16 However, in the oocyte, there is no reliable strict linear correlation between mRNA levels and the abundance of protein.17 In addition, oocyte proteins need to be activated by post-translational modifications. Maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK), for example, are both pivotal factors in driving the meiotic cell cycle progression of the oocyte and their activities are mediated by protein phosphoryaltion/dephosphoryaltion.18,19 In a word, RNA expression cannot represent protein expression in the mature oocyte. Proteomics provides a means of determining the proteins expressed in a cell. Recently, there have been several reports regarding mammalian oocyte proteomics, including the exploration of bovine, pig and murine oocyte proteomes.20-30 Proteomic technology has also been used to study protein changes during oocyte maturation (in vitro or in vivo) and early embryo development.20-23,25,26,30 However, through these studies in mature mouse oocytes, only a few Journal of Proteome Research 2008, 7, 4821–4830 4821 Published on Web 09/20/2008

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abundantly expressed proteins have been identified. In an effort to provide additional insight into the proteins involved in the processes of meiosis, oocyte activation, fertilization, epigenetic reprogramming and embryo development initiation, we have combined 2-DE, mass spectrometry, and phosphoprotein staining to generate a database of proteins expressed in the mouse oocyte.

Experimental Methods All experiments requiring the use of animals received prior approval from Nanjing Medical University and were performed according to USDA-approved protocols. Reagents. Urea (Cat. No. 17-1319-01), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Cat. No. 17-1314-01), iodoacetamide (Cat. No. RPN6302), Dithiothreitol (DTT) (Cat. No.17-1318-02), Immobilized pH gradient (IPG) buffer (pH 3-10, nonlinear) (Cat. No.17-6000-88), and IPG strips (pH 3-10, nonlinear) (Cat. No.17-6002-45) were from GE Healthcare (Uppsala, Sweden); Thiourea (Cat. No. T7875), acetonitrile (ACN) (Cat. No. 34851), ammonium bicarbonate (NH4HCO3) (Cat. No. A6141), trifluoracetic acid (TFA) (Cat. No. T0274), and ammonium phosphate monobasic (NH4H2PO4) (Cat. No. 467782) were from Sigma Chemical (St. Louis, MO); Protease inhibitor cocktail (Cat. No.78437) was purchased from Pierce Biotechnology (Rockford, IL); Peptide calibration standards (Part No. 206195) and the matrix material (R-cyano-4-hydroxy cinammic acid, R-HCCA) (Part No. 203072) were purchased from Bruker (Bruker Daltonik GmbH, Bremen, Germany). Sterile PBS was purchased from Qbiogene (Carlsbad, CA). Oocyte Collection. Mature, metaphase II-arrested mouse oocytes were collected from 8 weeks old female ICR mice. Intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) (10 IU) and human chorionic gonadotropin (HCG) (10 IU) were made 48 h apart. After 15-17 h, oocyte-cumulus complexes were released from the oviducts into Hepes-buffered Chatot, Ziomek, and Banister (CZB) medium.31 Cumulus cells were dispersed by treatment with 1 mg/mL hyaluronidase (H3506; Sigma Chemical, St. Louis, MO) in Hepes-CZB. Cumulus-free oocytes were washed with Hepes-CZB medium, and zona pellucida was removed by treating the eggs for 3 min in acid PBS (pH 2.5) followed by mechanical shearing. During this process, the status of oocyte was checked instantly and the oocytes with shape abnormalities or with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, and refractile body) were discarded. Only the denuded oocytes with normal morphology were chosen for proteomic analysis. Protein Extraction and Two-Dimensional Gel Electrophoresis. Oocytes were collected and extracted in lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 65 mM DTT, 2% (v/v) IPG buffer (pH 3-10, nonlinear) and 1% (v/v) inhibitor protease cocktail. A total of 80 µg of oocyte lysates was loaded by gel rehydration on 24-cm immobilized, pH 3-10, nonlinear gradient strips for 2-DE. Separation was performed as previously described.32 Isoelectric focusing was carried out in an IPGphor apparatus. Second-dimension separation was done in 12% polyacrylamide gels using an Ettan-Dalt six system (GE Healthcare, San Francisco, CA). Gels were visualized by silver staining according to the published procedure33 except that glutaraldehyde was omitted in the sensitizing solution. Silver-stained gels were scanned and analyzed using 2D Elite Image Master software (GE Healthcare, San Francisco, CA). 4822

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Phosphoprotein Separation and Detection. For phosphoprotein detection, 30 µg of oocyte protein extract was separated by 2-DE and stained with Pro-Q Diamond phosphoprotein gel stain (Cat. No. P21857, Molecular Probes, Eugene, OR). Briefly, the gel was fixed in 50% methanol/10% acetic acid overnight, washed three times with deionized water, incubated in Pro-Q Diamond phosphoprotein gel stain for 90 min, and destained with three washes of 20% acetonitrile (ACN) in 50 mM sodium acetate (pH 4.0) and two washes with deionized water. Images were acquired using Typhoon Variable Model Imagels 9400 (GE Healthcare, San Francisco, CA) with excitation at 532 nm and a 580 nm bandpass emission filter. After scanning, the same gel was silver stained to detect total protein spots and match against the micropreparative 2-DE gels. Enzymatic In-Gel Tryptic Digestion. Protein spots were excised, dehydrated in acetonitrile, and dried at room temperature. Proteins were reduced with 10 mM DTT/25 mM NH4HCO3 at 56 °C for 1 h and alkylated with 55 mM iodoacetamide/25 mM NH4HCO3 in the dark at room temperature for 45 min in situ. Gel pieces were thoroughly washed with 25 mM NH4HCO3, 50% ACN, and 100% ACN, and dried in a Speedvac. Dried gel pieces were reswollen with 2-3 µL of trypsin (Cat. No. V5111, Promega, Madison, WI) solution (10 ng/µL in 25 mM ammonium bicarbonate) at 4 °C for 30 min. Excess liquid was discarded and gel plugs were incubated at 37 °C for 12 h. TFA was added to a final concentration of 0.1% to stop the digestive reaction. Mass Spectrometry. Digests were immediately spotted onto 400 µm anchorchips (Part No. 209512, Bruker Daltonics, Bremen, Germany). Spotting was achieved by pipetting 1 µL of analyte onto the MALDI target plate in duplicate and then adding 0.05 µL of 2 mg/mL R-HCCA in 0.1% TFA/33% ACN which contained 2 mM NH4H2PO4. Bruker peptide calibration standards (containing Angiotensin_II, MH+ 1046.542; Angiotensin_I, MH+ 1296.685; Substance_P, MH+ 1347.735; Bombesin, MH+ 1619.822; Renin_Substate, MH+ 1758.932; ACTH 1-17 clip, MH+ 2093.086; ACTH 18-39 clip, MH+ 2465.198; Somatostatin 28 clip, MH+ 3147.471) was spotted down for external calibration. All samples were allowed to air-dry at room temperature, and 0.1% TFA was used for on-target washing. All samples were analyzed in a time-of-flight Biflex IV mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany); the spectrometer was run in positive ion reflectron mode with an accelerating voltage of 19 kV. Tandem mass spectra were provided by an Ultraflex II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) in LIFT (laser-induced forward transfer) mode. Database Queries and Protein Identifications. Mass spectra (m/z range 700-4000, resolution 10 000-20 000) were processed using the FlexAnalysis software (version 2.4, Bruker Daltonik GmbH). The parameters used were peak detection algorithm, SNAP (Sort Neaten Assign and Place); S/N threshold, 3.0; quality factor threshold, 50; and internal calibration, trypsin autodigestion peptides (trypsin_[108-115], MH+ 842.509; trypsin_[58-77], MH+ 2211.104). Masses detected frequently that arose from the matrix, trypsin, or known contaminants (e.g., keratins) were removed. The data were searched against the Swiss-Prot database (Release 48.7, 204 086 sequences, 74 182 688 residues, for Mus musculus 10 369 sequences) and the TrEMBL database (Release 31.7, 2 506 886 sequences, 805 901 253 residues, for M. musculus 51 916 sequences) by an in-house MASCOT (version 2.1, Matrix Science) search engine. For PMF, search conditions included mass accuracy set as 100

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Proteome and Phosphoproteome of Mouse Oocyte ppm, one missed cleavage allowed, M. musculus taxon, alkylation of cysteine by carbamidomethylation as a fixed modification, and oxidation of methionine as a variable modification. Proteins with a confidence limit of >95% and g4 peptides matching were considered to be identified. To eliminate redundancy caused by proteins appearing in both the Swiss-Prot and TrEMBL databases under different names and accession numbers, the Swiss-Prot nomenclature and accession numbers were adopted when the sequence was the same. Wherever more than one protein was identified as a match, they could be distinguished by first, the pI and Mr of the identified proteins could be compared to the gel location to confirm identity; second, a comparison of the peptides identified could show one matched protein contained a larger number of peptides or an extra peptide sequence not contained within the other. When the multiple matched proteins could not be distinguished by any of these methods, they were recorded as a physical redundancy or bioinformatics redundancy. If two or more proteins were identified from the spectrum and marked as a mixture (confidence value >95%) by Mascot Sever (details of the principle can be found at http:// www.matrixscience.com/pdf/2003WKSHP2.pdf), they were considered to be a physical redundancy (co-migrating proteins). If two or more proteins were found to share the same peptides, they were treated as a bioinformatics redundancy (proteins not able to be distinguished) and excluded from further bioinformatics analyses. Additionally, the Mascot Score and expectation of the first nonhomologous protein to the highest ranked hit was checked. Samples from PMF that gave no result or an ambiguous result were sequenced by tandem mass spectrometry. Some matched peaks chosen randomly from PMF were also sequenced by tandem mass spectrometry for verification. Each acquired MS/MS spectrum was also processed using the software FlexAnalysis v2.4 using a SNAP method set at a signalto-noise ratio threshold of 3.0. To search MS/MS spectra, the peak lists were submitted by Biotools (version 3.0, Bruker Daltonik GmbH) to Mascot Server for searching the Swiss-Prot 48.7 database and the TrEMBL 31.7 database. The search parameters for MS/MS data were 100 ppm for the precursor ion and 0.3 Da for the fragment ions. Cleavage specificity and covalent modifications were considered as described above. Confidence greater than 95% was considered significant. All significant MS/MS identifications by Mascot were manually verified for spectral quality and matched to y and b ion series. Construction of an Online Database. To construct a mouse oocyte proteome database, we used the Web-based system Make2D-DB II Package (ver. 2.50.1), an HTML generator for 2D images through ExPASy (http://www.expasy.org/ch2d/ make2ddb.html). We stored the identified protein spots in a relational database that was made accessible online via a common gateway interface (cgi) script on a Web server (http:// reprod.njmu.edu.cn/2d). We hyperlinked the individual protein entries to the relevant spots on the silver-stained and phosphorylated image maps created from the reference gel. Western Blot Analysis. Western Blot analysis was performed as previously described.32 Proteins extracted from about 200 oocytes were loaded per line. Antibodies against Annexin A7 (A4475, diluted 1:1000; Sigma Chemical, St. Louis, MO), Poly(rC)binding protein 1 (PCBP1) (SC-16504, diluted 1:100; Santa Cruz Technology, Inc., Santa Cruz, CA), suppressor of g2 allele of skp1 homologue (SUGT1) (ab30931, diluted 1:200; Abcam, Cambridge, MA), S-phase kinase-associated protein 1A (SKP1)

(SC-7163, diluted 1:200; Santa Cruz Technology, Inc., Santa Cruz, CA) and cofilin1 (ab11062, diluted 1:8000; Abcam, Cambridge, MA) were used, respectively. DAVID (Database for Annotation, Visualization, and Integrated Discovery) Analysis. Functional annotations were performed using the program DAVID 2007.34 DAVID is a Webbased, client/server application that allows users to access a relational database of functional annotation. Protein accession numbers were submitted to DAVID to analyze gene ontologies, protein domains, and biochemical and signal transduction pathways. The parameters used were GO (gene ontology) term, all levels; KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway in the pathways section; InterPro name, SMART name and Pfam name in the protein domains section; and SP_PIR_ Keywords in the functional categories section. Pathway Analysis by PathwayStudio. An analysis of cellular processes influenced by the protein profile obtained was performed using PathwayStudio (v4.00) software (Ariadne Genomics, Inc., Rockville, MD). PathwayStudio includes an automated text-mining tool which enables the software to generate pathways from the PubMed database and other public sources. Our gene list was imported into PathwayStudio to identify the cell processes influenced. Each identified cellular process was confirmed through the PubMed/Medline hyperlink embedded in each node. Chromosome Distribution Analysis. The chromosomal distribution of genes encoding the identified proteins in the mouse oocyte proteome were compared with known proportions of genes on mouse chromosomes using a standard χ2 contingency table, according to a previously published method.35 The chromosome distribution graph was generated by WebGestalt (WEB-based GEne SeT AnaLysis Toolkit, http:// bioinfo.vanderbilt.edu/webgestalt).36 Analysis of the Nuclear Localization Signal (NLS). LOCtree (available at http://cubic.bioc.columbia.edu/cgi-bin/var/nair/ loctree/query) is a system for predicting the subcellular localization of proteins and the DNA-binding propensity of nuclear proteins.37 The protein sequences were submitted to the LOCtree server to analyze the NLS of the mouse oocyte proteins.

Results Proteomic Survey and Construction of an Online Database. To clarify the protein composition of the mouse oocyte, mature mouse MII oocytes without zona pellucida were collected, and the status was checked severely to ensure only morphologically normal cells were chosen (as Figure 1 shows). Proteins were extracted and separated by 2-DE and silver-stained (Figure 2). Proteins were resolved on a pH 3-10 nonlinear gel resulting in the separation of 1478 spots. With the use of mass spectrometry, 58.8% of those spots were associated with 380 distinct proteins (Supplemental Table 1 in Supporting Information). An online, federated 2-DE database was generated for the 380 identified proteins with hyperlinks to the corresponding protein entries for each of the identified protein spots. Each protein entry includes accession number, protein name, description, spot ID number, organism, isoelectric point (pI), molecular weight, and mass spectrum data, as well as links to relevant entries in other online databases. This database can also be accessed at WORLD-2DPAGE (http://cn.expasy.org/ch2d/2dindex.html). We compared this database with the published mammalian oocyte proteomes,20,25,27,30 and the results showed 22 proteins previously reported in mammalian oocyte proJournal of Proteome Research • Vol. 7, No. 11, 2008 4823

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Figure 1. A representative picture showing mouse metaphase II oocytes collected for proteomic study. Only morphologically normal cells were chosen for further study.

Figure 2. Silver-staining of the 2-DE map of mouse oocyte proteins. Identified spots are indicated with gray asterisks.

teomic studies have also been identified in our study (detail information given in theSupplemental Table 2 in Supporting Information), and accounted for about 64.7% of the constructed mammalian oocyte proteomes. Further, to judge the quality of this protein expression profile, we have chosen 5 proteins which never reported in mammalian oocytes and perfomed Western blots (results shown in Supplemental Figure 1 in Supporting Information). The results showed these proteins were undoubtedly expressed in mouse MII oocytes, thus confirming our identification information. Annotation and Prediction of Proteins in the Mouse Oocyte. For an overview of the proteomic data collected, we analyzed the cellular component, biological processes and molecular functions of each of the proteins identified using gene ontology annotation from the DAVID database. Cellular component analysis revealed that the largest number of proteins are cytoplasmic (190), followed by mitochondrialassociated proteins (76), nuclear proteins (60), and membraneassociated proteins (49) (Figure 3A). Although there is no nucleus in the MII oocyte, and the nucleus-cytoplasm mixture forms after envelope breakdown, the nuclear proteins identified may contribute to this mixture. An analysis of the proteins identified according to their potential roles in biological processes indicated that more than a quarter of the proteins identified were involved in protein metabolism, while carbohydrate, lipid, and nucleotide metabolism had fewer numbers 4824

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Figure 3. Analysis of the mouse oocyte proteome according to Gene Ontology annotation. (A) Subcellular location analysis of the identified mouse oocyte proteins. (B) Analysis of the mouse oocyte proteins based on molecular function and biological process terms in Gene Ontology.

of associated proteins identified. With the use of molecular function classification, 100 proteins were associated with a protein binding function, followed by hydrolase activity (75), protein folding (34), RNA binding (15), translation regulator activity (11), proteasome endopeptidase activity (9) and Cacetyltransferase activity (3) (Figure 3B). Domain analysis using DAVID functional annotation tool of the identified oocyte proteins according to the Interpro, Pfam and SMART databases resulted in a dozen statistically significant domains, such as proteasome domain (Pfam: PF00227) (9 proteins contain the proteasome domain), AAA domain (SMART: SM00382) (8 proteins contain the AAA domain), KH_1 domain (Pfam: PF00013) (5 proteins contain the KH_1 domain), LSM domain (Pfam: PF01423) (4 proteins contain the LSM domain), HATPase_c domain (SMART: SM00387) (3 proteins contain the HATPase_c domain), ADF domain (SMART: SM00102) (3 proteins contain the ADF domain), and so on. These proteins are involved in events such as proteolysis, replication, RNA processing and motility. Other than the above data, the KEGG pathway analysis results indicate that many proteins are involved in the metabolism processes such as proteasome, pyruvate metabolism, glycolysis/gluconeogenesis, purine metabolism, and arginine and proline metabolism (Supplemental Table 3 in Supporting Information). Notably,

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Figure 4. Regulation pathways generated from mouse oocyte proteins using PathwayStudio software. Proteins are shown as red ovals, regulated processes are represented by yellow squares. Regulation events are displayed with arrows and documented by literature citations. (A) A large number of proteins identified participate in regulating cell cycle events, including meiosis and nearly all stages of mitosis. Proteins related to the process of reproduction and RNA/DNA processing are shown in panels B and C, respectively. Proteins with roles in early embryo development are shown in panel D (enlarged parts of this figure could be found at http://reprod.njmu.edu.cn/ data/index.htm).

there are 14 proteins involved in the composition of the proteasomes (this complex contains 30 components in total), indicating protein hydrolysis may be crucial for protein degradation during the complex molecular and morphological changes that occur during oocyte maturation and fertilization. A more detailed analysis of cellular processes influenced by the protein profile of the mouse oocyte was performed using PathwayStudio, an automated text-mining tool which enables the software to generate pathways from entries in the PubMed database, as well as other public sources. In Figure 4, each candidate protein related to cell cycle, reproduction, RNA/DNA processing, and embryo development is listed. The results are represented as a visualized graph, for example, within the events of reproduction, proteins involved in oogenesis, ovulation, fertilization and membrane fusion are set out. Since both meiosis and mitosis occur sequentially from oocyte maturation to embryogenesis, 111 proteins involved in cell cycle events including mitosis and meiosis are accumulated. This significant number of proteins relating to the cell cycle suggests that cell cycle events in oocytes are delicately regulated and may provide abundant material for rapid cell cleavage in early embryogenesis. Protein Heterogeneity of the Oocyte Proteome. Unexpectedly, a considerable number of proteins corresponded to more

than 2 spots on the gel. An analysis of proteins identified versus total number of spots detected showed that 152 proteins appeared more than once, with 12 proteins appearing not less than 10 times, resulting in an average of each protein corresponding to 2.3 spots. The protein LDH2 has been reported to be the most abundant protein in oocytes.38,39 LDH2 appears 54 times in our gel, which suggests its functional complexity in oocytes. According to the SP_PIR_Keywords Analysis of the proteins appearing more than once on the gel, for 14 proteins, their encoding genes have been reported to have alternative splicing forms. In addition, there is one protein that has been reported to have at least two isoforms due to the usage of alternative initiation codons in the same mRNA. In addition to mRNA regulation, PTM of proteins weighs heavily in protein heterogeneity. Our analysis found that 15 of the identified proteins could be phosphorylated, 22 proteins had the potential to be acetylated, and several more could be glycosylated (see Supplemental Table 4 in Supporting Information). Several of the proteins have the potential to be modified by multiple PTM forms, suggesting a multifunctional form of these proteins. On the basis of the unexpected heterogeneity of the oocyte proteomic analysis, and the result of the SP_PIR_Keywords Journal of Proteome Research • Vol. 7, No. 11, 2008 4825

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Figure 5. Phosphoproteome of the mouse oocyte. Phosphorylated forms of proteins stained by Pro-Q Diamond dye emitted green fluoresent light.

Analysis that indicated that phosphorylation was a common modification, we conducted a phosphoproteome analysis of the oocyte lysate. A 2-DE gel of the oocyte lysate was stained with a fluorescent Pro-Q Diamond dye that recognizes candidate phosphoproteins (Figure 5). There were 119 spots fluorescently labeled on the gel, 90 of those spots have been identified and correspond to 53 unique proteins (labeled in Supplemental Table 1 in Supporting Information). It is of note that 15 of these proteins correspond to more than one Pro-Q Diamond stained spots in the gel, with 7 proteins having more than two Pro-Q Diamond stained spots in the gel. We also analyzed the 147 proteins that appeared more than once on the silver-stained gel for phosphorylation modifications. We found that 35 of these proteins were consistent with proteins identified in the phosphoproteome study with Pro-Q Diamond dye detection. Since a single protein can have several phosphorylation patterns which can exist at the same time, we hypothesize that diverse phosphorylation states are an important mechanism in oocyte protein regulation. Chromosome Distribution. We mapped all genes associated with the proteins identified in this study onto the polytene chromosomes and observed the following distribution: X chromosome, 12 genes; chromosome 9, 25 genes; chromosome 11, 39 genes; and so on. Figure 6 shows the complete chromosome distribution of proteins identified in the proteomic analysis. A χ2 analysis of the distribution of genes identified from our proteomic screen versus the entire set of protein encoding genes in the mouse genome showed a significant difference between the proportion of genes located on each chromosome compared with the genomic distribution (χ2 ) 30.669, d.f. ) 19, P ) 0.044). No significant difference was observed when we excluded the genes matched with chromosome 11 (χ2 ) 21.868, d.f. ) 18, P ) 0.238), yet significant differences were observed when we compared the proportion of chromosome 11 genes to other chromosome associated genes with the genomic ratio (χ2 ) 9.753, d.f. ) 1, P ) 0.002). 4826

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Figure 6. Distribution of the mouse oocyte proteome in relation to the chromosomal organization of the mouse genome. Genes corresponding to the proteins expressed in the mouse oocyte proteome are marked with a “+”. Asterisks indicate a significant enrichment of 11-chromosome gene loci identified in the oocyte proteome (P < 0.05).

Therefore, deviation of the identified genes from the known proportion of genes per chromosome is primarily due to an enrichment of chromosome 11 gene loci identified in the oocyte proteome. This distribution suggests that deletion, duplication, or inversion at these loci will impair reproductive processes. Intersection between Oocytes and ESC Proteomics. The MII stage oocyte has been of interest developmentally for its ability to remodel the sperm nucleus and restore totipotency to a diploid zygote. In therapeutic cloning, somatic cell nuclear transfer into enucleated oocytes has been shown to erase the

Proteome and Phosphoproteome of Mouse Oocyte somatic epigenetic phenotype and return the nucleus to a totipotent state. In addition, reported somatic cell-ESC fusion experiments suggest that ESCs retain undefined components that can initiate the reprogramming of an introduced somatic nucleus.40 On the basis of these observations, it is hypothesized that the cytoplasmic environment of both oocytes and ESCs share the capacity to reprogram a somatic nucleus to a certain extent.41 Therefore, we analyzed the proteins in common between oocytes and ESCs in order to identify factors involved in epigenetic reprogramming. The oocyte proteins from our proteome were compared with recently published proteins that are expressed in mouse and human ESCs.42 The comparison identified an overlap of 256 proteins with mESCs and 243 with hESCs (Supplemental Table 5 in Supporting Information). A total of 218 proteins were found to be common between mouse oocyte, mESCs, and hESCs. Bioinformatic analysis of the 218 proteins showed 87 proteins among them had the potential to localize in the nucleus (Supplemental Table 6 in Supporting Information). In investigating the proteome of ESCs, Van et al. also compared the expressional profiles of human and mouse ESCs and their differentiated derivatives and found some ESCs enriched proteins (uniquely expressed in ESCs or highly expressed in ESCs than Dif-ESCs). The comparison of the ESCs enriched proteins with our data further showed an overlap of 69 proteins with mESCs_enriched proteins, 50 with hESCs_enriched, and 16 proteins were common between mouse oocytes, mESCs_enriched and hESCs_enriched (Supplemental Table 5 in Supporting Information).

Discussion Oocytes are responsible for the generation of offspring and, in vitro, can provide the signaling necessary for somatic nuclear reprogramming. Therefore, resolution of their proteins and associated signaling mechanisms are of great interest. Proteomic analysis of oocytes can identify the proteins present, providing clues to oocyte specific functions and signaling pathways. As shown in Figure 3B, we have distinguished between the molecular function and involvement in biological processes of these proteins in terms of their respective gene ontologies. Interestingly, proteins involved in protein metabolism are at the top of the list, while proteins involved in nucleotide metabolism are fewer in number. These observations are in contrast with previous findings associated with human oocyte transcripts.41 We speculate that, since MII stage oocytes appear to be transcriptionally silent, maternal proteins are the functional executers of the processes of fertilization and early embryo development. This results in the predominant role of protein metabolism. Nucleotide metabolism-related proteins might be deliberate to the ZGA until the early two-cell zygote stage. Our domain analysis indicates that several types of domains may function in oocytes. We focus on HATPase_c, KH_1 and LSM domains here. HATPase_c domain is found in histidine kinase, DNA gyrase B, topoisomerases, heat shock protein HSP90, phytochrome-like ATPases, and DNA mismatch repair proteins. It is speculated that all of these proteins are involved in DNA replication. By extension, the proteins identified in this study containing HATPase_c domain may also function during DNA replication after fertilization through zygote to the embryo. Our cluster analysis identified proteins Lsm1, Lsm2, Lsm3 and Lsm7. The LSM protein family contains Sm proteins as

research articles well as other related LSM (like Sm) proteins. These proteins form part of specific small nuclear ribonucleoproteins (snRNPs) that are involved in the processing of pre-mRNAs to mature mRNAs.43,44 Previous reports have demonstrated that in a complex of Lsm proteins, LSM 2-8 associates with U6 small nuclear RNA (snRNA) that is a component of spliceosome complexes in which pre-mRNA splicing occurs.45 Following completion of the splicing reaction, snRNPs must be recycled for subsequent rounds of splicing, and herein Lsm proteins promote the formation of U6-containing complexes.46 We hypothesize that Lsm proteins in oocytes may support the assembly or remodeling of RNP complexes involved in splicing which can generate various protein isoforms able to adapt to the intricate process of fertilization. In addition, proteins Lsm1-7 are predicted to localize to the cytoplasm and function in mRNA degradation which is responsible for the efficient removal of the 5′-cap from mRNA, thereby facilitating 5′ f 3′ degradation of mRNA in the deadenylation-dependent pathway of mRNA turnover.47 Identification of this class of proteins in our proteomic characterization of mouse MII oocytes provides a potential mechanism for maternal mRNA degradation after ZGA. In parallel with LSM proteins, KH_1 (K homology) domains also participate in RNA processing. The KH_1 domain was first identified in the human heterogeneous nuclear ribonucleoprotein (hnRNP) K. It is an evolutionarily conserved sequence that is present in a wide variety of nucleic acid-binding proteins and is suspected to bind RNA. On the basis of sequence homology, the KH_1 domain containing proteins Dppa5 (Esg1 as alias), Igf2bp2, Pcbp1, Pcbp2 and fubp1 would be predicted to assist in RNA processing and transport. Dppa5 has previously been reported to be associated with maintaining cell pluripotency, while Esg1 was originally found in mouse ESCs, mouse embryonic caricinoma cells (ECCs) and strongly expressed in mouse primordial germ cells (PGCs), and is assumed to serve as an informative marker.48 The expressional abundance of Dppa5 in differentiated ESCs was significantly lower than in ESCs. Furthermore, Esg1, in combination with Oct4 and Sox2, plays a conserved role in the pluripotent pathway of mouse and human stem and germ cells.49 The discovery of Esg1 protein in zygotes suggests its maternally inherited source, and our identification of Dppa5 in oocytes supports this hypothesis.49 Given the ability of mature oocytes to reprogram somatic nuclear cells into a totipotent state, the identification of Dppa5 in oocyte lysate provides a potential mechanism for this reprogramming event. In addition to the results given by DAVID analysis, another protein found in this database, ES cellassociated transcript 1 (Ecat1), has been characterized recently as having an atypical KH RNA binding domain.50 Ecat1 was initially identified by digital differential display of the expressed sequence tag (EST) libraries and found to be overrepresented in mouse ES cells in various somatic tissues.51 In addition to Oct4 and Sox2, Ecat1 was also considered an important candidate in reprogramming factors to generate induced pluripotent stem (iPS) cells.9 The detection of Ecat1 protein in mouse oocyte supports the suggestion of its role in inducing pluripotency. Signaling pathway analysis provides information about cell processes in which oocyte proteins could be involved. When considering proteins regulating the cell cycle, 111 proteins in total take part in meiosis and nearly all cycle phases of mitosis. Markedly large numbers of proteins are interrelated with mitosis, providing repertory for sequential rapid cleavages Journal of Proteome Research • Vol. 7, No. 11, 2008 4827

research articles during early embryogenesis. Furthermore, RNA/DNA processing related proteins provide considerable heterogeneity considering the opportunities for RNA editing, mRNA splicing, mRNA processing, and mRNA stabilization. Such processes will be responsible for the diverse isoforms in oocytes. Apart from proteins participating in oogenesis and fertilization, there are many additional proteins that are engaged in the processes that occur during embryo development, which include axis specification, gastrulation, neurogenesis, and so on. Thus, these maternal proteins are also essential in heterozygotes. In this proteome, 53 putative phosphoproteins have been examined using Pro-Q Diamond phosphoprotein stain, these proteins have been reported to participate in various processes, such as chromosome remodeling, maternal material storage and blastomere cleavage and so on. Epigenetic modifications of DNA and chromatin are important for genome function during development and in adults. The mammalian genome undergoes significant reprogramming of the modification patterns in the early embryo. Consequently, a deficiency in epigenetic reprogramming is a major reason for failed animal cloning. Proteins involved in chromosome remodeling would be expected to play a significant role in these processes. Coincidentally, some proteins in our profile were implicated in the processes of chromosome remodeling. One intriguing protein we identified is retinoblastoma binding protein-7 (RBBP7), also referred to as retinoblastoma-associated protein46 (Rbap46). It has been shown to be a core component of at least two major corepressor complexes, SIN3A and NuRD.52,53 Other closely related proteins include RBBP4 (Rbap48), and the histone deacetylases HDAC1 and HDAC2. These proteins form a core complex that is shared between SIN3A and NuRD to achieve transcriptional repression through global deacetylation of chromatin, thereby inhibiting the accessibility of transcriptional activators to DNA.54 Considering that NuRD has a role in DNA methylation and stable silencing mechanisms,55,56 our detection of Rbap46 in mouse oocytes hints at a possible mechanism for the silencing of transcription and genome methylation used for genetic imprinting of the mammalian oocyte. Rbap46 was also identified in our phosphoproteome analysis to correspond to one out of three identified spots. It indicates that phosphorylation maybe plays a role in regulating of the activity of Rbap46. However, Rbap46 has also been found to be expressed in ESCs, and a recent study has shown that Mbd3, another NuRD component, is required for pluripotency of embryonic stem cells.57 We hypothesize that the NuRD complex is related to epigenetic silencing in the cell-fate commitment of pluripotent cells. Furthermore, Rbap46 may have an important role in the EMT (epithelial-mesenchymal transition) during embryonic development,58 making Rbap46 essential for embryonic development after fertilization. The oocyte contains maternal molecules for oocyte maturation as well as for embryo development initiation. It is not known how oocyte mRNAs are masked and unmasked for accurate protein synthesis. There is a protein named Y-box binding protein 2 (YBX2 or MSY2) which maintains maternal mRNA stability in mouse oocytes.59 YBX2 has been shown to accumulate during oocyte growth, and then be completely degraded by the late two-cell stage, suggesting a mechanism for the global regulation of the stability and/or translation of maternal mRNA during oocyte growth.60,61 RNAi treatment of YBX2 has consistently resulted in an overall reduction in protein synthesis.62 In addition, phosphorylation of YBX2 can account for increased RNA binding affinity.63 Relative to these reports, 4828

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Ma et al. the information from our 2-DE gels not only demonstrates the expression of YBX2 in oocytes, but the 12 spots and 3 phosphorylated spots corresponding to YBX2 suggest variable splicing or PTMs, which may represent active/inactive forms with different binding affinities to maternal RNAs. At the point of fertilization, sperm nucleus enters the oocyte where it undergoes decondensation and paternal DNA is rapidly demethylated. The initiation of such sequential events is mainly attributed to maternal factors in the oocyte. Therefore, the identification of oocyte proteins is predicted to provide insights into the molecular basis for reprogramming. We identified nucleoplasmin-2 (NPM2) as an abundant protein based on its 19 spots, as well as 11 phosphoprotein spots. As previously reported, NPM2 plays an important role in sperm decondensation upon fertilization. It is compartmentalized to the oocyte nucleus before GVBD (germinal breakdown), but redistributes into the cytoplasm after GVBD. After fertilization, NPM2 is found in pronuclei and sequentially in the embryo nuclei until the eight-cell stage.64 NPM2 stores maternally derived histones due to its histone-binding capacity, especially histone H2A and H2B, and replaces sperm-specific basic proteins during sperm chromatin decondensation, resulting in an assembly of somatic-type nucleosomes onto sperm DNA.65,66 Additionally, hyperphosphorylation of NPM2, which occurs during maturation of the oocyte, facilitates its nuclear to cytoplasmic targeting along with histone replacement on sperm and decondensation.67 Eleven protein spots associated with NPM2 were stained by Pro-Q Diamond stain, suggesting that they represent various phosphorylated forms, which could affect the ability of NPM2 to exchange sperm nucleoprotein. In parallel, other modifications are likely to exist among the 19 isoforms of NPM2 identified from the protein spots, consistent with the diverse functional states of this protein. Among several blastomere cleavage related proteins, Nudc takes part in nuclear positioning during embryonic cell division.68 Depletion of Nudc results in defects in chromosome congression at the metaphase plate,69 indicating its potential role in spindle movement. Nudc appeared twice in the Pro-Q Diamond stained gel, which may represent different functional isoforms and underlie the rapid cleavages of embryos. In conclusion, 380 nonredundant proteins were detected in mouse oocyte. Because of the resolution limitations of 2-DE for the less-abundant, hydrophobic, very acidic or basic, and very large or small proteins,70 we believe the mouse oocyte proteome visualized here is incomplete. Some well-known oocyte proteins, such as Stella, Zar1, and Mater71-73 have not been detected. So the oocyte protein profile reported here is just preliminary. But we believe it should serve as a useful bridgehead for further characterization of the mechanisms involved in reproduction and early embryo development, especially during oocyte maturation, fertilization, reprogramming and blastomere cleavage. Mapping putative phosphoproteome using Pro-Q Diamond stain provides additional information in elucidating the regulation of these complex processes. We also anticipate that research on the components of the oocyte will uncover more factors that have the ablility to induce complete reprogramming and pluripotency, thus increasing the efficiency of SCNT and iPS cell generation and improving progress in the field of regenerative medicine.

Acknowledgment. This study was supported by grants from 973 program (No. 2006CB701503 and 2007CB948103).

Proteome and Phosphoproteome of Mouse Oocyte

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