Comparative Proteomic and Regulatory Network Analyses of the Elongating Pig Conceptus

2678 DOI 10.1002/pmic.200800776 Proteomics 2009, 9, 2678–2694

RESEARCH ARTICLE Comparative proteomic and regulatory network analyses of the elongating pig conceptus

Séverine A. Degrelle1*, Le Ann Blomberg1, Wesley M. Garrett1, Robert W. Li2 and Neil C. Talbot1

1 USDA Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD, USA 2 Bovine Functional Genomics Laboratory, Beltsville, MD, USA

Embryo loss during peri-implantation can approach 20% in swine following artificial insemina- Received: September 30, 2008 tion or natural mating and coincides with rapid conceptus elongation. The objective of the pres- Revised: December 31, 2008 ent study was to establish a comprehensive profile of the abundant proteins of the pig conceptus Accepted: January 13, 2009 at the time prior to implantation and identify stage-specific changes during elongation. The abundant proteins of a homogenous population of gestational day-11 ovoid (0.7–1 cm) and gestational day-12 filamentous (15–20 cm) porcine concepti were compared by extracting proteins from three independent conceptus pools and separating the proteins by 2-DE. Proteins in 305 spots were analyzed by MALDI-TOF or additionally by LC-MS/MS and 275 were positively identified representing 174 distinct proteins. The proteins could be classified into the following functional categories: cell proliferation/differentiation, cytoskeleton, metabolism, and stress response. Based on spot density, 35 proteins associated with cell proliferation, differentiation, apoptosis, and embryo/maternal signaling, were found to be differentially expressed between ovoid and filamentous concepti. A comparison of the protein expression profile with transcriptomic data from pig concepti of the same developmental stages identified similarities and dissimilarities between protein and mRNA expression profiles. This proteomic study helps to elucidate the biological mechanisms underlying the early embryonic development of the pig.

Keywords: Conceptus / Elongation / Pre-implantation / Proteome / Swine

Correspondence: Dr. Le Ann Blomberg, USDA, ARS, ANRI, Ani- 1 Introduction mal Biosciences and Biotechnology Laboratory, Bldg 200, Rm 22, BARC-East, Beltsville, MD 20705, USA In preparation for implantation, the porcine conceptus E-mail: [email protected] undergoes a rapid differentiation and expansion of the extra- Fax: 11-301-504-8414 embryonic tissues (primarily the trophectoderm and yolk-sac endoderm) between gestational day (gd)-11 and gd-12 [1, 2]. Abbreviations: ACTB, b-actin; ANXA, annexin; BP, biological pro- The conceptus increases in size and changes in shape be- cess; CALR, calreticulin; CBFGL, Computational Biology and Functional Genomics Laboratory; CLIC1, chloride intracellular tween gd-10 and gd-11 as they develop from a 1–2 mm channel 1; CTSB, cathepsin B; EEF1D, elongation factor-1-d; EZR, sphere to a 6–10 mm ovoid conceptus [2]. Over a 4 h period ezrin; gd, gestational day; GO, gene ontology; IL1B, interleukin 1 between gd-11 and gd-12, a tremendous expansion occurs b; IPA, ingenuity pathway analysis; KRT, cytokeratin; LGMN, legu- where the 10 mm ovoid conceptus rapidly elongates to form main MSN, moesin; MW, molecular weight; RA, retinoic acid; RBP, retinol binding protein; RPSA, ribosomal protein SA; SFN, stratifin; STAR, steroidogenic acute regulatory protein; TC, tenta- * Present address: INRA, UMR 1198 Biologie du Développement tive consensus; TPM3, tropomyosin 3 et Reproduction, Jouy en Josas, France

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2009, 9, 2678–2694 2679 a long thin filament (.100 mm); the early rapid elongation tractive hybridization (SSH) [14, 24], cDNA membrane array phase [2, 3]. The pig embryo continues to elongate beyond [25], and SAGE [5, 12] have allowed the simultaneous analy- gd-12 to ,gd-15 but this second phase is much slower. Aside sis of large numbers of transcripts. More than 300 genes from these obvious alterations in trophectoderm and endo- were found to be differentially expressed at the mRNA level derm morphology, microscopic changes occur within the during distinct stages of early rapid elongation in the porcine porcine embryonic disc, too, namely the beginning of gas- conceptus [5, 12]. These included factors known to regulate trulation [4–6]. For example, mesoderm-precursor cells the cytoskeleton (cytokeratin 8 (KRT8), cytokeratin 18 aggregate toward the posterior end of the embryonic disc and (KRT18), ezrin (EZR, aka., villin 2), moesin (MSN), b-actin also migrate extra-embryonically by gd-11 [4, 5]. Advance- (ACTB)) [5, 12, 24], E2 synthesis (steroidogenic acute reg- ment of gastrulation to the primitive streak stage can be ulatory protein (STAR), cytochrome p450scc (CYP11A), aro- found by the filamentous stage [5, 6], and at this point the pig matase (CYP19A)) [5, 12, 25], intracellular signaling/ concepti reach positions in the uterine horn where implan- immune response (IL1B, transforming growth factor b3 tation will be initiated and placentation will ensue. (TGFB3)) [5, 12, 23–25] and morphogenesis(cellular RA The progression and maintenance of implantation binding protein, RBP) [5, 13]. Nonetheless, a great deal of the involves closely integrated signals between the uterus and bioactivity of genes is exerted through their protein products, conceptus [3, 7, 8]. Hypothetically, elongation of the pig and, unfortunately, protein abundance or biological function embryo’s trophectoderm, i.e., primordial placental tissue, is is often not correlated with the mRNA level. The expression essential because it establishes an increased conceptus sur- of only a few of the genes from transcriptomic analyses has face area that enables adequate interaction with the maternal been validated at the protein level in porcine concepti (West- endometrium for attachment to the uterine surface epitheli- ern blot of RBP [26], STAR [27], and cytochromes P450 17 a- um. Although the degree of elongation required for adequate hydroxylase (CYP17A) and CYP19A [28]). Thus, considering implantation has not been proven, the minimal uterine sur- the central role proteins play in the physiology and structure face area necessary for efficient development has been of tissues/organisms it is imperative to explore protein determined [9]. The rapid transition in conceptus morphol- expression profiles as well. ogy followed by initiation of trophectoderm attachment to Proteomic studies have utilized 2-DE to identify secreted the uterine surface is the period of greatest embryonic loss in proteins from cultured concepti (gd-10.5–gd-18) or con- the pig [1, 10]. Several factors with potentially critical physi- ceptus explants (gd-15) and primordial endoderm cell lines ological functions have been shown to be differentially regu- derived from gd-11 concepti that could impact endometrial lated in the pig conceptus during this period. For example, responses [29–31]. Furthermore, changes in the uterine during the filamentous stage, concomitant with an increase luminal fluid protein profile in pregnant and non-pregnant in the mRNA of a cholesterol transporter and steroidogenic sows, prior and subsequent to early elongation, i.e., gd-10 enzymes that can regulate estrogen synthesis, the first sig- and gd-13, have also been examined [32]. However, little is nificant peak of estradiol (E2) is secreted by the conceptus known about global changes in the total cellular proteins of and studies have shown that E2 is a key signal for the the pig conceptus as it transitions through the peri-implan- maternal recognition of pregnancy in pigs [2, 11–13]. One of tation elongation phase. To establish a reference map of pro- the most abundant transcripts present in the conceptus dur- teins expressed during elongation of the porcine conceptus ing elongation, interleukin 1 b (IL1B) [5, 14], has been shown and to identify those proteins that are differentially expressed to regulate E2 synthesis [15] and potentially mediate at defined stages of this process, a global proteomic approach implantation [16, 17]. Though less abundant, the expression using 2-DE and MS was employed to establish the profile of of factors that regulate retinoid bioavailability and intracel- abundant proteins at the initiation (ovoid) and end (fila- lular signaling (retinoic acid (RA) receptors, retinol binding mentous) of the pig conceptus’ early rapid elongation phase protein (RBP), cellular RA binding protein), suggests that of development. The results provide new insights regarding vitamin A metabolites, which are potent developmental fac- proteome changes during embryonic pig development in tors, could have a role in the pig conceptus’ differentiation comparison to previous transcriptomic data. These findings and morphogenesis [18, 19]. Despite the identification of may be useful in the efforts to improve growth and survival some important factors, understanding the biology of con- rates of pig concepti so as to enhance swine production effi- ceptus elongation will require the elucidation of global gene ciency. expression to identify the many proteins and their interactions that regulate the conceptus’ transitional changes and survival. 2 Materials and methods Several research efforts have evaluated changes in gene expression at the mRNA level during the peri-implantation 2.1 Sample collection stages of embryonic pig development, initially by candidate gene approaches [13, 19–24], and, more recently by high All animal protocols were approved by the Beltsville Area through-put transcriptomic techniques [5, 12, 25]. For exam- Animal Care and Use Committee and met the United States ple, RNA arbitrarily primed-PCR [22, 23], suppression sub- Department of Agriculture and National Institutes of Health

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 2680 S. A. Degrelle et al. Proteomics 2009, 9, 2678–2694 guidelines for the care and use of animals. Concepti were form, and 3 vol. of ultrapure water were added. The mixture collected as described previously [5, 12]. Briefly, natural, nor- was vortexed and centrifuged at 12 000 rpm for 10 min at mal estrous cycling, hybrid gilts that were 6 months of age or 47C. The supernatant was discarded and the protein pellet older were mated using artificial insemination (AI) for in vivo was washed in methanol (4 vol.). To ensure a good pellet conceptus production. The day of AI was designated as gd 0. resuspension during the wash, the solution was sonicated Gilts were euthanized on the morning of gd-11 and gd-12. 15–30 s. After a second centrifugation, the protein pellet Reproductive tracts were excised from the gilts, and concepti was air dried for 15 min to remove residual methanol and were flushed from the oviduct end of each individual uterine finally resuspended in 300 mL lysis buffer. Protein con- horn into a petri dish using ice-cold PBS. Concepti that were centrations were determined by using the Compat-Able analyzed came from uterine flushes where all concepti had protein assay preparation reagent set and BCA protein assay the same morphology, i.e., the morphology and size of the kit (Pierce, Rockford, IL, USA) according to the manu- gd-11 and gd-12 concepti were ovoid (7–10 mm) and fila- facturer’s instruction. BSA was used as a standard. mentous (100–150 mm), respectively. Concepti were placed in cryovials, snap-frozen in liquid nitrogen and stored at 2.3 Separation of proteins by 2-DE 2807C. IEF was performed on an IPGphor unit using 11 cm IPG 2.2 Protein extraction strips pH 4–7, (GE Healthcare, Piscataway, NJ). Dry IPG strips were rehydrated in 200 mL rehydration buffer (7 M Proteins were extracted from three pools of ten individual urea, 2 M thiourea, 4% w/v CHAPS, 50 mM DTT, 0.5% v/v concepti collected from at least three gilts on three different IPG buffer pH 4–7 (carrier ampholytes, GE Healthcare)), dates using 1 mL lysis buffer (7 M urea, 2 M thiourea, 4% containing 400 mg protein at 207C for 15 h. The IEF was per- w/v CHAPS, 50 mM DTT) (Fig. 1). Samples were sonicated formed under the following conditions (i) 500 V, 1 h, (ii) 15 s and chilled on ice. Samples were desalted and con- 1000 V, 1 h, (iii) 8000 V, 2.5 h. Prior to second dimension centrated by methanol/chloroform precipitation; for one separation by SDS-PAGE, the IPG strip was equilibrated sample volume, 4 vol. of cold methanol, 1 vol. of chloro- 26in 5 mL equilibration buffer (6 M urea, 75 mM Tris-HCl,

Figure 1. Outline of 2-DE analysis of the proteins of porcine concepti during the elongation phase. The intracellular proteins of three distinct pools of ten pig concepti from ovoid and filamentous stages of development were separated by 2-DE with the isoelectrofocusing being performed on IPG strips with linear gradient of pH 4–7. Each sample was run in duplicate for a total of six replicates per stage to obtain sufficient numerical data for statistical analysis.

29.3% v/v glycerol, 2% w/v SDS, 1% w/v DTT, trace bromo- nate. Gel plugs were dehydrated with 100% ACN, dried phenol blue solution) for 15 min in a 15 mL tube at rt with under vacuum, and rehydrated in 20 mLof10mg/mL mod- gentle shaking. The SDS-PAGE (gel size 20 cm620 cm6 ified sequencing grade porcine trypsin (Promega, Madison, 1.5 mm) was performed using a 10% polyacrylamide gel. WI, USA) in 25 mM ammonium bicarbonate. An additional The IPG strips were loaded on top of the 10% gel and over- 20 mL of 25 mM ammonium bicarbonate buffer was added to laid with 1% w/v agarose. Gels were run in duplicate in a ensure the gel plug was completely immersed in the ammo- PROTEAN II xi Cell (BioRad, Hercules, CA, USA) at 157Cat nium bicarbonate buffer and the trypsin digestion was per- 15 mA per gel for 30 min followed by 25 mA per gel for 5 h formed overnight at 377C. The resulting tryptic fragments using Laemmli SDS electrophoresis buffer (25 mM Tris, were extracted with 50% ACN and 5% TFA with sonication. 192 mM Glycine, 0.1% w/v SDS). The peptide extract was dried to completeness by vacuum centrifugation and dissolved in 5 mL 50% ACN/0.1% TFA. 2.4 Imaging and data analysis 2.6 MS The 2-DE gels were stained in 100 mL of Gradipure colloidal CBB stain (Life gels, Clarkston, GA, USA) for 14–16 h. Gels For PMF, a Voyager DE-STR MALDI-TOF mass spectrome- were washed 46with 6% acetic acid for 1.5 h. The destained ter (Applied Biosystems, Framingham, MA, USA) operated gels were scanned using laser densitometry (Personal Den- in the positive ion reflector mode was used to analyze tryptic sitometer SI, GE Healthcare) and stored in 20% ammonium peptides. Samples of 0.5 mL were co-crystallized with 0.5 mL sulfate. The image analysis was performed with the Image- of CHCA matrix, and spectra were acquired with 50 shots of Master™ 2D Platinum 6.0 software (GE Healthcare). To cor- a 337 nm Nitrogen Laser operating at 20 Hz. Spectra were roborate the reproducibility of independent gels, the pooled calibrated using the trypsin autolysis peaks at m/z 842.51 embryo protein samples of the ovoid and filamentous stages and 2211.10 as internal standards. To analyze proteins that defined above were run in duplicate. Thus, three independ- were not positively identified by MALDI-TOF-MS, a tandem ent protein extractions of each embryonic stage yielded a LC mass spectrometer Thermo Finnigan LCQ Deca XP plus total of six gels that were utilized for proteome analysis. LC-MS/MS was used. Peptides were separated by reverse Briefly, protein spot contours were automatically detected by phase chromatography on a 100 mm60.18 mm BioBasic-18 ImageMaster™ software. A global spot image (master gel) column (ThermoFisher Scientific, San Jose, CA, USA) using was automatically created by matching a set of gels with a a 30 min. linear gradient from 5–40% ACN in 0.1% formic reference gel. One reference gel was used for each source of acid at a flow rate of 3 ml/min. The instrument was operated protein; ovoid or filamentous concepti. To avoid biological with a duty cycle that acquired MS/MS spectra on the three variation of embryo pools, spots not found in the reference most abundant ions identified by a survey scan from 400 to gel were disregarded. The ratio of the volume of each specific 1600 Da. Dynamic exclusion was employed to prevent the protein spot and the sum of the spot volume for all proteins continuous analysis of the same ions. Once two MS/MS in the gel were calculated (vol%) and used for quantitative spectra of any given ion had been acquired, the parent mass comparison. The coefficient of determination (r2) for the spot was placed on an exclusion list for the duration of 1.5 min. intensity was evaluated between replicates using the scatter The raw data were processed by Sequest [33] to generate DTA plot function of the ImageMaster software. To detect differ- files or MASCOT Distiller (http://www.matrixscience.com) entially expressed proteins during elongation of porcine to create peak lists for database searching. The merge.pl concepti, normalized spot volumes were compared between script from Matrix Science was used to convert multiple the two embryonic stages. For a protein spot to be included Sequest DTA files into a single MASCOTgeneric file suitable in the analysis, the spot had to be detected in at least one for searching in MASCOT search engine [34]. replicate of all three biological repeats and in a minimum four of six gels for each stage (ovoid or filamentous). The data 2.7 Protein identification obtained were evaluated statistically using the Student’s t- test p,0.01 (TIGR MeV 3.1 (MultiExperiment Viewer pro- Protein identification was performed by searching against gram), www.tm4.org/mev.html). the mammalian subset of the US National Center for Bio- technology Information (NCBI) nonredundant, SwissProt, 2.5 In-gel digestion of protein spots MSDB, and EST_others databases using the MASCOT search engine on the Matrix Science public domain server The destained 2-DE gels were washed three times with (www.matrixscience.com [34]). The following parameters 300 mL ultrapure deionized water and the gel plug contain- were used for searches: for MALDI-TOF spectrum: 25 ppm ing the protein of interest was cut out and stored at 2807C. mass accuracy, for MS/MS ions: 1 Da peptide tolerance, 1 Da Thawed gel plugs were washed with a destaining solution MS/MS tolerance and 11,21,31 peptide charge and for consisting of ultrapure deionized water/methanol/acetic both monoisotopic mass, trypsin as digesting enzyme with acid (4.5:4.5:1), followed by ultrapure deionized water and one missed cleavage allowed and oxidation of methionine, N- finally 50% ACN containing 25 mM ammonium bicarbo- terminal pyroglutamic acid from glutamic acid or glutamine

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 2682 S. A. Degrelle et al. Proteomics 2009, 9, 2678–2694 as allowable variable modifications. For MALDI-TOF data to BLAST comparisons of SAGE tags to the species-specific qualify as a positive identification, a protein’s score had to porcine gene index (SsGI, version 13.0) available through equal or exceed the minimum significant score threshold at Computational Biology and Functional Genomics Laborato- p,0.05. Positive identifications of proteins by MS/MS analy- ry (CBFGL) Database at the Dana Farber Cancer Institute sis required a minimum of two unique peptides with at least (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl? one peptide having a significant ion score. gudb = pig), formerly, housed at the Institute of Genome Research (Rockville, MD, USA). A perfect 14-bp match of 2.8 Gene ontology and network analyses the SAGE tag to TIGR target was required for inclusion in the final data set. Using the GI sequences from the anno- The 174 unique proteins identified were used for gene tated protein of the reference map (Supporting Information ontology (GO) and network analyses. The equivalent Human Table 1), a BLASTX on the same TIGR database was per- Swiss-Prot accession numbers were uploaded into the Gen- formed. The best CBFGL tentative consensus (TC) sequence eCodis web-based tool ([35], http://genecodis.dacya.ucm.es) hit was used as the common identifier between SAGE tag and GO (GOSlim) annotations of biological process (BP) and and protein sequences. The rank and profile (constitutive or cellular component (CC) were determined. The GOSlim differential expression between ovoid and filamentous feature is an abbreviated version of the in-depth GO content embryos) of the twenty-five most abundant proteins was that is able to provide a broad overview of the ontological determined and compared to the rank and profile of its content without the detail of specific fine grained terms. corresponding transcript. For a protein that appeared in Default parameters for the analysis were the hypergeometric multiple spots, the sum intensity of all the protein isoforms statistical test, no p-value correction, and a minimum of was used. three genes. Proteins with a known gene identifier (HUGO gene symbol) and their corresponding expression values were uploaded as the input dataset into the Ingenuity Path- 3 Results way Analysis version 5.0 (IPA) software (Ingenuity® Sys- tems, Redwood City, CA, USA; www.ingenuity.com). The 3.1 2-DE and protein identification: A reference map relative level of each protein was calculated based on the of porcine elongating concepti average intensity of replicate samples at each embryonic stage. The network analysis identified the biological func- To produce an overview of the porcine conceptus proteome, tions that were most significant to the genes in the network. proteins from ovoid and filamentous porcine concepti were The network genes associated with biological functions in extracted and separated by 2-DE (Fig. 1). The initial IEF was the Ingenuity Pathways Knowledge Base were considered for performed on IPG strips pH 3–10, but the majority of pro- the analysis. The Fischer’s exact test was used to calculate a p- tein detected spanned pH 4–8, thus all subsequent analyses value determining the probability that each biological func- were performed on IPG strips pH 4–7. Many of the visible tion and/or disease assigned to that network is due to chance spots on colloidal CBB stained gels were identified (Fig. 2). alone. A network is a graphical representation of the molec- The protein spots highlighted in Fig. 2 exhibited a similar ular relationships between genes/gene products. Genes or intensity between replicates (r2 = 0.88 for the ovoid stage and gene products are represented as nodes, and the biological r2 = 0.90 for the filamentous stage, Supporting Information relationship between two nodes is represented as an edge Fig. 1). A comparison of the three reference gels for each (line). All edges are supported by at least one reference from stage identified 380 spots that overlapped in the ovoid refer- the literature, from a textbook, or from canonical informa- ence gels and 310 spots that overlapped in the filamentous tion stored in the Ingenuity Pathways Knowledge Base. Hu- reference gels. A total of 305 protein spots were selected for man, mouse, and rat orthologs of a gene are stored as sepa- analysis by MALDI-TOF or LC-MS/MS and a significant rate objects in the Ingenuity Pathways Knowledge Base, but identification was made for 275 (90%) of the protein spots are represented as a single node in the network. The inten- using the MASCOT search engine (Supporting Information sity of the node color indicates the degree of up- (red) or Table 1); however 16 of the protein spots contained two down- (green) regulation. Nodes are displayed using various identifiable proteins. In most cases, the experimental pI and shapes that represent the functional class of the gene prod- MW of the proteins were in agreement with the theoretical uct. value that was determined using ProtParam (http://ca.expa sy.org/tools/protparam.html [36]). From the 275 protein 2.9 Comparison of proteomic and transcriptomic spots identified, 174 unique proteins (174/275, 63%) were data identified, and of those, 162 were annotated by the Geneco- dis GO software with subcellular and functional designa- Matching the tag and protein sequences had to be per- tions for 133 and 140 proteins, respectively (Fig. 3). Func- formed indirectly, through a common identifier. The tag tional grouping of the proteins included protein metabolism annotation of SAGE libraries from ovoid and filamentous (23%), cell death (11%), response to stress (11%), signal concepti already published [12] were updated by performing transduction (9%), and cell organization and biogenesis

Figure 2. Establishment of a reference map for the abundant proteins of the pig conceptus by 2-DE. The representative gel depicts proteins separated in the second dimension on a 12.5% SDS-PAGE gel and stained with colloidal CBB for protein visuali- zation. Spot numbers identify the 305 protein spots that were analyzed by MALDI-TOF or MALDI-TOF/LC-MS/MS. Protein identification was obtained for the 275 protein spots (black numerals; see Supporting Infor- mation Table 1 for identity) whereas protein identification was not obtained for 30 (black highlight/white numerals).

(7%). In addition, 30% of the proteins were associated with using the Student’s t-test (p,0.01). Thirty-five spots were the cytoplasm, 23% with the nucleus, and 18% with the determined to be either up- or down-regulated at a p,0.01 cytoskeleton. level between ovoid and filamentous concepti (Fig. 4, Table Thirty eight of the unique proteins (38/174, 22%) were 1). The main functional properties of the differentially detected in two or more spots (Supporting Information Table expressed proteins are given in Table 1. Eleven proteins 1). For most unique proteins that were identified in multiple were up-regulated in ovoid concepti whereas 24 were up- spots, the MW was typically the same but their pI differed. regulated in filamentous concepti. However, several spots However, in the case of some proteins, both the MW (6 10%) were identified as the same protein, and therefore 10 dis- and pI were different. These included KRT8 (29 spots), IL1B tinct proteins were found up-regulated in ovoid concepti (15 spots), EZR (5 spots), MSN (3 spots), keratin 9 (KRT9, 3 and 15 were up-regulated in filamentous concepti. Based on spots), and heat-shock 70 kDa protein 8 (HSPA8, 3 spots). the available GO annotations of the differentially expressed proteins, factors involved in cytoskeleton-organization-bio- 3.2 Differentially expressed proteins between ovoid genesis (cytokeratin 7 (KRT7), KRT8) and morphogenesis and filamentous stages (KRT18, EZR) were up-regulated at the ovoid stage whereas those modulating cell death (annexin 5 (ANXA5)), cell pro- Proteins differentially expressed between the ovoid and liferation and differentiation (stratifin (SFN), tumor protein, filamentous concepti were identified by statistical analysis Rho Family Cdc42), protein folding, and response to stimu- of the intensity of corresponding protein spots in the gel. lus proteins (RBP, IL1B) were predominantly up-regulated Proteins were statistically considered up- or down-regulated at the filamentous stage.

Figure 3. Subcellular distribution and functional annotation of the unique proteins. The subcellular and functional categor- ization of the 174 proteins in the pig conceptus reference map was performed using the Gene- codis GO database; 133 proteins were subcellularly annotated (A) and 140 were functionally annotated according to BPs (B).

3.3 Identification of seven regulatory networks position in the three merged primary networks (Supporting during the elongation process Information Fig. 5).

To further refine the characterization of the 174 unique pro- 3.4 Integration of proteomic and transcriptomic data teins from the reference map, biological interactions between the proteins identified were investigated using IPA Performing expression analysis at both the mRNA and pro- and 148 proteins were found to map to gene networks with tein levels can provide insight into the relationship between functional relationships. A total of 13 networks were built. the timing of the transcript and protein expression during Seven networks were found to be very significant, i.e., they the elongation phase of the porcine concepti. A BLAST had more identified proteins present than would be expected search against the CBFGL database porcine-specific gene by chance. Networks with high scores (.15) that contained index to update annotation of SAGE tags [18] followed by a differentially expressed proteins, are listed in the Table 2. comparison of the putative annotated transcripts (TC Those networks were associated with embryonic develop- sequences) with proteins identified by 2-DE, indicated that ment, cellular assembly and organization, cellular growth expression at both the mRNA and protein level coincided for and proliferation, cell death, and protein folding and PTM. 85 of the 174 genes. The expression (intensity) ratio of ovoid The three networks with the most significant p-value are il- versus filamentous concepti and rank of the 25 most abun- lustrated in Supporting Information Figs. 2, 3, and 4. A dant proteins identified by 2-DE was compared (Table 3) to functional overlap of those top networks is depicted in Sup- previous SAGE data mRNA expression ratio (frequency of porting Information Fig. 5. The highlighted (yellow) proteins SAGE tag (transcript)) and rank (tags from SAGE libraries are those identified in the reference map, red for increased ranked,2504; 4314 tags in ovoid and 4138 in filamentous expression in ovoid concepti and green for increased expres- [21]). Sixteen of the transcripts displayed a concordant ovoid sion in filamentous concepti. IL1B and RA occupied a focal to filamentous ratio with their protein counterpart (ACTB,

Figure 4. Comparison of 2-DE proteins profiles of porcine concepti at ovoid and filamentous stages. The spot intensity of reference gels for each developmental stage, ovoid (A) and filamentous (B) were compared and statistically analyzed using a Student’s t-test (p,0.01) to identify differentially expressed proteins. Proteins up-regulated at the ovoid stage (white arrows) and the filamentous stage (black arrows) are shown; spot numbers indicate the protein identification in Supporting Information Table 1. cytokeratin 19 (KRT19), heat-shock cognate 71 kDa protein rank 41, and 44 transcripts with a rank 44 met the cri- (HSPA8), tubulin subunit b-2 (TBB2), ATP synthase sub- terion for the filamentous and ovoid concepti, respectively. unit b, mitochondrial precursor (ATP5B), EZR, tubulin In comparison with the 25 most abundant proteins, only 8 subunit a-6 (TBA6), heat-shock 70 kDa protein 9 (HSPA9), transcripts could be categorized as abundant. Despite hav- IL1B, SFN, cathepsin B (CTSB), hexosaminidase (HEXA), ing a low transcript level (rank 765), LGMN, CALR, 40S ribosomal protein SA (RPSA), heat shock 60 kDa pro- CTSB, and ANXA6 were represented within the abundant tein 1 (aka., chaperonin; HSPD1), elongation factor-1-d protein population. (EEF1D), and ATPase, H1 transporting, lysosomal V1 subunit A (ATP6V1A)). Most were constitutively expressed at both the protein and RNA level, but EZR and HSPD1 were 4 Discussion increased in ovoid embryos and IL1B and SFN increased subsequent to elongation, i.e., in filamentous embryos. Of 4.1 Porcine conceptus development reference maps the remaining factors, the mRNA of KRT8, KRT18, pro- collagen-proline, 2-oxoglutarate 4-dioxygenase, b polypep- In the current study, the intracellular proteins of the porcine tide (P4HB), glucose-regulated protein, 78 kDa (HSPA5), conceptus at the initiation (ovoid) and the end (filamentous) and chloride intracellular channel 1 (CLIC1) was down- of the early rapid elongation phase were globally identified. regulated in the filamentous concepti while the protein The early, rapid elongation phase is exemplified by a marked expression remained constant. In contrast, calreticulin change in the length of the conceptus between gd-11 and gd- (CALR) and ANXA6 proteins were down-regulated, and 12 that is caused by a reorganization and expansion of the legumain (LGMN) and tropomyosin 3 (TPM3) proteins trophectoderm in preparation for implantation [1–5]. An in were up-regulated in the filamentous concepti while the silico functional analysis of the proteins identified in the mRNA remained constitutively expressed. The transcript present study pinpointed the regulation of metabolic, rank from the .4000 transcripts (SAGE tags) identified in growth, and death processes (Fig. 3; Table 2; Supporting each developmental stage [12] was used to determine the Information Fig. 5). Moreover, at the gd-11 ovoid stage just highly abundant transcripts, that is, the upper 1% based on preceding elongation of the conceptus, an up-regulation of transcript frequency [37, 38]. Forty-two transcripts with a proteins modulating cytoskeleton-organization/biogenesis

Table 1. Proteins differentially expressed (p,0.01) in the pig conceptus at ovoid and filamentous stages

Spot Ovoid Filamentous Protein identification [species] Acc. no. Biological function (GO) NCBInr n %Vol. 6 SD n %Vol. 6 SD

Up-regulated in ovoid embryos sd61 6 0.517 6 0.149 6 0.302 6 0.062 Keratin 8 [Bos taurus]gi)75812916 Cytoskeleton organization and biogenesis sd62 5 0.329 6 0.066 6 ND Keratin 8 [Bos taurus]gi)75812916 Cytoskeleton organization and biogenesis sd68 6 0.384 6 0.023 6 0.178 6 0.048 Unknown sd75 5 0.341 6 0.183 6 ND Keratin 18 [Bos taurus]gi)76617986 Morphogenesis sd80 6 0.110 6 0.009 6 0.087 6 0.010 Regucalcin [Sus scrofa]gi)116175265 Cell organization and biogenesis, protein metabolism sd86 5 0.075 6 0.020 6 ND Similar to Uridine phosphorylase 1 [Bos taurus]gi)149773558 Nucleotide catabolic process sd87 5 0.170 6 0.029 6 ND Palmitoyl-protein thioesterase 1 [Homo sapiens]gi)4506031 Protein metabolism sd88 6 0.126 6 0.018 6 ND Unknown sd241 4 0.047 6 0.018 6 ND Elongation factor G [Homo sapiens]gi)14285174 Protein biosynthesis sd263 6 0.439 6 0.269 6 0.130 6 0.049 Villin 2 [Canis familiaris]gi)73945744 Morphogenesis sd361 5 0.474 6 0.129 5 0.131 6 0.043 Keratin 7 [Homo sapiens]gi)67782365 Cytoskeleton organization and biogenesis Up-regulated in filamentous embryos sd93 6 ND 5 0.032 6 0.014 Rho Family Gtp-Binding Protein Cdc42 gi)7245833 Cell proliferation, cell differentiation, cell [Bos taurus] adhesion, cell motility, signal transduction sd104 6 0.337 6 0.117 6 0.599 6 0.141 Human elongation factor-1-delta gi)38522 Protein translation [Homo sapiens] sd105 6 ND 6 0.072 6 0.030 Translational elongation factor 1 delta gi)57164211 Protein translation [Ovis aries] sd106 6 ND 6 0.085 6 0.031 90kDa Heat shock protein [Homo sapiens]gi)306891 Protein folding sd107 6 ND 6 0.062 6 0.034 Annexin VIII [Homo sapiens]gi)113967 Protein folding, signal transduction sd108 6 0.096 6 0.007 6 0.218 6 0.089 Eukaryotic translation initiation factor 3 gi)114555316 Protein biosynthesis [Pan troglodytes] sd110 5 0.281 6 0.016 6 0.466 6 0.075 Legumain [Bos taurus]gi)27806555 Growth, proteolysis sd111 6 ND 6 0.165 6 0.034 Legumain [Bos taurus]gi)27806555 Growth, proteolysis sd114 6 ND 5 0.151 6 0.070 Tropomyosin 3 [Canis familiaris]gi)73961067 Cell motility sd115 6 0.224 6 0.039 6 0.325 6 0.053 Annexin V [Homo sapiens]gi)809185 Cell death, cell differentiation sd116 6 ND 5 0.098 6 0.020 Interleukin-1 beta [Sus scrofa]gi)30910830 Cell death, cell proliferation, signal transduction, inflammatory response sd118 4 0.107 6 0.017 6 0.249 6 0.040 Interleukin-1 beta [Sus scrofa]gi)30910830 Cell death, cell proliferation, signal transduction, inflammatory response sd119 6 ND 5 0.048 6 0.010 Interleukin-1 beta [Sus scrofa]gi)30910830 Cell death, cell proliferation, signal transduction, inflammatory response sd120 6 0.187 6 0.011 6 0.305 6 0.066 Tumor protein, translationally-controlled 1 gi)109120640 Cell death, cell differentiation, transport [Macaca mulatta] sd121 6 ND 5 0.113 6 0.021 Interleukin-1 beta [Sus scrofa]gi)52346210 Cell death, cell proliferation, signal transduction, inflammatory response sd122 6 ND 5 0.156 6 0.032 Retinol-binding protein (Rbp) [Sus scrofa]gi)2914422 Response to stimulus, transport sd123 6 ND 6 0.318 6 0.201 Retinol-binding protein (Rbp) [Sus scrofa]gi)2914422 Response to stimulus, transport sd124 6 ND 5 0.302 6 0.044 Stratifin [Sus scrofa]gi)113205700 Cell proliferation, cell differentiation, signal transduction, regulation of cell cycle sd285 4 0.079 6 0.016 6 0.303 6 0.208 Heat shock 70kDa protein 8 [Homo sapiens]gi)5729877 Protein folding sd376 5 0.036 6 0.004 5 0.051 6 0.006 Macrophage Capping Protein Cap G gi)21730367 Cell organization and biogenesis, protein [Homo sapiens] metabolism sd394 6 0.206 6 0.087 3 0.486 6 0.042 Tropomyosin 3 [Canis familiaris]gi)73961067 Cell motility sd415 5 0.057 6 0.009 4 0.091 6 0.013 Transaldolase 1 [Homo sapiens]gi)5803187 Metabolism sd511 4 0.083 6 0.045 4 0.433 6 0.145 Interleukin-1 beta [Sus scrofa]gi)30910830 Cell death, cell proliferation, signal transduction, inflammatory response sd512 4 0.057 6 0.023 5 0.252 6 0.113 Interleukin-1 beta [Sus scrofa]gi)30910830 Cell death, cell proliferation, signal transduction, inflammatory response

Protein names are from the NCBI database and spot numbers (sd) are from Supporting Information Table 1. The protein expression value represents the relative volume (intensity) of a spot to the overall volumes of all quantified spots.

Table 2. Statistically significant gene networks in porcine elongating concepti

Network Genetic in ingenuity networks Score Focus Top functions genes

1 ACTB, Actin, ANXA5, APOA1, ARHGDIA, ATPB, 66 31 Cellular assembly and organization; Calpain, CAPNS1, CCT2, CCT5, CCT8, CCT6A, cell Death; embryonic development CNN3, EEF1B2, F Actin, GSN, IL1B, KRT8, KRT18, KRT19, MSN, P4HB, PDCD6IP, PRDX2, Ras homolog, RPSA, RUVBL2, SFRS1, SLC9A3R1, TCP1, TPM3, TPT1, TUBB, VCL, EZR 2 14-3-3, Akt, ATPase, BAG2, CALR, Caspase, DDB1, 45 24 Protein degradation; post-translation EIF3S2 (includes EG:8668), HSP, Hsp70, Hsp90, modification; protein folding HSP90AA1, HSP90AB1, HSP90B1, HSPA4, HSPA5, HSPA8, HSPB1, Immunoproteasome Pa28/20s, LMNB1, MHC Class I, PPID, Proteasome PA700/20s, PSMA3, PSMB, PSMB2, PSMB4, PSMB6, PSMC3, PSMC4, RAD23B, SFN, STIP, VCP, YWHAE 3 AARS, AHCY, ALAS1, ANXA8, BCAR1, CALB1, CSH1, 28 17 Cellular growth and proliferation; cancer; DDIT3, EEF1D, EEF1G, EIF4E, ELN, G6PD, GARS, cell death HEXA, HNRPF, HNRPK, HSPD1, IGFBP4, INS1, KRAS, KRT16, LAP3, PCK1, PDHB, PDXK, PP1CA, PSME, retinoic acid, RPLP2, RPLP0 (includes EG:6175), RUVBL2, SPARC, TF, YWHAZ 4 ACTR3, ALB, ANXA6, Ap1, BTF3, Ck2, FTH1, 26 16 Cell morphology; skeletal and muscular Histone h3, HNRPK, HSPD1, Ikb, IL1, IL1F6,IL1RL1, system development and function; Jnk, Jnk dimer, LMNA, Mapk, NAP1L1, P38 MAPK, DNA replication, recombination PDGF BB, PI3K, Pkc(s), PPM1L, Ras, RBBP4, RBP4, and repair RNA polymerase II, SAFB, SET, SFRS7, SUGT1, TXNL2, UQCRC1, YBX1 5 ACY1, ADK, ANXA4, BCAR1, CASP4, CCL20, CD40LG, 24 15 Cell death; cancer; cellular growth and CLIC1, Cpla2, CTSB, CXCL1, DNTT, EGFR, ENO1, F2, proliferation GADD45A, GDI2, GHR, HP, HSP90AA1, IER3, IL1RAP, LGMN, MT1E, MYC, PPIA, PRDX3, PRDX6, PRPF19, RELB, RHOB, SNX6, SOD2, SUCLA2, YBX1 6 ATP5H (includes EG:10476), ATP6V1A, CKB, CTSD, 24 14 Connective tissue disorders; immunolog- Cyclin B, CYP2E1, DDX5, EEF2, ESR1, GAPDH ical disease; inflammatory disease (includes EG:2597), GDF15, GFM1, H1-transporting two-sector ATPase, HBEGF, HSP90AB1, HSPB1, hydrogen peroxide, IDH3A, MIF, MYCN, NACA, PGAM1, PHB, PLK3, PPA1, PPARGC1A, Protein- synthesizing GTPase, PTGS1, PTGS2, PTMA, Sod, TP53, TUFM, UPP1, ZYX 7 CCL27, CMPK, ECGF1, FLNB, FPRL1 (includes EG:2358), 16 11 Vitamin and mineral metabolism; FTL, FUT7, GBP1, GCLC, HAMP, HAS1, iron, KRT7, lipid metabolism; molecular transport KRT6A, MEFV, NADH2 dehydrogenase, NADH2 dehydrogenase (ubiquinone), NAPA, ND1, ND2, ND4, DUFS1, NDUFS3, NDUFV2, NFE2L2, PBEF1, Pka, SRM, TALDO1, TGFB1, TNF, TNFAIP2, TXN, UGDH, ZFP36

Proteins in bold are present in the input data list. Proteins in bold italics underlined or bold underlined are those statistically identified as up- or down-regulated at the ovoid stage, respectively.

and morphogenesis was observed (Fig. 3; Table 2). An exami- this period of development (Supporting Information Fig. 5). nation of known protein interactions, both direct and indi- Thus, we have generated a reference map and highlighted rect, suggests that intracellular signaling initiating from the potential physiological processes for some of the most abun- IL1B and RA pathways may be central to the regulation of a dant proteins present in the porcine conceptus during elon- large number of the genes involved in distinct BPs during gation.

4.2 Regulation through PTMs and differential proteins of the elongating porcine conceptus, the fact that expression the IL1B isoforms were readily detected is indicative of biological significance. IL1B signaling is normally required for In general, the observed MW and pI were in agreement successful implantation in mice [17, 48] and has been pro- with theoretical values for the majority of the proteins posed as the initiator of maternal-fetal communication at the identified in the study. However, a distinct protein spot time of implantation in humans [17, 49, 50]. Therefore, it is identified by 2-DE does not always correspond to one likely that the IL1B system is key to the implantation process unique protein. PTMs of precursor proteins by either pro- in pigs as well [16, 43]. teolytic processing or the addition of functional moieties, e.g., A number of proteins associated with the cytoskeleton phosphorylation or glycosylation, can result in a shift of pI, structure/remodeling, KRT8, KRT18, and KRT7, also exhib- or, in the case of latter, MW as well. For most proteins iden- ited multiple isoforms with distinct molecular weights and/ tified in multiple spots in this study, the change was a shift in or pIs, and, some isoforms were differentially expressed be- pI rather than MW (Supporting Information Table 1). A few tween ovoid and filamentous concepti (Table 1, Supporting proteins had isoforms that exhibited a distinct change in Information Table 1). In a prior study, we demonstrated that both MW (.10%) and pI, indicative of proteolytic cleavage multiple isoforms of KRT18 are present in ovoid, tubular, and the presence of additional moieties. These included and filamentous concepti, however, at the filamentous stage KRT8 (25 spots), IL1B (15 spots), KRT18 (9 spots), CTSB (4 the expression of KRT18 proteins was diminished [5]. Simi- spots), EZR (5 spots), MSN (2 spots), KRT9 (3 spots), and larly, in the present 2-DE analysis, specific isoforms of HSPA8 (3 spots). Variation in either MWand pI or both, may KRT18 along with another simple epithelia marker, KRT19, indicate post-translational alterations that are important in and the trophoblast marker KRT7 [51] were found in greater regulating protein’s biological activity. abundance at the ovoid stage. During mouse development, An important example of post-translational alterations KRT8 and KRT18 are the first Type I and II keratin proteins regulating biological activity is IL1B. IL1B is translated as a to be expressed and this is followed by the induction of precursor protein of ,35 kDa and processed by proteolytic KRT19 expression [52]. Additionally, the concurrent abroga- cleavage into an active form of ,18 kDa [39, 40]. However, tion of KRT19 and KRT18, or deletion of KRT8 alone, can various MW and pI of native or processed isoforms of IL1B induce trophoblast abnormalities that are lethal [52, 53]. have been described [41, 42]. The IL1B species detected in the Cytokeratins are important for the formation of lamellapodia porcine concepti by our 2-DE analysis ranged in size from and cell migration, the establishment of focal adhesions that ,36 to ,14 kDa (Supporting Information Table 1), and a few act as a signaling hub between cells, and cell cycle and of these were up-regulated at the filamentous stage (Table 1). apoptosis regulation [54–56]. Of the observed cytokeratins, A manual inspection of the LC-MS chromatograms to deter- all were found to be of slightly lower MW than their pre- mine the presence or absence of peptides in the N-terminal dicted MW. Since caspase cleaved cytokeratin products are prodomain of IL1B, revealed that three of the IL1B isolates reportedly much smaller than any of the observed cytoker- did not contain peptides spanning the prodomain. One of atin MWs seen here [57], our data implied that apoptosis was these three proteins had the expected 18 kDa MW of the a minor occurrence in the developing porcine conceptus over mature IL1B active form. This differential expression of the the time period examined. The varied MW/pI of cytokeratins putative mature IL1B isoform at the filamentous stage is observed in the study also implied that differentially phos- similar to the finding of a previous study where expression of phorylated and glycosylated species of KRT7, KRT8, and the active form of IL1B was first observed at the filamentous KRT18 are present, presumably to effect functional reorgan- stage [43]. This also coincides with the increased expression izations of the cell’s intermediate filaments during the rapid of IL1B mRNA and an increase of IL1B protein in the lumi- expansion of the porcine trophectoderm and/or promote nal fluid of the uterus [16]. The other two IL1B isoforms that interaction with the 14-3-3 proteins to enable mitosis [54, 56]. lacked the N-terminal IL1B prodomain had a lower MW, Perhaps as in other simple epithelial tissues, these changes ,14 kDa (sd490, sd492 – Supporting Information Table 1). in the cytokeratins help protect the developing conceptus These smaller isoforms, that were not differentially expres- from environmental stress and apoptosis [58] sed, were presumably too small to be active forms of IL1B, Another cytoskeleton protein, EZR, that binds actin fila- and they may have resulted from matrix metalloproteinase ments in the microvilli of polarized, absorptive cells, also cleavage of the protein [44]. Several other IL1B forms that demonstrated up-regulated protein expression at the ovoid were found, some differentially expressed, others not, con- stage compared to the filamentous stage (Table 1). Cell tained pro-IL1B sequences and were presumably artifacts of migration through the remodeling of the actin cytoskeleton the isolation technique [41] or represent alternative cleavage can be enabled through the cleavage of EZR by calpain I, in and/or processing products. However, alternate N-terminal turn, this proteolysis can be inhibited by the phosphorylation forms of biologically active IL1B have been reported and of EZR [59]. In gastric epithelial cells, cleavage of EZR by therefore might be expressed by the porcine conceptus or be calpain abrogates its apical membrane localization and taken up from the uterine environment [41, 45–47]. Since induces translocation to the cytoplasm [59]. Five unique our proteomic analysis detected the most abundant cellular spots for EZR were present in the porcine concepti with dis-

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2009, 9, 2678–2694 2689 tinct MWs and pIs. Most were in the range of 70 kDa similar early embryonic development is less clear. The remaining to the mature protein [59] and exhibited different pIs, which differentially expressed proteins, with the exception of one, may be indicative of phosphorylation or other modifications. were all characterized by up-regulation in the filamentous However, a significantly lower MW EZR protein of 43.7 kDa stage embryos. This in itself seems appropriate because of was also present, which may be similar to a calpain-cleaved the rapid changes in shape and cell number taking place 55 kDa species previously found in the rabbit [59]. Most of during the progression from the ovoid to the filamentous the EZR isoforms were constitutively expressed, however, a form. The differentially up-regulated proteins spanned a 70 kDa pI isoform was found to be down-regulated at the variety of functional categories and included heat-shock pro- filamentous stage. In some studies in the early mouse teins, endosome/lysosome processing proteins (LGMN and embryo, EZR has been found exclusively in the visceral ANXAs), translation factors, metabolic enzymes, cytoskeletal endoderm and was expressed particularly strongly in those elements, and signal transducers. A review of the literature endoderm cells in contact with the trophectoderm [60, 61]. In allows some limited comments. LGMN, ANXAs, and heat- contrast, other studies indicate that the expression of EZR shock proteins have been previously associated with pla- protein is predominant in the endoderm of developmentally centation or placental function [67–69]. LGMN, an endo- dormant mouse embryos and highly expressed in the tro- peptidase, is abundant in endosomes/lysosomes and is phectoderm, as opposed to endoderm, of normal mouse involved with protein processing [70, 71]. Additionally, embryos at the onset of implantation [62]. Likewise, human LGMN is highly expressed in the placenta [72], can modulate trophoblasts also express EZR, and its expression is under the expression of adhesion proteins like fibronectin [71], can the control of IL1B [63]. Whether this expression pattern is activate matrix metalloproteinases that are important in the same in the pig embryo’s trophectoderm/endoderm remodeling of the extracellular matrix [70], and is expressed bilayer is presently unknown and should be investigated in bovine trophectoderm in association with implantation further. [67]. ANXAs are phospholipid/calcium-binding membrane Among the proteins exhibiting the most extreme expres- proteins that are highly expressed in the placenta [68, 73, 74]. sion patterns, i.e., those detected in concepti of only one stage Their proposed functions in placental tissue include being of development, RBP was of particular interest because of its the cell surface-binding protein (ANXA2) for tissue-type importance in the sequestration of vitamin A metabolites, plasminogen activator (tPA), and thereby, modulation of the retinol and all trans RA derivatives, to control their transport extracellular matrix and cell invasiveness [75], anti-inflam- and bioavailability [18, 64]. Retinoids, especially RA deriva- matory properties through modulation of phospholipase A2 tives, are important embryonic morphogens that can be ter- and prostaglandin synthesis (ANXA1 and 2) [74], and pla- atogenic if not tightly controlled [18]. The RA metabolites are cental interface anti-thrombotic properties (ANXA5) [76, 77]. thought to form gradients along the embryo’s antero- Another ANXA that was up-regulated in the filamentous posterior axis during critical stages of embryogenesis where stage porcine embryos was ANXA8 which may be a minor they promote cellular differentiation and morphogenesis component of the pig’s placental ANXA complement [68]. In [18]. The secretion of RA metabolites by the porcine embryo, any case, ANXA8’s specific functions are not presently particularly the trophectoderm, is evident around the time of known. The other up-regulated ANXA at the filamentous elongation [65] and coincides with the appearance of RBP stage was ANXA5, and its expression has been shown to be [66]. In this study, the porcine embryo’s RBP protein was modulated in the chorion at parturition and to be critical to found to occur as two spots (sd122, 123 – Supporting Infor- embryo survival [74, 77]. Its modulation at implantation may mation Table 1) of similar MW but different pIs. The char- be similarly important. In general, however, the global sig- acteristics of the proteins were consistent with the size and nificance of the differential expression of these proteins is pI of RBP isoforms secreted by gd-15 porcine concepti [66]. not fully understood at this point in time. However, in contrast to the earlier radioimmunopreciptation study that identified the secretion of RBP from porcine con- 4.3 Comparison of proteomic and transcriptomic ceptus tissue at gd-11 and an increase by gd-12 [66], our col- expression loidal CBB staining of 2-DE gels detected expression of cellular RBP isoforms only in the gd-12 filamentous conceptus Of the 174 abundant proteins that were detected in the pres- (Table 1). Perhaps the increased sensitivity of the radioassay ent study, about 52% had been detected at the mRNA level in in comparison to protein staining or a ratio less than one for previous SAGE studies [5, 12]. Whether the inability to iden- cellular:secreted RBP may account for the difference be- tify a transcript for a greater number of the proteins is due to tween these findings. In any case, the up-regulation or dif- the incompleteness of the porcine genome still remains to be ferential expression of RBP shown here correlates with the determined. On the other hand, ,50% of transcripts are increased RBP transcription previously reported in elongat- present at 1 copy per cell, therefore it is also a possibility ing pig concepti [19]. that those proteins not detected at the mRNA level represent Several other proteins were found to be differentially rare transcripts, and the depth of the SAGE analysis was not expressed between the ovoid and filamentous stages al- sufficient to identify them [38, 78]. In SAGE, a transcript though their particular significance to this stage of porcine expression level is considered proportional to the frequency

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 2690 S. A. Degrelle et al. Proteomics 2009, 9, 2678–2694 with which a SAGE tag, representative of a distinct tran- being constitutively expressed at the protein level in ovoid or script, is detected within an experimental mRNA sample filamentous concepti and differentially regulated at the tran- [79]. The transcript (SAGE tag) frequency is considered script level or vice versa. Down-regulation of KRT8 and absolute, and therefore, it may be used directly to determine KRT18 mRNAs has been validated previously [5]. Ranking of differences in the abundance of specific transcripts within a the expression also indicated that some of the less abundant test sample or between different samples [80]. A comparison transcripts (LGMN, CALR, ANXA6, CTSB, and ATP6V1A) of the rank of the 25 most abundant proteins to mRNA indi- were some of the most abundant proteins. The disparity in cated that ,1/3 of the abundant proteins were also the most transcript versus protein expression, seen in other biological abundant transcripts, i.e., they were in the top 1% of all the systems (e.g., yeast in growth phase [81]; zebrafish ovarian unique mRNAs identified for at least one developmental follicules [82]), could be the result of asynchronous tran- stage (Table 3). Considering the biological importance of scription and translation or differences in the turnover of KRT8, KRT18, and IL1B in embryogenesis and implantation, mRNA or protein. Furthermore, alternative translation, it is noteworthy that these genes are very abundant at the novel splice variants, post-translational processing or accu- protein and RNA levels. An evaluation of the expression mulation of uterine luminal protein through endocytosis [83] profile (constitutive, up-regulated or down-regulated) at both may be additional phenomena that complicate the correla- levels indicated that .60% (16/25) had a similar profile be- tion of protein expression versus mRNA expression. tween ovoid and filamentous concepti; most being con- The proteomic repertoire found in this study lacked the stitutively expressed at both stages. However, nine factors expression of several genes previously noted at the mRNA (KRT8, KRT18, LGMN, CALR, CLIC1, TPM3, P4HB, whose functional roles are thought to be essential for porcine HSPA5, and ANXA6) exhibited discordant expression by embryo development, such as the estrogen regulating pro-

Table 3. Comparison between mRNA/protein expression levels of the 25 most abundant proteins

Gene TC Protein level (2-DE) RNA level (SAGE) a) name Rank GI Ovoid Fila- Ratio Tag Ovoid Fila- Ratio Best mentous mentous rank b)

KRT8 TC263700 1 gi)75812916 14.62 13.46 1.1 AGTATCCACA 209 104 2.0 9* KRT18 TC273630 2 gi)76617986 10.35 8.69 1.2 AGAAATCTGA 319 179 1.8 4* ACTB TC239011 3 gi)15277503 3.67 3.45 1.1 AGATGCATTG 51 42 1.2 95 KRT19 TC278551 4 gi)62751472 2.90 3.52 0.8 GACATCAAGA 151 146 1.0 13* HSPA8 TC267189 5 gi)5729877 1.71 1.99 0.9 GCAGTTGTAA 143 185 0.8 12* TUBB2 TC261945 6 gi)57209813 1.20 1.17 1.0 CTGTACAGAC 19 21 0.9 254 ATP5B TC246092 7 gi)89574051 1.05 0.95 1.1 AGAGCCTTGA 126 107 1.2 21* EZR TC259611 8 gi)73945744 1.03 0.48 2.2 CAGTGGATCA 57 12 4.8 81 TUBA1C TC298934 9 gi)76618145 0.99 1.16 0.9 TCTCAAAAAG 94 107 0.9 31* LGMN TC257922 10 gi)27806555 0.87 1.47 0.6 GACAAGGTGT 5 7 - 765 HSPA9 TC251561 11 gi)903309 0.68 0.70 1.0 TTGGGAAGTA 21 17 1.2 249 CALR TC270819 12 gi)1706140 0.68 0.38 1.8 GGGTGTTACA 3 0 - 897 IL1B TC271146 13 gi)30910830 0.67 1.49 0.4 GCACACTAGG 469 682 0.7 1* CLIC1 TC240636 14 gi)57094359 0.65 0.52 1.2 GTGCTCTGGC 50 28 1.8 97 SFN TC264138 15 gi)113205700 0.61 1.14 0.5 TTTCCTCTCA 72 225 0.3 9* CTSB TC265679 16 gi)309202 0.60 0.43 1.4 TTCCCACGGG 1 6 - 897 TPM3 TC239400 17 gi)73961067 0.52 1.09 0.5 TGCCCCTTAT 26 24 1.1 201 HEXA TC239720 18 gi)109081769 0.48 0.48 1.0 CCCCTGTGCC 9 10 - 536 P4HB TC286917 19 gi)27806501 0.47 0.43 1.1 GCTCAGTGAC 30 20 1.5 173 HSPA5 TC287881 20 gi)114626692 0.44 0.59 0.7 TGCATCTGGT 55 36 1.5 86 RPSA TC253810 21 gi)51766344 0.37 0.25 1.5 AACTCTTATT 84 75 1.1 45 HSPD1 TC252092 22 gi)114582388 0.35 0.16 2.2 CCTACAGATA 47 26 1.8 511 EEF1D TC240466 23 gi)38522 0.34 0.67 0.5 GCCCAGCTGG 9 10 - 536 ANXA6 TC254994 24 gi)13994159 0.32 0.15 2.1 GTCACTCCTG 3 6 - 198 ATP6V1A TC238401 25 gi)74002620 0.31 0.24 1.3 TTATCATTTG 2 1 - 2504 a) Data from Blomberg et al., 2005 [12] b) Best tag frequency rank from either ovoid (max. = 4314) or filamentous (max. = 4138) librairies. * Abundant transcripts (top 1%). Proteins in bold represent constitutively expressed proteins; differentially expressed mRNA. Proteins in bold/grey highlight represent differentially expressed proteins; mRNA not statistically different. Unbolded text represents proteins and mRNAs with similar expression patterns.

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2009, 9, 2678–2694 2691 teins (STAR, CYP17A, and CYP19A) and proteins involved in detected proteins found in this study represent only the most embryogenesis (MDK) or implantation (cyclooxygenase 2 abundant cellular proteins. (PTGS2)) [5, 13, 23, 25, 84, 85]. Proteins for STAR [27], CYP19A [28], MDK [5], and CYP17A [28] are expressed by the embryo. One explanation for not detecting STAR, MDK, and 4.5 Conclusions CYP17A is that their pIs of 9.17, 9.84, and 8.72, respectively, are outside the pI range used in the 2-DE analysis. Although A constant underlying criticism for transcriptomic studies the pIs of CYP19A and PTGS2 proteins are within the range has been that mRNA expression might not truly reflect the used in the 2-DE analysis, a plausible explanation for their expression profile of proteins or their relative biological ac- not being detected may be that their level of expression is tivity. This study indicates that proteins are present for many below the sensitivity range of colloidal CBB. If CYP19A has transcripts identified previously, and though only a small an expression profile similar to its transcript, this would number have been evaluated, many of them exhibited a likely be the case. Secondly, based on the solubilization tech- similar expression pattern. Within the panel of proteins niques used, intracellular proteins more so than membrane identified, several factors (EZR, TPM3, ANXA5, and ANXA8) proteins, such as CYP17A, CYP19A, PTGS2, and the mature along with their corresponding transcripts have not been isoform of STAR, would be detected. Overall, it is important reported previously in the pig conceptus and they could have to note that the proteins for a large number of previously important biological roles that enable morphogenesis of the described transcripts were expressed in the concepti exam- conceptus and its preparation for implantation. Thus, this ined. Also, the identification of the expression of additional work has provided some new insight into the maintenance genes at the protein level should help expand putative bio- and deviations in the expression of some potentially impor- logical mechanisms of importance during this period of tant developmental factors at two distinct levels, mRNA and development. protein. Furthermore, putative PTMs of some proteins (such as IL1B, the cytokeratins and EZR) may be indicative of the regulation of their bioactivity or alternative functions. All in 4.4 Caveats of proteomic technology all, a reference map of 174 unique proteins was established that provides a first glimpse of the porcine elongating It is important to keep in mind the limitations of proteomic embryo’s intracellular proteome repertoire. In the future, technology. In particular, with the 2-DE proteomic approach multidimensional protein identification technology (Mud- used here, detection of a large number of unique proteins PIT) analyses coupled with sub-cellular fractionation (e.g., can be severely limited by the over representation of just a nucleus and membrane) as illustrated recently by Cox et al. few or several proteins in the sample; the overwhelming [88], should increase the complexity of the proteomic map presence of albumin being a notorious example in serum/ and enable a more high-throughput protein analyses. The plasma proteomic analysis. In this study, the sum intensity identification of a greater number of proteins could help of KRT8 and KRT18 proteins accounted for ,19 and 11%, expand and refine the present networks identified into spe- respectively, of the total sum intensity of the intracellular cific mechanisms, such as cytokine/growth factor signaling, protein identified by 2-DE gels in ovoid and filamentous adhesion, etc., that are active in the embryo. The final chal- concepti. Furthermore, in the early hatched blastocysts, the lenge will be to differentiate the distinct mechanisms of ratio of the inner cell mass to extraembryonic tissue is ,0.3 conceptus development versus processes that promote physi- [86]. This disparity is amplified further as the blastocyst cal interaction with the maternal uterine compartment and grows and by gd-11, trophectoderm cells out number the their integration, which together are crucial for elongation, endoderm cells approximately five-fold (unpublished obser- implantation, placentation, and in utero survival of the con- vations). Thus, the majority of the proteins analyzed in the ceptus [3, 89–91]. present study are from the trophectoderm cells of the conceptus. The tremendously more complex proteome in relation to the mRNA transcriptome and distinct protein sub- We wish to acknowledge Lori Schreier for technical assistance cellular localization are additional factors that lessen the with animal management, Paul Graninger and Tom Caperna for relative depth of comprehensive protein analyses. Tens of advice on 2-DE and MALDI-TOF mass spectrometer sample thousands of gene products are interrogated with tran- preparations, and Larry Shade for bioinformatics assistance. This scriptomic methodologies simultaneously, whereas hun- work was supported by USDA-ARS Current Research Informa- dreds, or at most, a few thousand translated gene products tion System Project no. 1265-31000-082-00D. Mention of trade are detected by 2-DE technology. In addition, membrane name, proprietary product or vendor does not constitute a guar- proteins are underrepresented in 2-DE analysis primarily antee or warranty of the product by the U.S. Department of because they are in relatively low abundance, have alkaline Agriculture or imply its approval to the exclusion of other prod- pI’s and are not soluble in aqueous IEF media [87]. Without ucts or vendors that also may be suitable. the use of techniques to enrich for rarer proteins and that enable the efficient solubilization of the membrane, the The authors have declared no conflict of interest.

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