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 pro- teins 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 re- sponse. 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 tran- scriptomic 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 interac- tions 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