MOLECULAR REPRODUCTION AND DEVELOPMENT 73:1491–1502 (2006) Transcriptome Profiling of the Tubular Porcine Conceptus Identifies the Differential Regulation of Growth and Developmentally Associated Genes LE ANN BLOMBERG, 1 * WESLEY M. GARRETT, 1 MICHEL GUILLOMOT, 3 JEREMY R. MILES, 1 TAD S. SONSTEGARD, 2 CURTIS P. VAN TASSELL, 2 AND KURT A. ZUELKE 1 1 Biotechnology and Germplasm Laboratory, USDA Agricultural Research Service, Beltsville, MD 2 Bovine Functional Genomics Laboratory, USDA Agricultural Research Service, Beltsville, MD 3 JMR Biologie du De´veloppement et Reproduction, Institut National de la Recherche Agronomique, Jouy-en-Josas, France ABSTRACT Gastrulation and trophectoderm elongation of the porcine conceptus coincide with peak conceptus estrogen secretion from gestational day 11 to day 12. The current study aim was to identify genes required for elongation by defining the transcriptome profile of this dynamic tubular stage. The gastrulation and proliferative status of ovoid, tubular, and filamen- tous conceptuses were also examined. Polarization of the embryonic disc and growth throughout the con- ceptus were evident. An unamplified and two distinct amplified serial analysis of gene expression (SAGE) libraries were generated from tubular conceptus mRNA. Comparing the three libraries at 12,000 tags/ library indicated small-amplified RNA-SAGE was a reliable amplification procedure. The unamplified library was increased to 42,415 tags and statistical analyses of tag frequencies with previously generated ovoid and filamentous libraries revealed the differential expression (P < 0.05) of 483 and 364 tags between ovoid:tubular or tubular:filamentous libraries, respec- tively. Annotated transcripts known to be involved in development and also potentially regulated by estrogen (cytokeratins 8 and 18, stratifin, midkine, and glycolytic enzymes) were further analyzed by real-time PCR. The majority of glycolytic enzyme transcripts were constitutively expressed or downregulated at the filamentous stage. Likewise, cytokeratin mRNAs were less abundant in filamentous conceptuses, whereas stratifin and midkine were more abundant in tubular conceptuses. Analysis of protein revealed distinct expression patterns for cytokeratin 18, stratifin, and midkine. The function(s) of these factors and potential modulation by estrogen clearly needs to be elucidated to understand their physiological role in normal conceptus development. Mol. Reprod. Dev. 73: 1491–1502, 2006. ß 2006 Wiley-Liss, Inc. Key Words: peri-implantation; elongation; gastrula- tion; transcriptome; tubular conceptus INTRODUCTION Elongation of the pig conceptus during day 11 (D11) to day 12 (D12) is a critical peri-implantation period where the morphological remodeling of the trophectoderm from an ovoid (10 mm) to dynamic intermediate tubular states, and, ultimately, a long thin filament (>150 mm) occurs. Conceptus loss during this elonga- tion phase can approach 18% (Bennett and Leymaster, 1989). The expansion phase of the blastocyst’s trophec- toderm tissue during elongation is a very rapid process (30–40 mm/hr; Geisert et al., 1982; Stroband and Van der Lende, 1990; Geisert and Malayer, 2001). Cellular differentiation and remodeling rather than hyperplasia are thought to be responsible for the initial morpholo- gical transition in the pig blastocyst, and this differs from other ungulates where hyperplasia is a major component (Geisert et al., 1982; Pusateri et al., 1990; Geisert and Malayer, 2001). Asynchrony of trophecto- derm elongation among porcine conceptuses is evident, and precocious progression through this period is associated with conceptus competency (Bazer et al., 1993). Beyond D12 the filamentous conceptus continues to increase in length to 100 cm by day 16 (D16). Divergent from humans and mice, gastrulation in porcine, ovine, and bovine conceptuses is not dependent on implantation and commences around the time of elongation (Hue et al., 2001; Flechon et al., 2004; Guillomot et al., 2004). Early stages of ED polarization in ungulates is observed by the aggregation of meso- derm-like cells towards the posterior pole. In the pig, cow, and sheep conceptuses, primordial mesoderm cells intercalate in between the embryonic disc (ED; epiblast) and endoderm (hypoblast), migrate towards the extra- embryonic regions, and split to form the yolk sac membrane by fusion with the primitive endoderm (Stroband and Van der Lende, 1990; Maddox-Hyttel ß 2006 WILEY-LISS, INC. This article contains Supplementary Material available at http:// www.interscience.wiley.com/jpages/1040-452x/suppmat. Grant sponsor: USDA ARS CRIS; Grant number: 1265-31000-082. *Correspondence to: Le Ann Blomberg, Biotechnology and Germplasm Laboratory, USDA Agricultural Research Service, Animal and Natural Resources Institute, Bldg 200, Rm 101A, BARC-East, Beltsville, MD 20705. E-mail: lblomberg@anri.barc.usda.gov Received 30 December 2005; Accepted 12 February 2006 Published online 10 August 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20503