|Blomberg, Le Ann|
|Guillomot, Michael - INST NATL RECHERCHE|
|Van Tassell, Curtis|
Submitted to: Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 12, 2006
Publication Date: December 1, 2006
Citation: Blomberg, L., Garrett, W.M., Guillomot, M., Miles, J.R., Sonstegard, T.S., Van Tassell, C.P., Zuelke, K.A. 2006. Transcriptome profiling of the tubular porcine conceptus identifies the differential regulation of growth and developmentally associated genes. Molecular Reproduction and Development. 73(12):1491-1502. Interpretive Summary: Elongation of the pig conceptus, gestational day 11 through day 12, is a critical peri-implantation period that is realized by important morphological changes in the trophectoderm and embryonic disc. The foremost aim of this study was to elucidate the transcriptome profile of the intermediate tubular porcine conceptus via Serial Analysis of Gene Expression (SAGE) so as to identify molecular pathways whose regulation enable elongation. In addition, we examined the utility of small amplified RNA-SAGE (SAR-SAGE) for transcriptome analysis of porcine-derived tissues. We identified and performed further analysis of genes, potentially regulated by estrogen, that are involved in development and growth (cytokeratins 8 and 18, stratifin, midkine, and glycolytic enymes). The majority of glycolytic enzyme transcripts were constitutively expressed or down-regulated at the filamentous stage. Likewise, cytokeratin mRNAs were decreased in filamentous conceptuses, however, stratifin and midkine were increased in tubular conceptuses. Analysis of protein revealed distinct expression patterns for cytokeratin 18, stratifin, and midkine. Our results suggest that if glycolytic transcript levels reflect their protein level, increased glucose-dependent energy production is not required during elongation. On the other hand, the differential regulation of stratifin and midkine protein as well as its spatial localization, indicate a potential role for these gene products in the development of the conceptus during elongation. Furthermore, SAR-SAGE proved to be a more reliable amplification method than PCR, a finding that will highly impact future analyses of earlier stages of conceptus development with small sample sizes.
Technical Abstract: Gastrulation and trophectoderm elongation of the porcine conceptus coincide with peak conceptus estrogen secretion from gestational day 11 through 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 states of ovoid, tubular, and filamentous conceptuses were also examined. Embryonic disc polarization and growth were examined in the conceptus. 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 more reliable amplification procedure than PCR. 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, respectively. Annotation of the tags, identified transcripts involved in development and growth (cytokeratins 8 and 18, stratifin, midkine, and glycolytic enymes), that are potentially regulated by estrogen, and they were analyzed by real-time PCR. The majority of glycolytic enzyme transcripts were constitutively expressed or down-regulated at the filamentous stage. Likewise, cytokeratin mRNAs were decreased in filamentous conceptuses, whereas stratifin and midkine were increased in tubular conceptuses. Analysis of protein revealed distinct expression patterns for cytokeratin 18, stratifin, and midkine. This is the first report of a potential role for stratifin and midkine in porcine conceptus development.