Transcriptome profiling of the tubular porcine conceptus identifies the differential regulation of growth and developmentally associated genes

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 filamentous conceptuses were also examined. Polarization of the embryonic disc and growth throughout the conceptus 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, respectively. 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.