122 UNIQUE EXPRESSION PATTERNS OF DIFFERENTIATION, GROWTH, AND CELL STRUCTURE FACTORS IN THE ELONGATING PORCINE CONCEPTUS

Abstract

Elongation of the trophectoderm and gastrulation of the embryonic disc, observed during gestational Days 11 (D11) through 12 (D12), denote a critical period of porcine conceptus development. Serial analysis of gene expression identified genes involved in cellular differentiation/structure (cytokeratin-8 and -18) and growth/cell migration/mesoderm-epithelial interaction (stratifin and midkine), which could potentially be regulated by steroids such as estrogen. Characterization of these factors is lacking in porcine conceptuses, therefore, the current study investigated mRNA expression of these factors in elongating conceptuses and primordial tissues as well as protein expression and cellular localization to better define their biological significance. Conceptuses examined were of ovoid (D11; 6-10 mm), tubular (D11; 11-50 mm), or filamentous (D12; >100 mm) morphology. Cells of the conceptus were highly proliferative at all stages and the embryonic disc of the ovoid conceptus was already polarized as indicated by the protein expression of Ki67 and brachyury. Real-time PCR was utilized to determine the transcript expression profiles. Differential expression of cytokeratin-18 and midkine were not apparent; however, cytokeratin-8 was clearly down-regulated in filamentous compared to ovoid conceptuses. In contrast, stratifin mRNA levels were greatest in tubular conceptuses of 42-50 mm size. Transcripts for cytokeratin-8 and -18, stratifin, and midkine were detected in both cell types (endoderm and trophoblast) of the trophoectoderm. Western blotting and/or immunohistochemistry were utilized to examine protein expression and cellular localization. The embryonic disc of ovoid conceptuses was almost devoid of cytokeratin-18 protein, however, its distribution was uniform throughout the trophectoderm at all stages of elongation. Stratifin and midkine proteins demonstrated more unique expression patterns within the conceptus. Distinct cell populations of the embryonic disc and the trophoectoderm contained stratifin; cellular localization was predominantly cytoplasmic but occasional nuclear translocation was evident. Furthermore, total protein levels of stratifin were not different among ovoid, tubular, and filamentous conceptuses, but proteolysis of the protein was apparent at the filamentous stage. Midkine protein expression was prominent in the embryonic disc of ovoid conceptuses. In tubular conceptuses, midkine was associated with cells that appeared to be migrating away from embryonic disc as well as some concentrating in the tips of the trophectoderm. Our findings suggested that cytokeratin-8 and -18 are associated primarily with the trophectoderm, as seen in other species. Furthermore, the distribution and localization of stratifin and midkine proteins could reflect attributed functions of these factors, minimal anti-proliferative activity in the rapidly growing conceptuses and cell migration important for gastrulation/trophectoderm elongation, respectively.