The placental expression of the porcine pregnancy-associated glycoprotein (pPAG) gene family examined in situ and in vitro

Abstract

The objective of the study was to define the expression of the porcine pregnancy-associated glycoprotein (pPAG) gene family, including pPAG1, pPAG2, pPAG3, pPAG4 and pPAG6 that belong to the aspartic proteinase family. Porcine pPAG2, PAG4 and pPAG6 are members of a subfamily (pPAG2-like), which all have highly conserved sequences to pepsins, within two catalytic domains, suggesting enzymatic activity of these molecules. In contrast, pPAG1 and pPAG3 have catalytic sites with critical amino acid substitutions that likely render these molecules enzymatically inactive. The expression of pPAG mRNA was examined by using in situ hybridisation (ISH) in placental tissues or cultured cells and by ribonuclease protection assay (RPA). The pPAG protein family, secreted in vitro during long-term cultures, was examined using Western blotting. The trophoblastic pPAG mRNA expression starts around implantation and is continued in chorionic epithelium (trophectoderm) throughout pregnancy. ISH performed on porcine placental sections with pPAG antisense cDNA probes revealed an expression of pPAG transcripts, locally restricted only to trophectoderm. The pPAG2-like mRNA expression occurred in different trophectoderm cells. Some trophoblast cells were bigger than others and were involved in local rearrangements of maternal epithelium layer, especially in developing placental folds. A high similarity of dominating pPAG2-like transcript expression was confirmed by RPA analysis. Cultures of trophoblast cells revealed their differentiation to multinucleated forms that were not observed in situ. This confirms a strong inhibitory effect of the maternal microenvironment of uterine lumen on mononuclear trophoblast within porcine placental units that was not present during the development of multinucleated trophoblast cells in vitro. Long-term cultures of chorionic explants revealed a very efficient system of pPAG protein production in vitro. Western blotting of secretory pPAG proteins indicated similar immunologic epitope(s) of these molecules and pregnancy-stage dependent profile of chorionic secretion. Thus, some of the subpopulation(s) of porcine trophoblast cells expressing pPAG2-like transcripts and their secretory products can play an important role(s) in the mechanism(s) of the confrontation between trophoblast/trophectoderm cells and maternal endometrial epithelium during implantation, placenta formation and successful pregnancy maintenance in the pig.