Cultured bovine trophoblast cells differentially express genes encoding key steroid synthesis enzymes

Placental trophoblasts are an important source of endocrine, paracrine and autocrine acting hormones. The aim of the present study was to establish and evaluate a tissue culture model for bovine trophoblasts to study regulation of key genes of steroid hormone synthesis. Trophoblast cells were isolated from cotyledons by collagenase disaggregation and subsequent percoll density gradient centrifugation. The cells were seeded on collagen coated dishes and incubated for up to seven days. The cells were characterized for the presence of mesenchymal vimentin and epithelial cytokeratin filaments and for Dolichos biflorus agglutinin (DBA) binding, a marker for differentiated trophoblast giant cells. Transcripts of Hsd3b, Cyp17 and Cyp19 encoding 3beta-HSD, P450c17 and P450arom, the key enzymes of progesterone, androgen, and oestrogen biosynthesis, respectively, and of Csh1 encoding the trophoblast-specific hormone placental lactogen (PL) were measured by qPCR. Uninucleate cotyledonary epithelial cells and bi- and trinucleate trophoblast giant cells efficiently formed a dense cell layer on the collagen coated dishes within 24 h. Bi- and trinucleate cells showed DBA binding and weak or undetectable cytokeratin immunoreactivity. Vimentin-positive, fibroblast-like cells were found on top of this cell layer. Cyp19 transcripts were found in freshly dissociated but not in cultured cells. Cyp17 expression continuously increased, Hsd3b transcripts largely and rapidly increased during the first days in culture, followed by a decline after three days, whereas Csh1 decreased towards day seven. Serum free culture conditions significantly enhanced Cyp17 and Csh1 but not Hsd3b expression. The data indicate that collagen is a favourable substrate for cultured binucleate trophoblast giant cells. The cells represent an in vitro model to study the regulation of key genes of placental progesterone and androgen but not of oestrogen biosynthesis.