Bovine trophoblastic cell differentiation on collagen substrata: formation of binucleate cells expressing placental lactogen

The differentiation of trophectoderm in ruminants is marked by the appearance of binucleate cells in cytotrophoblasts. Binucleate cells are produced by the acytokinesis of cytotrophoblasts and undergo endoreduplication. They secrete hormones such as placental lactogen, and exhibit migratory behavior to transfer their hormones into maternal circulations. In this study, we showed that a bovine trophoblastic cell line (BT-1) established from in vitro fertilized blastocysts differentiated into binucleate cells on collagen gel. BT-1 had cytotrophoblastic epithelial characteristics in that it expressed cytokeratin, E-cadherin and interferon-tau. It spontaneously formed multicellular spherical vesicles floating in the medium. We cultured these vesicles on type I collagen substrata. Most vesicles attached to the collagen substrata, and exhibited cell outgrowth and proliferation. We found that after more than 10 days, clusters of binucleate cells appeared in the cell colonies on the collagen gel, but not on the collagen film. These binucleate cells have features characteristic of those in vivo, including an increased nuclear DNA content and the expression of placental lactogen. BT-1 is a useful model with which to study trophoblast differentiation in ruminants.