Secretion of Biologically Active InterferoNτ by in Vitro-Derived Bovine Trophoblastic Tissue1

Abstract

Secretion of interferon tau (IFNτ) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Ménézo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFNτ secretion by in vitro-derived trophoblastic tissue. IFNτ activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-μl drops of BRL cell-conditioned medium, mean IFNτ secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFNτ secretion by trophoblastic tissue increased to mean levels of > 10⁵ antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10³ antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFNτ was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFNτ. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFNτ.