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

Abstract

Secretion of interferon tau (IFN tau) 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 IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau 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-microliters drops of BRL cell-conditioned medium, mean IFN tau 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. IFN tau secretion by trophoblastic tissue increased to mean levels of > 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.

Relationship Between IFN-t Production by Bovines Embryos Derived Ex Vivo and Completely Produced In Vitro

“…In this study, the embryos which maintained in good and excellent quality had a better production of IFN- compared with those which turned to fair quality. This can be explained by a higher number of degenerating cells; in others the present study shows that the embryo quality is associated with IFN- production and confirms earlier studies (Kubish et al 1998; Hernández-Ledezma et al; 1993; Stojkovic et al 1995 and. Finally this study leads to the conclusion that significant differences in the production of IFN- were found in relationship with embryo origin, ex vivo and completely produced in www.intechopen.com vitro.…”

“…Nowadays, after years of development, these techniques have reached a level of stability. The analysis of IFN- production by in vitro blastocysts confirms earlier studies (Kubish et al 2004(Kubish et al , 1998Hernández-Ledezma et al 1993;Stojkovic et al 1995Stojkovic et al , 1999 and strongly suggests that expression of IFN- is constitutive and partly non dependent on factors from the uterine environment. Nevertheless, it is known that IFN- expression and secretion level might be modulated in vivo by growth factors and cytokines (Ko et al 1991;Imakawa et al 1993;Martal et al 1998;Spencer and Bazer 2002;Miyazaki et al 2002;Emond et al 2004).…”

“…The induction of IFN- expression appeared genetically determined because IFN- secretion has been evidenced from hatched blastocysts completely produced in vitro (Hernández-Ledezma et al 1992Stojkovic et al 1995). Therefore, the uterine environment is not necessary for the induction of IFN- expression but it could play an essential role in the control of IFN- secretion.…”

Maintenance or regression of the corpus luteum during multiple decisive periods of bovinepregnancy

“…However, IFNT is likely to no longer be a major factor in maintaining the CL during the second month of pregnancy since IFNT secretion from the developing conceptus peaks by day 23 of pregnancy and then dramatically decreases during the next few weeks (Godkin et al, 1988;Stojkovic et al, 1995). This provokes the obvious question: What maintains the CL after day 30 of pregnancy when IFNT is no longer present?…”

Derivation, growth and applications of human embryonic stem cells

“…In addition, day 8 blastocysts possess significantly more ICM cells (, 50) than their day 6 counterparts (, 30 ICM cells; Fig. 1) which is probably due to the presence of hyaluronic acid (HA) in the second step and different growth factors such as LIF, insulin-like growth factor-II (IGF-II), and transforming growth factor b (TGFb) in BRL-medium (Stojkovic et al 1995). HA plays a very important role in early embryogenesis (Stojkovic et al 2003) and, when added to culture medium, HA increases the cell number of mouse (Gardner et al 1999, Figueiredo et al 2002 and bovine blastocysts (Stojkovic et al 2002).…”