Isolation, Characterization and Immunocytochemical Localization of Bovine Trophoblast Protein-11

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.

Section: Discussionsupporting

confidence: 91%

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

Stojković

Wolf

Büttner

et al. 1995

“…Total RNA was also isolated from an in vivo 17-day bovine conceptus as a positive control for bIFN-expression [18] by the method of Chomczynski and Sacchi [34]. RNA transcripts were reverse transcribed to first-strand cDNA using the GeneAmp RNA PCR kit (Perkin Elmer, Foster City, CA) essentially as specified by the manufacturer's instructions.…”

Section: Reverse Transcription-polymerase Chain Reaction (Rt-pcr) Anamentioning

confidence: 99%

“…For example, a specific interferon, interferon tau, is produced specifically by the trophectoderm cells of the ruminant conceptus during the establishment of pregnancy, and it was demonstrated to be of importance in maintenance of the corpus luteum [15][16][17][18]. Some of the biology of bovine interferon tau (bIFN-) is difficult to assess without a culture system for bovine trophectoderm maintenance and propagation [19,20].…”

Section: Introductionmentioning

confidence: 99%

Bovine Blastocyst-Derived Trophectoderm and Endoderm Cell Cultures: Interferon Tau and Transferrin Expression as Respective In Vitro Markers

Talbot

Caperna

Edwards

et al. 2000

127

105

Continuous cultures of bovine trophectoderm (CT-1 and CT-5) and bovine endoderm (CE-1 and CE-2) were initiated and maintained on STO feeder cells. CT-1 and CT-5 were derived from the culture of intact, 10- to 11-day in vitro-produced blastocysts. CE-1 and CE-2 were derived from the culture of immunodissected inner cell masses of 7- to 8-day in vitro-produced blastocysts. The cultures were routinely passaged by physical dissociation. Although morphologically distinct, the trophectoderm and endoderm both grew as cell sheets of polarized epithelium (dome formations) composed of approximately cuboidal cells. Both cell types, particularly the endoderm, grew on top of the feeder cells for the most part. Trophectoderm cultures grew faster, relative to endoderm, in large, rapidly extending colonies of initially flat cells with little or no visible lipid. The endoderm, in contrast, grew more slowly as tightly knit colonies with numerous lipid vacuoles in the cells at the colony centers. Ultrastructure analysis revealed that both cell types were connected by desmosomes and tight junctional areas, although these were more extensive in the trophectoderm. Endoderm was particularly rich in rough endoplasmic reticulum and Golgi apparatus indicative of cells engaged in high protein production and secretion. Interferon tau expression was specific to trophectoderm cultures, as demonstrated by reverse transcription-polymerase chain reaction, Western blot, and antiviral activity; and this property may act as a marker for this cell type. Serum protein production specific to endoderm cultures was demonstrated by Western blot; this attribute may be a useful marker for this cell type. This simple coculture method for the in vitro propagation of bovine trophectoderm and endoderm provides a system for assessing their biology in vitro.

“…By Day 22, when placenta formation is initiated, IFNT is no longer detected [5]. Bovine and caprine conceptuses exhibit a similar cell-specific and temporal pattern of IFNT expression [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Induction of Endogenous Interferon Tau Gene Transcription by CDX2 and High Acetylation in Bovine Nontrophoblast Cells1

Sakurai

Sakamoto

Muroi

et al. 2009

Self Cite

Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.