Annual Report 2003

the cells grew sparsely. These findings suggestthat the cell population comprising aFig. 3Detection of PL in BT-1 cells by immunofluorescence.(a) Hoechst 33342 staining shows binucleate cells (arrow,magnified in the inset). Bar = 30µm(b) Immunodetection of PLcolony is morphologically and functionallyheterologous. The BT-1 cells express severaltrophoblast-specific proteins. RT-PCR analysisrevealed that the BT-1 expressed mRNAsencoding Placental lactogen (PL), Interferon- (IFN-), Prolactin-Related Protein-I (PRP-1), and Pregnancy-Associated Glycoprotein-1(PAAG-1). In the 8 tested genes (see the legendof Fig. 2), their expression profiles ofBT-1 were comparable to those of fetal membranedeveloped in vivo (Fig. 2). BoneMorphogenetic protein and transcription factorOct 4 were also expressed. The presenceof IFN- protein in culture medium was confirmedby Western blotting. A small numberof cells were shown to be PL-positive andbinucleated (Fig. 3). The present series ofstudies established a novel cell line derivedfrom bovine blastocyst, characterized itsmorphological and functional properties, andproposed a powerful model system for thestudy of trophoblast cell lineage and placentalfunction.Regeneration of a bovine endometrial modelin vitro by a unique three-dimensionalcell culture system using ascorbateKazuyoshi Hashizume, Toru Takahashi,Kanako KaneyamaDevelopmental Biology DepartmentFig. 1Process for formation of mutli-cellularspheroid using AsA-P.BES were cultured for 6 days in mediumwith (B) or without (A) AsA-P. Cells culturedwithout AsA-P detached and separatedinto single cells after EDTAsolution treatment (C). Cells culturedwith AsA-P did not disperse into singlecells after EDTA solution treatment (D).They gradually detached from the cultureplate edge after agitating withpipette (E) and finally floated as a wavycell sheet (F). Scale bars represent 500µm (A-D), 1mm (E), and 2mm (F).The endometrium is one of the most complextissues, undergoing dynamic changes asa result of tissue remodeling in response toimplantation and pregnancy processes. Theimplantation mechanism remains unclearbecause of complex interactions by variousfactors inherent in that process such ascytokines, growth factors, hormones andadhesion molecules. An in vitro model mayprovide a tool for clarifying the compleximplantation process. If multicellular spher-18 Annual Report 2003