Dynamic Expression of Vimentin in Bovine Blastocysts in Extended In Vitro Culture

Contents In mammalian blastocysts, the inner cell mass (ICM) differentiates into the epiblast and the hypoblast. In vitro culture of bovine pre-implantation embryos is generally carried out for a limited duration at most up to the blastocyst formation, therefore, it is unclear whether and when the differentiation of ICM occurs in vitro. In bovine species, epiblast formation is characterized by the expression of the intermediate filament protein vimentin. In the present study, the spatial and temporal expression of vimentin in bovine blastocysts maintained under extended in vitro culture (EIVC) was investigated. Bovine blastocysts produced by in vitro maturation, fertilization, and culture were further cultured in vitro in modified synthetic oviduct fluid up to day 12 (fertilization = day 0). Early, expanded, or hatched blastocysts on day 8 and hatched blastocysts on days 9 and 12 were individually subjected to indirect immunofluorescence analysis with an anti-vimentin monoclonal antibody and to semi-quantitative reverse transcription-polymerase chain reaction analysis to examine the vimentin expression. Most of the blastocysts (25/26) did not exhibit any significant immunostaining for the vimentin protein on day 8. On the other hand, the vimentin protein was observed in 59.1% (13/22) and 86.4% (19/22) of the hatched blastocysts on days 9 and 12, respectively. Vimentin was detected as filaments localized within a portion of the ICM, and its expression levels varied among the embryos. Vimentin transcript could not be detected in 50% (3/6) of the blastocysts on day 8, whereas it was detected in all the blastocysts on days 9 and 12. Examination of the day 8 blastocysts revealed that compared with the slow developers, the fast developers (blastocysts which had already expanded by 168 h post-insemination) had significantly higher levels of vimentin transcripts. These results indicate that the ICM differentiates dynamically in bovine blastocysts maintained under EIVC, as reflected by the expression of vimentin and that the higher vimentin expression may reflect the higher developmental competence of embryos.