Ultrastructural and immunohistochemical characterization of the bovine epiblast

The epiblast represents the final embryonic founder cell population with the potential for giving rise to all cell types of the adult body. The pluripotency of the epiblast is lost during the process of gastrulation. Large animal species have a lack of specific markers for pluripotency. The aim of the present study was to characterize the bovine epiblast cell population and to provide such markers. Bovine Day 12 and Day 14 embryos were processed for transmission-electron microscopy or immunohistochemistry. In Day 12 embryos, two cell populations of the epiblast were identified: one constituting a distinctive basal layer apposing the hypoblast, and one arranged inside or above the former layer, including cells apposing the Rauber layer. Immunohistochemically, staining for the octamer-binding transcription factor 4 (OCT4, also known as POU5F1), revealed a specific and exclusive staining of nuclei of the complete epiblast. Colocalization of vimentin and OCT4 was demonstrated. Only trophectodermal cells stained for alkaline phosphatase. Staining for the proliferation marker Ki-67 was localized to most nuclei throughout the epiblast. A continuous staining for zonula occludens-1 protein was found between cells of the trophectoderm and hypoblast but was not evident in the epiblast. A basement membrane, detected by staining for laminin, formed a "cup-like" structure in which the epiblast was located. The ventrolateral sides of the cup appeared to be incomplete. In conclusion, the bovine epiblast includes at least two cell subpopulations, and OCT4 was shown, to our knowledge for the first time, to be localized exclusively to epiblast cells in this species.