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BOR - Papers in Press, published online ahead of print November 10, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.034348
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Submitted July 12, 2004
Returned for revision August 25, 2004
Accepted October 25, 2004

Embryo


Ultrastructural and Immunohistochemical Characterization of the Bovine Epiblast

Morten Vejlsted *, Birthe Avery , Mette Schmidt , Torben Greve , Natalie Alexopoulos , and Poul Maddox-Hyttel

* To whom correspondence should be addressed. E-mail: mov{at}kvl.dk.

Abstract
The epiblast represents the final embryonic founder cell population with potential for giving rise to all cell types of the adult body. The pluripotency of the epiblast is lost during the process of gastrulation. In large animal species there is a lack of specific markers for pluripotency. The aim of the present study was to characterize the bovine epiblast cell population and 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 Rauber's layer. Immunohistochemically, staining for the octamer-binding transcription factor-4 (Oct-4) revealed a specific and exclusive staining of nuclei of the complete epiblast. Coexpression of vimentin and Oct-4 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 ventro-lateral sides of the "cup" appeared to be incomplete. In conclusion, the bovine epiblast includes at least two cell subpopulations and Oct-4 was shown, for the first time, to exclusively stain epiblast cells in this species.

Key words: Embryo • Developmental biology • Early development


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