181 Oct-4: A POTENTIAL MARKER FOR PLURIPOTENCY IN CATTLE

Abstract

The POU (Pit-Oct-Unc) domain transcription factor Oct-4 is one of the most acknowledged markers for pluripotency in murine and primate embryonic cells. At the blastocyst stage, expression of Oct-4 has been shown to remain high in the inner cell mass (ICM) while being rapidly down regulated in the trophectoderm (TE). Furthermore, in these species, expression of Oct-4 is maintained in pluripotent derivatives of the ICM and in embryonic stem (ES) cells, but lost upon differentiation. In the bovine embryo, a marker with similar qualities has long been sought. The aim of this study was to investigate, using a commercially available antibody for immunohistochemistry (IHC), whether Oct-4 might serve this role. In vitro produced (IVP) embryos were transferred to synchronized recipients at Day 6 post insemination (p.i) and flushed at Day 12; in vivo-derived embryos were flushed at Day 14. Day 8 IVP embryos (n = 20) were fixed and processed for IHC in paraffin sections together with Day 12 (n = 18) and Day 14 (n = 3) embryos. From Day 8 IVP embryos, outgrowth colonies (OCs) were formed by intact blastocyst culture on mouse SNL feeder cells. OCs were photographed using a stereomicroscope on Days 12, 14, and 16 p.i., and were examined for Oct-4 expression by in situ IHC at Day 16 p.i. (n = 94). From isolated embryonic discs of Day 12 embryos, OCs were derived by similar culture and were either processed for IHC on paraffin sections at Days 16, 18, and 20 p.i. (n = 9) or used for establishment of ES-like cell lines. Of colonies formed, representative specimens from each of the initial 5 passages (n = 18) were examined for Oct-4 expression either in paraffin sections or in situ. In Day 8 embryos, Oct-4 expression was demonstrated in all nuclei of both ICM and TE cells except for presumptive apoptotic ones. Approximately one-fifth of the OCs presented a substantial amount of Oct-4 positive cells of putative ICM, but also of TE origin. Apparently, the formation of Oct-4 positive OCs was favored by initial attachment of the embryonic pole to the feeder cells. In Day 12 and 14 embryos, specific and exclusive Oct-4 staining of nuclei of the complete epiblast, but not the hypoblast and the TE, was revealed. All OCs derived from Day 12 embryonic discs showed specific staining for Oct-4 in nuclei of putative epiblast origin only. On subsequent culture of these isolated epiblast derivatives, loss of Oct-4 staining from colonies was observed by passage 3. This study has, for the first time, shown expression of Oct-4 to be limited to pluripotent cells of bovine Day 12 and 14 embryos. Compared with murine and primate embryos, down-regulation of Oct-4 expression in bovine TE cells appears to be delayed. Findings indicate that Oct-4 may be used as a marker for pluripotency in bovine ES-like cells, although TE derivatives may maintain Oct-4 expression when isolated from Day 8 embryos.