Expression of Pluripotency-Related Genes during Bovine Inner Cell Mass Explant Culture

Abstract

Abstract

Recent findings identifying the transcription factors involved in the regulation of pluripotency and self-renewal in embryonic stem cells (ESC) may provide keys that enable the derivation of ESC in domestic species. In this study we monitored the expression of pluripotency-related genes in bovine inner cell mass (ICM) explants during the critical first steps in establishment of primary cultures. The expression of NANOG and POU5F1 transcripts and proteins in intact, in vitro produced (IVP) blastocysts was confirmed by quantitative RT-PCR and fluorescent immunocytochemistry. NANOG was localized to the nucleoplasm as well as the nucleoli in the ICM, whereas it appeared to be restricted to the nucleoli in trophectoderm cells. POU5F1 was localized in the nuclei of ICM and trophectoderm cells. ICM explants were analyzed by quantitative PCR and semiquantitative RT-PCR. The three major pluripotency-related transcription factors, NANOG, POU5F1, and SOX2, were expressed initially in the ICM explants, but were downregulated with subsequent culture. Markers of differentiation (BMP4, HNF4, NCAM, CDX2) and genes involved in LIF, BMP, and WNT signaling pathways were also expressed. ICM explants were cultured in the presence of various concentrations of cytokines belonging to the TGF-β superfamily. Noggin, a cytokine inhibiting the BMP4 pathway, successfully upregulated the relative expression of NANOG mRNA in the ICM explants with respect to controls.