Developmental expression of pluripotency determining factors in caprine embryos: Novel pattern of NANOG protein localization in the nucleolus

Abstract

Transcription factors, POU5F1/OCT4 and NANOG, whose expression is restricted to the inner cell mass (ICM) in mouse and human blastocysts, are used to characterize undifferentiated embryonic stem cells (ESC) in vitro. However, POU5F1 may not be a useful marker in domestic animals due to its expression in both ICM and trophectoderm (TE), while NANOG mRNA and protein expression have only been described fully in mice. In an effort to identify ESC markers for domestic animals, expression patterns of NANOG, POU5F1, and the cell surface markers (SSEA1, SSEA4, TRA-1-60, TRA-1-81) were examined in preimplantation goat embryos, a species that has proven to be a superior choice for the production of transgenic proteins in milk (biopharming). Our results indicate that while goat embryos express POU5F1, SSEA1, and SSEA4 proteins, their expression is not strictly restricted to the ICM. In a unique staining pattern, NANOG protein was localized to the nucleoplasm and nucleoli in ICM cells, but was localized strictly to nucleoli in TE. This pattern may reflect down-regulation of protein by sequestration/degradation utilizing a nucleolar mechanism known to operate in stem cells. Furthermore, NANOG mRNA in TE was also significantly down-regulated as compared with that in ICM. Taken together, this novel expression pattern of NANOG in goat preimplantation embryos suggests that NANOG could serve as marker of pluripotency in goats and may be useful in derivation and characterization of caprine ESC. This study is the first to characterize both NANOG mRNA and protein expression in any species other than the mouse.

Three‐dimensional localisation of NANOG, OCT4, and E‐cadherin in porcine pre‐ and peri‐implantation embryos

“…In this study, we clearly demonstrated that OCT4 protein expression is restricted to the epiblast in the hatched porcine blastocyst. This finding is contrasted by other reports on expression of OCT4 protein and mRNA in the trophectoderm at this stage of development in both porcine and caprine blastocysts (He et al, 2006;Keefer et al, 2007). This contradiction may be explained by the possible existence of two isoforms of OCT4.…”

“…Antibodies are available that recognise either the individual isoforms (N-terminal) or both isoforms (C-terminal). The antibody used in the present study recognises exclusively the human isoform of OCT4A, whereas the antibody used by Keefer et al (2007) recognises a human 265-aa isoform of OCT4, which corresponds to isoform B, and the antibody used by He et al (2006) is likely to recognise both isoforms, since it is raised against the C-terminal of OCT4, which is shared by the two isoforms. In the present study, we clearly show that OCT4 is expressed exclusively in the epiblast until the first OCT4-expressing mesoderm is formed, just prior to the appearance of the primitive streak.…”

“…It has been stated several times that the maintenance of the pluripotent cell population must be regulated by alternative mechanisms in other mammalian species, such as man, goat, cow, and pig. One fact that has sparked this opinion is controversy that OCT4, in addition to being expressed in the ICM, is also expressed in the trophectoderm of the pre-hatching blastocyst in these non-rodent species (Cauffman et al, 2006;He et al, 2006). Interestingly, some data indicate that trophectoderm expression of this transcription factor is actually also found in the mouse (Palmieri et al, 1994;Silva et al, 2009).…”

Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines

“…NANOG transcripts have been localized both in the ICM and TE of the bovine and caprine blastocyst [8,26], which is consistent with our results. Human and mouse ESC strongly express Nanog [19], in accordance with the positive immunostaining found for NANOG in our bESC-like colonies.…”

Candidate gene expression patterns in rabbit preimplantation embryos developed in vivo and in vitro

“…In mouse, the NANOG expression was observed from compact morula onwards [57], in human from pronuclear stage onwards [58], in bovine embryos from 8-cell onwards [59], in caprine embryos from 8-to 16-cell onwards [60], and in porcine from 4-cell embryo onwards [61,62]. In this study, transcripts of NANOG were detected in all embryonic stages similar to the observation in human embryos.…”