153 IMMUNOCYTOCHEMICAL STUDIES OF OCT-4, SOX-2, AND β-TUBULIN III EXPRESSION IN EARLY PORCINE EMBRYOS

Abstract

In the mouse, the transcription factor SOX-2 is known to have at least 2 roles: (1) it acts as a co-factor of the transcription factor OCT-4, the key regulator of pluripotency essential for the development of the inner cell mass/epiblast; and (2) it is involved in the direction of neural development. In this study, we elucidate the localization of SOX-2 in early porcine embryos in relation to that of OCT-4 and the early neuronal marker β-tubulin III. Embryos were flushed from uteri, fixed in 4% paraformaldehyde, and processed for paraffin sectioning. Sections (5 µm) were stained with anti-OCT-3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the ABC-AEC-method and counterstained with hematoxylin, or processed for double immunofluorescent staining using antibodies against SOX-2 (R&D Systems Europa, Ltd., Abington, Oxon, UK) and β-tubulin III (Sigma-Aldrich Denmark A/S, Copenhagen, Denmark) and counterstaining with Hoechst. The embryos were classified as pre-streak (n = 8), primitive streak (n = 4), neural groove (n = 5), and somite (n = 4) stage (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). At the early pre-streak stage, SOX-2 and OCT-4 staining was found in the nuclei and a weak β-tubulin III staining in the cytoplasm of all epiblast cells. At the late pre-streak and the primitive streak stage, SOX-2 staining became polarized to the nuclei in the anterior epiblast region, whereas OCT-4 staining was found in all nuclei of the epiblast and of the forming meso- and endoderm. The β-tubulin III staining was restricted to the epiblast and showed no anterior-posterior polarization. At the primitive streak, when cells were involuting to form the meso- and endoderm, SOX-2 staining of nuclei was absent. At the neural groove stage, the SOX-2 and β-tubulin III staining was localized to nuclei and cytoplasm, respectively, of the same cells and observed in the neural plate and groove. A polarization in SOX-2 staining was observed in an anterior-posterior direction. At the somite stage, the SOX-2 and β-tubulin III staining was again localized to the same cells and observed in the neuropores and neural tube. The SOX-2 staining of the neural tube was polarized in a dorso-ventral direction. At the neural groove and somite stage, the OCT-4 staining gradually disappeared from the epiblast, mesoderm, and endoderm except from scattered cells, presumably primordial germ cells, localized in the endoderm. Our results suggest that also in the porcine embryo SOX-2 plays a dual role, being involved in regulation of both pluripotency and neural development.