147 IDENTIFICATION OF PRIMORDIAL GERM CELLS IN PORCINE EMBRYOS FROM THE PRIMITIVE STREAK STAGE

Abstract

In embryonic stem cell research, Oct-4 is one of the most widely used markers of pluripotency. Moreover, at least in the mouse, this marker is restricted to primordial germ cells (PGCs) after gastrulation. Vimentin is often used as a marker of mesoderm/mesenchyme in embryonic tissues and appears to localize to the same embryonic cells as Oct-4, at least in the bovine epiblast. The expression of neither of these markers has been completely addressed in the pig. Therefore, the purpose of the present study was to examine the expression of Oct-4 and vimentin in the porcine epiblast during differentiation and establishment of the three germ layers, i.e. the process of gastrulation. A total of 410 porcine embryos were collected at 8 to 17 days post-insemination from 29 sows of the Danish Landrace breed. Embryos were categorized based on stereo-microscopic observations into the following stages: pre-streak stages 1 and 2, primitive streak stage, neural groove stage, and somite stage. Specimens were fixed at all stages, dehydrated and embedded in paraffin wax. Selected embryos at each stage (n = 28) were completely cut into serial sections for immunohistochemical evaluation of Oct-4 and vimentin. Pre-streak stage 1 embryos were defined by lack of polarization of the embryonic disk, whereas in pre-streak stage 2 embryos a crescent shaped thickening was seen at the posterior pole of the disk. This thickening, marking the first morphological anterior-posterior polarization of the embryo proper, was shown to be a site of incipient ingression of cells from the epiblast. Immunohistochemical analyses localized Oct-4 to nuclei and vimentin to cytoplasm of both founding and ingressing epiblast cells. During formation of mesoderm and endoderm, at the primitive streak stage, solitary Oct-4 positive cells, i.e. potential PGCs, were seen scattered in the endoderm. Cells of the epiblast displayed positive labeling for Oct-4 until specification for the ectoderm cell lineage at the subsequent neural groove stage. In mesoderm, Oct-4 likewise disappeared by the time of formation of the first somites, defining the following somite stage. Thus, at this stage the only cells labeled for Oct-4, i.e. potential PGCs, were seen solitarily scattered in the endoderm. By the 15-somite stage, such cells were no longer visible in the endoderm but were seen located in the mesoderm, spreading from the stalk of the yolk sac and allantois and extending through the mid- and hindgut areas into the incipient genital ridge. Vimentin localized to the mesenchyme and most other derivatives of neural crest and mesodermal origin. In conclusion, based on Oct-4 labeling and distribution pattern, we strongly believe that we have identified the porcine PGCs from the primitive streak stage.