Cloning and expression of pluripotent factors around the time of gastrulation in the porcine conceptus

Abstract

Early in embryonic development a series of events occur whereby pluripotent cells undergo differentiation to give rise to the three germ layers and extraembryonic tissues of the developing conceptus. Nanog, Sox-2, and Oct-4 genes have been identified as having key roles in maintaining pluripotency in undifferentiated human and mouse cells but recent evidence suggests they may have different roles in farm animals. We cloned the coding sequence for porcine Nanog including 452 base pairs of the Nanog promoter, and partial coding sequences of Oct-4 and Sox-2. Embryos were flushed from sows 10, 12, 15, and 17 days post insemination. RNA was isolated from whole d-10 and -12 conceptuses, d-15 embryonic disk, distal and proximal extraembryonic tissue, and d-17 embryonic disk, distal and proximal extraembryonic tissue, and allantois for real-time PCR. RNA from d-40 maternal myometrium and endometrium, fetal placenta, and liver were also used in real-time PCR. The homeodomain and c-terminal tryptophan repeats are highly conserved in porcine Nanog compared to the mouse, human and bovine. In the promoter, the highly conserved Octamer and Sox binding sequences are also present. The Nanog expression pattern was different when compared to Oct-4 and Sox-2. Day-40 tissues demonstrated the highest expression including endometrium (7 fold) fetal liver (27 fold), placenta (40 fold) and myometrium (72 fold) when compared to day 15 distal extraembryonic tissue. Oct-4 and Sox-2 expression was lowest in d-40 tissues except for fetal liver which was 20 and 71 fold, respectively, higher than endometrium. Oct-4 levels were consistent in d-10, -12, and -15 conceptuses and disk but dropped 3 fold in d-17 disk. On the other hand, Sox-2 was upregulated a 1000 fold in the d-15 disk and 2000 fold in the d-17 disk when compared to the d-12 conceptus. Nanog may have other roles in than maintenance of pluripotency including a possible role in multipotent or progenitor stem cells. Expression of all 3 markers in fetal liver suggests a more primitive cell type is present such as hematopoietic stem cells.