105 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS Oct-4 AND NANOG IN PRE-IMPLANTATION GOAT EMBRYOS

Abstract

The objective of this study was to determine the expression patterns of the pluripotency-determining factors, Oct-4 and Nanog, in pre-implantation goat embryos. The POU octamer-binding domain transcription factor Oct-4 and the homeobox transcription factor Nanog have been shown to play key roles in the maintenance of pluripotency in the inner cell mass (ICM) of pre-implantation mouse embryos and in embryonic stem cells. As Oct-4 protein has been observed in human, monkey, bovine, and porcine pre-implantation embryos, its role in embryonic development and differentiation may be conserved across these species. The patterns of mRNA expression for Oct-4 and Nanog have not been reported for ruminant embryos. In this study, total RNA was extracted from 10 in vivo-derived goat embryos at each stage (8-cell, morula, and blastocyst) using an Absolutely RNA Nanoprep Kit (Stratagene, La Jolla, CA, USA). The first-strand cDNAs were synthesized using Superscript III (Invitrogen, Carlsbad, CA, USA) and cDNAs were amplified with PfuUltra hotstart PCR master mix (Stratagene). Oct-4 primers were designed based on bovine Oct-4 open-reading sequence, while Nanog primers were designed based on the human Nanog open-reading sequence. Expression screening by PCR was performed. Oct-4 mRNA expression was detected at the 8-cell, morula and blastocyst stages. Sequencing of the 1.1-kb PCR product with Oct-4 primers revealed 87% homology to human cDNA sequence and 96% homology to the bovine sequence. Protein localization of Oct-4 as observed by immunocytochemistry was diffuse at the morula stage, but moved to a more nuclear location at the blastocyst stage. Oct-4 protein and mRNA expression were detected in both the ICM and trophectoderm of expanded blastocysts. This pattern of protein expression is similar to that reported by others in the pig and cow. As caprine, bovine, and porcine embryos all show extensive proliferation and elongation of the trophectoderm, continued expression of Oct-4 protein in the trophectoderm may be necessary to prevent premature differentiation of the trophectoderm. Nanog mRNA was detected at the morula and blastocyst stages. Nanog mRNA was detected in the ICM but not the trophectoderm of expanded goat blastocysts, a pattern that follows the expression observed in mice. Sequencing of the 698 bp PCR product obtained by RT-PCR from goat blastocysts confirmed that the mRNA detected was Nanog. Sequence alignment (ClustalW) showed that the cDNA sequence identities were 96% between goat and human and 70% between goat and mouse. The amino acid identities were 93% between goat and human and 52% between goat and mouse. To our knowledge this is the first report of detection of Nanog in domestic animals. These results are supportive of the premise that core components involved in the control of pluripotency are analogous across vertebrate species.