NANOG regulates pluripotency in the early primate embryo

Abstract

OBJECTIVE: NANOG is a transcription factor critical for the formation and maintenance of the pluripotent inner cell mass (ICM) lineage and embryonic stem (ES) cells in the mouse. However, little is known about NANOG in primate. The objective of this study was to define the role of NANOG during preimplantation embryo development in the rhesus monkey (Macaca mulatta).DESIGN: Oocytes and fertilized embryos at various stages were sampled and analyzed for NANOG expression by immunocytochemistry and real-time PCR. Further, NANOG transcripts were up-regulated by microinjection of RNA into oocytes followed by fertilization.MATERIALS AND METHODS: Oocytes were retrieved following controlled ovarian stimulation. For in vitro RNA synthesis, NANOG cDNA was cloned and transcribed as a complementary (c)RNA. cRNA was microinjected directly into mature MII oocyte cytoplasm followed by fertilization.RESULTS: Maternal NANOG transcripts and protein were present in unfertilized, mature MII oocytes. After fertilization, embryonic expression of NANOG was first detected in some nuclei of the 4-cell stage embryos. At the 8-cell, morula and compact morula stage embryos strong signal was confined. In blastocysts, NANOG expression became restricted to the ICM but absent in the trophectoderm. NANOG overexpression resulted in improved blastocyst development (51% and 42% blastocyst rates in NANOG and controls groups, respectively). Further, NANOG-injected blastocysts contained significantly higher ICM numbers compared to controls (41±21 and 25±11, respectively; P<0.05).CONCLUSIONS: Our results suggest that NANOG is an important maternal factor with a critical role in formation of the pluripotent ICM lineage in primates. We also demonstrate that supplementation of NANOG prior to fertilization improves embryo development with larger ICMs. OBJECTIVE: NANOG is a transcription factor critical for the formation and maintenance of the pluripotent inner cell mass (ICM) lineage and embryonic stem (ES) cells in the mouse. However, little is known about NANOG in primate. The objective of this study was to define the role of NANOG during preimplantation embryo development in the rhesus monkey (Macaca mulatta). DESIGN: Oocytes and fertilized embryos at various stages were sampled and analyzed for NANOG expression by immunocytochemistry and real-time PCR. Further, NANOG transcripts were up-regulated by microinjection of RNA into oocytes followed by fertilization. MATERIALS AND METHODS: Oocytes were retrieved following controlled ovarian stimulation. For in vitro RNA synthesis, NANOG cDNA was cloned and transcribed as a complementary (c)RNA. cRNA was microinjected directly into mature MII oocyte cytoplasm followed by fertilization. RESULTS: Maternal NANOG transcripts and protein were present in unfertilized, mature MII oocytes. After fertilization, embryonic expression of NANOG was first detected in some nuclei of the 4-cell stage embryos. At the 8-cell, morula and compact morula stage embryos strong signal was confined. In blastocysts, NANOG expression became restricted to the ICM but absent in the trophectoderm. NANOG overexpression resulted in improved blastocyst development (51% and 42% blastocyst rates in NANOG and controls groups, respectively). Further, NANOG-injected blastocysts contained significantly higher ICM numbers compared to controls (41±21 and 25±11, respectively; P<0.05). CONCLUSIONS: Our results suggest that NANOG is an important maternal factor with a critical role in formation of the pluripotent ICM lineage in primates. We also demonstrate that supplementation of NANOG prior to fertilization improves embryo development with larger ICMs.