Study On Molecular Markers Of Embryonic Stem Cells In Goat Embryos

Embryonic stem cells (ES cells) are derived from ICM (inner cell mass) in development of blastula, and in vitro culturing of these cells can finally maintain the characteristics of self-renewal and pluripotency on development. The transcription factors, POU class 5 homeobox 10 (OCT4/POU5F1), NANOG and sex determining region-Y-box2 (SOX2), play crucial roles in the maintenance of self-renewal and pluripotency in animal ESCs, are expressed in preimplantation mammalian development with different space (cellular locali- zation ) time(along a developmental timeline). Understanding the system of transcription control to ES cells is the foundation on understanding the process of embryonic development and creating of transgenic animals. In this study, we obtained early in vivo-derived embryos by superovulation and in vitro-derived embryos Parthenogenetic activation, Qualitative and quantitative PCR and immunocytochemical detected expression of OCT4, NANOG and SOX2 in all early developmental stages of the goat embryos. The results obtained were as follows:1. Guanzhong Gairy Goats were superovulated with a result of total 231 in vivo-derived embryos, including 49 embryos of 2-cell, 12 4-cell, 38 8~16-cell, 102 morulae, 30 blastocyst. We also got 32 mature oocytes. In the study, we obtained 13.5 embryos from per donor goat. And we obtained 242 parthenogenesis embryos by the parthenogenesis of in vitro maturation (IVM) goat oocytes and parthenogenetic activation, including 87 embryos of 2-cell, 53 4-cell, 46 8-cell, 40 morulae, 16 blastocyst, and 15 mature oocytes.2. Expression was examined by Qualitative and quantitative PCR and indirect immuno- cytochemistry in all in vivo-derived goat embryos. Oocytes and the early stage embryos revealed a variable OCT4, NANOG and SOX2 expression pattern. The mRNA of OCT4 was expressed from oocyte to morula stage and the expression level fluctuated during embryonic development. We got higher level of expression in oocyte, 4-cell and blastocyst than 2-cell, 8-cell and morula. OCT4 expressed continually concomitant with cytoplasmic localization of the protein in oocyte, 2-cell, 4-cell, 8-cell, blastomere at central embryo in compact morula stage, ICM in blastocyst, but nuclear and cytoplasmic localization in blastomere which was around embryo during compact morula stage and trophectoderm, which implied that OCT4 exerted its biological function at morula stage and was related to compaction of morula and embryonic differentiation. The expression level of NANOG mRNA rose up from 2-cell gradually, achieved peak value at morula stage and declined slightly in blastocyst. NANOG protein located in cytoplasm before 8-cell, in nuclei after 8-cell stage and was similar as the location of OCT4 in morula stage and blastocyst. The CDX2 protein was detected from the blastocyst stage and expressed in cytoplasm and nuclei of blastular trophectoderm during blastocyst stage.3. The expression of OCT4, NANOG and SOX2 in vivtro-derived embryos were similar as in vivo-derived. However, all transcription levels of mRNA were lower in vivtro than in vi- vo. Temporal and spatial expression of their protein was examined in vivtro-derived embryos. Both OCT4 and NANOG protein were located in nuclei from the 8-cell stage to the blastocyst stage, however, SOX2 was located in nuclei from the morula stage to the blastocyst stage. Additional, CDX2 protein was detected in the ICM cell and TE cell at the blastocyst stage, but not in the ICM cell in blastocyst in vivo-derived embryos.