The Transcriptome Research Of The Tibetan Antelope-Bovine And The Przewalski’s Gazelle-Bovine Interspecies Cloned Embryos

The Tibetan antelope (Pantholopshodgsonii) and the Przewalski’s gazelle (Procapraprzewalskii) were endangered species on the Tibetan Plateau. They belong to Antelope Bovidae, Artiodactyla, which is national class I protected animals in china. Interspecies somatic cell nuclear transfer (iSCNT) has been regarded as a potential alternative for rescuing highly endangered species and could be used as a model for studying nuclear-cytoplasmic interactions. However, iSCNT embryos often failed to produce viable offspring. The reasons may be attributed to the mechanisms associated with embryos development. In this study, the terminal differentiation cells of the Tibetan antelope and Przewalski’s Gazelle used as donor cells and the bovine oocytes as recipient cytoplasm. The study found that in the interspecies cloned embryos(ZBNT and PBNT) has low blastocyst rate(less than 1.5%), embryos development blocked in the period of 8-16 cells. At first, we did the analysis of the transcription group from the arrest period of embryonic development, and tried to find out the reason of the developmental block of the cloned embryos. At the same time, the somatic cell reprogramming technology-induced pluripotent stem cells technology were be used to induce the Tibetan antelope somatic cells in vitro. The induced cells (iPS-ZLY cells) were then used as donors to transfer to the enucleated bovine oocytes.The purpose of this study was founded another way to improve the development efficiency of the inter-species cloned embryos.1. The Przewalski’s gazelle fibroblast nuclear donor cells transferred into enucleated bovine oocytes (PBNT) was investigated. The percentage of embryos that developed to morula/blastocyst stages was extremely low even with the use of various treatments that included different SCNT protocols and the treatment of embryos with small molecules. Transcript microarray analyses of the cloned embryos showed that the up-regualtion reprogramming-associated genes in BBNT embryos were significantly higher than observed in PBNT embryos(1527:643). 139 transcripts related to various transcription regulation factors (TF) were unsuccessfully activated in the iSCNT embryos. Maternal degradation profiles showed that 1,515 genes were uniquely down-regulated in the BBNT (bovine-bovine NT) embryos, while 343 genes were down-regulated in the PBNT embryos. The incompatibilities between mitochondrial DNA (mtDNA) and nuclear DNA revealed that the TOM/TIM complex associated genes in BBNT embryos had the highest expression level, while the PBNT embryos exhibited a much lower expression rate. Conclusions:Improper degradation of maternal transcripts, down-regulation of transcription regulation factors and abnormal expression of genes associated with mitochondrial function in PBNT embryos likely contributed to incomplete reprogramming of the donor cell nuclei and therefore lead to the developmental failure of the cloned embryos.2. On the basis of the above research, we also redesigned and reintroducted the Tibetan antelope-bovine interspecies cloned embryos ZBNT, tried to identify universals in interspecies cloned embryos(ZBNT and PBNT)development, further revealing the reasons for the development failure of the cloned embryos. We compared comprehensive transcriptome microarray analysis of different group(ZBNT, PBNT, BBNT). Analysis results showed that:after compared with the intraspeceis cloned embryos(BBNT), there were 411 genes were down regulated genes in the interspecies cloned embryos(ZBNT, PBNT). GO analysis of these genes has focused on the functional areas of the cell membrane system, such as:nuclear lumen, intracellular organelle lumen,membrane-enclosed lumen, organelle lumen, et al. Also in ribosomal regions, including:complex biogenesis ribosome, biogenesis ribonucleoprotein and other functional areas. We analyzed TOMM (translocase of outer mitochondrial membrane)/TIMM (translocase of inner mitochondrial membrane) complex gene families, it was found that the whole down regulated expression in interspecies cloned embryos which was consistent with the previous conclusions.In interspecies cloned embyos, transcription co-activator family members mediator complex were down regulated expression, which was very obvious, the mediator family was an important structural protein transcription initiation. The ribosome biogenesis gene family, including:ribosomal protein large subunit (RPL family) and ribosomal protein small subunit (RPS family), in interspecies cloned embryos when the expression was generally lower than in intra-species cloned embryos. Similarly, when compared with BBNT, with upregulation of the expression of 729 genes in interspecies cloned embryos. Through GO analysis, the main function of these genes in the nucleotide binding, purine nucleotide binding, ribonucleotide binding, purine ribonucleotide binding and other related pathways. On the pathway of HSP (Heat Shock Protein) family gene expression in interspecies cloned embyos was significantly higher than in intra-species cloned embryos. In summary, the ZBNT and PBNT cloned embryos, abnormal expression were exsited not only in the expression of mitochondrial related gene abnormalities, but also in the transcription initiation and cytoplasmic ribosomal and mitochondrial ribosomal associated gene. The abnormal expression of these genes, lead to low efficiency development of cloned embryos, an important failure cause of the reprogramming process. In addition, in ZBNT and PBNT cloned embryos, HSP (Heat Shock Protein) family gene expression were significantly up-regulated, including HSP70, HSP90 important subunit, there would exist certain stress mechanism in the interspecies cloned embryos development.3. In this study, the Tibetan antelope fibroblasts were treated by the four classic mouse(Mus musculus) transcription factors Oct-4 (POU Class 5 Homeobox 1, POU5F1), Sox2 (Sex Determining Region Y-Box 2), Klf4 (Kruppel-Like Factor 4) and c-Myc (V-Myc Avian Myelocytomatosis Viral Oncogene Homolog) to induce the cell reprogramming. The pluripotent gene expression, cell growth curve and cell cycle of the induced cells were detected in this study. The induced cells (iPS-ZLY cells) were then used as donors to transfer to the enucleated bovine oocytes. The results showed that the induced Tibetan antelope fibroblast cells (iPS-ZLY) had numerically higher cell mass in the G2-M stage than the Tibetan antelope fibroblasts(ZLY)(21.5%vs16.7%).Cell growth curve indicated that iPS-ZLY cells proliferated faster than the ZLY cells, iPS-ZLY cells gone into the growth of the plateau, in 4 days, while the ZLY cells gone into a plateau in 6 days. The karyotypes of both iPS-ZLY and ZLY cells were similar (68.3%vs69.6%). The pluripotent gene Oct-4 expressed only in the iPS-ZLY. When transfer of the iPS-ZLY and ZLY cells, irrespectively to enucleated bovine oocytes. the iPS-ZLY cells resulted in 2.4% cloned blastocyst development, while the ZLY cells yielded 0.95% cloned blasatocysts. These results suggested treatment of Tibetan antelope fibroblast cells with transcriptional factors enhance cell proliferation, induce pluripotent gene expression and improve interspecies cloned embryo development. To our knowledge, this is the first report that using the four mouse transcription factors to induce the Tibetan antelope somatic cells, and then to evaluate the potential development of the SCNT reconstructed embryos resulted from the induced cells. This experiment provides an alternative method to increase the reprogramming of endangered animal cells and probably are beneficial for endangered animal protection.