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Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells
Bong-Seok Song, Sang-Hee Lee, Sun-Uk Kim, Ji-Su Kim, Jung Park, Cheol-Hee Kim, Kyu-Tae Chang, Yong-Mahn Han, Kyung-Kwang Lee, Dong-Seok Lee, Deog-Bon Koo
BMC Developmental Biology , 2009, DOI: 10.1186/1471-213x-9-44
Abstract: No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB)-iSCNT, and bovine-bovine (BB)-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA) at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM), we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively.The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.The derivation of human embryonic stem cells (hESCs) from somatic cell nuclear transfer (SCNT) blastocysts represents an innovative strategy for overcoming immune rejection during transplantation. However, autologous human therapeutic cloning using human donor cells and oocytes has been continuously faced with legal and moral quandaries. Thus, monkey primary cells and bovine oocytes have been used as alternative donor and recipient cells for SCNT, respectively. In addition, interspecies SCNT (iSCNT) shows promise as a technique for examining nucleocytoplasmic interactions [1], stem cells [2], and the cloning of endangered animals whose oocytes are difficult to obtain [3,4]. The most important application of iSCNT lies in its potential to facilitate the reprogrammin
Effect of Mitochondrial Dysfunction in Nuclear Donor and Ooplasmic Recipient on Early Development of Goat-Sheep Interspecies Somatic Nuclear Transfer Embryos
Junwei Cao,Libing Ma,Hua Song,Huanmin Zhou,Yanru Zhang
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2012.4079.4085
Abstract: Mitochondria is a kind of important organelle for producing and supplying energy in eukaryotes. The interspecies somatic nuclear transfer could result in coexisting of nuclear donor and ooplasmic recipient mitochondria in reconstructed embryos. In the present study, after nuclear donor and/or ooplasmic recipient mitochondria damaged by photoirradiating rhodamine-123, examination of the goat-sheep interspecies somatic nuclear transfer embryos early development ability in vitro was performed. The result shows that sheep ooplasmic recipient mitochondria damaged could result in reconstructed embryos development ability declined in 2-cell and morulae/blastocyst stage but does not affect 2-cells stage to 8-cells stage development; goat nuclear donor cells mitochondria damaged can not affect reconstructed embryos development ability before morulae/blastocyst stage; after both nuclear donor and ooplasmic recipient mitochondria are damaged, the reconstructed embryos development pattern is same as that of only ooplasmic recipient mitochondrial damaged. The result indicated that before interspecies somatic nuclear transfer reconstructed embryo development to morulae/blastocyst stage, ooplasmic recipient mitochondrial is dominant over nuclear donor mitochondrial in affecting reconstructed embryos development ability.
Serial nuclear transfer improves the development of interspecies reconstructed giant panda (Aluropoda melanoleuca) embryos
Jinsong Li,Dayuan Chen,Zhiming Han,Ziyu Zhu,Duancheng Wen,Qingyuan Sun,Zhonghua Liu,Minkang Wang,Li Lian,Jun Du,Pengyan Wang,Hemin Zhang
Chinese Science Bulletin , 2002, DOI: 10.1360/02tb9108
Abstract: Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonquiescent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blastomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial II and serial III were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups were 19.4%, 13.5% and 10.3%, respectively, which are significantly higher than that in somatic NT group. These results indicate that the nuclei from nonquiescent somatic cells can support early development of reconstructed embryos and serial NT can improve the development rate of interspecies reconstructed embryos.
Construction of Human-bovine Interspecies Embryos and Investigation of Interspecies Embryonic Mitochondrial Source
人-牛异种克隆胚构建及其线粒体来源

Lu Yang,Dong Zhang,Yongsheng Wang,Daquan Sun,Yong Zhang,
杨鹭
,张东,王勇胜,孙达权,张涌

生物工程学报 , 2008,
Abstract: Obtaining human blastocysts is a prerequisite for cell replacement therapy using embryonic stem cells. We established an interspecies somatic cell nuclear transfer (iSCNT) technique for producing blastocysts without sacrificing human oocytes. Human foetal fibroblasts were used as donor cells injected into the enucleated bovine oocytes in nuclear transfer, whereas bovine foetal fibroblasts were used to produce intraspecies embryos. We also examined the fate of human and bovine mitochondrial DNA (mtDNA) during preimplantation development after nuclear transfer by PCR. PCR analysis for the detection of human and bovine mtDNA was done at the 2,8-morula, and blastocyst stages of the embryos. Result: 2.8% interspecies embryos developed to blastocysts after cultured in an SOF medium, while blastocyst rate of intraspecies embryos were 10.1%. Both human and bovine mtDNAs existed until the morula stage, whereas only the bovine mtDNA was found at the blastocyst stage. These results indicated that interspecies cloning without using human oocytes could generate human blastocysts. Because of the incoordination between bovine mtDNA and human nuclear gene, developmental rate of interspecies embryos was significantly lower than intraspecie. Whether the embryonic stem cell could be used for cell replacement therapy need further research.
Serial nuclear transfer improves the development of interspecies reconstructed giant panda (Aluropoda melanoleuca) embryos

LI Jinsong,

科学通报(英文版) , 2002,
Abstract: Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonquiescent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blastomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial II and serial III were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups were 19.4%, 13.5% and 10.3%, respectively, which are significantly higher than that in somatic NT group. These results indicate that the nuclei from nonquiescent somatic cells can support early development of reconstructed embryos and serial NT can improve the development rate of interspecies reconstructed embryos. These authors contributed equally to this work.
Interspecies Somatic Cell Nuclear Transfer Is Dependent on Compatible Mitochondrial DNA and Reprogramming Factors  [PDF]
Yan Jiang,Richard Kelly,Amy Peters,Helena Fulka,Adam Dickinson,Daniel A. Mitchell,Justin C. St. John
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0014805
Abstract: Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2′,3′-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.
Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos
Nelida Rodriguez-Osorio, Zhongde Wang, Poothappillai Kasinathan, Grier P Page, James M Robl, Erdogan Memili
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-190
Abstract: Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping.The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.The process of early embryonic development is determined by activation of the embryonic genome, which for bovine embryos begins as a "minor genome activation" at the 1-cell stage [1] ascending to a "major genome activation" during the 8-cell to 16-cell stage [2]. In the absence of proper genome activation, the developing embryo will die because it can no longer support its essential developmental functions [3,4]. In the case of embryos produced by somatic cell nuclear transfer (SCNT) the somatic nucleus has to be reprogrammed in order to restart and continue the developmental process. It is believed that, guided by the ooplasm, the somatic nucleus aborts its own program of somatic gene expression and re-establishes a particular
Reconstruction of human embryos derived from somatic cells
Changfu Lu,Ge Lin,Changqing Xie,Fei Gong,Hong Zhou,Yueqiu Tan,Guangxiu Lu
Chinese Science Bulletin , 2003, DOI: 10.1007/BF03184065
Abstract: Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into M II oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethy laminopurine (6-DMAP). After oocyte activation and 2PN formation, we removed the female PN. By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation, and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into the blastocyst stagein vitro.
Transcriptional Differences between Rhesus Embryonic Stem Cells Generated from In Vitro and In Vivo Derived Embryos  [PDF]
Alexandra J. Harvey, Shihong Mao, Claudia Lalancette, Stephen A. Krawetz, Carol A. Brenner
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043239
Abstract: Numerous studies have focused on the transcriptional signatures that underlie the maintenance of embryonic stem cell (ESC) pluripotency. However, it remains unclear whether ESC retain transcriptional aberrations seen in in vitro cultured embryos. Here we report the first global transcriptional profile comparison between ESC generated from either in vitro cultured or in vivo derived primate embryos by microarray analysis. Genes involved in pluripotency, oxygen regulation and the cell cycle were downregulated in rhesus ESC generated from in vitro cultured embryos (in vitro ESC). Significantly, several gene differences are similarly downregulated in preimplantation embryos cultured in vitro, which have been associated with long term developmental consequences and disease predisposition. This data indicates that prior to derivation, embryo quality may influence the molecular signature of ESC lines, and may differentially impact the physiology of cells prior to or following differentiation.
Induction of somatic embryogenesis in soybean: physicochemical factors influencing the development of somatic embryos
Bonacin, Gisele Aparecida;Di Mauro, Antonio Orlando;Oliveira, Roberto Carlos de;Perecin, Dilermando;
Genetics and Molecular Biology , 2000, DOI: 10.1590/S1415-47572000000400027
Abstract: the embryogenic capability of five soybean cultivars (renascen?a, ias-5, iac-17, br-16 and ft-cometa) was studied at different auxin concentrations (8, 10 and 12 mg/l naphthalene acetic acid, naa), at different phs (5.8 and 7.0) and at low (8-12 mem-2 s-1) and high (27-33 mem-2 s-1) light intensities. the experimental design was completely randomized with four replications. immature cotyledons 4-6 mm in length were placed in the six induction mediums evaluated and submitted to two light intensities. twenty immature cotyledons per cultivar were placed on each petri dish, which was considered to be one replication. the number of somatic embryos per treatment per replication was counted. the results showed genotype influence on somatic embryogenic capability of each cultivar, with the most embryogenic cultivars being br-16, ft-cometa and ias-5. auxin concentration and ph value also influenced somatic embryo production, with 10 mg/l naa being the best auxin concentration and 7.0 the best ph value. the interactions cultivar x auxin, auxin x ph and ph x light were significant, while other double interactions were not. all triple and quadruple interactions were significant, except cultivar x ph x light. no significant differences in somatic embryo production were observed in medium with different phs or when the petri dishes containing immature cotyledons were exposed to the two light intensities evaluated. however, a higher number of somatic embryos was produced when the medium ph was adjusted to 7.0.
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