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Sample records for hybrid embryo cells

  1. Hybrids of sugar pine by embryo culture

    Science.gov (United States)

    E. C. Stone; J. W. Duffield

    1950-01-01

    A modified embryo culture technique was used to facilitate germination of seed obtained after pollinating sugar pine with pollen from blister rust- resistant Armand and Korean pines. Resulting seedlings appear to be hybrids.

  2. Cell adhesion in embryo morphogenesis.

    Science.gov (United States)

    Barone, Vanessa; Heisenberg, Carl-Philipp

    2012-02-01

    Visualizing and analyzing shape changes at various scales, ranging from single molecules to whole organisms, are essential for understanding complex morphogenetic processes, such as early embryonic development. Embryo morphogenesis relies on the interplay between different tissues, the properties of which are again determined by the interaction between their constituent cells. Cell interactions, on the other hand, are controlled by various molecules, such as signaling and adhesion molecules, which in order to exert their functions need to be spatiotemporally organized within and between the interacting cells. In this review, we will focus on the role of cell adhesion functioning at different scales to organize cell, tissue and embryo morphogenesis. We will specifically ask how the subcellular distribution of adhesion molecules controls the formation of cell-cell contacts, how cell-cell contacts determine tissue shape, and how tissue interactions regulate embryo morphogenesis.

  3. Cytogenetic characterization and fluorescence in situ hybridization of (GATA)10 repeats on established primary cell cultures from Indian water snake (Natrix piscator) and Indian mugger (Crocodylus palustris) embryos.

    Science.gov (United States)

    Rao, L; Turlapati, R; Patel, M; Panda, B; Tosh, D; Mangalipalli, S; Tiwari, A; Orunganti, V P; Rose, D; Anand, A; Kulashekaran, M K; Priya, S R; Mishra, R K; Majumdar, K; Aggarwal, R K; Singh, L

    2009-01-01

    Sex determination among reptiles has continued to draw the attention of geneticists and the mechanisms involved have been extensively studied and documented in the past 3 decades. The setting up of primary cell lines of reptilian tissues is an important tool in the present study which is a unique aspect not applied in earlier studies. Establishing the cell lines from various species of reptiles would help in our understanding of the mechanisms of evolution and differentiation of sex chromosomes. Therefore, in the present study, we have established for the first time primary cell cultures from Indian water snake (Natrix piscator) and Indian mugger (Crocodylus palustris) embryos. In the preliminary growth stage, 2 types of cells, fibroblast- and epithelial-like, were found to be attached and proliferating in vitro. These fibroblast-like cell cultures were later overtaken by epithelial cells. The cell lines were grown in minimal essential medium supplemented with fetal bovine serum and subcultured for a period of 8-10 months. The morphology of cell types was kept under constant observation microscopically. Interestingly, at a subsequent passage of the cells sporadically scattered neuronal-like and beating cells were observed. The suitable temperature for growth of these cell cultures was 28-30 degrees C. Chromosome analysis was performed from the actively proliferating cells, which revealed 5 pairs of macrochromosomes and 15 pairs of microchromosomes in Natrix piscator, and 15 pairs of only macrochromosomes in Crocodylus palustris. (GATA)(n) repeats are well known to be associated with sex chromosomes. Fluorescence in situ hybridization performed with (GATA)(10) repeats delineated the W chromosome in the cells of Natrix piscator which has so far not been reported. This cell culture method has presently only been applied to water snakes and crocodile embryos in the current study, but it will be employed in other reptilian species and could go a long way to being a

  4. Embryo development and corresponding factors affecting in vitro germination of Cymbidium faberi × C. sinense hybrid seeds

    OpenAIRE

    Li Fengtong; Bao Jianzhong; Sun Ye; Liu Chungui; Ma Hui; Zhang Tian; Chen Xiulan

    2016-01-01

    A better understanding of embryo development would provide insights into seed quality and subsequent germination events in the interspecific hybridization of Cymbidium faberi ‘Jiepeimei’ × C. sinense ‘Qijianheimo’. At the mature stage, 26.1% of the ovules were abnormal. Most of the hybrid embryos could develop normally. Abortions mainly occurred at the zygote (9.5%) and 2-4-celled embryo (15.1%) stages. No germination was observed at 90 and 105 days after p...

  5. Comparison of tetraploid blastocyst microinjection of outbred Crl:CD1(ICR), hybrid B6D2F1/Tac, and inbred C57BL/6NTac embryos for generation of mice derived from embryonic stem cells.

    Science.gov (United States)

    Kirchain, Sharron M; Hayward, Alison M; Mkandawire, John M; Qi, Peimin; Burds, Aurora A

    2008-04-01

    Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% +/- 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%).

  6. Hybridization between Gossypium herbaceum and Gossypium stochsii through embryo rescue

    Institute of Scientific and Technical Information of China (English)

    GILLMANJEETS; YPSBAJAJ

    1996-01-01

    Nutrient media and culture conditions have been defined for ovules 3 and 5 d after pollination and embryos of Gossypium herbaceum respectively.The technique was then used to produce interspecific hybrids between a cultivated Gossypium herbaceum and a wild species,G.stocksii.The hybrid plants were transferred to field and they exhibited most of the characters of the pollen parent i.e.G.stocksii.

  7. Detection of Embryo Sex Chromosome by Dual Color Fluorescent In-Situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    刘群; 朱桂金

    2003-01-01

    Summary: In order to evaluate the effects of sex chromosomal mosaicism on the accuracy of single-cell gender diagnosis, sex chromosomes of 21 normal fertilized embryos were detected by dual colorfluorescent in-situ hybridization (FISH). The results showed that 4 embryos had sex chromosomalmosaicism (19%) and the remaining 17 showed uniformly XX or XY signals in all blastomeres. Inconclusion, identification of sex by dual color FISH analysis of a single cell was accurate and efficient,and sex chromosomal mosaicism would not affect preimplantation gender diagnosis.

  8. Induction and Histology of Primary and Secondary Somatic Embryos of Phalaenopsis Hybrid Classic Spotted Pink (Orquidaceae

    Directory of Open Access Journals (Sweden)

    Cláudia Ulisses de Carvalho Silva

    2016-09-01

    Full Text Available The present work had as objectives to induce the formation of somatic embryos in vitro on Phalaenopsis hybrid Classic Spotted Pink, using different nutrient medium and assess the internal morphology of these embryos by means of histological and histochemical analysis. Young leaves of plants grown in vitro were used as explants for induction of somatic embryos in different nutrient medium: New Dogashima Medium, containing ANA (0.537 mM and BAP (4.440 mM plus phytagel and with pH 5.8 (NDM and the Murashige & Skoog with half the concentration of salts, plus NNA (0.537 mM and TDZ (13.621 mM, jellied with gelrite and pH 5.2 (0.5 MS. Primary somatic embryos were obtained to 90 days of cultivation in half MS and have been transferred to the same means for obtaining of secondary embryos. The primary and secondary somatic embryos were subcultived for MS with half the concentration of salts, without fitoregulator subjected to photoperiod of 16 hours, which stimulated the production of chlorophyll in primary embryos as secondary, promoting the development of those in protocorms and later in plants. The histological analysis showed that the somatic embryos were formed directly from the epidermal layers of the explants, without going through the phase of callus, featuring direct somatic embryogenesis. The histochemical methods used made it possible to highlight the deposition of starch and lipids in cells embriogenics as a result of physiological mechanisms, enabling the development of primary and secondary embryos in plants. Therefore, the medium 0.5 MS Plus ANA (0.537 mM and TDZ (13.621 mM, jellied with gelrite and pH 5.2 promoted to obtain primary and secondary embryos with ability to regenerate plants showing morphological similar the mother plant.

  9. Embryo forming cells in carrot suspension cultures.

    OpenAIRE

    Toonen, M.A.J.

    1997-01-01

    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earli...

  10. Culture of Cells from Amphibian Embryos.

    Science.gov (United States)

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  11. Embryo development and corresponding factors affecting in vitro germination of Cymbidium faberi × C. sinense hybrid seeds

    Directory of Open Access Journals (Sweden)

    Li Fengtong

    2016-01-01

    Full Text Available A better understanding of embryo development would provide insights into seed quality and subsequent germination events in the interspecific hybridization of Cymbidium faberi ‘Jiepeimei’ × C. sinense ‘Qijianheimo’. At the mature stage, 26.1% of the ovules were abnormal. Most of the hybrid embryos could develop normally. Abortions mainly occurred at the zygote (9.5% and 2-4-celled embryo (15.1% stages. No germination was observed at 90 and 105 days after pollination (DAP, when the embryo was at the early globular stage, with abundant organelles but no storage materials. During 110-130 DAP, the globular embryo was formed and the starch grains began to accumulate in plastids. The hybrid seeds collected at 120 DAP showed initiation of germination. Germination significantly increased at 135 DAP and was maximal at 150 DAP, during which period the hybrid embryos developed into the late globular stage. The storage materials, i.e. lipid and protein bodies, began to accumulate and the filamentary structures derived from suspensor cells still persisted. After the seeds matured (160 DAP, the germination percentage declined sharply. Safranin staining revealed that the outer seed coat was totally cuticularized and the inner seed coat appeared as a cuticle layer enclosing the embryo proper tightly, which may be the main factor inhibiting the subsequent germination of hybrid seeds. In conclusion, 150 DAP should be the opportune time for the in vitro germination of C. faberi ‘Jiepeimei’ × C. sinense ‘Qijianheimo’ hybrid seeds.

  12. Pavia symposium on embryos and stem cells

    Directory of Open Access Journals (Sweden)

    M Boiani

    2009-08-01

    Full Text Available Stem cells occur in very small numbers in adult tissues, in higher numbers in the fetus and its annexes and they can be derived from whole embryos or parts thereof. For a couple of years, stem cells can also be derived straight from somatic cells by retrovirus-mediated transfer of selected genes in culture. Stem cells have varying capacities for self-renewal and differentiation according to their origin, up to the point that they can be propagated in vitro for years and give rise to a wide range of cell types. This makes them suitable means for cell and tissue-replacement therapies. Gaining access to stem cells involves manipulation of living organisms i.e. human beings or animal models. The extent of manipulation ranges from the mere biopsy and culture of body cells in vitro, to their genetic manipulation and back-grafting in vivo.This raises bioethical issues as to whether we should manipulate animal and in particular human life. Many scientific meetings have been organized to present research findings on stem cells and on their potency, differentiation and therapeutic applications for treating disease. In many of these meetings, the ‘mother’ of all stem cells - the embryo - has often been ignored, because embryonic stem cells do not exist as such in the embryo and because the manipulation of the embryo brings up ethical concerns.To make up for this omission, the theme of our meeting was ‘Pluripotency and differentiation in embryos and stem cells’.With a strong sense of purpose and commitment, we managed to hold a two-day symposium concerned with three major topics: 1 natural and induced pluripotency; 2 mechanisms of cell fate control; 3 adult and cancer stem cells. The meeting took place on January 17th-18th 2008 in the beautiful, frescoed halls of Collegio Ghislieri and Borromeo, two foremost colleges in the Pavia campus. A small but prominent group of scientists took part in the meeting – James Adjaye (Germany, Anne Grete Byskov

  13. Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

    Science.gov (United States)

    Lee, W-J; Lee, J-H; Jeon, R-H; Jang, S-J; Lee, S-C; Park, J-S; Lee, S-L; King, W-A; Rho, G-J

    2017-02-13

    Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

  14. Fearing a non-existing Minotaur? The ethical challenges of research on cytoplasmic hybrid embryos.

    Science.gov (United States)

    Camporesi, S; Boniolo, G

    2008-11-01

    In this paper we address the ethical challenges of research on cytoplasmic hybrid embryos, or "cybrids". The controversial pronouncement of the UK's Human Embryology and Fertilisation Authority of September 2007 on the permissibility of this area of research is the starting point of our discussion, and we argue in its favour. By a rigorous definition of the entities at issue, we show how the terms "chimera" and "hybrid" are improper in the case of cybrids, and how their use can bias the debate creating moral prejudices. After analysing the scientific aspects of cybrids research and sketching out current alternatives, we enter the ethical debate, starting from the premise that research on early human embryos is ethically permissible under some circumstances. We emphasise how research on cybrids has positive consequences in terms of scientific and therapeutic applications, since it allows the derivation of human embryonic stem cells genetically tailored to the somatic cell donor. Such cell lines offer a unique in vitro model both for studies of human pathogenesis and for drug screening and discovery. Research on cybrids also circumvents the problem of the scarcity of human oocytes and their ethically dubious donation. Finally, we object to the most common arguments against cybrids research, that is, moral repugnance, the slippery slope argument, the appeal to "nature", and the unfair distribution of economical resources.

  15. The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation.

    Science.gov (United States)

    Li, Xiangyun; Wei, Wei; Yong, Jun; Jia, Qing; Yu, Yuansong; Di, Keqian

    2005-07-01

    The aim of this paper was to determine whether the genetic background of tetraploid embryos contributed to the survival of mice derived from embryonic stem (ES) cells by tetraploid embryo complementation. Twenty-five newborns were produced by aggregation of hybrid ES cells and tetraploid embryos with different genetic backgrounds. These newborns were entirely derived from ES cells judged by microsatellite DNA (A specific sequence of DNA bases or nucleotides that contains mono, di, tri or tetra repeats) and coat colour phenotype and germline transmission. Fifteen survived to adulthood while seven died of respiratory failure. All newborns were derived from outbred or hybrid tetraploid aggregates and no newborns were from the inbreds. Our results demonstrate that the genetic heterozygosity, fitness of tetraploid embryos and fitness of ES cells are crucial parameters influencing survival of mice derived from ES cells by tetraploid embryo aggregation. In addition, this method represents a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.

  16. Identification of the Sex of Earlier Embryos from Generic Hybrids of Chicken-Quail by Wpkci

    Institute of Scientific and Technical Information of China (English)

    QIAO Ai-jun; MA Wen-xia; LI Da-quan; MENG Qing-mei

    2008-01-01

    In this study,a protocol was deveolped the sex of earlier embryos of chicken(♂)-quail(♀)hybrids and successfully tested the sex proportion of each period (66-120 h). We acquired cross bred eggs by artificial insemination, hatched them in the same batch according to the standard hatching condition of chicken, and collected earlier living embryos at 66,72,78, 84,90,96,102,108,114, and 120 h randomly. We adopted RT-PCR protocol and multiple PCR, made the known sex quail as the external control, employed β-actin as the internal control, and used primers that were designed according to conservative area of gene Wpkci of quail to identify the sex of earlier hybrid embryos. The results indicated that the primer of Wpkci can be used to identify the sex of hybrid embryos accurately; there were more male than female in earlier embryos, the sex proportion of earlier embryos compared with academic numerical value was significantly different (P0.05). In the present study, we concluded that a simple, fast, credible and stable protocol to identify the sex of earlier hybrids embryos had been established by using primer of Wpkci; in earlier embryos, the death rate of female was higher than that of male and there was no fluctuant peak.

  17. Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos

    DEFF Research Database (Denmark)

    Viuff, Dorthe; Greve, Torben; Holm, Peter

    2002-01-01

    electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only...

  18. Chromosomal mosaicism in mouse two-cell embryos after paternal exposure to acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Bishop, Jack; Lowe, Xiu; Wyrobek, Andrew J

    2008-10-14

    Chromosomal mosaicism in human preimplantation embryos is a common cause ofspontaneous abortions, however, our knowledge of its etiology is limited. We used multicolor fluorescence in situ hybridization (FISH) painting to investigate whether paternally-transmitted chromosomal aberrations result in mosaicism in mouse 2-cell embryos. Paternal exposure to acrylamide, an important industrial chemical also found in tobacco smoke and generated during the cooking process of starchy foods, produced significant increases in chromosomally defective 2-cell embryos, however, the effects were transient primarily affecting the postmeiotic stages of spermatogenesis. Comparisons with our previous study of zygotes demonstrated similar frequencies of chromosomally abnormal zygotes and 2-cell embryos suggesting that there was no apparent selection against numerical or structural chromosomal aberrations. However, the majority of affected 2-cell embryos were mosaics showing different chromosomal abnormalities in the two blastomeric metaphases. Analyses of chromosomal aberrations in zygotes and 2-cell embryos showed a tendency for loss of acentric fragments during the first mitotic division ofembryogenesis, while both dicentrics and translocations apparently underwent propersegregation. These results suggest that embryonic development can proceed up to the end of the second cell cycle of development in the presence of abnormal paternal chromosomes and that even dicentrics can persist through cell division. The high incidence of chromosomally mosaic 2-cell embryos suggests that the first mitotic division of embryogenesis is prone to missegregation errors and that paternally-transmitted chromosomal abnromalities increase the risk of missegregation leading to embryonic mosaicism.

  19. Plant Regeneration and Somatic Embryogenesis from Immature Embryos Derived through Interspecific Hybridization among Different Carica Species

    Directory of Open Access Journals (Sweden)

    Latifah Amin

    2012-12-01

    Full Text Available Plant regeneration and somatic embryogenesis through interspecific hybridization among different Carica species were studied for the development of a papaya ringspot virus-resistant variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv. Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days after pollination and the embryos were aborted at 150 days after pollination. The immature hybrid embryos were used for plant regeneration and somatic embryogenesis. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora showed the highest percentage of germination, as well as plant regeneration on growth regulators free culture medium after 7 days pre-incubation on half-strength MS medium supplemented with 0.2 mg/L BAP, 0.5 mg/L NAA and 60 g/L sucrose. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora produced maximum callus, as well as somatic embryos when cultured on half-strength MS medium containing 5 mg/L 2,4-D, 100 mg/L glutamine, 100 mg/L casein hydrolysate and 60 g/L sucrose. The somatic embryos were transferred into half-strength MS medium containing 0.5 mg/L BAP and 0.2 mg/L NAA and 60 g/L sucrose for maturation. The highest number of regenerated plants per hybrid embryo (10.33 was recorded from the cross of C. papaya cv. Shahi × C. cauliflora. Isoenzyme and dendrogram cluster analysis using UPGMA of the regenerated F1 plantlets confirmed the presence of the hybrid plantlets.

  20. Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos

    DEFF Research Database (Denmark)

    Viuff, Dorthe; Greve, Torben; Holm, Peter

    2002-01-01

    In porcine embryos, nucleoli are first observed during the third postfertilization cell cycle, i.e., at the 4-cell stage. However, direct studies of the initiation of rRNA transcription have not been reported. This transcription was investigated in the present study by simultaneous visualization...... of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of argyrophilic nucleolar proteins by silver staining of extracted and fixed nuclei from in vivo-derived porcine embryos (n = 229). Nucleologenesis was observed by transmission...... electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only...

  1. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...... nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four......-cell stage (n = 45), 38% of the embryos contained 1-3 nuclei with signs of rRNA transcription, indicating an asynchronous transcription initiation. This pattern continued in the following stages, as 78% (n = 47), 47% (n = 42) and 83% (n = 37) of the embryos revealed a mixture of transcriptionally inactive...

  2. Interspecific hybridization of Trifolium alexandrinum with T. constantinopolitanum using embryo rescue.

    Science.gov (United States)

    Roy, A K; Malaviya, D R; Kaushal, P; Kumar, B; Tiwari, A

    2004-04-01

    The embryo rescue technique was successfully used to raise hybrids between Trifolium alexandrinum and T. constantinopolitanum. As a result of its narrow genetic base, genetic improvement in Egyptian clover (syn. Berseem; T. alexandrinum), an important fodder crop in tropical and subtropical countries, is hampered, thereby making it imperative to introduce alien genes from related species. In a conventional interspecific hybridization program, hybrids could not be raised due to post-fertilization barriers. Of the several combinations tried, pollination 2 days after emasculation was found to be the best. Globular embryos were observed 5-7 days after pollination (DAP), followed by heart-shaped embryos 10-12 DAP. Embryos excised at the heart-shaped stage responded well to EC3 culture medium. Of 612 crosses, 33 healthy embryos could be excised and cultured on EC3 medium. The plumule emerged 8-12 days following inoculation. The embryo-rescued plants were hardened, inoculated with Rhizobium and transferred to the field. The hybrids showed intermediate morphological features with reduced pollen fertility (55-65%) and a chromosomal complement of 2n = 16. Biochemical characterization using isozymes confirmed hybridity.

  3. Embryogenic calli induced in interspecific (Elaeis guineensis x E. oleifera hybrid zygotic embryos

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    Paula Cristina da Silva Angelo

    2009-01-01

    Full Text Available The hybridization between oil palm (Elaeis guineensis and caiaué (E. oleifera plants is directed to obtainprogenies presenting high yields like oil palm but with reduced shoot height and resistance to lethal yellowing like caiaué.Cloning F1, BC1 and BC2 progenies can make the replication of selection trials easier. The objective of this work was to inducesomatic embryogenesis in interspecific zygotic embryos collected 100 days after pollination. Three progenies were cultivatedin an induction medium developed for Tenera (E. guineensis tp. dura x pisifera embryos. The number of embryos bearing calliand germinating was recorded and submitted to the Z test. Calli were weighted and submitted to histological analysis.Progenies differed in the number of embryos presenting plumules and calli simultaneously. By the ninth month, the apices ofincompletely developed somatic embryos were observed protruding from the surfaces of nodular calli. Highly embryogenicand friable secondary calli producing globular somatic embryos were not observed.

  4. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

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    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  5. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    Science.gov (United States)

    Mehta, Rajvi H

    2014-11-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of 'discarding' embryos. What would be the criteria for discarding embryos and the potential 'use' of ESC derived from the 'abnormal appearing' embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  6. Creating New Germplasm by Distant Hybridization in Stone Fruits:Ⅱ-Embryo Rescue and Hybrid Identification Between Plum and Apricot

    Institute of Scientific and Technical Information of China (English)

    YANG Hong-hua; CHEN Xue-sen; FENG Bao-chun; LIU Huan-fang; ZHENG Zhou

    2004-01-01

    Embryo abortion stage and rescue system of hybrids were studied in the distant hybridization between plum and apricot. Identification of the hybrids was also made. The results showed:(1)Embryo abortion started from three weeks after pollination.(2)The germination and growth of embryos were different at different growth stages,which could germinate and grow with PF value> 0.5,but failed with PF value< 0.5. In embryo rescue system of hybrids,the best germination and differentiation medium was MS + 6-BA 2 mg L-1+ IAA 0.3 mg L-1,the rate of germination and differentiation reached up to 80%,bud induction and multiplication medium was MS + 6-BA 1.5 mg L-1+ IAA 0.3 mg L-1,rooting medium was 1/2 MS + IAA0.8mgL-1. Some hybrids were transplanted into the field successfully.(3)Leaf shape investigation and identification by S allele-specific PCR and RAPDs showed that the hybrids were true ones.

  7. Producing fully ES cell-derived mice from eight-cell stage embryo injections.

    Science.gov (United States)

    DeChiara, Thomas M; Poueymirou, William T; Auerbach, Wojtek; Frendewey, David; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    In conventional methods for the generation of genetically modified mice, gene-targeted embryonic stem (ES) cells are injected into blastocyst-stage embryos or are aggregated with morula-stage embryos, which are then transferred to the uterus of a surrogate mother. F0 generation mice born from the embryos are chimeras composed of genetic contributions from both the modified ES cells and the recipient embryos. Obtaining a mouse strain that carries the gene-targeted mutation requires breeding the chimeras to transmit the ES cell genetic component through the germ line to the next (F1) generation (germ line transmission, GLT). To skip the chimera stage, we developed the VelociMouse method, in which injection of genetically modified ES cells into eight-cell embryos followed by maturation to the blastocyst stage and transfer to a surrogate mother produces F0 generation mice that are fully derived from the injected ES cells and exhibit a 100% GLT efficiency. The method is simple and flexible. Both male and female ES cells can be introduced into the eight-cell embryo by any method of injection or aggregation and using all ES cell and host embryo combinations from inbred, hybrid, and outbred genetic backgrounds. The VelociMouse method provides several unique opportunities for shortening project timelines and reducing mouse husbandry costs. First, as VelociMice exhibit 100% GLT, there is no need to test cross chimeras to establish GLT. Second, because the VelociMouse method permits efficient production of ES cell-derived mice from female ES cells, XO ES cell subclones, identified by screening for spontaneous loss of the Y chromosome, can be used to generate F0 females that can be bred with isogenic F0 males derived from the original targeted ES cell clone to obtain homozygous mutant mice in the F1 generation. Third, as VelociMice are genetically identical to the ES cells from which they were derived, the VelociMouse method opens up myriad possibilities for creating mice with

  8. New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Lorena Rodrigo

    2014-01-01

    Full Text Available The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS using array comparative genomic hybridization (aCGH. The study included 1420 CCS cycles for recurrent miscarriage (n=203; repetitive implantation failure (n=188; severe male factor (n=116; previous trisomic pregnancy (n=33; and advanced maternal age (n=880. CCS was performed in cycles with fresh oocytes and embryos (n=774; mixed cycles with fresh and vitrified oocytes (n=320; mixed cycles with fresh and vitrified day-2 embryos (n=235; and mixed cycles with fresh and vitrified day-3 embryos (n=91. Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2% and pregnancy rates per transfer (range: 46.0–62.9% were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1% due to the higher percentage of aneuploid embryos (85.3% and lower number of cycles with at least one euploid embryo available per transfer (40.3%. We concluded that aneuploidy is one of the major factors which affect embryo implantation.

  9. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    OpenAIRE

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF...

  10. Proteomic Analysis Reveals Different Involvement of Embryo and Endosperm Proteins during Aging of Yliangyou 2 Hybrid Rice Seeds

    Science.gov (United States)

    Zhang, Ying-Xue; Xu, Heng-Heng; Liu, Shu-Jun; Li, Ni; Wang, Wei-Qing; Møller, Ian M.; Song, Song-Quan

    2016-01-01

    Seed aging is a process that results in a delayed germination, a decreased germination percentage, and finally a total loss of seed viability. However, the mechanism of seed aging is poorly understood. In the present study, Yliangyou 2 hybrid rice (Oryza sativa L.) seeds were artificially aged at 100% relative humidity and 40°C, and the effect of artificial aging on germination, germination time course and the change in protein profiles of embryo and endosperm was studied to understand the molecular mechanism behind seed aging. With an increasing duration of artificial aging, the germination percentage and germination rate of hybrid rice seeds decreased. By comparing the protein profiles from the seeds aged for 0, 10 and 25 days, a total of 91 and 100 protein spots were found to show a significant change of more than 2-fold (P cell defense and rescue (28%), and with storage protein (18%). In endosperms, most of the identified proteins were involved in metabolism (37%), in energy (27%), and in protein synthesis and destination (11%). The most marked change was the increased abundance of many glycolytic enzymes together with the two fermentation enzymes pyruvate decarboxylase and alcohol dehydrogenase in the embryos during aging. We hypothesize that the decreased viability of hybrid rice seeds during artificial aging is caused by the development of hypoxic conditions in the embryos followed by ethanol accumulation.

  11. Transgenic manipulation of plant embryo sacs tracked through cell-type-specific fluorescent markers: cell labeling, cell ablation, and adventitious embryos.

    Science.gov (United States)

    Lawit, Shai J; Chamberlin, Mark A; Agee, April; Caswell, Eric S; Albertsen, Marc C

    2013-06-01

    Expression datasets relating to the Arabidopsis female gametophyte have enabled the creation of a tool set which allows simultaneous visual tracking of each specific cell type (egg, synergids, central cell, and antipodals). This cell-specific, fluorescent labeling tool-set functions from gametophyte cellularization through fertilization and early embryo development. Using this system, cell fates were tracked within Arabidopsis ovules following molecular manipulations, such as the ablation of the egg and/or synergids. Upon egg cell ablation, it was observed that a synergid can switch its developmental fate to become egg/embryo-like upon loss of the native egg. Also, manipulated was the fate of the somatic ovular cells, which can become egg- and embryo-like, reminiscent of adventitious embryony. These advances represent initial steps toward engineering synthetic apomixis resulting in seed derived wholly from the maternal plant. The end goal of applied apomixis research, fixing important agronomic traits such as hybrid vigor, would be a key benefit to agricultural productivity.

  12. In vitro embryo rescue of interspecific hybrids of oil palm (Elaeis oleifera x Elaeis guineensis)

    OpenAIRE

    Alves, Sergio Augusto Oliveira; de Lemos, Oriel Filgueira; Filho,Benedito Gomes dos Santos; da Silva, André Luís Lopes

    2011-01-01

    The oil palm (Elaeis guineennsis Jacq.) is the plant with major oil yield in tropical areas over the world. Pará state is the major producer of Brazil. However, the production of resistant hybrids (Elaeis oleifera x Elaeis guinennsis) to several diseases is necessary, in spite of less productive, is a viable alternative for areas where occur more aggressive diseases. The aim of this research was to rescue in vitro embryos of four varieties of interspecific hybrids of oil palm (Elaeis oleifera...

  13. Developmental potential and behavior of tetraploid cells in the mouse embryo.

    Science.gov (United States)

    Eakin, Guy S; Hadjantonakis, Anna-Katerina; Papaioannou, Virginia E; Behringer, Richard R

    2005-12-01

    Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.

  14. New mechanism for neural stem cell maintenance in early embryos

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Teamning up with co-workers from Japan, UK and US,CAS biochemists have revealed a novel mechanism for maintaining neural stem cells in early embryos. Their work was published on the 6 August issue of Cell Development.

  15. Admixed human embryos and stem cells: legislative, ethical and scientific advances.

    Science.gov (United States)

    Bahadur, G; Iqbal, M; Malik, S; Sanyal, A; Wafa, R; Noble, R

    2008-01-01

    This paper examines the regulatory framework currently governing the creation of animal-human hybrids and chimera embryos in stem cell research, and some of the ethical implications of such research. It discusses the findings of a recent government select committee that considered the topic. It considers the debate around the precise definition of a human embryo, and whether such hybrids therefore fall within the remit of the Human Fertilisation and Embryology Authority. It outlines the advantages of such hybrids, in lessening the need for human egg donors, as well as the moral objections to species boundary violation. It calls for an examination of the scientific benefits of such research to inform debate on the question, and argues for the need to take genuine account of the public's views on this matter.

  16. Development of hematopoietic stem cell activity in the mouse embryo.

    NARCIS (Netherlands)

    A.M. Müller (Albrecht); A. Medvinsky; J. Strouboulis (John); F.G. Grosveld (Frank); E.A. Dzierzak (Elaine)

    1994-01-01

    textabstractThe precise time of appearance of the first hematopoietic stem cell activity in the developing mouse embryo is unknown. Recently the aorta-gonad-mesonephros region of the developing mouse embryo has been shown to possess hematopoietic colony-forming activity (CFU-S) in irradiated recipie

  17. Cell adhesion in zebrafish embryos is modulated by March 8.

    Science.gov (United States)

    Kim, Mi Ha; Rebbert, Martha L; Ro, Hyunju; Won, Minho; Dawid, Igor B

    2014-01-01

    March 8 is a member of a family of transmembrane E3 ubiquitin ligases that have been studied mostly for their role in the immune system. We find that March 8 is expressed in the zebrafish egg and early embryo, suggesting a role in development. Both knock-down and overexpression of March 8 leads to abnormal development. The phenotype of zebrafish embryos and Xenopus animal explants overexpressing March 8 implicates impairment of cell adhesion as a cause of the effect. In zebrafish embryos and in cultured cells, overexpression of March 8 leads to a reduction in the surface levels of E-cadherin, a major cell-cell adhesion molecule. Experiments in cell culture further show that E-cadherin can be ubiquitinated by March 8. On the basis of these observations we suggest that March 8 functions in the embryo to modulate the strength of cell adhesion by regulating the localization of E-cadherin.

  18. Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos.

    Science.gov (United States)

    Li, Xiangyun; Yu, Yuansong; Wei, Wei; Yong, Jun; Yang, Jie; You, Jiefang; Xiong, Xiaoran; Qing, Tingting; Deng, Hongkui

    2005-06-01

    Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.

  19. Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation.

    Science.gov (United States)

    Li, Xiang-Yun; Jia, Qing; Di, Ke-Qian; Gao, Shu-Min; Wen, Xiao-Hui; Zhou, Rong-Yan; Wei, Wei; Wang, Li-Ze

    2007-03-01

    The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3-11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells.

  20. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    Science.gov (United States)

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  1. Comparative proteomic analysis of embryos between a maize hybrid and its parental lines during early stages of seed germination.

    Directory of Open Access Journals (Sweden)

    Baojian Guo

    Full Text Available In spite of commercial use of heterosis in agriculture, the molecular basis of heterosis is poorly understood. It was observed that maize hybrid Zong3/87-1 exhibited an earlier onset or heterosis in radicle emergence. To get insights into the underlying mechanism of heterosis in radicle emergence, differential proteomic analysis between hybrid and its parental lines was performed. In total, the number of differentially expressed protein spots between hybrid and its parental lines in dry and 24 h imbibed seed embryos were 134 and 191, respectively, among which 47.01% (63/134 and 34.55% (66/191 protein spots displayed nonadditively expressed pattern. Remarkably, 54.55% of nonadditively accumulated proteins in 24 h imbibed seed embryos displayed above or equal to the level of the higher parent patterns. Moreover, 155 differentially expressed protein spots were identified, which were grouped into eight functional classes, including transcription & translation, energy & metabolism, signal transduction, disease & defense, storage protein, transposable element, cell growth & division and unclassified proteins. In addition, one of the upregulated proteins in F1 hybrids was ZmACT2, a homolog of Arabidopsis thaliana ACT7 (AtACT7. Expressing ZmACT2 driven by the AtACT7 promoter partially complemented the low germination phenotype in the Atact7 mutant. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of proteins, and it is concluded that the altered pattern of gene expression at translational level in the hybrid may be responsible for the observed heterosis.

  2. The embryo rescue derived intergeneric hybrid between chrysanthemum and Ajania przewalskii shows enhanced cold tolerance.

    Science.gov (United States)

    Deng, Yanming; Chen, Sumei; Chen, Fadi; Cheng, Xi; Zhang, Fei

    2011-12-01

    Five intergeneric hybrids between the chrysanthemum cultivar 'Zhongshanjingui' (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While 'Zhongshanjingui' bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids' flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2n = 45, of which GISH analysis was able to establish that 27 were inherited from 'Zhongshanjingui' and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content.

  3. Ethical questions concerning research on human embryos, embryonic stem cells and chimeras.

    Science.gov (United States)

    Bobbert, Monika

    2006-12-01

    Research using human embryos and embryonic stem cells is viewed as important for various reasons. Apart from questions concerning legal regulations, numerous ethical objections are raised pertaining to the use of surplus embryos from reproductive medicine as well as the creation of embryos and stem cells through cloning. In the hopes of avoiding ethical problems, alternatives have been proposed including the extraction of egg cells from "dead" embryos derived from in vitro fertilization procedures, the extraction of pluripotent stem cells from blastocysts, technologies such as "altered nuclear transfer" (ANT) and "oocyte-assisted reprogramming" (ANT-OAR) as well as parthenogenesis. Initial ethical assessments show that certain questions pertaining to such strategies have remained unanswered. Furthermore, with the help of new or more differentiated biotechnological procedures, it is possible to create chimeras and hybrids in which human and non-human cells are combined. Human-animal chimeras, in which gametes or embryonic tissue have been mixed with embryonic or adult stem cells, demonstrate a different "quality" and "degree of penetration" from those produced in previous experiments. Not only does this have consequences regarding questions of patentability, this situation also raises fundamental questions concerning the human being's self image, the concept of person, identity and species and the moral rights and duties that are connected with such concepts. There is a need for legal regulation, on the national as well as the international level.

  4. Embryo development and corresponding factors affecting in vitro germination of Cymbidium faberi × C. sinense hybrid seeds

    National Research Council Canada - National Science Library

    Li, Fengtong; Bao, Jianzhong; Sun, Ye; Liu, Chungui; Ma, Hui; Zhang, Tian; Chen, Xiulan

    2016-01-01

    A better understanding of embryo development would provide insights into seed quality and subsequent germination events in the interspecific hybridization of Cymbidium faberi ‘Jiepeimei’ × C. sinense ‘Qijianheimo...

  5. Effect of donor cell type on nuclear remodelling in rabbit somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Tian, J; Song, J; Li, H; Yang, D; Li, X; Ouyang, H; Lai, L

    2012-08-01

    Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.

  6. Introduction of DT40 cells into chick embryos

    Institute of Scientific and Technical Information of China (English)

    Mariko Toba; Fumio Ebara; Hiroki Furuta; Yuichi Matsushimal; Yasuo Kitagawa; Noboru Fujihara

    2001-01-01

    To examine the transfection of exogenous genes into chick embryos, applying the characteristics of avian leukosis virus (ALV)-induced chicken B cell line DT40 to the production of chimeric birds. Methods: The DT40cells incorporated with exogenous gene (lacZ constructs encoding Escherichia coli β-galactosidase: β-gal) were introduced into chick embryos by the injection of cells into stage X blastoderm. Manipulated eggs were incubated for 3 (trial 1 ) or 6 (trial 2) days, and the expression of lacZ DNA was detected by a histochemical staining method of β-galactosidase and polymerase chain reaction (PCR) analysis. Results: The survival rates of the manipulated embryos incubated for 3 days (stage 18-20: trial 1) and 6 days (stage 28, 30: trial 2) were about 42% and 38%, respectively.The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60% and 23%, respectively, for the survived embryos. Conclusio: The rate of embryonic viability and expression rate of introduced genes were not so high, but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chick embryos.

  7. Differentially expressed genes implicated in embryo abortion of mango identified by suppression subtractive hybridization.

    Science.gov (United States)

    He, J H; Ma, F W; Chen, Y Y; Shu, H R

    2012-11-14

    Embryo abortion in mango severely damages mango production worldwide. The mechanisms by which the mango embryos abort have long been an intriguing question. We used subtractive suppression hybridization to investigate the differentially expressed genes involved in this process. We generated 2 cDNA libraries from normal seed and aborted seed embryos of mango cultivar 'Jinhuang'. One thousand five hundred and seventy-two high-quality expressed sequence tags (ESTs) were obtained, with 1092 from the normal seed tester library and 480 from the aborted seed tester library. These ESTs were assembled into 783 unigenes, including 147 contigs and 636 singletons in contigs; 297 singletons in gene ontology (GO) indicated coverage of a broad range of GO categories. Seven candidate genes from different categories were selected for semi-quantitative PCR analysis, and their possible functions in embryo abortion are discussed. These data provide new insight into the genetic regulation of embryo abortion in mango and may aid in further identification of novel genes and their functions.

  8. Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos

    DEFF Research Database (Denmark)

    Jakobsen, Anne Sørig; Avery, Birthe; Dieleman, Steph J.

    2006-01-01

    of the embryonic genome. In the present study, ribosomal RNA (rRNA) transcription was investigated by visualization of the rRNA by fluorescent in situ hybridization, and subsequent visualization of the argyrophilic nucleolar proteins by silver staining. A total of 145 in vivo developed and 200 in vitro produced...... bovine embryos were investigated to allow comparison of transcription initiation. Signs of active transcription of rRNA were observed in the third cell cycle in 29% of the in vitro produced embryos (n=35) and in 58% of the in vivo developed embryos (n=11). Signs of active transcription of rRNA were...... not apparent in the early phase of the fourth cell cycle but restarted later on. All embryos in the fifth or later cell cycles were all transcribing rRNA. The signs of rRNA synthesis during the third and fourth embryonic cell cycles could be blocked by actinomycin D, which is a strong inhibitor of RNA...

  9. The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells

    Directory of Open Access Journals (Sweden)

    Masood Bazrgar

    2016-12-01

    Full Text Available Background: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies. Materials and Methods: In this cohort study, we used fluorescence in situ hybridization (FISH to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization. Results: Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%, while 2(6.7% were diploid, and 2(6.7% were triploid. Of those with mosaicism, 23(88.5% were determined to be diploid-aneuploid and 3(11.5% were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05; however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality. Conclusion: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells.

  10. Assessment of early cleaving in vitro fertilized human embryos at the 2-cell stage before transfer improves embryo selection.

    Science.gov (United States)

    Sakkas, D; Percival, G; D'Arcy, Y; Sharif, K; Afnan, M

    2001-12-01

    To determine the most viable embryos for transfer. Study 1: Preselection of early-cleaving 2-cell embryos for transfer. Study 2: Alternating weeks during which preselection was performed and not performed. ART program, Birmingham Women's Hospital, Birmingham, United Kingdom. Patients undergoing IVF or ICSI cycles with transfer on day 2. Culture of all fertilized embryos. Number of fertilized embryos cleaving to the 2-cell stage on day 1, embryo quality, implantation rates, and pregnancy rates. Patients with early-cleaving 2-cell embryos had significantly higher pregnancy and implantation rates (45 of 100 [45.0%] and 58 of 219 [25.5%], respectively) than did patients without early-cleaving 2-cell embryos (31 of 130 [23.8%] and 43 of 290 [14.8%], respectively). In weeks during which preselection was used, the overall pregnancy and implantation rates of the clinic improved. The presence of early-cleaving 2-cell embryos improves a patient's chance of achieving pregnancy. Use of more stringent embryo selection criteria can improve overall pregnancy rates.

  11. Hybrid Fuel Cell Technology Overview

    Energy Technology Data Exchange (ETDEWEB)

    None available

    2001-05-31

    For the purpose of this STI product and unless otherwise stated, hybrid fuel cell systems are power generation systems in which a high temperature fuel cell is combined with another power generating technology. The resulting system exhibits a synergism in which the combination performs with an efficiency far greater than can be provided by either system alone. Hybrid fuel cell designs under development include fuel cell with gas turbine, fuel cell with reciprocating (piston) engine, and designs that combine different fuel cell technologies. Hybrid systems have been extensively analyzed and studied over the past five years by the Department of Energy (DOE), industry, and others. These efforts have revealed that this combination is capable of providing remarkably high efficiencies. This attribute, combined with an inherent low level of pollutant emission, suggests that hybrid systems are likely to serve as the next generation of advanced power generation systems.

  12. Control of oriented cell division in the Arabidopsis embryo

    NARCIS (Netherlands)

    Dop, van M.; Liao, C.Y.; Weijers, D.

    2015-01-01

    Multicellular plant development requires strict control of cell division orientation. A key unanswered question is how developmental regulators interact with the generic cell division machinery to trigger oriented divisions. We discuss the Arabidopsis embryo as a model for addressing this question.

  13. Live-cell imaging of mitosis in Caenorhabditis elegans embryos.

    Science.gov (United States)

    Powers, James A

    2010-06-01

    Caenorhabditis elegans is a wonderful model system for live imaging studies of mitosis. A huge collection of research tools is readily available to facilitate experimentation. For imaging, C. elegans embryos provide large clear cells, an invariant pattern of cell division, only six chromosomes, a very short cell cycle, and remain healthy and happy at room temperature. Mitosis is a complicated process and the types of research questions being asked about the mechanisms involved are continuously expanding. For each experiment, the details of imaging methods need to be tailored to the question. Specific imaging methods will depend on the microscopy hardware and software available to each researcher. This article presents points to consider when choosing a microscope, designing an imaging experiment, or selecting appropriate worm strains for imaging. A method for mounting C. elegans embryos and guidelines for fluorescence and differential interference contrast imaging of mitosis in live embryos are presented.

  14. Developmental potential of embryonic cells in a nucleocytoplasmic hybrid formed using a goldfish haploid nucleus and loach egg cytoplasm.

    Science.gov (United States)

    Fujimoto, Takafumi; Saito, Taiju; Sakao, Suzu; Arai, Katsutoshi; Yamaha, Etsuro

    2010-01-01

    In teleosts, viable nucleocytoplasmic hybrids, formed by combining a nucleus from one species with the egg cytoplasm of another, have been used as one of the methods for breed improvement in aquaculture, but have been little exploited for developmental biology studies. Here, we used an artificial androgenesis technique to form nucleocytoplasmic hybrids comprising a goldfish haploid nucleus and loach egg cytoplasm. These hybrids were used to investigate interactions between the nucleus and cytoplasm during embryonic development. Additionally, the developmental characteristics of embryonic cells of nucleocytoplasmic hybrids were examined in chimeras produced by transplantation of blastomeres into recipient loach or goldfish embryos. We found that the nucleocytoplasmic hybrids arrested at the dome stage of embryonic development and did not form any gastrula structures. The goosecoid (gsc) and no tail (ntl) genes were expressed normally before gastrulation in nucleocytoplasmic hybrids, similar to diploid loach. However, expression of the gsc and ntl genes was not maintained in nucleocytoplasmic hybrids. In chimeric embryos, blastomeres derived from nucleocytoplasmic hybrids were found to mix with the cells of recipient loach embryos at the gastrula stage. The transplanted blastomeres formed small clusters at the somitogenesis stage and, finally, small spots at the hatching stage. In contrast, when the blastomeres were transplanted into goldfish embryos, the transplanted blastomeres aggregated in the chimeric embryos. Thus, embryonic cells from nucleocytoplasmic hybrids that arrest before gastrulation could survive beyond the somitogenesis stage depending on the cytoplasmic environment in the recipient embryos.

  15. Early embryo and larval development of inviable intergeneric hybrids derived from Crassostrea angulata and Saccostrea cucullata

    Science.gov (United States)

    Su, Jiaqi; Wang, Zhaoping; Zhang, Yuehuan; Yan, Xiwu; Li, Qiongzhen; Yu, Ruihai

    2016-06-01

    To detect the intergeneric hybridization between the oyster Crassostrea angulata and Saccostrea cucullata coexisting along the southern coast of China, reciprocal crosses were conducted between the two species. Barriers for sperm recognizing, binding, penetrating the egg, and forming the pronucleus were detected by fluorescence staining. From the results, although fertilization success was observed in hybrid crosses, the overall fertilization rate was lower than that of intraspecific crosses. A large number of hybrid larvae died at 6-8 d after hatching, and those survived could not complete metamorphosis. C. angulata ♀× S. cucullata ♂ larvae had a growth rate similar to that of the maternal species, whereas S. cucullata ♀ × C. angulata ♂ larvae grew the slowest among all crosses. Molecular genetics analysis revealed that hybrid progeny were amphimixis hybrids. This study demonstrated that hybrid embryos generated by crossing C. angulata and S. cucullata could develop normally to the larval state, but could not complete metamorphosis and then develop to the spat stage. Thus, there is a post-reproductive isolation between C. angulata and S. cucullata.

  16. Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras.

    Science.gov (United States)

    Kruglova, A A; Kizilova, E A; Zhelezova, A I; Gridina, M M; Golubitsa, A N; Serov, O L

    2008-12-01

    Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76-80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11-13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.

  17. Sulfated polysaccharides and cell differentiation in the sea urchin embryo.

    Science.gov (United States)

    Løvtrup-Rein, H; Løvtrup, S

    1984-01-01

    The synthesis of sulfated polysaccharides during the embryonic development of Paracentrotus lividus has been investigated by incorporation of radioactive sulfate, glucose, glucosamine and fucose. The following substances become labelled: fucan sulfate (approximately 60%), heparan sulfate (approximately 20%) and dermatan sulfate (approximately 20%), and possibly a very slight amount of chondroitin sulfate. In animalized and vegetalized embryos, the rate of incorporation is significantly reduced, and furthermore dermatan sulfate is almost absent in animalized embryos. It is concluded that this substance is associated with the differentiation of vegetative cells, possibly the mesenchyme cells.

  18. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    Science.gov (United States)

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  19. Nuclear reprogramming by interphase cytoplasm of 2-cell mouse embryos

    Science.gov (United States)

    Kang, Enugu; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P.; Schöler, Hans; Mitalipov, Shoukhrat

    2014-01-01

    Summary Successful mammalian cloning employing somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II-arrested (MII) oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing pluripotency in somatic cell nuclei1-3. However, these poorly defined maternal factors presumably decline sharply after fertilization since cytoplasm of pronuclear stage zygotes is reportedly inactive4, 5. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase (M-phase) can also support derivation of embryonic stem cells (ESCs) following SCNT6-8, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in M-phase but not in interphase cytoplasm are “trapped” inside the nucleus during interphase and effectively removed during enucleation9. Here, we investigated the presence of reprogramming activity in the interphase cytoplasm of 2-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated M-phase and interphase zygotes and 2-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Then, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ESC, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ESCs capable of contributing to traditional germline and tetraploid chimeras. In addition, direct transfer of cloned embryos, reconstructed with ESC nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to utilize interphase cytoplasm in SCNT could impact efforts to generate autologous human ESCs for

  20. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.

    Science.gov (United States)

    Hartshorn, Cristina; Rice, John E; Wangh, Lawrence J

    2003-01-01

    Xist gene expression begins at the late 2-cell stage in female mouse embryos and by the third division results in the accumulation of an average 100 copies of Xist RNA per cell, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). In the blastocyst, the trophectoderm maintains the paternally imprinted pattern of Xist expression present during early development, while either the maternal or the paternal X chromosome can express Xist among cells of the inner mass. Fluorescent in situ hybridization (FISH) has previously established that Xist transcripts are localized on the silenced X chromosome, forming aggregates of variable dimensions in blastomeres of 8-cell embryos. This observation and the fact that Xist RNA accumulation per cell sharply decreases after morula stage raise the possibility that cells of cleaving embryos contain different levels of Xist RNA, perhaps linked to their subsequent developmental fates. We show here that Xist RNA is efficiently recovered from single blastomeres isolated from 8-cell embryos following laser zona drilling. Sexing of the samples and simultaneous quantification of Xist RNA in individual cells is achieved with a multiplex Xist/Sry real-time RT-PCR assay sensitive to the single-copy level. This analysis reveals that Xist RNA is indeed accumulated to substantially different levels in individual blastomeres of the same 8-cell embryo and that two blastomeres contain most of the molecules per embryo. These results support the conclusion that cells of the early mammalian embryo are not all functionally equivalent. Differential Xist gene expression could arise from differences in DNA methylation, or the order in which cells divide. Copyright 2003 Wiley-Liss, Inc.

  1. Production of Buffalo Embryonic Stem Cell from HMC Embryos

    Directory of Open Access Journals (Sweden)

    M. Zandi

    2016-06-01

    Full Text Available Embryonic stem cells (ESCs are derived from the inner cell mass (ICM of blastocyst and differentiate into all three embryonic germ layers: ectoderm, endoderm, and mesoderm. In this study, ESCs are derived from Hand Made Cloning (HMG blastocysts and their efficiencies compared to ESCs derived from In Vitro Fertilization (IVF embryos. Feeder layer was used for ESCs culture, and culture medium consisting of Knockout- Dulbecco’s Modified Eagle’s Medium (Ko-DMEM supplemented with Knockout Serum Replacement (KSR, Leukemia Inhibitory Factor (LIF, Basic Fibroblast Growth Factor-2 (FGF-2, L-glutamine, nonessential amino acids and gentamicin. The cell surface antigens used for characterization were the SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81 and the pluripotency markers were NANOG, OCT3/4 and SOX2. Results showed that, the growth rate of ESCs colonies in ESCs from IVF embryos was significantly higher than ESCs from HMG embryos (120% compared with 65%, respectively. Not only real-time PCR results revealed the same expression level of SOX2, OCT3/4 and cMYC between them, but also ESCs from HMG embryos resulted to higher expression of NANOG. Both of ESCs groups maintain in pluripotency state for more than two years and differentiated to the different types of cells like neuron, epithelial, lipid and muscle cells.

  2. Parameters influencing derivation of embryonic stem cells from murine embryos.

    Science.gov (United States)

    Batlle-Morera, Laura; Smith, Austin; Nichols, Jennifer

    2008-12-01

    The derivation of ES cells is poorly understood and varies in efficiency between different strains of mice. We have investigated potential differences between embryos of permissive and recalcitrant strains during diapause and ES cell derivation. We found that in diapause embryos of the recalcitrant C57BL/6 and CBA strains, the epiblast failed to expand during the primary explant phase of ES cell derivation, whereas in the permissive 129 strain, it expanded dramatically. Epiblasts from the recalcitrant strains could be expanded by reducing Erk activation. Isolation of 129 epiblasts facilitated very efficient derivation of ES cell lines in serum- and feeder-free conditions, but reduction of Erk activity was required for derivation of ES cells from isolated C57BL/6 or CBA epiblasts. The results suggest that the discrepancy in ES cell derivation efficiency is not attributable merely to variable prodifferentiative effects of the extra-embryonic lineages but also to an intrinsic variability within the epiblast to maintain pluripotency.

  3. Microfluidics for gametes, embryos, and embryonic stem cells.

    Science.gov (United States)

    Smith, G D; Swain, J E; Bormann, C L

    2011-01-01

    Microfluidics is a young but established field that holds significant potential for scientific discovery. The utility of microfluidics can improve our knowledge of basic biology as well as expand our understanding in specialized areas such as assisted reproduction and stem cell developmental biology. This review describes the technology of microfluidics and discusses applications within assisted reproduction technology and embryonic stem cell growth and directed differentiation. Development of an integrated microfluidic platform for assisted reproduction, which can manipulate gametes, embryos, embryonic stem cells, their culture environment, and incorporate biomarker analysis, could have a dramatic impact on the basic understanding of embryo/embryonic stem cell development, as well as provide significant improvements in current technologies used to treat infertility, preserve fertility, and derive therapeutic cells from stem cells.

  4. Embryonic stem cells from blastomeres maintaining embryo viability.

    Science.gov (United States)

    Klimanskaya, Irina

    2013-01-01

    A wide variety of cell and tissue types that are sought in regenerative medicine can be generated from embryonic stem cells (ESCs), and currently two derivatives of human embryonic stem cells (hESCs) have entered human clinical trials. However, the ethical controversy surrounding this technology, which uses preimplantation human embryos to generate cell lines, is limiting research and the development of new therapies. Several new technologies such as induced pluripotent cells or parthenogenetically derived pluripotent cells hold great promise, but more research is needed before their derivatives can be proven to be safe and functional for use in human patients. The blastomere biopsy-based technique allows the derivation of human ESClines without sacrificing a human embryo and was shown to be robust and produce safe and functional derivatives of therapeutic value.

  5. Hypoxia, hormones, and red blood cell function in chick embryos.

    Science.gov (United States)

    Dragon, Stefanie; Baumann, Rosemarie

    2003-04-01

    The red blood cell function of avian embryos is regulated by cAMP. Adenosine A(2A) and beta-adrenergic receptor activation during hypoxic conditions cause changes in the hemoglobin oxygen affinity and CO(2) transport. Furthermore, experimental evidence suggests a general involvement of cAMP in terminal differentiation of avian erythroblasts.

  6. Microspore embryogenesis: reprogramming cell fate from pollen to embryo development

    NARCIS (Netherlands)

    Hui Li,

    2014-01-01

    Microspore embryogenesis is an expression of plant cell totipotency that leads to the production of haploid embryos. Besides being a widely exploited plant breeding tool, microspore embryogenesis is also a fascinating system that can be used to obtain a deeper mechanistic understanding of plant toti

  7. Rayleigh instability of the inverted one-cell amphibian embryo

    NARCIS (Netherlands)

    Nouri, Comron; Luppes, Roel; Veldman, Arthur E.P.; Tuszynski, Jack A.; Gordon, Richard

    2008-01-01

    The one-cell amphibian embryo is modeled as a rigid spherical shell containing equal volumes of two immiscible fluids with different densities and viscosities and a surface tension between them. The fluids represent denser yolk in the bottom hemisphere and clearer cytoplasm and the germinal vesicle

  8. Reconstruction of human embryos derived from somatic cells

    Institute of Scientific and Technical Information of China (English)

    LU Changfu; LIN Ge; XIE Changqing; GONG Fei; ZHOU Hong; TAN Yueqiu; LU Guangxiu

    2003-01-01

    Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into MⅡ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (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 theblastocyst stage in vitro.

  9. Transcriptome analysis of mouse stem cells and early embryos.

    Directory of Open Access Journals (Sweden)

    Alexei A Sharov

    2003-12-01

    Full Text Available Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

  10. Transcriptome Analysis of Mouse Stem Cells and Early Embryos

    Science.gov (United States)

    Sharov, Alexei A; Piao, Yulan; Matoba, Ryo; Dudekula, Dawood B; Qian, Yong; VanBuren, Vincent; Falco, Geppino; Martin, Patrick R; Stagg, Carole A; Bassey, Uwem C; Wang, Yuxia; Carter, Mark G; Hamatani, Toshio; Aiba, Kazuhiro; Akutsu, Hidenori; Sharova, Lioudmila; Tanaka, Tetsuya S; Kimber, Wendy L; Yoshikawa, Toshiyuki; Jaradat, Saied A; Pantano, Serafino; Nagaraja, Ramaiah; Boheler, Kenneth R; Taub, Dennis; Hodes, Richard J; Longo, Dan L; Schlessinger, David; Keller, Jonathan; Klotz, Emily; Kelsoe, Garnett; Umezawa, Akihiro; Vescovi, Angelo L; Rossant, Janet; Kunath, Tilo; Hogan, Brigid L. M; Curci, Anna; D'Urso, Michele; Kelso, Janet; Hide, Winston

    2003-01-01

    Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine. PMID:14691545

  11. Natural selection of human embryos: decidualizing endometrial stromal cells serve as sensors of embryo quality upon implantation.

    Directory of Open Access Journals (Sweden)

    Gijs Teklenburg

    Full Text Available BACKGROUND: Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1beta, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. CONCLUSIONS: Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits

  12. Parental exposure to environmental concentrations of diuron leads to aneuploidy in embryos of the Pacific oyster, as evidenced by fluorescent in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Barranger, Audrey, E-mail: audrey.barranger@ifremer.fr [Ifremer, SG2M, Laboratory of Genetics and Pathology of Marine Molluscs, Avenue de Mus du Loup, 17390 La Tremblade (France); Ifremer, Department of Biogeochemistry and Ecotoxicology, Laboratory of Ecotoxicology, Rue de l’Ile d’Yeu, BP 21105, 44311 Nantes Cedex 03 (France); Benabdelmouna, Abdellah, E-mail: abdellah.benabdelmouna@ifremer.fr [Ifremer, SG2M, Laboratory of Genetics and Pathology of Marine Molluscs, Avenue de Mus du Loup, 17390 La Tremblade (France); Dégremont, Lionel [Ifremer, SG2M, Laboratory of Genetics and Pathology of Marine Molluscs, Avenue de Mus du Loup, 17390 La Tremblade (France); Burgeot, Thierry; Akcha, Farida [Ifremer, Department of Biogeochemistry and Ecotoxicology, Laboratory of Ecotoxicology, Rue de l’Ile d’Yeu, BP 21105, 44311 Nantes Cedex 03 (France)

    2015-02-15

    Highlights: • FISH was realized on oyster embryos from diuron-exposed genitors. • rDNA genes were used as probes on the interphase nuclei of embryo preparations. • Higher aneuploidy level was observed in embryos from diuron-exposed genitors. • Hypo- and hyperdiploid (triploid) nuclei were detected. - Abstract: Changes in normal chromosome numbers (i.e. aneuploidy) due to abnormal chromosome segregation may arise either spontaneously or as a result of chemical/radiation exposure, particularly during cell division. Coastal ecosystems are continuously subjected to various contaminants originating from urban, industrial and agricultural activities. Genotoxicity is common to several families of major environmental pollutants, including pesticides, which therefore represent a potential important environmental hazard for marine organisms. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to short-term exposure to the herbicide diuron at environmental concentrations during gametogenesis. In this paper, Fluorescent in situ hybridization (FISH) was used to further characterize diuron-induced DNA damage at the chromosomal level. rDNA genes (5S and 18-5.8-28S), previously mapped onto Crassostrea gigas chromosomes 4, 5 and 10, were used as probes on the interphase nuclei of embryo preparations. Our results conclusively show higher aneuploidy (hypo- or hyperdiploidy) level in embryos from diuron-exposed genitors, with damage to the three studied chromosomal regions. This study suggests that sexually developing oysters are vulnerable to diuron exposure, incurring a negative impact on reproductive success and oyster recruitment.

  13. Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

    Directory of Open Access Journals (Sweden)

    Li Xianyao

    2010-07-01

    Full Text Available Abstract Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1 causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi. Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB, cell cycle regulation (cyclin B2, CDK1, and CKI3, matrix metalloproteinases (MMPs and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR. A bioinformatics tool (Ingenuity Pathway Analysis used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections.

  14. Embryo quality predictive models based on cumulus cells gene expression

    Directory of Open Access Journals (Sweden)

    Devjak R

    2016-06-01

    Full Text Available Since the introduction of in vitro fertilization (IVF in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.

  15. Feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells by whole embryo freezing.

    Science.gov (United States)

    Higaki, Shogo; Mochizuki, Kentaro; Baba, Hiroko; Akashi, Yuichiro; Yamaha, Etsuro; Katagiri, Seiji; Takahashi, Yoshiyuki

    2009-08-01

    We investigated the feasibility of cryopreservation of zebrafish (Danio rerio) blastomeres and primordial germ cells (PGCs) by rapid freezing of dechorionated whole embryos at the blastula, gastrula and segmentation stages. Initially we examined the glass-forming properties and embryo toxicities of 5 cryoprotectants: methanol (MeOH), ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (1,3-BG). Embryos at the blastula and gastrula stages had high sensitivities to cryoprotectant toxicities and were fragile against mechanical damage. Thus the segmentation stage embryos, the PGCs of which were visualized by injecting green fluorescence protein-nos1 3'UTR mRNA, were frozen using solutions containing each cryoprotectant at 6 M (first trial) and 2 types of cryoprotectants at 3 M each (second trial). In the first trial, live PGCs were recovered from most of the embryos frozen with EG (about 2 cells/embryo); however, a few embryos had live PGCs when embryos were frozen with other cryoprotectants. In the second trial, a mixture of EG + PG better preserved the viability of PGCs in frozen embryos. Live PGCs were recovered from all embryos frozen with EG + PG (about 3 cells/embryo), and the survival rate of PGCs was estimated to be about 25% based on the number of live PGCs in fresh embryos (about 12 cells/embryo). The present study indicates that we can utilize rapid freezing of dechorionated whole embryos at the segmentation stage for the cryopreservation of PGCs.

  16. Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

    Institute of Scientific and Technical Information of China (English)

    YAN Jie; ZHU Guijin; LIU Jianxin; AI Jihui

    2000-01-01

    An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0.4%bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73% developed to the morula stage and 67.21% cavitated to blastocysts with 59.74 % hatching, as compared with 61.34% to morula stage, 48.47% to blastocysts and none hatching in the controls,respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

  17. Use of "excess" human embryos for stem cell research: protecting women's rights and health.

    Science.gov (United States)

    Cohen, C B

    2000-01-01

    Proposed National Institutes of Health guidelines for stem cell research are too narrowly drawn and do not adequately protect the freedom of choice and health of women who donate embryos. They need to be expanded to cover not only the point of embryo donation, but also that of embryo creation. Guidelines are provided to ensure that donors undergoing hyperstimulation and egg retrieval gave voluntary informed consent to the production of embryos that might later prove in excess. A standard for determining when embryos have been overproduced is presented to address the possibility that additional embryos will be created for stem cell research in violation of the guidelines and at risk to women's health.

  18. Embryo splitting

    Directory of Open Access Journals (Sweden)

    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

  19. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.

    Science.gov (United States)

    Eggan, K; Akutsu, H; Loring, J; Jackson-Grusby, L; Klemm, M; Rideout, W M; Yanagimachi, R; Jaenisch, R

    2001-05-22

    To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

  20. In vivo assay for the developmental competence of embryo-derived zebrafish cell lines

    NARCIS (Netherlands)

    Speksnijder, JE; Hage, WJ; Lanser, PH; Collodi, P; Zivkovic, D

    1997-01-01

    We have produced chimeric zebrafish embryos by transplanting permanent embryo-derived cell lines into blastula-stage embryos. Furthermore, we have established a fluorescent in vivo assay to monitor the developmental effects and fate of such transplanted cells using confocal laser scanning microscopy

  1. Factors affecting the cryosurvival of mouse two-cell embryos.

    Science.gov (United States)

    Critser, J K; Arneson, B W; Aaker, D V; Huse-Benda, A R; Ball, G D

    1988-01-01

    A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Transcript profiling of individual twin blastomeres derived by splitting two-cell stage murine embryos.

    Science.gov (United States)

    Roberts, R Michael; Katayama, Mika; Magnuson, Scott R; Falduto, Michael T; Torres, Karen E O

    2011-03-01

    In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition.

  3. Comprehensive embryo analysis of advanced maternal age-related aneuploidies and mosaicism by short comparative genomic hybridization.

    Science.gov (United States)

    Rius, Mariona; Daina, Gemma; Obradors, Albert; Ramos, Laia; Velilla, Esther; Fernández, Sílvia; Martínez-Passarell, Olga; Benet, Jordi; Navarro, Joaquima

    2011-01-01

    The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

  4. Organic and hybrid solar cells

    CERN Document Server

    Huang, Hui

    2014-01-01

    This book delivers a comprehensive evaluation of organic and hybrid solar cells and identifies their fundamental principles and numerous applications. Great attention is given to the charge transport mechanism, donor and acceptor materials, interfacial materials, alternative electrodes, device engineering and physics, and device stability. The authors provide an industrial perspective on the future of photovoltaic technologies.

  5. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  6. Osteoclast-like Cells in Early Zebrafish Embryos

    Directory of Open Access Journals (Sweden)

    Faiza Sharif

    2014-06-01

    Full Text Available Objective: Genes involved in bone and tissue remodelling in the vertebrates include matrix metalloproteinase-9 (mmp-9, receptor activator of necrosis factor κ-β (rank, cathepsin-k (Ctsk and tartrate-resistant acid phosphatase (TRAcP. We examine whether these markers are expressed in cells of zebrafish embryos of 1-5 days post fertilization. We also examine adult scales, which are known to contain mature osteoclasts, for comparison. Materials and Methods: In this experimental study, in situ hybrdisation, histochemistry and serial plastic and paraffin sectioning were used to analyse marker expression. Results: We found that mmp-9 mRNA, TRAcP enzyme and Ctsk YFP protein were expressed in haematopoietic tissues and in the cells scattered sparsely in the embryo. Ctsk and rank mRNA were both expressed in the branchial skeleton and in the developing pectoral fin. In these skeletal structures, histology showed that the expressing cells were located around the developing cartilage elements, in the parachondral tissue. In a transgenic zebrafish line with YFP coupled to Ctsk promoter, Ctsk expressing cells were found around pharyngeal skeletal elements. To see whether we could activate osteoclasts, we exposed prim-6 zebrafish embryos to a mixture of 1 μM dexamethasone and 1 μM vitaminutes D3. These compounds, which are known to trigger osteoclastogenensis in cell cultures, lead to an increase in intensity of Ctsk YFP expression around the skeletal elements. Conclusion: Our findings show that cells expressing a range of osteoclast markers are present in early larvae and can be activated by the addition of osteoclastogenic compounds.

  7. Stem cell research on other worlds, or why embryos do not have a right to life.

    Science.gov (United States)

    Blackford, R

    2006-03-01

    Anxieties about the creation and destruction of human embryos for the purpose of scientific research on embryonic stem cells have given a new urgency to the question of whether embryos have moral rights. This article uses a thought experiment involving two possible worlds, somewhat removed from our own in the space of possibilities, to shed light on whether early embryos have such rights as a right not to be destroyed or discarded (a "right to life"). It is argued that early embryos do not have meaningful interests or any moral rights. Accordingly, claims about the moral rights of embryos do not justify restrictions on stem cell research.

  8. Embryos, Clones, and Stem Cells: A Scientific Primer

    Directory of Open Access Journals (Sweden)

    Kenyon S. Tweedell

    2004-01-01

    Full Text Available This article is intended to give the nonspecialist an insight into the nuances of “clones”, cloning, and stem cells. It distinguishes embryonic and adult stem cells, their normal function in the organism, their origin, and how they are recovered to produce stem cell lines in culture. As background, the fundamental processes of embryo development are reviewed and defined, since the manipulation of stem cell lines into desired specialized cells employs many of the same events. Stem cells are defined and characterized and shown how they function in the intact organism during early development and later during cell regeneration in the adult. The complexity of stem cell recovery and their manipulation into specific cells and tissue is illustrated by reviewing current experimentation on both embryonic and adult stem cells in animals and limited research on human stem cell lines. The current and projected use of stem cells for human diseases and repair, along with the expanding methodology for the recovery of human embryonic stem cells, is described. An assessment on the use of human embryonic stem cells is considered from ethical, legal, religious, and political viewpoints.

  9. Preferential recruitment of the maternal centromere-specific histone H3 (CENH3) in oat (Avena sativa L.) × pearl millet (Pennisetum glaucum L.) hybrid embryos.

    Science.gov (United States)

    Ishii, Takayoshi; Sunamura, Naohiro; Matsumoto, Ayaka; Eltayeb, Amin Elsadig; Tsujimoto, Hisashi

    2015-12-01

    Chromosome elimination occurs frequently in interspecific hybrids between distantly related species in Poaceae. However, chromosomes from both parents behave stably in a hybrid of female oat (Avena sativa L.) pollinated by pearl millet (Pennisetum glaucum L.). To analyze the chromosome behavior in this hybrid, we cloned the centromere-specific histone H3 (CENH3) genes of oat and pearl millet and produced a pearl millet-specific anti-CENH3 antibody. Application of this antibody together with a grass species common anti-CENH3 antibody revealed the dynamic CENH3 composition of the hybrid cells before and after fertilization. Despite co-expression of CENH3 genes encoded by oat and pearl millet, only an oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo. Oat CENH3 enables a functional centromere in pearl millet chromosomes in an oat genetic background. Comparison of CENH3 genes among Poaceae species that show chromosome elimination in interspecific hybrids revealed that the loop 1 regions of oat and pearl millet CENH3 exhibit exceptionally high similarity.

  10. Generation and developmental characteristics of porcine tetraploid embryos and tetraploid/diploid chimeric embryos.

    Science.gov (United States)

    He, Wenteng; Kong, Qingran; Shi, Yongqian; Xie, Bingteng; Jiao, Mingxia; Huang, Tianqing; Guo, Shimeng; Hu, Kui; Liu, Zhonghua

    2013-10-01

    The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P<0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

  11. Abnormalities occurring during female gametophyte development result in the diversity of abnormal embryo sacs and leads to abnormal fertilization in indica/japonica hybrids in rice.

    Science.gov (United States)

    Zeng, Yu-Xiang; Hu, Chao-Yue; Lu, Yong-Gen; Li, Jin-Quan; Liu, Xiang-Dong

    2009-01-01

    Embryo sac abortion is one of the major reasons for sterility in indica/japonica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indica/japonica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.

  12. Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indicaljaponica Hybrids in Rice

    Institute of Scientific and Technical Information of China (English)

    Yu-Xiang Zeng; Chao-Yue Hu; Yong-Gen Lu; Jin-Quan Li; Xiang-Dong Liu

    2009-01-01

    Embryo sac abortion is one of the major masons for sterility in indicaljaponica hybrids In rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indicaljaponica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagamatogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucallus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.

  13. The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

    Science.gov (United States)

    Kong, Xiangyi; Yang, Shuting; Gong, Fei; Lu, Changfu; Zhang, Shuoping; Lu, Guangxiu; Lin, Ge

    2016-01-01

    Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI) treatment cycles, n = 799) were classified as follows: less than 5 cells (10C; n = 42). Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In 10C embryos increased compared to 7-8C embryos (45.8%, 33.3% vs. 11.1%, respectively). In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (10C). In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

  14. Towards stable silicon nanoarray hybrid solar cells.

    Science.gov (United States)

    He, W W; Wu, K J; Wang, K; Shi, T F; Wu, L; Li, S X; Teng, D Y; Ye, C H

    2014-01-16

    Silicon nanoarray hybrid solar cells benefit from the ease of fabrication and the cost-effectiveness of the hybrid structure, and represent a new research focus towards the utilization of solar energy. However, hybrid solar cells composed of both inorganic and organic components suffer from the notorious stability issue, which has to be tackled before the hybrid solar cells could become a viable alternative for harvesting solar energy. Here we show that Si nanoarray/PEDOT:PSS hybrid solar cells with improved stability can be fabricated via eliminating the water inclusion in the initial formation of the heterojunction between Si nanoarray and PEDOT:PSS. The Si nanoarray hybrid solar cells are stable against rapid degradation in the atmosphere environment for several months without encapsulation. This finding paves the way towards the real-world applications of Si nanoarray hybrid solar cells.

  15. Generation of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos.

    Science.gov (United States)

    Artus, Jérôme; Hadjantonakis, Anna-Katerina

    2011-01-01

    From the hybrid creatures of the Greek and Egyptian mythologies, the concept of the chimera has evolved and, in modern day biology, refers to an organism comprises of at least two populations of genetically distinct cells. Mouse chimeras have proven an invaluable tool for the generation of genetically modified strains. In addition, chimeras have been extensively used in developmental biology as a powerful tool to analyze the phenotype of specific mutations, to attribute function to gene products and to address the question of cell autonomy versus noncell autonomy of gene function. This chapter describes a simple and economical technique used to generate mouse chimeras by embryo aggregation. Multiple aggregation combinations are described each of which can be tailored to answer particular biological questions.

  16. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...

  17. Research on Growth Behavior of Embryos for Bovine and Murine on Primary Murine Embryos Fibroblast Cell Feeder Layer

    Institute of Scientific and Technical Information of China (English)

    AN Li-long; XIAO Mei; FENG Xiu-Liang; DOU Zhong-ying; QIU Huai; YANG Qi; LEI An-min; YANG Chun-rong; GAO Zhi-min

    2002-01-01

    The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the mixture-like colonies. Compared with Murine ICM, the bovine ICM grew more fast. So, the bovine ICM was passaged at first after a culture of approximately 5 - 6 days in vitro, but murine ICM was passaged at first after an attachment of 3 - 4 days on PMEF feeder layer. The mixture colonies of bovine ICM differentiated very early, while the others differentiated very late. Most ICM-like mass of Bovine grew in a defined spot, but bovine ICMs like stream and ICMs like net proliferated fast and dispersed quickly. We found that the single blastomeres derived from late bovine morula and late murine morula formed sub-blastophere; moreover, the bovine ICM cell would differentiate rapidly if the trophoblast was removed.

  18. Endocardial tip cells in the human embryo - facts and hypotheses.

    Directory of Open Access Journals (Sweden)

    Mugurel C Rusu

    Full Text Available Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43-56 days were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin, CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.

  19. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Yeoman, Richard R; Nusser, Kevin D; Wolf, Don P

    2002-05-01

    Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

  20. Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

    Institute of Scientific and Technical Information of China (English)

    JIA JING QIU; WU WEN ZHANG; ZHI LI WU; YI HONG WANG; MIN QIAN; YI PING LI

    2003-01-01

    One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos,which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is oftenassumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in culturedembryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of[35S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strainwere compared in their development both in vitro and in vivo. The detection of TRC expression, a markerof ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even inthe 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as comparedwith normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG,TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo.But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidencessuggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to thedisability in initiating ZGA, but to a delay of ZGA.

  1. Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

    DEFF Research Database (Denmark)

    Svarcova, Olga; Dinnyes, A.; Polgar, Z.

    2009-01-01

    displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

  2. Embryo and endosperm development in caryopses of hybrids from crosses between tetraploid wheats and their alloplasmic lines with rye

    Directory of Open Access Journals (Sweden)

    Wijciech Sodkiewicz

    2014-01-01

    Full Text Available Data concerning the embryo and endosperm development in twenty-day-old caryopses of hybrids obtained as the result of pollination with rye pollen of tetraploid wheats (Triticum dicoccoides, T. dicoccum, T. durum and T. polonicum, their alloplasmic lines with T. timopheevi plasma and aIlaplasmic T. timopheevi lines with cytoplasma of the above mentioned tetraploid wheats and hexaploid wheat (T. macha were analysed. A high variability was noted between the tetraploid wheats as regards the degree of development of the embryo and of the endosperm in the hybrid caryopses and a decisive influence of the wheat genotype on these characters. The data for alloplasmic lines showed that the cytoplasm may have a modifying effect on the expression of these genotype characters.

  3. Functional evaluation of ES-somatic cell hybrids in vitro and in vivo.

    Science.gov (United States)

    Sumer, Huseyin; Kim, Kitai; Liu, Jun; Ng, Kitwa; Daley, George Q; Verma, Paul J

    2014-06-01

    Embryonic stem cells (ESCs) have previously been reported to reprogram somatic cells following fusion. The resulting ES-somatic cell hybrids have been shown to adopt the transcriptional profile of ESCs, suggesting that the pluripotent program is dominant. ES-somatic cell hybrids have most characteristics of pluripotent cells in vitro; however, it remains unclear whether the somatic genome is an active partner in the hybrid cells or simply retained predominately as silent cargo. Furthermore, the functional properties of ES-somatic cell hybrids in vivo have been limited to studies on their contribution to teratomas and developing embryos/chimeras. The extent of their pluripotency remains largely unclear. Here we determined that the somatic genome is actively transcribed by generating ES-somatic cell hybrids using Rag2-deficient ESCs fused to autologous wild-type somatic cells. Rag2 expression was detected during in vitro differentiation, suggesting that the somatic genome follows the correct temporal cues during differentiation. Furthermore, ES-somatic cell hybrids maintain their tetraploid state following 4 weeks of differentiation in vivo and are immune tolerated when transferred into matched individuals. The ES-somatic cell hybrids can efficiently differentiate into hematopoietic precursors in both myeloid and lymphoid lineages in vitro, suggesting that the somatic genome is actively transcribed following cell fusion based reprogramming. However, the ES-somatic cell hybrids showed an altered hematopoietic potential following in vitro differentiation and were unable to show hematopoietic engraftment in a mouse model.

  4. Development of resistant tomato population with bacterial canker resistance genes from interspecific hybrids by the support of embryo rescue

    Directory of Open Access Journals (Sweden)

    Aylin KABAŞ

    2016-06-01

    Full Text Available Bacterial canker is one of the most important diseases causing economic yield loss in tomato production areas in the world. The best way to control for this disease is to use resistant varieties. However, there are few studies on variety breeding studies of this disease compared with other disease resistant breeding studies. In this study we aimed to improve inbred lines carrying bacterial canker resistance genes to use in the breeding of resistant varieties. Susceptible inbred line AK1 (S. esculentum and resistant LA2157 (S. peruvianum were crossed. Embryo rescue and ovule culture techniques were applied in 30 fruits to get F1 hybrids. Rescued embryos and immature ovules were cultured in petri dishes containing solidified MS medium without hormone. 30 healty embryos were excised and cultured from 30 fruits 27-61 day old (1 embryo fruit-1 in embryo rescue method. The two surviving plants from acclimatization were transferred to the greenhouse to get their BC1 progenies. Resistance tests were performed according to the stem inoculation method in the BC1 and BC2 progenies. The mixture of 14 aggressive Turkish Cmm strains were used to confirm the resistance. The plants were valued by 0-4 scale. Plants with 0 and 1 scale values were used to obtain next progenies. A total of 80 BC3 resistant progenies were transferred to our variety breeding programme.

  5. Use of fluorescence in situ hybridization to assess the chromosomal status of embryos obtained from cryopreserved oocytes.

    Science.gov (United States)

    Cobo, A; Rubio, C; Gerli, S; Ruiz, A; Pellicer, A; Remohí, J

    2001-02-01

    To analyze the chromosomal status of human embryos obtained from frozen-thawed oocytes. Fluorescence in situ hybridization analysis of embryos obtained after oocyte cryopreservation. Department of Obstetrics and Gynecology at the University of Perugia, Italy, and the Instituto Valenciano de Infertilidad, Spain. Oocyte donors (n = 43). Fertilization, development, and chromosomal status of the embryos were compared with a control group (n = 18) of patients undergoing preimplantation genetic diagnosis for sex chromosome-linked diseases. Collection of oocytes after conventional ovarian stimulation and cryopreservation using propanediol as the cryoprotectant and a slow freezing procedure. Microinjection of surviving metaphase II oocytes and evaluation of fertilization and embryo development up to blastocyst stage. Chromosomal analysis after embryo biopsy. Survival, fertilization, and blastocyst rates. Embryo chromosomal analysis employing specific probes for chromosomes 13,18,21, X and Y. The overall survival rate was 59.4%. There was no difference between cryopreservation and control groups in fertilization rates (76.5% vs. 90.5%) or blastocyst development (29.6% vs. 35%). The percentage of blastocysts from the original number of cryopreserved oocytes was only 5.6%, comparable to the 5.9% obtained in the control group. The percentage of embryos with abnormal number of chromosomes in the cryopreservation group (28.6%) was comparable to the 26% observed in the controls. Fertilization and cleavage rates after oocyte freezing are acceptable. Survival is, however, still poor, leading to overall results that make the technique clinically inefficient. There is no increase in the rate of chromosomal abnormalities, indicating that the technique is, nevertheless, safe enough to be further explored and improved.

  6. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization.

    Science.gov (United States)

    Cheng, Christina N; Li, Yue; Marra, Amanda N; Verdun, Valerie; Wingert, Rebecca A

    2014-07-17

    The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

  7. Genomic imprinting in primate embryos and embryonic stem cells.

    Science.gov (United States)

    Mitalipov, Shoukhrat M

    2006-01-01

    Embryonic stem (ES) cells hold promise for cell and tissue replacement approaches to treating human diseases. However, long-term in vitro culture and manipulations of ES cells may adversely affect their epigenetic integrity including imprinting. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. We demonstrated aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ES cell lines while SNRPN and NDN were normally imprinted and expressed from the paternal allele. In contrast, expanded blastocyst-stage embryos, from which these ES cells were derived, exhibited normal paternal expression of IGF2 and maternal expression of H19. To test the possibility that aberrant methylation at an imprinting centre (IC) upstream of H19 accounts for the relaxed imprinting of IGF2 and H19, we performed comprehensive methylation analysis by investigating methylation profiles of CpG sites within the IGF2/H19 IC. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analysed ES cell lines consistent with biallelic expression of these genes. Cellular overproliferation and tumour formation resulting from tissue or cell transplantation are potential problems that must be addressed before clinical trials of ES cell-based therapy are initiated.

  8. Nanostructured organic and hybrid solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Weickert, Jonas; Dunbar, Ricky B.; Hesse, Holger C.; Wiedemann, Wolfgang; Schmidt-Mende, Lukas [Department of Physics and Center for NanoScience (CeNS), Ludwig-Maximilians University (LMU) Munich, Amalienstr. 54, 80799 Munich (Germany)

    2011-04-26

    This progress report highlights recent developments in nanostructured organic and hybrid solar cells. The authors discuss novel approaches to control the film morphology in fully organic solar cells and the design of nanostructured hybrid solar cells. The motivation and recent results concerning fabrication and effects on device physics are emphasized. The aim of this review is not to give a summary of all recent results in organic and hybrid solar cells, but rather to focus on the fabrication, device physics, and light trapping properties of nanostructured organic and hybrid devices. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  9. DNA replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos.

    Science.gov (United States)

    Encalada, S E; Martin, P R; Phillips, J B; Lyczak, R; Hamill, D R; Swan, K A; Bowerman, B

    2000-12-15

    In early Caenorhabditis elegans embryos, asymmetric cell divisions produce descendants with asynchronous cell cycle times. To investigate the relationship between cell cycle regulation and pattern formation, we have identified a collection of embryonic-lethal mutants in which cell divisions are delayed and cell fate patterns are abnormal. In div (for division delayed) mutant embryos, embryonic cell divisions are delayed but remain asynchronous. Some div mutants produce well-differentiated cell types, but they frequently lack the endodermal and mesodermal cell fates normally specified by a transcriptional activator called SKN-1. We show that mislocalization of PIE-1, a negative regulator of SKN-1, prevents the specification of endoderm and mesoderm in div-1 mutant embryos. In addition to defects in the normally asymmetric distribution of PIE-1, div mutants also exhibit other losses of asymmetry during early embryonic cleavages. The daughters of normally asymmetric divisions are nearly equal in size, and cytoplasmic P-granules are not properly localized to germline precursors in div mutant embryos. Thus the proper timing of cell division appears to be important for multiple aspects of asymmetric cell division. One div gene, div-1, encodes the B subunit of the DNA polymerase alpha-primase complex. Reducing the function of other DNA replication genes also results in a delayed division phenotype and embryonic lethality. Thus the other div genes we have identified are likely to encode additional components of the DNA replication machinery in C. elegans.

  10. B1 SOX coordinate cell specification with patterning and morphogenesis in the early zebrafish embryo.

    Directory of Open Access Journals (Sweden)

    Yuichi Okuda

    2010-05-01

    Full Text Available The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization, RT-PCR, and microarray analyses. Importantly, these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: (1 early dorsoventral patterning by controlling bmp2b/7; (2 gastrulation movements via the regulation of pcdh18a/18b and wnt11, a non-canonical Wnt ligand gene; (3 neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively and ascl1a (negatively, and regional transcription factor genes, e.g., hesx1, zic1, and rx3; and (4 neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX. We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate

  11. Cryopreservation of primordial germ cells by rapid cooling of whole zebrafish (Danio rerio) embryos.

    Science.gov (United States)

    Higaki, Shogo; Mochizuki, Kentaro; Akashi, Yuichiro; Yamaha, Etsuro; Katagiri, Seiji; Takahashi, Yoshiyuki

    2010-04-01

    The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EG and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EG and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs.

  12. Feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells by whole embryo freezing

    OpenAIRE

    HIGAKI, SHOGO; Mochizuki, Kentaro; Baba, Hiroko; Akashi, Yuichiro; Yamaha, Etsuro; Katagiri, Seiji; Takahashi, Yoshiyuki

    2009-01-01

    We investigated the feasibility of cryopreservation of zebrafish (Danio rerio) blastomeres and primordial germ cells (PGCs) by rapid freezing of dechorionated whole embryos at the blastula, gastrula and segmentation stages. Initially we examined the glass-forming properties and embryo toxicities of 5 cryoprotectants: methanol (MeOH), ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (1,3-BG). Embryos at the blastula and gastrula stages had high se...

  13. Donating embryos for human embryonic stem cell (hESC) research: a committee opinion.

    Science.gov (United States)

    2013-10-01

    hESC research is an ethically acceptable use of human embryos that are in excess of those needed to meet the fertility goals of patients. The ethical basis for this view and issues to be considered during the informed consent process for the donation of embryos are developed in this document. This report replaces the Committee's 2009 report, "Donating spare embryos for stem cell research" (Fertil Steril 2009;91:667-70).

  14. Dechorionation of medaka embryos and cell transplantation for the generation of chimeras.

    Science.gov (United States)

    Porazinski, Sean R; Wang, Huijia; Furutani-Seiki, Makoto

    2010-12-22

    Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out (1). Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species (2), thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps (3) so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

  15. Dissection of Cell Division Processes in the One Cell Stage Caenorhabditis elegans Embryo by Mutational Analysis

    OpenAIRE

    Gönczy, Pierre; Schnabel, Heinke; Kaletta, Titus; Amores, Ana Duran; Hyman, Tony; Schnabel, Ralf

    1999-01-01

    To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III wi...

  16. Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

    Directory of Open Access Journals (Sweden)

    Svetlana Gavrilov

    2011-01-01

    Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

  17. Type II cytokeratin gene expression is indicative of early cell differentiation in the chick embryo

    Energy Technology Data Exchange (ETDEWEB)

    Charlebois, T.S.

    1988-01-01

    Embryonic development in vertebrates appears to involve a series of inductive tissue interactions that lead to regional specializations, which eventually become elaborated in the basic body plan of the embryo. The inductive interactions leading to early regionalization of the embryo are often particularly difficult to evaluate because of the absence of available morphological or biochemical evidence that such events have occurred. In the 36 hour chick embryo, the regional subdivision of the early ectoderm is evidence by a marked lens-forming bias in the head ectoderm, which is absent in the presumptive dorsal epidermis of the trunk region. As a strategy for isolating genes whose differential expression might reflect this regional subdivision, a cDNA library from 36 hour embryos was prepared and screened for differential hybridization to ({sup 32}P)cDNA probes synthesized using template RNA isolated from 36 hour head ectoderm and trunk ectoderm. A cDNA clone (T4) was isolated which hybridizes to transcripts present at much higher levels in trunk ectoderm than in head ectoderm. Partial nucleotide and deduced amino acid sequences of this clone indicate that it represents a gene encoding a type II cytokeratin. The distribution of transcripts complementary to the T4 probe was evaluated in early embryos using RNA gel blot analysis and in situ hybridization to tissue sections.

  18. Programmed cell death and hybrid incompatibility.

    Science.gov (United States)

    Frank, S A; Barr, C M

    2003-01-01

    We propose a new theory to explain developmental aberrations in plant hybrids. In our theory, hybrid incompatibilities arise from imbalances in the mechanisms that cause male sterility in hermaphroditic plants. Mitochondria often cause male sterility by killing the tapetal tissue that nurtures pollen mother cells. Recent evidence suggests that mitochondria destroy the tapetum by triggering standard pathways of programmed cell death. Some nuclear genotypes repress mitochondrial male sterility and restore pollen fertility. Normal regulation of tapetal development therefore arises from a delicate balance between the disruptive effects of mitochondria and the defensive countermeasures of the nuclear genes. In hybrids, incompatibilities between male-sterile mitochondria and nuclear restorers may frequently upset the regulatory control of programmed cell death, causing tapetal abnormalities and male sterility. We propose that hybrid misregulation of programmed cell death may also spill over into other tissues, explaining various developmental aberrations observed in hybrids.

  19. Fresh or frozen? Classifying 'spare' embryos for donation to human embryonic stem cell research.

    Science.gov (United States)

    Ehrich, Kathryn; Williams, Clare; Farsides, Bobbie

    2010-12-01

    United Kingdom (UK) funding to build human embryonic stem cell (hESC) derivation labs within assisted conception units (ACU) was intended to facilitate the 'In-vitro fertilisation (IVF)-stem cell interface', including the flow of fresh 'spare' embryos to stem cell labs. However, in the three sites reported on here, which received this funding, most of the embryos used for hESC research came from long term cryopreservation storage and/or outside clinics. In this paper we explore some of the clinical, technical, social and ethical factors that might help to explain this situation. We report from our qualitative study of the ethical frameworks for approaching women/couples for donation of embryos to stem cell research. Members of staff took part in 44 interviews and six ethics discussion groups held at our study sites between February 2008 and October 2009. We focus here on their articulations of social and ethical, as well as scientific, dimensions in the contingent classification of 'spare' embryos, entailing uncertainty, fluidity and naturalisation in classifying work. Social and ethical factors include acknowledging and responding to uncertainty in classifying embryos; retaining 'fluidity' in the grading system to give embryos 'every chance'; tensions between standardisation and variation in enacting a 'fair' grading system; enhancement of patient choice and control, and prevention of regret; and incorporation of patients' values in construction of ethically acceptable embryo 'spareness' ('frozen' embryos, and embryos determined through preimplantation genetic diagnosis (PGD) to be genetically 'affected'). We argue that the success of the 'built moral environment' of ACU with adjoining stem cell laboratories building projects intended to facilitate the 'IVF-stem cell interface' may depend not only on architecture, but also on the part such social and ethical factors play in configuration of embryos as particular kinds of moral work objects.

  20. Dynamic Properties of Electrotonic Coupling between Cells of Early Xenopus Embryos.

    Science.gov (United States)

    Dicaprio, R A; French, A S; Sanders, E J

    1974-05-01

    Frequency response functions were measured between the cells of Xenopus laevis embryos during the first two cleavage stages. Linear systems theory was then used to produce electronic models which account for the electrical behavior of the systems. Coupling between the cells may be explained by models which have simple resistive elements joining each cell to its neighbors. The vitelline, or fertilization, membrane which surrounds the embryos has no detectable resistance to the passage of electric current. The electrical properties of the four-cell embryo can only be explained by the existence of individual junctions linking each pair of cells. This arrangement suggests that electrotonic coupling is important in the development of the embryos, at least until the four-cell stage.

  1. Radiation-induced emission from golden hamster embryo cells

    Science.gov (United States)

    Miyazaki, Tetsuo; Nagasaka, Shigeru; Maeda, Isao; Matsumoto, Takuro; Koyama, Shinji; Kodama, Seiji; Watanabe, Masami

    1996-06-01

    Emission from high-energy-electron-irradiated golden hamster embryo (GHE) cells has been studied over the temperature range 12-300 K both by a one-shot-single-photon-counting method and by photocurrent measurements with an oscilloscope. Emission from the irradiated phosphate buffered saline (PBS) also has been studied. The emission spectra from PBS at 12 and 77 K show a maximum around 330 and 380 nm, respectively, which are the same spectra as those from irradiated pure H 2O. The emission from irradiated GHE consists of the new band at 480 nm in addition to the emission from H 2O. The 480 nm emission is observed at the temperature range of 12-300 K, though the emission at 300 K is much lower than that at low temperature. The 480 nm emission is ascribed to the transition from excited organic substances in GHE cells. The intensity of 480 nm emission at 300 K increases linearly with increasing irradiation-dose in the range of 11-600 Gy.

  2. HYBRID FUEL CELL-SOLAR CELL SPACE POWER SUBSYSTEM CAPABILITY.

    Science.gov (United States)

    This report outlines the capabilities and limitations of a hybrid solar cell- fuel cell space power subsystem by comparing the proposed hybrid system...to conventional power subsystem devices. The comparisons are based on projected 1968 capability in the areas of primary and secondary battery, fuel ... cell , solar cell, and chemical dynamic power subsystems. The purpose of the investigation was to determine the relative merits of a hybrid power

  3. Abnormalities in centrosome number in human embryos and embryonic stem cells.

    Science.gov (United States)

    Gu, Yi-Fan; OuYang, Qi; Dai, Can; Lu, Chang-Fu; Lin, Ge; Gong, Fei; Lu, Guang-Xiu

    2016-05-01

    Chromosomal abnormalities are common in human embryos. Previous studies have suggested links between centrosome number and chromosome abnormalities, but information regarding abnormalities in centrosome number in human embryos is limited. We analyzed abnormalities in centrosome number in human embryos and embryonic stem cells (hESCs). Following normal fertilization, supernumerary centrosomes were present at rates of 7.3% in two-pronucleus (2PN)-stage zygotes and 6.5% in first-cleavage zygotes. Supernumerary centrosomes were also detected in 24.4% of blastomeres from 60% of embryos derived from 2PN zygotes. Conversely, in mono- (1PN) and tri-pronucleus (3PN) zygotes, the frequency of abnormal centrosome number increased substantially at first cleavage. Rates in blastomeres of Day-3 embryos, however, were about the same between embryos derived from 1PN and 2PN zygotes, whereas abnormalities in centrosome number were higher in those from 3PN zygotes. By comparison, the rate of abnormal centrosome numbers in hESCs was 1.5-11.2%. Thus, abnormalities in centrosome number existed in human zygotes and cleaved embryos-especially those resulting from aberrant fertilization-but the frequency of such abnormalities was lower in hESCs derived from these embryos. These findings identify a source of the chromosomal instability in human embryos and hESCs, and highlight new safety issues for human assisted reproductive technology. Mol. Reprod. Dev. 83: 392-404, 2016. © 2016 Wiley Periodicals, Inc.

  4. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

    Science.gov (United States)

    Galli, C; Colleoni, S; Duchi, R; Lagutina, I; Lazzari, G

    2007-03-01

    Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

  5. Dissection of Cell Division Processes in the One Cell Stage Caenorhabditis elegans Embryo by Mutational Analysis

    Science.gov (United States)

    Gönczy, Pierre; Schnabel, Heinke; Kaletta, Titus; Amores, Ana Duran; Hyman, Tony; Schnabel, Ralf

    1999-01-01

    To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes. PMID:10085292

  6. Teratogenic and cytotoxic effects of VOsalen complex on chicken embryos, hepatic and fibroblastic- cell cultures

    Directory of Open Access Journals (Sweden)

    Abdolmaleki A

    2013-04-01

    Full Text Available Background: Salen metal complexes are used successfully in a wide range of asymmet-ric reactions and important in the pharmaceutical and industry. On the toxicity of salen vanadium oxide (VOsalen on embryo and cell cultures, little information is available. In the present study, the toxic and teratogenic effects of VOsalen was evaluated against chicken embryos as a animal model and liver and fibroblast cell cultures which was derived from the embryo.Methods: The VOsalen compound was synthesized. The compound solution was inject-ed in triplicate examination, in the air sac of the eggs, at third day of incubation. Treat-ed and control eggs, on day 19 of incubation opened and embryos were weighted, then mortality rate was recorded. The liver and fibroblast cell culture were treated by this and survival fraction was recorded.Results: The survived fraction of the embryos depends on the compound concentration. In concentration of 300μM/egg, 36/32% of the embryos survived and the Lethal dose 50% (LD50 was 226/37 μM/egg. Morphological study of the treated embryos showed retarded growth, and skeletal staining showed the deletion of caudal vertebrate. The compound was inhibited liver and fibroblast cells growth with IC50 1047/25 and 1036/82μM respectively. The cytoplasm of treated cells became dense and their interco-nnections were loosed.Conclusion: The VOsalen compound had low toxic effects against the embryos and the cultured cells at the concentrations. Significant cytotoxic effect was not observed in the treated cells. However the proliferative cells were affected significantly in comparison with the cells which their growth was stopped. The effect of VOsalen compound against replication of liver cells were lower than fibroblast cells.

  7. Improved Method for Ex Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

    Directory of Open Access Journals (Sweden)

    Timo Schomann

    2013-01-01

    Full Text Available The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6 of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

  8. Polarity and cell division orientation in the cleavage embryo: from worm to human

    Science.gov (United States)

    Ajduk, Anna; Zernicka-Goetz, Magdalena

    2016-01-01

    Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells—blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos. PMID:26660321

  9. Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos.

    Science.gov (United States)

    Higaki, Shogo; Eto, Yoshiki; Kawakami, Yutaka; Yamaha, Etsuro; Kagawa, Noriko; Kuwayama, Masashige; Nagano, Masashi; Katagiri, Seiji; Takahashi, Yoshiyuki

    2010-04-01

    This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.

  10. Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo.

    Science.gov (United States)

    Onjiko, Rosemary M; Moody, Sally A; Nemes, Peter

    2015-05-26

    Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo.

  11. Generating porcine chimeras using inner cell mass cells and parthenogenetic preimplantation embryos.

    Directory of Open Access Journals (Sweden)

    Kazuaki Nakano

    Full Text Available BACKGROUND: The development and validation of stem cell therapies using induced pluripotent stem (iPS cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. METHODOLOGY/SIGNIFICANT PRINCIPAL FINDINGS: In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4-8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3% was similar to that after the injection of ICMs into morulae (24/29, 82.8%. We also found that 4-8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%. After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. CONCLUSION/SIGNIFICANCE: Our findings indicate that the aggregation method using parthenogenetic morulae or 4-8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated

  12. Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

    Science.gov (United States)

    Nakano, Kazuaki; Watanabe, Masahito; Matsunari, Hitomi; Matsuda, Taisuke; Honda, Kasumi; Maehara, Miki; Kanai, Takahiro; Hayashida, Gota; Kobayashi, Mirina; Kuramoto, Momoko; Arai, Yoshikazu; Umeyama, Kazuhiro; Fujishiro, Shuh-hei; Mizukami, Yoshihisa; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Background The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. Methodology/Significant Principal Findings In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. Conclusion/Significance Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such

  13. Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage.

    Science.gov (United States)

    Zhao, Q; Qiu, Y G; Tian, J T; Wang, C S; An, T Z

    2016-11-17

    The cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P M-embryos were octoploid before the first cleavage. Furthermore, 81.25% of the blastomeres of blastocysts developed from the M-embryos showed abnormal ploidy compared with those developed from the G1-embryos (22.55%). However, some of the blastomeres remained diploid in all the M-embryos tested. A portion of the blastomeres restored normal diploidy in some of the M-embryos at the blastocyst stage. This finding provides an explanation for M-embryos developing to term.

  14. Stem cells from residual IVF-embryos - Continuation of life justifies isolation.

    Science.gov (United States)

    Bongaerts, Ger P A; Severijnen, René S V M

    2007-01-01

    Embryonic stem cells are undifferentiated pluripotent cells that can indefinitely grow in vitro. They are derived from the inner mass of early embryos. Because of their ability to differentiate into all three embryonic germ layers, and finally into specialized somatic cell types, human embryonic stem cells represent important material for studying developmental biology and cell replacement therapy. They are usually isolated from excess human IVF-embryos. Since many people regard isolation of human stem cells as intentional killing of the embryo, it is a very difficult ethical problem. Similar feelings concern medical or scientific use of these stem cells. Is this feeling correct, or does it arise from a sentimental view? The problem encloses two aspects: (i) use of stem cells for medical therapy and scientific research and (ii) isolation of stem cells from human IVF-embryos. Worldwide human tissues are cultured, transplanted and used for medical and scientific research. Therefore, it may be concluded that factual use of human embryonic stem cells cannot be a real ethical problem. The main key of the problem seems to be hidden in the exact definition of 'death'; in other words: is there nothing between 'death' and 'life'? Bacterial spores, lyophilised bacteria and other micro-organisms, micro-organisms stored in glycerol mixtures at -80 degrees C and tissue cultures and sperm cells stored in liquid nitrogen, they are all neither dead nor alive, but still viable. From this point it is clear that there is more than the antithesis 'dead' versus 'alive'. In addition, we think that there is still another alternative: partial death. The present view concerning isolation of stem cells implies that residual embryos and thus new human lives are killed, and that therefore these embryos must be (passively) destroyed. However, it is especially the very well planned IVF-procedure that makes that passive destruction of not-implanted embryos means intentional killing. By isolation

  15. Design and fabrication of an integrated cell processor for single embryo cell manipulation.

    Science.gov (United States)

    Park, Jungyul; Jung, Seng-Hwan; Kim, Young-Ho; Kim, Byungkyu; Lee, Seung-Ki; Park, Jong-Oh

    2005-01-01

    This paper presents an integrated cell processor for the automatic handling of individual embryo cells. The integrated processor can perform various functions such as cell transport, isolation, orientation, and immobilization. These functions are indispensable and frequently used for the manipulation of single cells, but can only be carried out by a skillful operator. The purpose of this study was the integration and automation of these functions for effective cell manipulation, using a MEMS approach. The isolation of a cell was performed using polypyrrole (PPy) valves in a microchannel into which cells were transported. The orientation of cells was controlled by electrorotation (ER), and the target cell was immobilized by suction from a microhole. All of these functions were seamlessly realized on a single chip. Excellent experimental results with mouse (B6CBA) embryo cells showed that this device could substitute for routine and cumbersome manual work. It is expected that the integrated chip will contribute significantly to faster and more reliable manipulation of cells.

  16. Pollen Viability, Pistil Receptivity, and Embryo Development in Hybridization of Nelumbo nucifera Gaertn

    Directory of Open Access Journals (Sweden)

    Yan-Li Wang

    2012-01-01

    Full Text Available Seed set is usually low and differs for different crosses of flower lotus (Nelumbo nucifera Gaertn.. The reasons remain unknown, and this has a negative impact on lotus breeding. To determine the causes, we carried out two crosses of flower lotus, that is, “Jinsenianhua” × “Qinhuaihuadeng” and “Qinhuaihuadeng” × “Jinsenianhua” and pollen viability, pistil receptivity, and embryo development were investigated. The pollen grains collected at 05:00-06:00 hrs had the highest viability, and the viabilities of “Jinsenianhua” and “Qinhuaihuadeng” were 20.6 and 15.7%, respectively. At 4 h after artificial pollination, the number of pollen grains germinating on each stigma reached a peak: 63.0 and 17.2 per stigma in “Jinsenianhua” × “Qinhuaihuadeng” and “Qinhuaihuadeng” × “Jinsenianhua”, respectively. At 1 d after artificial pollination, the percentages of normal embryos in the two crosses were 55.0 and 21.9%, respectively; however, at 11 d after pollination, the corresponding percentages were 20.8 and 11.2%. Seed sets of the two crosses were 17.9 and 8.0%, respectively. The results suggested that low pistil receptivity and embryo abortion caused low seed set in “Qinhuaihuadeng” × “Jinsenianhua”, whereas low fecundity of “Jinsenianhua” × “Qinhuaihuadeng” was mainly attributable to embryo abortion.

  17. Embryonic stem cell as nuclear donor could promote in vitro development of the heterogeneous reconstructed embryo

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The nucleus of a somatic cell could be dedifferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellucida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic stem cells was 16.1%, which was significantly higher than that of both the adult mouse fibroblast cells (0%-3.1%, P < 0.05) and fetus mouse fibroblast cells (2.1%-3.7%, P < 0.05). Chromosome analysis confirmed that blastocyst cells were derived from ES donor cell. These observations show that reprogramming is easier in interspecific embryos reconstructed with ES cells than that reconstructed with somatic cells, and that ES cells have the higher ability to direct the reconstructed embryos development normally than fibroblast cells.

  18. Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis

    Science.gov (United States)

    Vera-Rodriguez, Maria; Chavez, Shawn L.; Rubio, Carmen; Pera, Renee A. Reijo; Simon, Carlos

    2015-01-01

    Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT–qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n=85). Our data indicate that the chromosomal status of aneuploid embryos (n=26), including those that are mosaic (n=3), correlates with significant differences in the duration of the first mitotic phase when compared with euploid embryos (n=28). Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 h of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature. PMID:26151134

  19. Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo.

    Science.gov (United States)

    Lombard-Banek, Camille; Moody, Sally A; Nemes, Peter

    2016-02-12

    We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

  20. Cell membrane and cell junctions in differentiation of preimplanted mouse embryos.

    Science.gov (United States)

    Izquierdo, L; Fernández, S; López, T

    1976-12-01

    Cell membrane and cell junctions in differentiation of preimplanted mouse embryos, (membrana celular y uniones celulares en la diferenciación del embrión de ratón antes de la implantación). Arch. Biol. Med. Exper. 10: 130-134, 1976. The development of cell junctions that seal the peripheral blastomeres could be a decisive step in the differentiation of morulae into blastocysts. The appearance of these junctions is studied by electron microscopy of late morulae and initial blastocysts. Zonulae occludentes as well as impermeability to lanthanum emulsion precedes the appearance of the blastocel and hence might be considered as one of its necessary causes.

  1. High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Esther K. Kieserman, Chanjae Lee, Ryan S. Gray, Tae Joo Park and John B. Wallingford Corresponding author ([]()). ### INTRODUCTION Embryos of the frog *Xenopus laevis* are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than in...

  2. MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

    Science.gov (United States)

    Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

    2011-02-15

    At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

  3. Stem cells from residual IVF-embryos - Continuation of life justifies isolation.

    NARCIS (Netherlands)

    Bongaerts, G.P.A.; Severijnen, R.S.V.M.

    2007-01-01

    Embryonic stem cells are undifferentiated pluripotent cells that can indefinitely grow in vitro. They are derived from the inner mass of early embryos. Because of their ability to differentiate into all three embryonic germ layers, and finally into specialized somatic cell types, human embryonic ste

  4. Stem cells from residual IVF-embryos - Continuation of life justifies isolation.

    NARCIS (Netherlands)

    Bongaerts, G.P.A.; Severijnen, R.S.V.M.

    2007-01-01

    Embryonic stem cells are undifferentiated pluripotent cells that can indefinitely grow in vitro. They are derived from the inner mass of early embryos. Because of their ability to differentiate into all three embryonic germ layers, and finally into specialized somatic cell types, human embryonic

  5. Seminal fluid and the generation of regulatory T cells for embryo implantation

    NARCIS (Netherlands)

    Robertson, Sarah A; Prins, Jelmer R; Sharkey, David J; Moldenhauer, Lachlan M

    2013-01-01

    T regulatory (Treg) cells are essential mediators of the maternal immune adaptation necessary for embryo implantation. In mice, insufficient Treg cell activity results in implantation failure, or constrains placental function and fetal growth. In women, Treg cell deficiency is linked with unexplaine

  6. ES cells derived from cloned embryos in monkey - a jump toward human therapeutic cloning

    Institute of Scientific and Technical Information of China (English)

    Xiangzhong Yang; Sadie L Smith

    2007-01-01

    @@ Therapeutic cloning refers to the derivation of embryonic stem cells (ntESC) from embryos derived from somatic cell nuclear transfer (SCNT) also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus (DNA) removed.

  7. Fuel cell hybrid drive train test facility

    NARCIS (Netherlands)

    J. Bruinsma; Edwin Tazelaar; Bram Veenhuizen; I. Zafina; H. Bosma

    2009-01-01

    Fuel cells are expected to play an important role in the near future as prime energy source on board of road-going vehicles. In order to be able to test all important functional aspects of a fuel cell hybrid drive train, the Automotive Institute of the HAN University has decided to realize a

  8. Fuel cell hybrid drive train test facility

    NARCIS (Netherlands)

    J. Bruinsma; Edwin Tazelaar; Bram Veenhuizen; I. Zafina; H. Bosma

    2009-01-01

    Fuel cells are expected to play an important role in the near future as prime energy source on board of road-going vehicles. In order to be able to test all important functional aspects of a fuel cell hybrid drive train, the Automotive Institute of the HAN University has decided to realize a station

  9. Reproductive semi-cloning respecting biparental embryo origin: embryos from syngamy between a gamete and a haploidized somatic cell.

    Science.gov (United States)

    Tesarik, J

    2002-08-01

    Embryos formed by somatic cell nuclear transfer to enucleated oocytes (cloning) have given rise to viable offspring in several mammalian species. The possibility of future application of this technique to human assisted reproduction (reproductive cloning) has been widely debated. On this background there is current discussion of the potential for a cloning-derived technique, which aims at syngamy between a gamete nucleus from one parent and a somatic cell nucleus from the other. Critical analysis of the clinical indications, the current state of the art, biological concerns and ethical considerations relative to this technique, called here reproductive semi-cloning, are presented. Such a technique requires validation by further research before it can be considered as a treatment option. This debate explores issues raised by the technique.

  10. Sox17-Mediated XEN Cell Conversion Identifies Dynamic Networks Controlling Cell-Fate Decisions in Embryo-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Angela C.H. McDonald

    2014-10-01

    Full Text Available Little is known about the gene regulatory networks (GRNs distinguishing extraembryonic endoderm (ExEn stem (XEN cells from those that maintain the extensively characterized embryonic stem cell (ESC. An intriguing network candidate is Sox17, an essential transcription factor for XEN derivation and self-renewal. Here, we show that forced Sox17 expression drives ESCs toward ExEn, generating XEN cells that contribute to ExEn when placed back into early mouse embryos. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. To orchestrate this conversion process, Sox17 acts in autoregulatory and feedforward network motifs, regulating dynamic GRNs directing cell fate. Sox17-mediated XEN conversion helps to explain the regulation of cell-fate changes and reveals GRNs regulating lineage decisions in the mouse embryo.

  11. No relationship between embryo morphology and successful derivation of human embryonic stem cell lines.

    Directory of Open Access Journals (Sweden)

    Susanne Ström

    Full Text Available BACKGROUND: The large number (30 of permanent human embryonic stem cell (hESC lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002-2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. METHODS: We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. RESULTS: Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. CONCLUSION: Even very poor quality embryos with few cells in the ICM can give origin to hESC lines.

  12. A comparative study on efficiency of adult fibroblast, putative embryonic stem cell and lymphocyte as donor cells for production of handmade cloned embryos in goat and characterization of putative ntES cells obtained from these embryos.

    Science.gov (United States)

    Dutta, Rahul; Malakar, Dhruba; Khate, Keviletsu; Sahu, Shailendra; Akshey, Yogesh; Mukesh, Manishi

    2011-09-15

    The main purpose of the experiment was to compare the efficiency of three cell types, namely adult fibroblast, putative embryonic stem (ES) cell, and lymphocyte, as donor cells for somatic cell nuclear transfer by handmade cloning in goats. The outcome clearly shows that putative embryonic stem cells, with a cleavage and blastocyst production rate of 74.69% ± 3.92 and 39.75% ± 3.86, respectively, performs better in comparison to adult fibroblast cell and lymphocyte. Between adult fibroblast cell and lymphocyte no statistically significant difference exists at P II DRB genes of cloned embryos and three donor cells were performed to verify the cloned embryos. The amplified PCR products were subjected to SSCP to confirm their genetic identity. The karyotyping of the cloned embryos showed normal chromosomal status as expected in goat. Significantly, in the second stage of the experiment, the produced cloned embryos were successfully used to derive ntES-like cells. The rate of primary colony formation rate was 62.50% ± 4.62 for fibroblast donor cell derived embryos. The same was 60.60% ± 4.62 for putative ES donor cell derived embryos and 66.66% ± 4.62 for lymphocyte donor cell derived embryos, respectively. The putative ntES colonies were positively characterized for alkaline phosphatase, Oct-4, TRA-1-60, TRA-1-81, Sox-2, and Nanog by Immunocytochemistry and Reverse Transcription PCR. To further validate the stem ness, the produced putative ntES colonies were differentiated to embryoid bodies. Immunocytochemistry revealed that embryoid bodies expressed NESTIN specific for ectodermal lineage; GATA-4 for endodermal lineage and smooth muscle actin-I, and troponin-I specific for mesodermal lineage. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. It could be of substantial significance as patient specific ntES cells have proven therapeutic significance.

  13. Attitudes of patients, healthcare professionals and ethicists towards embryonic stem cell research and donation of gametes and embryos in Germany.

    Science.gov (United States)

    Krones, T; Neuwohner, E; Bock, K; Manolopoulos, K; Tinneberg, H R; Richter, G

    2006-11-01

    Due to the Embryo Protection Act, creation of supernumerary embryos, as well as egg and embryo donation, is prohibited in Germany. Human stem cell research is regulated through the Act on stem cells that came into force in 2002. A cross-sectional survey of 101 IVF couples (n=202) in two fertility centres, and representative samples of healthcare professionals and ethicists (n=879), was carried out, and their attitudes towards embryonic stem cell research and donation of gametes and embryos compared. A clear majority of IVF couples favoured legalization of egg and embryo donation and embryonic stem cell research for various purposes. The willingness of couples to donate was related to purpose and to other independent influences. The majority of physicians voted for legalization of embryonic stem cell production from surplus embryos. Most human geneticists and obstetricians approved egg, but not embryo, donation to other couples. Ethicists and midwives were opposed to every kind of donation and research on surplus embryos. The IVF couples surveyed have positive attitudes towards donation and research using surplus embryos, whereas the healthcare professionals and ethicists are predominantly sceptical about most research activities destroying human embryos. This difference should be considered carefully in legal and ethical discussions on reprogenetics.

  14. In Vivo Differentiation Potential of Epiblast Stem Cells Revealed by Chimeric Embryo Formation

    Directory of Open Access Journals (Sweden)

    Yali Huang

    2012-12-01

    Full Text Available Chimera formation after blastocyst injection or morula aggregation is the principal functional assay of the developmental potential of mouse embryonic stem cells (ESCs. This property, which demonstrates functional equivalence between ESCs and the preimplantation epiblast, is not shared by epiblast stem cell (EpiSC lines. Here, we show that EpiSCs derived either from postimplantation embryos or from ESCs in vitro readily generate chimeras when grafted to postimplantation embryos in whole embryo culture. EpiSC derivatives integrate and differentiate to derivatives of all three embryonic germ layers and primordial germ cells. In contrast, grafted ESCs seldom proliferate in postimplantation embryos, and fail to acquire the identity of their host-derived neighbors. EpiSCs do not incorporate efficiently into embryonic day 8.5 embryos, a stage by which pluripotency has been lost. Thus, chimera formation by EpiSCs requires a permissive environment, the postimplantation epiblast, and demonstrates functional equivalence between this cell type and EpiSCs.

  15. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Jin Li

    Full Text Available Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf. Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

  16. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    Science.gov (United States)

    Li, Jin; Zeng, Guofang; Qi, Yawei; Tang, Xudong; Zhang, Jingjing; Wu, Zeyong; Liang, Jie; Shi, Lei; Liu, Hongwei; Zhang, Peihua

    2015-01-01

    Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

  17. Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

  18. Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos.

    Science.gov (United States)

    Kang, Eunju; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P; Schöler, Hans R; Mitalipov, Shoukhrat

    2014-05-01

    Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative

  19. Production of transgenic canine embryos using interspecies somatic cell nuclear transfer.

    Science.gov (United States)

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2012-02-01

    Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that

  20. Pulsatile cell-autonomous contractility drives compaction in the mouse embryo.

    Science.gov (United States)

    Maître, Jean-Léon; Niwayama, Ritsuya; Turlier, Hervé; Nédélec, François; Hiiragi, Takashi

    2015-07-01

    Mammalian embryos initiate morphogenesis with compaction, which is essential for specifying the first lineages of the blastocyst. The 8-cell-stage mouse embryo compacts by enlarging its cell-cell contacts in a Cdh1-dependent manner. It was therefore proposed that Cdh1 adhesion molecules generate the forces driving compaction. Using micropipette aspiration to map all tensions in a developing embryo, we show that compaction is primarily driven by a twofold increase in tension at the cell-medium interface. We show that the principal force generator of compaction is the actomyosin cortex, which gives rise to pulsed contractions starting at the 8-cell stage. Remarkably, contractions emerge as periodic cortical waves when cells are disengaged from adhesive contacts. In line with this, tension mapping of mzCdh1(-/-) embryos suggests that Cdh1 acts by redirecting contractility away from cell-cell contacts. Our study provides a framework to understand early mammalian embryogenesis and original perspectives on evolutionary conserved pulsed contractions.

  1. Hybrid Arc Cell Studies: Status Report

    Energy Technology Data Exchange (ETDEWEB)

    Berg J. S.

    2012-09-28

    I report on the status, at the end of FY12, of the studies of an arc cell for a hybrid synchrotron accelerating from 375 GeV/c to 750 GeV/c in momentum. Garren produced a complete lattice that gives a good outline of the structure of a hybrid synchrotron lattice. It is, however, lacking in some details: it does not maintain a constant time of flight, it lacks chromaticity correction, its cell structure is not ideal for removing aberrations from chromaticity correction, and it probably needs more space between magnets. I have begun studying cell structures for the arc cells to optimize the lattice performance and cost. I present some preliminary results for two magnets per half cell. I then discuss difficulties encountered, some preliminary attempts at resolving them, and the future plans for this work.

  2. Structure and implied functions of truncated B-cell receptor mRNAs in early embryo and adult mesenchymal stem cells: Cdelta replaces Cmu in mu heavy chain-deficient mice.

    Science.gov (United States)

    Lapter, Smadar; Livnat, Idit; Faerman, Alexander; Zipori, Dov

    2007-03-01

    Stem cells exhibit a promiscuous gene expression pattern. We show herein that the early embryo and adult MSCs express B-cell receptor component mRNAs. To examine possible bearings of these genes on the expressing cells, we studied immunoglobulin mu chain-deficient mice. Pregnant mu chain-deficient females were found to produce a higher percentage of defective morulae compared with control females. Structure analysis indicated that the mu mRNA species found in embryos and in mesenchyme consist of the constant region of the mu heavy chain that encodes a recombinant 50-kDa protein. In situ hybridization localized the constant mu gene expression to loose mesenchymal tissues within the day-12.5 embryo proper and the yolk sac. In early embryo and in adult mesenchyme from mu-deficient mice, delta replaced mu chain, implying a possible requirement of these alternative molecules for embryo development and mesenchymal functions. Indeed, overexpression of the mesenchymal-truncated mu heavy chain in 293T cells resulted in specific subcellular localization and in G(1) growth arrest. The lack of such occurrence following overexpression of a complete, rearranged form of mu chain suggests that the mesenchymal version of this mRNA may possess unique functions.

  3. Hybrid emitter all back contact solar cell

    Science.gov (United States)

    Loscutoff, Paul; Rim, Seung

    2016-04-12

    An all back contact solar cell has a hybrid emitter design. The solar cell has a thin dielectric layer formed on a backside surface of a single crystalline silicon substrate. One emitter of the solar cell is made of doped polycrystalline silicon that is formed on the thin dielectric layer. The other emitter of the solar cell is formed in the single crystalline silicon substrate and is made of doped single crystalline silicon. The solar cell includes contact holes that allow metal contacts to connect to corresponding emitters.

  4. Survival Assessment of Mouse Preimplantation Embryos After Exposure to Cell Phone Radiation

    Science.gov (United States)

    Safian, Fereshteh; Khalili, Mohammad Ali; Khoradmehr, Arezoo; Anbari, Fatemeh; Soltani, Saeedeh; Halvaei, Iman

    2016-01-01

    Background: Using cellular phone has rapidly increased all over the world. Also, the concern on the possible health hazards of electromagnetic fields (EMF) induced from cell phones to reproduction has been growing in many countries. The aim of this study was to assess the consequences and effects of exposure to the cell phone radiation on the quality and survival rates of preimplantation embryos in mice. Methods: A total of 40 mice (20 females and 20 males), 6 weeks old and sexually mature BALB/c, were used for control and experimental groups. The ovary burses were removed and the zygotes were dissected in the morning after mating. Next, 2-cell embryos were divided into two groups of control (n=150) and experimental (n=150). EMF (900–1800 MHz) was used for four days in experimental group for 30 min/day in culture at 37°C in a CO 2 incubator. The quality of embryos was recorded daily and the fluorescent staining was used for identification of viable blastocysts. All data were compared by Student’s t-test and Mann-Whitney test (p<0.05). Results: The rate of embryo survival to the blastocysts stage was similar in both groups. However, the percentage of dead embryos at the 2-cell stage was significantly higher in EMF-exposed group compared with controls (p=0.03). Also, the loss of cell viability significantly increased in experimental blastocysts (p=0.002). Conclusion: The normal embryonic development up to the blastocyst stage indicates that EMF-exposure commonly did not have adverse effect on embryo development in mice. But, it caused loss of blastocysts cell viability. PMID:27478766

  5. Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

    Directory of Open Access Journals (Sweden)

    Tony Y-C Tsai

    2014-02-01

    Full Text Available During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min and the subsequent 11 cycles are short (∼30 min and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

  6. The influence of interspecies somatic cell nuclear transfer on epigenetic enzymes transcription in early embryos

    Directory of Open Access Journals (Sweden)

    Martin Morovic

    2016-10-01

    Full Text Available One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a genes in early embryonic stages of interspecies (bovine, porcine nuclear transfer embryos (iSCNT by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly infl uenced by the ooplasmic environment.

  7. An integrated modelling framework from cells to organism based on a cohort of digital embryos

    Science.gov (United States)

    Villoutreix, Paul; Delile, Julien; Rizzi, Barbara; Duloquin, Louise; Savy, Thierry; Bourgine, Paul; Doursat, René; Peyriéras, Nadine

    2016-12-01

    We conducted a quantitative comparison of developing sea urchin embryos based on the analysis of five digital specimens obtained by automatic processing of in toto 3D+ time image data. These measurements served the reconstruction of a prototypical cell lineage tree able to predict the spatiotemporal cellular organisation of a normal sea urchin blastula. The reconstruction was achieved by designing and tuning a multi-level probabilistic model that reproduced embryo-level dynamics from a small number of statistical parameters characterising cell proliferation, cell surface area and cell volume evolution along the cell lineage. Our resulting artificial prototype was embedded in 3D space by biomechanical agent-based modelling and simulation, which allowed a systematic exploration and optimisation of free parameters to fit the experimental data and test biological hypotheses. The spherical monolayered blastula and the spatial arrangement of its different cell types appeared tightly constrained by cell stiffness, cell-adhesion parameters and blastocoel turgor pressure.

  8. Hybrid solar cells : Perovskites under the Sun

    NARCIS (Netherlands)

    Loi, Maria Antonietta; Hummelen, Jan C.

    2013-01-01

    Mixed-halide organic–inorganic hybrid perovskites are reported to display electron–hole diffusion lengths over 1 μm. This observation provides important insight into the charge-carrier dynamics of this class of semiconductors and increases the expectations for highly efficient and cheap solar cells.

  9. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek;

    2016-01-01

    One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

  10. Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

    Science.gov (United States)

    Behboodi, E; Bondareva, A; Begin, I; Rao, K; Neveu, N; Pierson, J T; Wylie, C; Piero, F D; Huang, Y J; Zeng, W; Tanco, V; Baldassarre, H; Karatzas, C N; Dobrinski, I

    2011-03-01

    Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.

  11. Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

    2013-01-01

    therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs...... from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month...

  12. Brazilian hybrid electric fuel cell bus

    Energy Technology Data Exchange (ETDEWEB)

    Miranda, P.E.V.; Carreira, E.S. [Coppe-Federal Univ. of Rio de Janeiro (Brazil). Hydrogen Lab.

    2010-07-01

    The first prototype of a hybrid electric fuel cell bus developed with Brazilian technology is unveiled. It is a 12 m urban-type, low-floor, air-conditioned bus that possesses three doors, air suspension, 29 seats and reversible wheelchair site. The bus body was built based on a double-deck type monoblock vehicle that is able to sustain important load on its roof. This allowed positioning of the type 3 hydrogen tanks and the low weight traction batteries on the roof of the vehicles without dynamic stabilization problems. A novel hybrid energy configuration was designed in such a way that the low-power (77 kWe) fuel cell works on steady-state operation mode, not responding directly to the traction motor load demand. The rate of kinetic energy regeneration upon breaking was optimized by the use of an electric hybrid system with predominance of batteries and also by utilizing supercapacitors. The electric-electronic devices and the security control softwares for the auxiliary and traction systems were developed in-house. The innovative hybrid-electric traction system configuration led to the possibility to decrease the fuel cell power, with positive impact on weight and system volume reduction, as well as to significantly decrease the hydrogen consumption. (orig.)

  13. Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (

    Directory of Open Access Journals (Sweden)

    Minghui Zhao

    2017-07-01

    Full Text Available Objective Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT. Methods Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3 in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR. Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

  14. Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Peura, T.T.; Hartwich, K.M.;

    2005-01-01

    The processes of cellular differentiation were studied in somatic cell nuvlear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos...... were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients...... embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas...

  15. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell...... intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body...... (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures...

  16. Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

    Science.gov (United States)

    Duncan, S A

    2005-12-01

    Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

  17. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  18. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...... stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high...

  19. Involvement of insulin in early development of mouse one-cell stage embryos

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.

  20. Involvement of insulin in early development of mouse one-cell stase embryos

    Institute of Scientific and Technical Information of China (English)

    YU BingZhi; YU DaHai; ZHANG Zhe; DENG Xin; XU XiaoYan; FENG Chen; LI YanXiao; CUI Cheng; SU WenHui; ZHAO HongMei

    2008-01-01

    Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-suits suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.

  1. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.

    Science.gov (United States)

    Martin, G R

    1981-12-01

    This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

  2. Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen.

    Science.gov (United States)

    Liu, Jie; Westhusin, Mark; Long, Charles; Johnson, Gregory; Burghardt, Robert; Kraemer, Duane

    2010-12-01

    Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos.

  3. The influence of early embryo traits on human embryonic stem cell derivation efficiency.

    Science.gov (United States)

    O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

    2011-05-01

    Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

  4. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  5. Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

    NARCIS (Netherlands)

    T. Yokomizo (Tomomasa); T. Yamada-Inagawa (Tomoko); A.D. Yzaguirre (Amanda); M.J. Chen (Michael); N.A. Speck (Nancy); E.A. Dzierzak (Elaine)

    2012-01-01

    textabstractWe describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development

  6. Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo.

    NARCIS (Netherlands)

    M.F.T.R. de Bruijn (Marella); N.A. Speck; M.C. Peeters (Marian); E.A. Dzierzak (Elaine)

    2000-01-01

    textabstractThe aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site within the mammalian embryo body, and the first place from which hematopoietic stem cells (HSCs) emerge. Within the complex embryonic vascular, excretory and reproductive tissues of the

  7. CAS, INRA to Set Up Sino-French Lab for Mammal-embryo Cell-biology

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ CAS Vice President CHEN Zhu and Dr. Marion Guillou, chairman of Institut National de la Recherche Agronomique (INRA) in France,signed on Nov. 4 in Beijing an agreement on the founding of a SinoFrench lab for mammal-embryo cell biology (LABIOCEM).

  8. Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus

    DEFF Research Database (Denmark)

    Li, Yiping; Handberg, K.J.; Juul-Madsen, H.R.

    2007-01-01

    -host interaction, we measured steady-state levels of transcripts from 28 cellular genes of chicken embryo (CE) cell cultures infected with IBDV vaccine stain Bursine-2 during a 7-day infection course by use of the quantitative real-time RT-PCR SYBR green method. Of the genes tested, 21 genes (IRF-1, IFN 1...

  9. ABA inhibits embryo cell expansion and early cell division events during coffee (Coffea arabica 'Rubi') seed germination.

    Science.gov (United States)

    Da Silva, E A Amaral; Toorop, Peter E; Van Lammeren, André A M; Hilhorst, Henk W M

    2008-09-01

    Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes. Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against beta-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy. The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of beta-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of beta-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion. Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.

  10. Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

    Science.gov (United States)

    Xi, Yongmei; Nada, Yoich; Soh, Tomoki; Fujihara, Noboru; Hattori, Masa-Aki

    2004-02-01

    This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras. Copyright 2004 Wiley-Liss, Inc.

  11. Production of nuclear transfer embryos by using somatic cells isolated from milk in buffalo (Bubalus bubalis).

    Science.gov (United States)

    Golla, K; Selokar, N L; Saini, M; Chauhan, M S; Manik, R S; Palta, P; Singla, S K

    2012-10-01

    Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos.

  12. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    Directory of Open Access Journals (Sweden)

    Alexander Krivokharchenko

    Full Text Available Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  13. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    Science.gov (United States)

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  14. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

    Science.gov (United States)

    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  15. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos

    Directory of Open Access Journals (Sweden)

    Daniela Ávila-González

    2015-09-01

    Full Text Available Data from the literature suggest that human embryonic stem cell (hESC lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1 from poor-quality (PQ embryos derived and maintained on human amniotic epithelial cells (hAEC. This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  16. In vitro development competence of bovine nuclear transfer embryos derived from Nanog-overexpressing fibroblast cells

    Directory of Open Access Journals (Sweden)

    Xi-bang Zheng, Yan Yun, Yong-ce Hu, Yong Li, Hua-yan Wang, Xiao-ling Ma, Jin-qiang Sui, An-min Lei and Zhong-ying Dou

    2014-04-01

    Full Text Available The purpose of this study was to establish Nanog-expressing cell lines that can be used as donor cells to construct transgenic cloned embryos, and to investigate their in vitro development competence. By reverse transcription-polymerase chain reaction (RT-PCR, the cDNA of Nanog gene was cloned from fetal bovine primordial genital ridge tissues. The gene was inserted into PMD18-T vector using recombination techniques and then subcloned into vector pEGFP-C1. After confirmation by restrictive endonuclease digestion and sequencing, the recombinant plasmid pEGFP-Nanog was transfected into skin fibroblast cells. A stable transfected cell line was successfully established after two months of selection with neomycine (G418. Fluorescence microscopy, RT-PCR, and Western Blotting assays indicated that Nanog mRNA and EGFP-Nanog fusion protein were expressed in these cells. The EGFP-Nanog expressing fibroblast cells and the intact fibroblast cells (BEF422 were respectively used to construct cloned embryos. The results showed that the cleavage rate of recombinant embryos in BEF422 cells was significantly (P<0.05 higher than in EGFP-Nanog expressing cells (82.14 vs 40.38 %, but the blastocyst development rate in the latter was slightly higher than in the former (17.30 vs 14.29% (P<0.05, indicating that Nanog-overexpressed fibroblasts may be a better candidate of donor cells. To our knowledge, this is the first time that Nanog gene has been introduced into fibroblast cells to produce cloned embryos in bovine.

  17. Experimental risk assessment of bovine viral diarrhea virus transmission via in vitro embryo production using somatic cell nucleus transfer.

    Science.gov (United States)

    Gregg, K; Chen, S H; Sadeghieh, S; Guerra, T; Xiang, T; Meredith, J; Polejaeva, I

    2009-07-01

    The objective of this study was to perform a comprehensive risk assessment on infectious disease transmission in the system of in vitro embryo production via somatic cell nucleus transfer (SCNT) technology using bovine viral diarrhea virus (BVDV) as a model. The risks of BVDV transmission in each step of the SCNT embryo production procedure, from donor cells to preimplantation SCNT embryo culture, were carefully examined using a sensitive real-time polymerase chain reaction assay. The identified primary source of BVDV transmission in SCNT embryo production was donor cell infection, most likely caused by contaminated fetal bovine serum in the culture medium. The risk of disease transmission through contaminated oocytes from an abattoir was relatively low, and it can be greatly minimized by cumulus cell removal and adequate oocyte washing procedures. Of the 200 cumulus-oocyte complexes (COCs) and more than 1500 cumulus cell-free oocyte (CFO) samples collected from multiple sources over a course of 7 months, only 2.5% of the COCs were BVDV positive, and all of the CFOs (100%) were BVDV negative. To evaluate the risk of BVDV introduction during in vitro SCNT embryo culture, 324 SCNT embryos were produced from 18 different cell lines using oocytes from 26 different batches collected over a course of 9 months. The embryos were cultured in vitro for 7 days and then tested for BVDV. All of the 324 SCNT embryos (100%) were negative, indicating that the embryo culture system is virtually risk-free for BVDV transmission. Based on these results, a standard operational protocol (SOP) for SCNT embryo production was proposed to greatly minimize the risk of BVDV transmission through the SCNT embryo production system. This SOP could be a starting point to produce a SCNT system that is virtually risk-free for disease transmission in general.

  18. Hybrid Silicon Nanocone–Polymer Solar Cells

    KAUST Repository

    Jeong, Sangmoo

    2012-06-13

    Recently, hybrid Si/organic solar cells have been studied for low-cost Si photovoltaic devices because the Schottky junction between the Si and organic material can be formed by solution processes at a low temperature. In this study, we demonstrate a hybrid solar cell composed of Si nanocones and conductive polymer. The optimal nanocone structure with an aspect ratio (height/diameter of a nanocone) less than two allowed for conformal polymer surface coverage via spin-coating while also providing both excellent antireflection and light trapping properties. The uniform heterojunction over the nanocones with enhanced light absorption resulted in a power conversion efficiency above 11%. Based on our simulation study, the optimal nanocone structures for a 10 μm thick Si solar cell can achieve a short-circuit current density, up to 39.1 mA/cm 2, which is very close to the theoretical limit. With very thin material and inexpensive processing, hybrid Si nanocone/polymer solar cells are promising as an economically viable alternative energy solution. © 2012 American Chemical Society.

  19. Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

  20. β-catenin functions pleiotropically in differentiation and tumorigenesis in mouse embryo-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Noriko Okumura

    Full Text Available The canonical Wnt/β-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. β-Catenin, encoded by the Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if β-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established β-catenin-deficient (β-cat(Δ/Δ mouse embryo-derived stem cells and showed that β-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs. However, teratomas formed from embryo-derived β-cat(Δ/Δ ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional β-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that β-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight β-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.

  1. Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed

    Energy Technology Data Exchange (ETDEWEB)

    Jegla, D.E.; Sussex, I.M.

    1989-01-01

    We mapped the fate of cells in the shoot meristem of the dry-seed embryo of sunflower, Helianthus annuus L. cv. Peredovic, using irradiation-induced somatic sectors. We analyzed 249 chlorophyll-deficient or glabrous (hairless) sectors generated in 236 plants. Most sectors observed in the inflorescence extended into vegetative nodes. Thus cell lineages that ultimately gave rise to reproductive structures also contributed to vegetative structures. No single sector extended the entire length of the shoot. Thus the shoot is not derived from one or a few apical initials. Rather, the position, vertical extent, and width of the sectors at different levels of the shoot suggest that the shoot is derived from three to four circumferential populations of cells in each of three cell layers of the embryo meristem. Sectors had no common boundaries even in plants with two or three independent sectors, but varied in extent and overlapped along the length of the shoot. Thus individual cells in a single circumferential population behaved independently to contribute lineages of different vertical extents to the growing shoot. The predicted number of circumferential populations of cells as well as the apparent cell number in each population was consistent with the actual number of cells in the embryo meristem observed in histological sections.

  2. Production, morphology, and cytogenetics of Triticum aestivum (L.) Thell × Elymus scabrus (R. Br.) Love intergeneric hybrids obtained by in ovulo embryo culture.

    Science.gov (United States)

    Ahmad, F; Comeau, A

    1991-06-01

    Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD), and an apomictic Triticeae species, Elymus scabrus (syn. Agropyron scabrum) (2n=6x=42, HHSSSS), the first successful report of this cross. Nine tiny, underdeveloped, and structureless embryos were obtained in vitro only by in ovulo embryo culture at 4 days after pollination, which gave rise to five mature hybrid plants. All the hybrid plants were vigorous and possessed a phenotype intermediate to the two parents. There were 2n=6x=42 (ABDHSS) somatic chromosomes in the hybrids. There was little or no homology between the parental genomes, as shown by an overall meiotic chromosome association of 32.83 I + 4.08 rod II + 0.21 ring II + 0.18 III + 0.02 IV. The hybrids were completely sterile and so far backcrosses to wheat parent have not been successful. Alternate approaches to induce gene transfer(s) from E. scabrus to wheat are being attempted.

  3. Effect of Different High CO2 Concentrations on the Development of 2-cell Mouse Embryos in vitro

    Institute of Scientific and Technical Information of China (English)

    Li-hua LU; Wei-jie ZHU

    2003-01-01

    Objective To investigate effects of different high CO2 concentrations on the development of 2-cell mouse embryos in vitroMethods At levels of 5% CO2 (control group), 5.7% CO2, 6.0% CO2 and 15% CO2, embryos were incubated in drops with CZB medium, respectively, and the drops were covered by paraffin oil which was treated with three-distilled water. In addition, at the level of 15% CO2, there were another two groups, in which paraffin oil was treated with phosphate-buffered saline (PBS) solution or the drops were uncovered. The development of embryos in all stages was noted.Results The developmental rates of blastocysts in five experimental groups were significantly lower than that of the control group (P0.05). At the level of 15% CO2, 15% embryos developed in the 4-cell stage with irregular blastomere and degenerated quickly in the group which paraffin oil was treated with distilled water; 2.2% embryos developed in the 4-cell stage in the group which paraffin oil was treated with PBS and the rest stagnated in the 2-cell stage. Conclusions High CO2 concentrations had toxic effect on the in vitro development of 2-cell mouse embryos, and was responsible for the inhibition of the embryos. It is important for the development of embryos in vitro to detect strictly CO2 concentration.

  4. Analysis of fuel cell hybrid locomotives

    Science.gov (United States)

    Miller, Arnold R.; Peters, John; Smith, Brian E.; Velev, Omourtag A.

    Led by Vehicle Projects LLC, an international industry-government consortium is developing a 109 t, 1.2 MW road-switcher locomotive for commercial and military railway applications. As part of the feasibility and conceptual-design analysis, a study has been made of the potential benefits of a hybrid power plant in which fuel cells comprise the prime mover and a battery or flywheel provides auxiliary power. The potential benefits of a hybrid power plant are: (i) enhancement of transient power and hence tractive effort; (ii) regenerative braking; (iii) reduction of capital cost. Generally, the tractive effort of a locomotive at low speed is limited by wheel adhesion and not by available power. Enhanced transient power is therefore unlikely to benefit a switcher locomotive, but could assist applications that require high acceleration, e.g. subway trains with all axles powered. In most cases, the value of regeneration in locomotives is minimal. For low-speed applications such as switchers, the available kinetic energy and the effectiveness of traction motors as generators are both minimal. For high-speed heavy applications such as freight, the ability of the auxiliary power device to absorb a significant portion of the available kinetic energy is low. Moreover, the hybrid power plant suffers a double efficiency penalty, namely, losses occur in both absorbing and then releasing energy from the auxiliary device, which result in a net storage efficiency of no more than 50% for present battery technology. Capital cost in some applications may be reduced. Based on an observed locomotive duty cycle, a cost model shows that a hybrid power plant for a switcher may indeed reduce capital cost. Offsetting this potential benefit are the increased complexity, weight and volume of the power plant, as well as 20-40% increased fuel consumption that results from lower efficiency. Based on this analysis, the consortium has decided to develop a pure fuel cell road-switcher locomotive

  5. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

    Science.gov (United States)

    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

  6. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  7. Inhibition of cell migration in sea urchin embryos by beta-D-xyloside.

    Science.gov (United States)

    Solursh, M; Mitchell, S L; Katow, H

    1986-12-01

    This investigation examines the effect of exogenous xylosides on primary mesenchyme cell behavior in Strongylocentrotus purpuratus embryos. In confirmation of studies in some other species the addition of 2 mM p-nitrophenyl-beta-D-xylopyranoside blocks the migration but not the initial ingression of primary mesenchyme cells. The blastocoel matrix of treated embryos appears deficient in a 15- to 30-nm-diameter granular component that is observed extensively on the basal lamina and on filopodia of migrating primary mesenchyme cells in untreated embryos. Other blastocoel components appear unaffected by ultrastructural criteria. The incorporation of 35SO4(2-) per embryo into ethanol precipitates of isolated blastocoel matrices was reduced significantly after xyloside treatment but the distribution of 35SO4(2-) after polyacrylamide gel electrophoresis or the glycosaminoglycan composition was unaffected. Chromatography on Sepharose CL-2B demonstrates a reduction in size of sulfated components of the blastocoel. While over 60% of the 35S-labeled material from the blastocoel of normal mesenchyme blastulae is voided from a Sepharose CL-2B column run in a dissociative solvent, only 10% from xyloside treated embryos is voided. Instead, there is a large included peak with Kav of 0.33. This material is acid soluble but cetylpyridinium chloride precipitable. It apparently consists largely of free glycosaminoglycan chains. Based on analysis of chondroitinase ABC digestion products this material consists of 41% chondroitin-6-sulfate and 58% dermatan sulfate. These results are consistent with a role in cell migration for intact chondroitin sulfate/dermatan sulfate proteoglycans in the sea urchin blastocoel matrix.

  8. Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

    Directory of Open Access Journals (Sweden)

    Hartung Odelya

    2007-12-01

    Full Text Available Abstract Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when

  9. Efficient production of intersubspecific hybrid mice and embryonic stem cells by intracytoplasmic sperm injection.

    Science.gov (United States)

    Shinmen, Akie; Honda, Arata; Ohkawa, Mika; Hirose, Michiko; Ogonuki, Narumi; Yuzuriha, Misako; Miki, Hiromi; Mochida, Keiji; Inoue, Kimiko; Abe, Kuniya; Ito, Masao; Ogura, Atsuo

    2007-09-01

    Recently, mice and embryonic stem (ES) cells with allelic polymorphisms have been used extensively in the field of genetics and developmental biology. In this study, we examined whether intersubspecific hybrid mice and ES cells with these genotypes can be efficiently produced by intracytoplasmic sperm injection (ICSI). Frozen-thawed spermatozoa from wild-derived strains, JF1 (Mus musculus molossinus), MSM (M. m. molossinus), HMI (M. m. castaneus), and SWN (M. m. spp.), were directly injected into mature oocytes from laboratory mice ([C57BL/6 x DBA2]F1; M. m. domesticus). The in vitro and in vivo developmental capacity of F1 embryos was not significantly different among the groups (P > 0.05), and term offspring were efficiently obtained in all groups (27%-34% of transferred embryos). However, the mean body and placental weights of the offspring differed significantly with genotype (P cell lines. The ES cell lines were established at a high efficiency (9 lines from 20 blastocysts) and their allelic polymorphisms were confirmed. Thus, ICSI using cryopreserved spermatozoa allows the efficient and immediate production of a number of F1 hybrid mice and ES cell lines, which can be used for polymorphic analysis of mouse genetics.

  10. Techno-economic comparison of series hybrid, plug-in hybrid, fuel cell and regular cars

    NARCIS (Netherlands)

    van Vliet, O.P.R.|info:eu-repo/dai/nl/288519361; Kruithof, T.; Turkenburg, W.C.|info:eu-repo/dai/nl/073416355; Faaij, A.P.C.|info:eu-repo/dai/nl/10685903X

    2010-01-01

    We examine the competitiveness of series hybrid compared to fuel cell, parallel hybrid, and regular cars. We use public domain data to determine efficiency, fuel consumption, total costs of ownership and greenhouse gas emissions resulting from drivetrain choices. The series hybrid drivetrain can be

  11. Techno-economic comparison of series hybrid, plug-in hybrid, fuel cell and regular cars

    NARCIS (Netherlands)

    van Vliet, O.P.R.; Kruithof, T.; Turkenburg, W.C.; Faaij, A.P.C.

    2010-01-01

    We examine the competitiveness of series hybrid compared to fuel cell, parallel hybrid, and regular cars. We use public domain data to determine efficiency, fuel consumption, total costs of ownership and greenhouse gas emissions resulting from drivetrain choices. The series hybrid drivetrain can be

  12. The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

    Institute of Scientific and Technical Information of China (English)

    XUXIN; SUMIOSUGANO; 等

    1999-01-01

    Primordial germ cells (PGCs),as precursors of mammalian germ lineage,have been gaining more attention as a new resource of pluripotent stem cells,which bring a great possibility to study developmental events of germ cell in vitro and at animal level.EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.With an attempt to study the differentiation capability of EG4 cells with a reporter protein:green fluorescence protein,and the possible application of EG4 cells in the research of germ cell development,we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells.Then,the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied.The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies.

  13. In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana.

    Science.gov (United States)

    Kurihara, Daisuke; Kimata, Yusuke; Higashiyama, Tetsuya; Ueda, Minako

    2017-09-11

    In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

  14. Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x x 2x sexual hybridisation and its application to extensive breeding programs.

    Science.gov (United States)

    Aleza, P; Juárez, J; Cuenca, J; Ollitrault, P; Navarro, Luis

    2010-09-01

    Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x x 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x x 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders' rights.

  15. Embryo as epiphenomenon: some cultural, social and economic forces driving the stem cell debate.

    Science.gov (United States)

    Green, R M

    2008-12-01

    Our human embryonic stem cell debates are not simply about good or bad ethical arguments. The fetus and the embryo have instead become symbols for a larger set of value conflicts occasioned by social and cultural changes. Beneath our stem cell debates lie conflicts between those who would privilege scientific progress and individual choice and others who favour the sanctity of family life and traditional family roles. Also at work, on both the national and international levels, is the use of the embryo by newly emergent social groups to express resentment against cultural elites. The organisational needs of religious groups have also played a role, with the issue of protection of the embryo and fetus serving as a useful means of rallying organisational allegiance in the Roman Catholic and evangelical communities. Because the epiphenomenal moral positions on the status and use of the embryo are driven by the powerful social, cultural or economic forces beneath them, they will most likely change only with shifts in the underlying forces that sustain them.

  16. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

    Directory of Open Access Journals (Sweden)

    Jeongwoo Kwon

    Full Text Available ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10 is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ, and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR, supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

  17. Development of interspecies nuclear transfer embryos reconstructed with argali (Ovis ammon) somatic cells and sheep ooplasm.

    Science.gov (United States)

    Pan, Xiaoyan; Zhang, Yanli; Guo, Zhiqin; Wang, Feng

    2014-02-01

    Interspecies nuclear transfer has already achieved success in several species, which shows great potential in recovery and conservation of endangered animals. The study was conducted to establish an efficient system for in vitro argali (Ovis ammon)-sheep embryo reconstruction via interspecies somatic cell nuclear transfer (iSCNT). The competence of domestic sheep cytoplasts to reprogram the adult argali fibroblast nuclei was evaluated, and the effects of enucleation methods and donor cell passage and cell state on the in vitro development of argali-sheep cloned embryos were also examined. Sheep oocytes could support argali and sheep fibroblast cell nuclei transfer and develop to blastocysts in vitro. Oocytes matured for 21–23 h and enucleated by chemically assisted enucleation (CAE) had a higher enucleation rate than blind enucleation (BE), but the development rate of iSCNTembryos was the same (P>0.05). Moreover, passage numbers of fibroblast cells cell cycle stages did not affect the development rate of iSCNT reconstructed embryos. Thus sheep cytoplasm successfully supports argali nucleus development to blastocyst stage after optimising the nuclear transfer procedure, which indicates that iSCNT can be used to conserve endangered argali in the near future.

  18. Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

    Directory of Open Access Journals (Sweden)

    Tong Zhang

    Full Text Available BACKGROUND: Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported. METHODOLOGY AND PRINCIPAL FINDINGS: To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures. CONCLUSION AND SIGNIFICANCE: Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

  19. In Situ Microprobe Single-Cell Capillary Electrophoresis Mass Spectrometry: Metabolic Reorganization in Single Differentiating Cells in the Live Vertebrate (Xenopus laevis) Embryo.

    Science.gov (United States)

    Onjiko, Rosemary M; Portero, Erika P; Moody, Sally A; Nemes, Peter

    2017-07-05

    Knowledge of single-cell metabolism would provide a powerful look into cell activity changes as cells differentiate to all the tissues of the vertebrate embryo. However, single-cell mass spectrometry technologies have not yet been made compatible with complex three-dimensional changes and rapidly decreasing cell sizes during early development of the embryo. Here, we bridge this technological gap by integrating capillary microsampling, microscale metabolite extraction, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to enable direct metabolic analysis of identified cells in the live frog embryo (Xenopus laevis). Microprobe CE-ESI-MS of cell content allowed us to detect ∼230 different molecular features (positive ion mode), including 70 known metabolites, in single dorsal and ventral cells in 8-to-32-cell embryos. Relative quantification followed by multivariate and statistical analysis of the data found that microsampling enhanced detection sensitivity compared to whole-cell dissection by minimizing chemical interferences and ion suppression effects from the culture media. In addition, higher glutathione/oxidized glutathione ratios suggested that microprobed cells exhibited significantly lower oxidative stress than those dissected from the embryo. Fast (5 s/cell) and scalable microsampling with minimal damage to cells in the 8-cell embryo enabled duplicate and triplicate metabolic analysis of the same cell, which surprisingly continued to divide to the 16-cell stage. Last, we used microprobe single-cell CE-ESI-MS to uncover previously unknown reorganization of the single-cell metabolome as the dorsal progenitor cell from the 8-cell embryo formed the neural tissue fated clone through divisions to the 32-cell embryo, peering, for the first time, into the formation of metabolic single-cell heterogeneity during early development of a vertebrate embryo.

  20. T cell precursor migration towards beta 2-microglobulin is involved in thymus colonization of chicken embryos

    DEFF Research Database (Denmark)

    Dunon, D; Kaufman, J; Salomonsen, J;

    1990-01-01

    beta 2-microglobulin (beta 2m) attracts hemopoietic precursors from chicken bone marrow cells in vitro. The cell population responding to beta 2m increases during the second period of thymus colonization, which takes place at days 12-14 of incubation. The precursors from 13.5 day old embryos were...... isolated after migration towards beta 2m in vitro and shown to be able to colonize a 13 day old thymus in ovo, where they subsequently acquire thymocyte markers. In contrast these beta 2m responsive precursors did not colonize embryonic bursa, i.e. differentiate into B lymphocytes. During chicken...... embryogenesis, peaks of beta 2m transcripts and of free beta 2m synthesis can only be detected in the thymus. The peak of free beta 2m synthesis in the thymus and the increase of beta 2m responding bone marrow cells both occur concomitantly with the second wave of thymus colonization in chicken embryo, facts...

  1. A Neuroblastoma × Glioma Hybrid Cell Line with Morphine Receptors

    Science.gov (United States)

    Klee, Werner A.; Nirenberg, Marshall

    1974-01-01

    A neuroblastoma × glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 × 106 receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors. PMID:4530316

  2. In Vitro Developmental Potential of Cloned Embryos Derived from Bovine Somatic Cells and Rabbits Oocyte

    Institute of Scientific and Technical Information of China (English)

    LIU Ya; LI Bin; ZHAO Huan; CHENG Li-zi; ZHANG Xiao-rong; CHEN Da-yuan; ZHANG Yun-hai; ZHANG Zhi-guo; JING Ren-tao; WANG Cun-li; ZHANG Mei-lin; LI Dong-wei

    2003-01-01

    180 reconstituted embryos were produced by nuclear transplantation using bovine ear fibroblasts at G0 or non-G0 stage as donor nuclei and oocytes collected from superovulated multiparous or young rabbits as recipients. After cultivation in two kinds of medium M199+ 10%FBS or RD+ 10%FBS, 112 of them developed to 2-cell stage (62.2%) and 26 to morula stage (14.4%) and 20 of them eventually developed to blastocyst stage (11. 1% ). There is no significant difference for the cleavage rates in two groups of reconstituted embryos derived from G0-stage and non-G0 stage donor cells respectively. However, G0-stage donor cells could result in higher rate of 8-cell - 16-cell stage embryos significantly (P<0.05), as well as higher rate of blastocysts (P<0.01). It seems that using two different culture systems had no significant effects on the cleavage rate, morula rate or blastocyst rate (P>0.05).

  3. High-Throughput Screening for Bioactive Molecules Using Primary Cell Culture of Transgenic Zebrafish Embryos

    Directory of Open Access Journals (Sweden)

    Haigen Huang

    2012-09-01

    Full Text Available Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

  4. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  5. Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Peura, T.T.; Hartwich, K.M.

    2005-01-01

    The processes of cellular differentiation were studied in somatic cell nuvlear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos w...

  6. Splitting the cell, building the organism: Mechanisms of cell division in metazoan embryos.

    Science.gov (United States)

    Kumar, Megha; Pushpa, Kumari; Mylavarapu, Sivaram V S

    2015-07-01

    The unicellular metazoan zygote undergoes a series of cell divisions that are central to its development into an embryo. Differentiation of embryonic cells leads eventually to the development of a functional adult. Fate specification of pluripotent embryonic cells occurs during the early embryonic cleavage divisions in several animals. Early development is characterized by well-known stages of embryogenesis documented across animals--morulation, blastulation, and morphogenetic processes such as gastrulation, all of which contribute to differentiation and tissue specification. Despite this broad conservation, there exist clearly discernible morphological and functional differences across early embryonic stages in metazoans. Variations in the mitotic mechanisms of early embryonic cell divisions play key roles in governing these gross differences that eventually encode developmental patterns. In this review, we discuss molecular mechanisms of both karyokinesis (nuclear division) and cytokinesis (cytoplasmic separation) during early embryonic divisions. We outline the broadly conserved molecular pathways that operate in these two stages in early embryonic mitoses. In addition, we highlight mechanistic variations in these two stages across different organisms. We finally discuss outstanding questions of interest, answers to which would illuminate the role of divergent mitotic mechanisms in shaping early animal embryogenesis.

  7. Primordial germ cells and amnion development in the avian embryo

    NARCIS (Netherlands)

    De Melo Bernardo, Ana

    2016-01-01

    Primordial germ cells (PGCs) are the progenitors of the gametes, responsible for transmitting genetic information from generation to generation. Although there is a long history of gamete biology research, there is still a lot to be learned about many of the mechanisms underlying germ cell

  8. Primordial germ cells and amnion development in the avian embryo

    NARCIS (Netherlands)

    De Melo Bernardo, Ana

    2016-01-01

    Primordial germ cells (PGCs) are the progenitors of the gametes, responsible for transmitting genetic information from generation to generation. Although there is a long history of gamete biology research, there is still a lot to be learned about many of the mechanisms underlying germ cell developme

  9. Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

    Directory of Open Access Journals (Sweden)

    Amparo Galán

    Full Text Available Blastomere fate and embryonic genome activation (EGA during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM (n = 120, stemness (n = 190 and Trophectoderm (TE (n = 45, were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1, stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT, and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR. The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92 such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2 and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4, as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

  10. Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

    Science.gov (United States)

    Galán, Amparo; Montaner, David; Póo, M. Eugenia; Valbuena, Diana; Ruiz, Verónica; Aguilar, Cristóbal; Dopazo, Joaquín; Simón, Carlos

    2010-01-01

    Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented. PMID:21049019

  11. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

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    LiBing Ma

    Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

  12. Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as follows: SCNT embryos were reconstructed by using fetal fibroblasts of Chinese miniature pig as donors and in vitro matured oocytes of prepubertal gilts as recipients. Reconstructed embryos were induced by electrical fusion/activation and cultured in BSA-containing North Carolina State University 23 medium (NCSU-23) or Porcine Zygote Medium (PZM-3) at the gas condition of 5% CO2, 7% O2, 88% N2. A total of 230 cloned embryos were transferred to three surrogate sows, producing three piglets. One of them is apparently healthy. The clonal provenance of the piglet was indicated by its coat color and confirmed by DNA microsatellite analysis. These results indicate that the use of in vitro matured oocytes from prepubertal gilts as recipient, combined with cloned embryos cultured at low oxygen tension is an effective way to produce cloned pigs.

  13. Molecular biology of the stress response in the early embryo and its stem cells.

    Science.gov (United States)

    Puscheck, Elizabeth E; Awonuga, Awoniyi O; Yang, Yu; Jiang, Zhongliang; Rappolee, Daniel A

    2015-01-01

    Stress is normal during early embryogenesis and transient, elevated stress is commonplace. Stress in the milieu of the peri-implantation embryo is a summation of maternal hormones, and other elements of the maternal milieu, that signal preparedness for development and implantation. Examples discussed here are leptin, adrenaline, cortisol, and progesterone. These hormones signal maternal nutritional status and provide energy, but also signal stress that diverts maternal and embryonic energy from an optimal embryonic developmental trajectory. These hormones communicate endocrine maternal effects and local embryonic effects although signaling mechanisms are not well understood. Other in vivo stresses affect the embryo such as local infection and inflammation, hypoxia, environmental toxins such as benzopyrene, dioxin, or metals, heat shock, and hyperosmotic stress due to dehydration or diabetes. In vitro, stresses include shear during handling, improper culture media and oxygen levels, cryopreservation, and manipulations of the embryo to introduce sperm or mitochondria. We define stress as any stimulus that slows stem cell accumulation or diminishes the ability of cells to produce normal and sufficient parenchymal products upon differentiation. Thus stress deflects downwards the normal trajectories of development, growth and differentiation. Typically stress is inversely proportional to embryonic developmental and proliferative rates, but can be proportional to induction of differentiation of stem cells in the peri-implantation embryo. When modeling stress it is most interesting to produce a 'runting model' where stress exposures slow accumulation but do not create excessive apoptosis or morbidity. Windows of stress sensitivity may occur when major new embryonic developmental programs require large amounts of energy and are exacerbated if nutritional flow decreases and removes energy from the normal developmental programs and stress responses. These windows correspond

  14. The chick embryo as an experimental system for melanoma cell invasion.

    Directory of Open Access Journals (Sweden)

    Christian Busch

    Full Text Available BACKGROUND: A primary cutaneous melanoma will not kill the patient, but its metastases. Since in vitro studies on melanoma cells in 2-D cultures do often not reflect reality, 3-D models might come closer to the physiological situation in the patient during cancer initiation and progression. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe the chick embryo model for in vivo studies of melanoma cell migration and invasion. After transplantation of neural crest-derived melanoma cells into the neural tube, the melanoma cells resume neural crest cell migration along the medial and lateral pathways and finally undergo apoptosis in the target areas. Upon transplantation into ectopic areas such as the hindbrain or the optic cup malignant invasion and local tissue destruction occurs. In contrast, melanocytes are not able to spontaneously resume neural crest cell migration. However, malignant invasion can be induced in melanocytes by pre-treatment with the TGF-beta family members bone morphegenetic protein-2 or nodal. Transplantation of MCF7 breast cancer cells yields a different growth pattern in the rhombencephalon than melanoma cells. CONCLUSIONS/SIGNIFICANCE: The chick embryo model is a feasible, cost-effective in vivo system to study invasion by cancer cells in an embryonic environment. It may be useful to study invasive behavior induced by embryonic oncogenes and for targeted manipulation of melanoma or breast cancer cells aiming at ablation of invasive properties.

  15. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

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    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  16. Hybrid solar cell on a carbon fiber

    Science.gov (United States)

    Grynko, Dmytro A.; Fedoryak, Alexander N.; Smertenko, Petro S.; Dimitriev, Oleg P.; Ogurtsov, Nikolay A.; Pud, Alexander A.

    2016-05-01

    In this work, a method to assemble nanoscale hybrid solar cells in the form of a brush of radially oriented CdS nanowire crystals around a single carbon fiber is demonstrated for the first time. A solar cell was assembled on a carbon fiber with a diameter of ~5-10 μm which served as a core electrode; inorganic CdS nanowire crystals and organic dye or polymer layers were successively deposited on the carbon fiber as active components resulting in a core-shell photovoltaic structure. Polymer, dye-sensitized, and inverted solar cells have been prepared and compared with their analogues made on the flat indium-tin oxide electrode.

  17. Canine Distemper Virus Utilizes Different Receptors to Infect Chicken Embryo Fibroblasts and Vero cells

    Institute of Scientific and Technical Information of China (English)

    Jun Chen; Xiu Liang; Pei-fu Chen

    2011-01-01

    Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF)is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV)also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PERTM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.

  18. Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer.

    Science.gov (United States)

    Cong, Peiqing; Zhu, Kongju; Ji, Qianqian; Zhao, Haijing; Chen, Yaosheng

    2013-09-01

    Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

  19. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

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    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results 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. Conclusion 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.

  20. Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

    Science.gov (United States)

    Pang, Yun-Wei; Sun, Ye-Qing; Sun, Wei-Jun; Du, Wei-Hua; Hao, Hai-Sheng; Zhao, Shan-Jiang; Zhu, Hua-Bin

    2016-03-01

    Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions.

  1. Effects of seawater acidification on cell cycle control mechanisms in Strongylocentrotus purpuratus embryos.

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    Sean P Place

    Full Text Available Previous studies have shown fertilization and development of marine species can be significantly inhibited when the pH of sea water is artificially lowered. Little mechanistic understanding of these effects exists to date, but previous work has linked developmental inhibition to reduced cleavage rates in embryos. To explore this further, we tested whether common cell cycle checkpoints were involved using three cellular biomarkers of cell cycle progression: (1 the onset of DNA synthesis, (2 production of a mitotic regulator, cyclin B, and (3 formation of the mitotic spindle. We grew embryos of the purple sea urchin, Strongylocentrotus purpuratus, in seawater artifically buffered to a pH of ∼7.0, 7.5, and 8.0 by CO(2 infusion. Our results suggest the reduced rates of mitotic cleavage are likely unrelated to common cell cycle checkpoints. We found no significant differences in the three biomarkers assessed between pH treatments, indicating the embryos progress through the G(1/S, G(2/M and metaphase/anaphase transitions at relatively similar rates. These data suggest low pH environments may not impact developmental programs directly, but may act through secondary mechanisms such as cellular energetics.

  2. Production of wild buffalo (Bubalus arnee) embryos by interspecies somatic cell nuclear transfer using domestic buffalo (Bubalus bubalis) oocytes.

    Science.gov (United States)

    Priya, D; Selokar, N L; Raja, A K; Saini, M; Sahare, A A; Nala, N; Palta, P; Chauhan, M S; Manik, R S; Singla, S K

    2014-04-01

    The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin-18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p cell proliferation rate was significantly (p cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open-pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re-expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified-warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.

  3. Single-Cell Profiling of Epigenetic Modifiers Identifies PRDM14 as an Inducer of Cell Fate in the Mammalian Embryo

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    Adam Burton

    2013-11-01

    Full Text Available Cell plasticity or potency is necessary for the formation of multiple cell types. The mechanisms underlying this plasticity are largely unknown. Preimplantation mouse embryos undergo drastic changes in cellular potency, starting with the totipotent zygote through to the formation of the pluripotent inner cell mass (ICM and differentiated trophectoderm in the blastocyst. Here, we set out to identify and functionally characterize chromatin modifiers that define the transitions of potency and cell fate in the mouse embryo. Using a quantitative microfluidics approach in single cells, we show that developmental transitions are marked by distinctive combinatorial profiles of epigenetic modifiers. Pluripotent cells of the ICM are distinct from their differentiated trophectoderm counterparts. We show that PRDM14 is heterogeneously expressed in 4-cell-stage embryos. Forced expression of PRDM14 at the 2-cell stage leads to increased H3R26me2 and can induce a pluripotent ICM fate. Our results shed light on the epigenetic networks that govern cellular potency and identity in vivo.

  4. Techno-economic comparison of series hybrid, plug-in hybrid, fuel cell and regular cars

    Science.gov (United States)

    van Vliet, Oscar P. R.; Kruithof, Thomas; Turkenburg, Wim C.; Faaij, André P. C.

    We examine the competitiveness of series hybrid compared to fuel cell, parallel hybrid, and regular cars. We use public domain data to determine efficiency, fuel consumption, total costs of ownership and greenhouse gas emissions resulting from drivetrain choices. The series hybrid drivetrain can be seen both as an alternative to petrol, diesel and parallel hybrid cars, as well as an intermediate stage towards fully electric or fuel cell cars. We calculate the fuel consumption and costs of four diesel-fuelled series hybrid, four plug-in hybrid and four fuel cell car configurations, and compared these to three reference cars. We find that series hybrid cars may reduce fuel consumption by 34-47%, but cost €5000-12,000 more. Well-to-wheel greenhouse gas emissions may be reduced to 89-103 g CO 2 km -1 compared to reference petrol (163 g km -1) and diesel cars (156 g km -1). Series hybrid cars with wheel motors have lower weight and 7-21% lower fuel consumption than those with central electric motors. The fuel cell car remains uncompetitive even if production costs of fuel cells come down by 90%. Plug-in hybrid cars are competitive when driving large distances on electricity, and/or if cost of batteries come down substantially. Well-to-wheel greenhouse gas emissions may be reduced to 60-69 g CO 2 km -1.

  5. Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

    Institute of Scientific and Technical Information of China (English)

    Wang ErYao; YU Yang; Li XueMei; JIAO LiHong; Wang Liu

    2007-01-01

    A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse activation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.

  6. The importance of carbonic anhydrase II in red blood cells during exposure of chicken embryos to CO2.

    Science.gov (United States)

    Everaert, N; Willemsen, H; Hulikova, A; Brown, H; Decuypere, E; Swietach, P; Bruggeman, V

    2010-07-31

    The importance of carbonic anhydrase (CA) during exposure of chicken embryos to CO(2) during the second half of incubation was investigated. The protein abundance and activity of CAII in erythrocytes was significantly higher in CO(2)-exposed embryos compared to normal conditions. Daily injections of acetazolamide (ATZ), an inhibitor of CA, increased blood P(CO2) and decreased blood pH in both control and CO(2)-incubated embryos. ATZ increased blood bicarbonate concentration in embryos exposed to normal atmosphere and in day-12 embryos exposed to high CO(2). The tendency of an increased blood potassium concentration in ATZ-injected embryos under standard atmospheric conditions might indicate that protons were exchanged with intracellular potassium. However, there was no evidence for such an exchange in CO(2)-incubated ATZ-treated embryos. This study shows for the first time that chicken embryos adapt to CO(2) during the second half of incubation by increasing CAII protein expression and function in red blood cells. This response may serve to "buffer" elevated CO(2) levels.

  7. Relationship Between Development, Metabolism, and Mitochondrial Organization in 2-Cell Hamster Embryos in the Presence of Low Levels of Phosphate

    Science.gov (United States)

    Ludwig, Tenneille E.; Squirrell, Jayne M.; Palmenberg, Ann C.; Bavister, Barry D.

    2016-01-01

    The effect of low concentrations of inorganic phosphate (Pi) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 µM KH2PO4. Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with Pi did not alter TCA-cycle activity. However, culture with ≥2.5 µM Pi significantly increased (P organization was significantly (P culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence. PMID:11717124

  8. An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes.

    Science.gov (United States)

    Patel, Hiren; Chougule, Shruti; Chohan, Parul; Shah, Naval; Bhartiya, Deepa

    2014-10-01

    Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 microM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

  9. A Bicontinuous Double Gyroid Hybrid Solar Cell

    KAUST Repository

    Crossland, Edward J. W.

    2009-08-12

    We report the first successful application of an ordered bicontinuous gyroid semiconducting network in a hybrid bulk heterojunction solar cell. The freestanding gyroid network is fabricated by electrochemical deposition into the 10 nm wide voided channels of a self-assembled, selectively degradable block copolymer film. The highly ordered pore structure is ideal for uniform infiltration of an organic hole transporting material, and solid-state dye-sensitized solar cells only 400 nm thick exhibit up to 1.7% power conversion efficiency. This patterning technique can be readily extended to other promising heterojunction systems and is a major step toward realizing the full potential of self-assembly in the next generation of device technologies. © 2009 American Chemical Society.

  10. Hybrid nanorod-polymer solar cells.

    Science.gov (United States)

    Huynh, Wendy U; Dittmer, Janke J; Alivisatos, A Paul

    2002-03-29

    We demonstrate that semiconductor nanorods can be used to fabricate readily processed and efficient hybrid solar cells together with polymers. By controlling nanorod length, we can change the distance on which electrons are transported directly through the thin film device. Tuning the band gap by altering the nanorod radius enabled us to optimize the overlap between the absorption spectrum of the cell and the solar emission spectrum. A photovoltaic device consisting of 7-nanometer by 60-nanometer CdSe nanorods and the conjugated polymer poly-3(hexylthiophene) was assembled from solution with an external quantum efficiency of over 54% and a monochromatic power conversion efficiency of 6.9% under 0.1 milliwatt per square centimeter illumination at 515 nanometers. Under Air Mass (A.M.) 1.5 Global solar conditions, we obtained a power conversion efficiency of 1.7%.

  11. Cell-free DNA in human follicular fluid as a biomarker of embryo quality.

    Science.gov (United States)

    Scalici, E; Traver, S; Molinari, N; Mullet, T; Monforte, M; Vintejoux, E; Hamamah, S

    2014-12-01

    Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for in vitro fertilization (IVF) outcomes? CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes. CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated. One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (n = 26) or ICSI (n = 17). CfDNA level was quantified in each individual follicular fluid sample. At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers. Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean ± SD, 1.62 ± 2.08 ng/µl). CfDNA level was significantly higher in small follicles (8-12 mm in diameter) than in large ones (>18 mm) (mean ± SD, 2.54 ± 0.78 ng/µl versus 0.71 ± 0.44 ng/µl, respectively, P = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (r = -0.34; P = 0.003). A weak significant negative correlation between DNA integrity and 17β-estradiol level in follicular fluid samples at oocyte collection day was observed (r = -0

  12. Lipid-mediated gene transfection into chick embryo retinal cells in ovo and in vitro.

    Science.gov (United States)

    Toy, J; Bradford, R L; Adler, R

    2000-12-15

    Several lipofection reagents were tested on chick embryo retinal cultures using green fluorescent protein (GFP) as a reporter gene; best results were obtained with the GenePORTER (GP) reagent, which yielded approximately 4.4% of the cells with intense GFP fluorescence. Cell survival and structural differentiation appeared normal, but one of the immunocytochemical markers studied (visinin) was less frequently observed in GP-treated cultures. When similar plasmid-GP mixtures were injected into chick embryo eyes in ovo, bright GFP-fluorescent cells were observed in different retinal layers, without detectable detrimental effects on retinal morphology. Particularly extensive reporter gene expression was obtained upon intraocular injection of GP plus naked DNA from a RCAS retrovirus, which resulted in the development of abundant radial columns of alkaline phosphatase-positive cells, separated by columns of negative cells. We conclude that lipid-based transfection offers a quick, simple and fairly innocuous means for gene delivery into proliferating and postmitotic retinal cells, in vitro as well as in the developing eye in ovo, and that transfection of naked retroviral DNA can lead to extensive expression of foreign genes by retinal cells, bypassing the time-consuming steps required for the generation of high-titer virion stocks.

  13. Programming Pluripotent Precursor Cells Derived from Xenopus Embryos to Generate Specific Tissues and Organs

    Directory of Open Access Journals (Sweden)

    Annette Borchers

    2010-11-01

    Full Text Available Xenopus embryos provide a rich source of pluripotent cells that can be differentiated into functional organs. Since the molecular principles of vertebrate organogenesis appear to be conserved between Xenopus and mammals, this system can provide useful guidelines for the directional manipulation of human embryonic stem cells. Pluripotent Xenopus cells can be easily isolated from the animal pole of blastula stage Xenopus embryos. These so called “animal cap” cells represent prospective ectodermal cells, but give rise to endodermal, mesodermal and neuro-ectodermal derivatives if treated with the appropriate factors. These factors include evolutionary conserved modulators of the key developmental signal transduction pathways that can be supplied either by mRNA microinjection or direct application of recombinant proteins. This relatively simple system has added to our understanding of pancreas, liver, kidney, eye and heart development. In particular, recent studies have used animal cap cells to generate ectopic eyes and hearts, setting the stage for future work aimed at programming pluripotent cells for regenerative medicine.

  14. Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

    Science.gov (United States)

    Wang, Chao-Jie; Wang, Guang; Wang, Xiao-Yu; Liu, Meng; Chuai, Manli; Lee, Kenneth Ka Ho; He, Xiao-Song; Lu, Da-Xiang; Yang, Xuesong

    2016-06-15

    Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

  15. Influences of different factors on the germination of encapsulated somatic embryos of Saccharum spp hybrid var C 87-51

    Directory of Open Access Journals (Sweden)

    Elisa Quiala

    2001-04-01

    Full Text Available With the objective to regenerate plants from sugarcane somatic embryos encapsulated in sodium alginate hidrogel different factors was studied. The addition of coconut water, GA3 (0.5 mg.l-1 and 6-BAP (0.2 mg.l-1 to the synthetic endosperm increased the velocity and germination percentage of somatic embryos encapsulated. The immersion treatment in 50mM solution of KNO3 during five hours improved the encapsulated somatic embryos germination percentage. ABBREVIATOR: AG3- Giberelic Acid; 6-BAP- 6-Bencilaminopurine; MS- Murashige and Skoog (1962 Key Words: sodium alginate, sugar cane, synthetic seed

  16. Effect of roscovitine-treated donor cells on development of porcine cloned embryos.

    Science.gov (United States)

    Park, H J; Koo, O J; Kwon, D K; Kang, J T; Jang, G; Lee, B C

    2010-12-01

    Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72 h for serum starvation or with 15 μM roscovitine for 24 h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively p roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis-related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine-treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production. © 2009 Blackwell Verlag GmbH.

  17. Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos

    Directory of Open Access Journals (Sweden)

    Daniel Concepcion

    2017-07-01

    Full Text Available Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos.

  18. Effects of developmental stages of tetraploid embryos on mouse chimeras from embryonic stem cells%四倍体胚胎发育阶段对胚胎干细胞嵌合体小鼠制备的影响

    Institute of Scientific and Technical Information of China (English)

    吴兴龙; 张鹏; 赵虎; 王鹏博; 胡春超; 李相运

    2011-01-01

    目的 探讨四倍体胚胎发育阶段对胚胎干细胞(ES)嵌合体小鼠制备的影响.方法 通过2-细胞胚胎电融合法制备四倍体胚胎,采用显微注射方法将ES细胞分别注入1-细胞、4-细胞、囊胚3个发育阶段的四倍体胚胎中.所用ES细胞分别为杂交系B6D2F1×129/Sv和近交系C57BL/6J,经胚胎移植和剖腹产以获得ES小鼠.结果 实验表明,2-细胞胚胎电融合率为92.45%,4-细胞胚胎发育率为93.51%,囊胚发育率为90.42%.杂交ES细胞注射四倍体囊胚获得22只ES小鼠,效率显著高于近交系ES细胞以及其他发育阶段的四倍体胚胎,ES小鼠与B6D2F1×129/Sv杂交小鼠毛色一致且具有正常生殖能力.结论 四倍体胚胎的发育阶段显著影响ES小鼠的制备.%Objective To investigate the effects of developmental stages of tetraploid embryos on mouse chimeras from embryonic stem cells ( ES ).Methods Tetraploid embryo complementation and microinjection were utilized to produce ES mice that derived completely from ES cells.Tetraploid embryos were firstly prepared by electrofusion of 2-cell mouse embryos, and then embryonic stem ( ES ) cells with different genetic background ( hybrid or inbred ) were injected into tetraploid 1-cell, 4-cell, and blastocyst stage embryos.The injected embryos were transferred into uterine horns of pseudopregnant 2.5days female CDl mice.The CD1 mice were suhjected into cesarean sections after 16 days.Results Our data showed that 92.45% of 2-cell embryos were electrofused, 93.51% and 90.42% of the electrofused embryos developed to 4-cell and blastocyst stages, respectively.Blastocysts were injected with hybrid ES cells and 22 ES mice were obtained by Cesarean section.Blastocyst was more effective than 1-cell and 4-cell.ES mice had normal germline transmission capahility and the same coat color as the hybrid mice which hybrid ES cells were drived from.Conclusion The tetraploid embryo developmental stage is able to affect the ES mouse

  19. Cell-in-Shell Hybrids: Chemical Nanoencapsulation of Individual Cells.

    Science.gov (United States)

    Park, Ji Hun; Hong, Daewha; Lee, Juno; Choi, Insung S

    2016-05-17

    Nature has developed a fascinating strategy of cryptobiosis ("secret life") for counteracting the stressful, and often lethal, environmental conditions that fluctuate sporadically over time. For example, certain bacteria sporulate to transform from a metabolically active, vegetative state to an ametabolic endospore state. The bacterial endospores, encased within tough biomolecular shells, withstand the extremes of harmful stressors, such as radiation, desiccation, and malnutrition, for extended periods of time and return to a vegetative state by breaking their protective shells apart when their environment becomes hospitable for living. Certain ciliates and even higher organisms, for example, tardigrades, and others are also found to adopt a cryptobiotic strategy for survival. A common feature of cryptobiosis is the structural presence of tough sheaths on cellular structures. However, most cells and cellular assemblies are not "spore-forming" and are vulnerable to the outside threats. In particular, mammalian cells, enclosed with labile lipid bilayers, are highly susceptible to in vitro conditions in the laboratory and daily life settings, making manipulation and preservation difficult outside of specialized conditions. The instability of living cells has been a main bottleneck to the advanced development of cell-based applications, such as cell therapy and cell-based sensors. A judicious question arises: can cellular tolerance against harmful stresses be enhanced by simply forming cell-in-shell hybrid structures? Experimental results suggest that the answer is yes. A micrometer-sized "Iron Man" can be generated by chemically forming an ultrathin (cell. Since the report on silica nanoencapsulation of yeast cells, in which cytoprotective yeast-in-silica hybrids were formed, several synthetic strategies have been developed to encapsulate individual cells in a cytocompatible fashion, mimicking the cryptobiotic cell-in-shell structures found in nature, for example

  20. Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos

    DEFF Research Database (Denmark)

    Alexopoulos, Natalie I.; Maddox-Hyttel, Poul; Tveden-Nyborg, Pernille Yde

    2008-01-01

    the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy...... was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining...

  1. Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos

    DEFF Research Database (Denmark)

    Alexopoulos, Natalie I.; Maddox-Hyttel, Poul; Tveden-Nyborg, Pernille Yde

    2008-01-01

    the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy...... recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast...

  2. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results 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. Conclusion 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.

  3. Mitosis and cell death in the tail of the chick embryo.

    Science.gov (United States)

    Mills, C L; Bellairs, R

    1989-01-01

    Although somites develop from the mesoderm in the tail of the chick embryo, they do not form to the tip of the tail. Previous work has shown that this terminal mesoderm possesses many of the characteristics of the segmental plate mesoderm which gives rise to the somites in the trunk. This investigation is aimed therefore at understanding why the terminal mesoderm fails to form somites. Mitotic and pyknotic rates have been obtained for the tail region of chick embryos between stages 13 and 27. Embryos were treated with colchicine, so that the mitoses were blocked in metaphase, and counts were made on serial sections. The overall mitotic rates were highest between stages 15 and 18. Regions of high mitotic rate, which are an indication of cell synchrony, were found in the tail bud mesoderm though not in a consistent location, and only infrequently near the anterior end of the tail segmental plate. In the trunk however (Stern and Bellairs 1984) a single peak of cell synchrony was routinely found near the cranial end of the segmental plate. It is concluded that the cells of the tail mesoderm are less synchronised in preparation for somitogenesis than are the corresponding mesoderm cells in the trunk. A further conclusion is that the tail bud is not per se a region of high proliferation, though there are patches of high mitotic rate. The overall pyknotic rate reached a maximum at stage 25; peaks of pyknosis corresponded initially with the mitotic peaks and were associated with the ventral ectodermal ridge and the tail gut. By stage 25 however, the high levels of cell death were restricted mainly to the tip of the tail.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Time course of programmed cell death, which included autophagic features, in hybrid tobacco cells expressing hybrid lethality.

    Science.gov (United States)

    Ueno, Naoya; Nihei, Saori; Miyakawa, Naoto; Hirasawa, Tadashi; Kanekatsu, Motoki; Marubashi, Wataru; van Doorn, Wouter G; Yamada, Tetsuya

    2016-12-01

    PCD with features of vacuolar cell death including autophagy-related features were detected in hybrid tobacco cells, and detailed time course of features of vacuolar cell death were established. A type of interspecific Nicotiana hybrid, Nicotiana suaveolens × N. tabacum exhibits temperature-sensitive lethality. This lethality results from programmed cell death (PCD) in hybrid seedlings, but this PCD occurs only in seedlings and suspension-cultured cells grown at 28 °C, not those grown at 36 °C. Plant PCD can be classified as vacuolar cell death or necrotic cell death. Induction of autophagy, vacuolar membrane collapse and actin disorganization are each known features of vacuolar cell death, but observed cases of PCD showing all these features simultaneously are rare. In this study, these features of vacuolar cell death were evident in hybrid tobacco cells expressing hybrid lethality. Ion leakage, plasma membrane disruption, increased activity of vacuolar processing enzyme, vacuolar membrane collapse, and formation of punctate F-actin foci were each evident in these cells. Transmission electron microscopy revealed that macroautophagic structures formed and tonoplasts ruptured in these cells. The number of cells that contained monodansylcadaverine (MDC)-stained structures and the abundance of nine autophagy-related gene transcripts increased just before cell death at 28 °C; these features were not evident at 36 °C. We assessed whether an autophagic inhibitor, wortmannin (WM), influenced lethality in hybrid cells. After the hybrid cell began to die, WM suppressed increases in ion leakage and cell deaths, and it decreased the number of cells containing MDC-stained structures. These results showed that several features indicative of autophagy and vacuolar cell death were evident in the hybrid tobacco cells subject to lethality. In addition, we documented a detailed time course of these vacuolar cell death features.

  5. Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator.

    Science.gov (United States)

    Talukder, Anup K; Yousef, Mohamed S; Rashid, Mohammad B; Awai, Kensuke; Acosta, Tomas J; Shimizu, Takashi; Okuda, Kiyoshi; Shimada, Masayuki; Imakawa, Kazuhiko; Miyamoto, Akio

    2017-08-19

    Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus.

  6. Ethical and policy issues surrounding the donation of cryopreserved and fresh embryos for human embryonic stem cell research.

    Science.gov (United States)

    Cohen, Cynthia B

    2009-06-01

    The use of human embryos in human embryonic stem cell (hESC) research raises significant ethical and policy issues associated with their donation. Recent research conducted in several countries assesses the percent of persons with cryopreserved and fresh supernumerary embryos willing to donate them for research, their reasons for considering this option, and the concerns they raise about its personal import. Such research provides new insights into rising ethical and policy questions associated with embryo donation for hESC research that should be addressed. In response to such questions, it is argued here that consent to the donation of supernumerary embryos for hESC research should be sought in two or three stages, depending on whether fresh or frozen embryos are at issue, in order to provide patients and their partners with sufficient time and information before they make a final decision. In addition, steps should be taken to support the voluntariness of their decisions by having personnel other than the treating reproductive specialist or stem cell investigators solicit their consent. Prospective embryo donors should also be given a choice about the uses to which hESCs derived from their donated embryos will be put in order to honor their ethical convictions and ensure that there are sufficient embryos for this research. The well-being and rights of those who donate embryos for this research require the sort of support and protection that can be provided by an ethical and policy framework that allows hESC investigations to move forward according to standards that are transparent and that resound with public values.

  7. ABA-Mediated Inhibition of Germination Is Related to the Inhibition of Genes Encoding Cell-Wall Biosynthetic and Architecture:Modifying Enzymes and Structural Proteins in Medicago truncatula Embryo Axis

    Institute of Scientific and Technical Information of China (English)

    Christine Gimeno-Gilles; Eric Lelièvre; Laure Viau; Mustafa Malik-Ghulam; Claudie Ricoult; Andreas Niebel; Nathalie Leduc; Anis M. Limami

    2009-01-01

    Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid (ABA) but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.

  8. Growing an Embryo from a Single Cell: A Hurdle in Animal Life.

    Science.gov (United States)

    O'Farrell, Patrick H

    2015-08-07

    A requirement that an animal be able to feed to grow constrains how a cell can grow into an animal, and it forces an alternation between growth (increase in mass) and proliferation (increase in cell number). A growth-only phase that transforms a stem cell of ordinary proportions into a huge cell, the oocyte, requires dramatic adaptations to help a nucleus direct a 10(5)-fold expansion of cytoplasmic volume. Proliferation without growth transforms the huge egg into an embryo while still accommodating an impotent nucleus overwhelmed by the voluminous cytoplasm. This growth program characterizes animals that deposit their eggs externally, but it is changed in mammals and in endoparasites. In these organisms, development in a nutritive environment releases the growth constraint, but growth of cells before gastrulation requires a new program to sustain pluripotency during this growth.

  9. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  10. The mitochondrial function was impaired in APP knockout mouse embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    SHENG BaiYang; NIU Ying; ZHOU Hui; YAN JiaXin; ZHAO NanMing; ZHANG XiuFang; GONG YanDao

    2009-01-01

    The amyloid precursor protein (APP) is recognized as the source of Aβ, which plays an important role in Alzheimer's disease. However, the biological function of APP is obscure. Previous studies showed that mitochondria could be a target of APP. In this work, APP knockout mouse embryo fibroblast (MEF) cells were used to test if APP plays any role in maintaining the mitochondrial function. As the result, APP knockout MEF cells (APP-/- cells) showed the abnormal mitochondrial function, including slower cell proliferation, lower mitochondrial membrane potential, lower intracellular ROS, higher mitochon-drial membrane fluidity and lower cytochrome c oxidase activity than their wild-type counterparts. However, no change was found in the amount of mitochondria in MEF APP-/- cells.

  11. Mechanochemical Pattern Formation in the Polarization of the One-Cell C. Elegans Embryo

    Science.gov (United States)

    Bois, Justin S.; Grill, Stephan W.

    2013-12-01

    Cellular polarity refers to the uneven distribution of certain proteins and nucleic acids on one half of a cell versus the other. Polarity establishment is often an essential process in the development, being responsible for cell differentiation upon division of the polarized cell. The one cell embryo of the nematode Caenorhabditis elegans is a classic model system for the study of polarity. Interestingly, distribution of polarity proteins is accompanied by directional movements of the cell cytoskeleton in this system. In addition to undergoing diffusion, the polarity proteins are transported by these movements. Thus, polarization is achieved by both mechanical and chemical means. We discuss our current understanding of this process in the C. elegans model system. We also discuss more general consequences of mechanochemical coupling in morphogenesis.

  12. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  13. Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    JI Jingjuan; GUO Tonghang; TONG Xianhong; LUO Lihua; ZHOU Guixiang; FU Yingyun; LIU Yusheng

    2007-01-01

    Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P<0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.

  14. Perkembangan Praimplantasi Embrio Mencit dengan Materi Genetik yang Berasal dari Parental, Maternal, dan Inti Sel Somatik (PRE-IMPLANTATION DEVELOPMENT OF MOUSE EMBRYO WITH GENETIC MATERIAL DERIVED FROM PARENTAL, MATERNAL AND SOMATIC CELL NUCLEUS

    Directory of Open Access Journals (Sweden)

    Harry Murti

    2014-05-01

    Full Text Available Cloned embryo and parthenogenetic embryo are a potential source of stem cells for regenerativemedicine. Stem cells derived from those embryos are expected to overcome the ethical issues to the use offertilization embryos for therapeutic purposes. The pre-implantation development is a critical step fordeveloping embryos reach the blastocyst stage. The objectives in vivo of this research are to produce mousecloned embryo, parthenogenetic embryo, and fertilized embryo and to study stages of  in vitro pre-implantation development culture. In vivo fertilized embryos, mouse oocytes, and cumulus cells were usedin this study. Treatment was performed on female mice superovulated with PMSG and hCG injections.Two-cell stage of in vivo fertilized embryos were collected on the second day post hCG injection. Clonedembryos were produced through Somatic Cell Nuclear Transfer (SCNT, which included enucleation, nucleartransfer and artificial activation. Parthenogenetic embryos were produced with artificial activationtechnique. The result of the research indicated that SCNT application was able to produce cloned embryos which could develop to blastocyst stage (3,2%. In addition, artificial activation of oocytes could produceparthenogenetic embryos which were able to develop up to the blastocyst stage (8,6%. In conclusion,efficiency level of parthenogenetic embryos that is able to reach the blastocyst stage was higher than in thecloned embryos. Fertilized embryos shows a better development and more efficient compared to in vitrocloned embryos and parthenogenetic embryos cultures.

  15. Risk of equine infectious anemia virus disease transmission through in vitro embryo production using somatic cell nuclear transfer.

    Science.gov (United States)

    Gregg, K; Polejaeva, I

    2009-08-01

    Prevention and regulation of equine infectious anemia virus (EIAV) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. Embryo production via the somatic cell nuclear transfer (SCNT) technology uses oocytes collected mainly from untested animals, which creates a potential risk of EIAV transmission through infected embryos. The current review examines the risk of EIAV disease transmission through SCNT embryo production and transfer. Equine infectious anemia virus is a lentivirus from the family Retroviridae. Because of a lack of direct reports on this subject, relevant information gathered from close relatives of EIAV, such as human immunodeficiency virus (HIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), and small ruminant lentiviruses (SRLVs), is summarized and used to predict the biological plausibility of EIAV disease transmission through transfers of the equine SCNT embryos. Based on published information regarding interaction of oocytes with lentiviruses and the sufficiency of oocyte and embryo washing procedures to prevent lentivirus transmission from in vitro-produced embryos of various species, we predicted the risk of EIAV transmission through SCNT embryo production and transfer to be very small or absent.

  16. Hybrid Organic/Inorganic Nanocomposites for Photovoltaic Cells

    Directory of Open Access Journals (Sweden)

    Ruchuan Liu

    2014-04-01

    Full Text Available Inorganic/organic hybrid solar cells have attracted a lot of interest due to their potential in combining the advantages of both components. To understand the key issues in association with photoinduced charge separation/transportation processes and to improve overall power conversion efficiency, various combinations with nanostructures of hybrid systems have been investigated. Here, we briefly review the structures of hybrid nanocomposites studied so far, and attempt to associate the power conversion efficiency with these nanostructures. Subsequently, we are then able to summarize the factors for optimizing the performance of inorganic/organic hybrid solar cells.

  17. Hybrid Organic/Inorganic Nanocomposites for Photovoltaic Cells.

    Science.gov (United States)

    Liu, Ruchuan

    2014-04-02

    Inorganic/organic hybrid solar cells have attracted a lot of interest due to their potential in combining the advantages of both components. To understand the key issues in association with photoinduced charge separation/transportation processes and to improve overall power conversion efficiency, various combinations with nanostructures of hybrid systems have been investigated. Here, we briefly review the structures of hybrid nanocomposites studied so far, and attempt to associate the power conversion efficiency with these nanostructures. Subsequently, we are then able to summarize the factors for optimizing the performance of inorganic/organic hybrid solar cells.

  18. Recombinant Mouse Canstatin Inhibits Chicken Embryo Chorioallantoic Membrane Angiogenesis and Endothelial Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Wei-Hong HOU; Tian-Yun WANG; Bao-Mei YUAN; Yu-Rong CHAI; Yan-Long JIA; Fang TIAN; Jian-Min WANG; Le-Xun XUE

    2004-01-01

    Human canstatin, a 24 kD fragment of the α2 chain of type Ⅳ collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M 15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

  19. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

    Science.gov (United States)

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  20. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    Directory of Open Access Journals (Sweden)

    Binghua Xue

    Full Text Available Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

  1. Transplantation of mammalian embryonic stem cells and their derivatives to avian embryos.

    Science.gov (United States)

    Goldstein, Ronald S

    2010-09-01

    Xenografting of normal and transformed mammalian tissues and cells to chick embryos has been performed for almost 100 years. Embryonic stem cells, derived more than 25 years ago from murine, and more than 10 years ago from human blastocysts, have transformed many fields of biological research. There is a growing body of studies combining these two widely-used experimental systems. This review surveys those reports in which murine or human embryonic stem cells, or differentiated derivatives of these pluripotent stem cells, were transplanted to embryonated chick eggs. Many of these studies have utilized the unique characteristics of both experimental models to obtain answers to developmental questions that are difficult or impossible to approach with xenografting to adult rodents or tissue culture-only techniques.

  2. The forces that shape embryos: physical aspects of convergent extension by cell intercalation

    Science.gov (United States)

    Keller, Ray; Shook, David; Skoglund, Paul

    2008-03-01

    We discuss the physical aspects of the morphogenic process of convergence (narrowing) and extension (lengthening) of tissues by cell intercalation. These movements, often referred to as 'convergent extension', occur in both epithelial and mesenchymal tissues during embryogenesis and organogenesis of invertebrates and vertebrates, and they play large roles in shaping the body plan during development. Our focus is on the presumptive mesodermal and neural tissues of the Xenopus (frog) embryo, tissues for which some physical measurements have been made. We discuss the physical aspects of how polarized cell motility, oriented along future tissue axes, generate the forces that drive oriented cell intercalation and how this intercalation results in convergence and extension or convergence and thickening of the tissue. Our goal is to identify aspects of these morphogenic movements for further biophysical, molecular and cell biological, and modeling studies.

  3. Cofilin and Vangl2 cooperate in the initiation of planar cell polarity in the mouse embryo.

    Science.gov (United States)

    Mahaffey, James P; Grego-Bessa, Joaquim; Liem, Karel F; Anderson, Kathryn V

    2013-03-01

    The planar cell polarity (PCP; non-canonical Wnt) pathway is required to orient the cells within the plane of an epithelium. Here, we show that cofilin 1 (Cfl1), an actin-severing protein, and Vangl2, a core PCP protein, cooperate to control PCP in the early mouse embryo. Two aspects of planar polarity can be analyzed quantitatively at cellular resolution in the mouse embryo: convergent extension of the axial midline; and posterior positioning of cilia on cells of the node. Analysis of the spatial distribution of brachyury(+) midline cells shows that the Cfl1 mutant midline is normal, whereas Vangl2 mutants have a slightly wider midline. By contrast, midline convergent extension fails completely in Vangl2 Cfl1 double mutants. Planar polarity is required for the posterior positioning of cilia on cells in the mouse node, which is essential for the initiation of left-right asymmetry. Node cilia are correctly positioned in Cfl1 and Vangl2 single mutants, but cilia remain in the center of the cell in Vangl2 Cfl1 double mutants, leading to randomization of left-right asymmetry. In both the midline and node, the defect in planar polarity in the double mutants arises because PCP protein complexes fail to traffic to the apical cell membrane, although other aspects of apical-basal polarity are unaffected. Genetic and pharmacological experiments demonstrate that F-actin remodeling is essential for the initiation, but not maintenance, of PCP. We propose that Vangl2 and cofilin cooperate to target Rab11(+) vesicles containing PCP proteins to the apical membrane during the initiation of planar cell polarity.

  4. Hybrid Perovskite/Perovskite Heterojunction Solar Cells.

    Science.gov (United States)

    Hu, Yinghong; Schlipf, Johannes; Wussler, Michael; Petrus, Michiel L; Jaegermann, Wolfram; Bein, Thomas; Müller-Buschbaum, Peter; Docampo, Pablo

    2016-06-28

    Recently developed organic-inorganic hybrid perovskite solar cells combine low-cost fabrication and high power conversion efficiency. Advances in perovskite film optimization have led to an outstanding power conversion efficiency of more than 20%. Looking forward, shifting the focus toward new device architectures holds great potential to induce the next leap in device performance. Here, we demonstrate a perovskite/perovskite heterojunction solar cell. We developed a facile solution-based cation infiltration process to deposit layered perovskite (LPK) structures onto methylammonium lead iodide (MAPI) films. Grazing-incidence wide-angle X-ray scattering experiments were performed to gain insights into the crystallite orientation and the formation process of the perovskite bilayer. Our results show that the self-assembly of the LPK layer on top of an intact MAPI layer is accompanied by a reorganization of the perovskite interface. This leads to an enhancement of the open-circuit voltage and power conversion efficiency due to reduced recombination losses, as well as improved moisture stability in the resulting photovoltaic devices.

  5. Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos.

    Science.gov (United States)

    Chatfield, Jodie; O'Reilly, Marie-Anne; Bachvarova, Rosemary F; Ferjentsik, Zoltan; Redwood, Catherine; Walmsley, Maggie; Patient, Roger; Loose, Mathew; Johnson, Andrew D

    2014-06-01

    A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates.

  6. Nuclear-mitochondrial incompatibility in interorder rhesus monkey-cow embryos derived from somatic cell nuclear transfer.

    Science.gov (United States)

    Kwon, Daekee; Koo, Ok-Jae; Kim, Min-Jung; Jang, Goo; Lee, Byeong Chun

    2016-10-01

    Monkey interorder somatic cell nuclear transfer (iSCNT) using enucleated cow oocytes yielded poor blastocysts development and contradictory results among research groups. Determining the reason for this low blastocyst development is a prerequisite for optimizing iSCNT in rhesus monkeys. The aim of this study was to elucidate nuclear-mitochondrial incompatibility of rhesus monkey-cow iSCNT embryos and its relationship to low blastocyst development. Cytochrome b is a protein of complex III of the electron transport chain (ETC). According to meta-analysis of amino acid sequences, the homology of cytochrome b is 75 % between rhesus monkeys and cattle. To maintain the function of ETC after iSCNT, 4n iSCNT embryos were produced by fusion of non-enucleated cow oocytes and rhesus monkey somatic cells. The blastocyst development rate of 4n iSCNT embryos was higher than that of 2n embryos (P monkey iSCNT embryos reconstructed with cow oocytes have nuclear-mitochondrial incompatibility due to fundamental species differences between rhesus monkeys and cattle. Nuclear-mitochondrial incompatibility seems to correlate with low ETC activity and extremely low blastocyst development of rhesus monkey-cow iSCNT embryos.

  7. Effect of Ultrahigh Frequency Radiation Emitted from 2G Cell Phone on Developing Lens of Chick Embryo: A Histological Study

    Directory of Open Access Journals (Sweden)

    Mary Hydrina D'Silva

    2014-01-01

    Full Text Available A Mobile phone in operation emits a pulsed radiofrequency electromagnetic field which is absorbed into the user’s body particularly the head region. Contradictory scientific reports on the health effect of nonionizing radiations on biological tissues have prompted to undertake the present study to evaluate the damage in the developing lens of a chick embryo following exposure to radiation emitted from a 2G cell phone. Fertilized chick embryos were incubated in two groups in a standard egg incubator. The experiment group was exposed to radiation emitted from a 2G cell phone. On completion of scheduled duration, the embryos were collected and processed for routine histological studies. The 9th to 12th day chick embryo eyes were processed for assessment of DNA damage using the alkaline comet assay technique. The lens thickness and the equatorial diameter were measured using oculometer and statistically compared for both groups. In the present study, the exposure of chick embryos to a 2G cell phone caused structural changes in lens epithelial cells, formation of cystic cells and spaces, distortion of lens fibers, and formation of posterior aberrant nuclear layer. The DNA damage in the developing eyes of the experiment group assessed by comet assay was highly significant.

  8. Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Yunhai; PAN; Dengke; SUN; Xiuzhu; SUN; Guojie; WANG; Xiaobo; LIU; Xiaohui; LI; Yan; DAI; Yunping; LI; Ning

    2006-01-01

    In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O2) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O2) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.3%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MⅡ oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.

  9. From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo.

    Science.gov (United States)

    Sardet, Christian; Paix, Alexandre; Prodon, François; Dru, Philippe; Chenevert, Janet

    2007-07-01

    The dorsoventral and anteroposterior axes of the ascidian embryo are defined before first cleavage by means of a series of reorganizations that reposition cytoplasmic and cortical domains established during oogenesis. These domains situated in the periphery of the oocyte contain developmental determinants and a population of maternal postplasmic/PEM RNAs. One of these RNAs (macho-1) is a determinant for the muscle cells of the tadpole embryo. Oocytes acquire a primary animal-vegetal (a-v) axis during meiotic maturation, when a subcortical mitochondria-rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER-mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization at metaphase of meiosis I initiates a series of dramatic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. The first major phase depends on sperm entry which triggers a calcium wave leading in turn to an actomyosin-driven contraction wave. The contraction concentrates the cER-mRNA domain and myoplasm in and around a vegetal/contraction pole. The precise localization of the vegetal/contraction pole depends on both the a-v axis and the location of sperm entry and prefigures the future site of gastrulation and dorsal side of the embryo. The second major phase of reorganization occurs between meiosis completion and first cleavage. Sperm aster microtubules and then cortical microfilaments cause the cER-mRNA domain and myoplasm to reposition toward the posterior of the zygote. The location of the posterior pole depends on the localization of the sperm centrosome/aster attained during the first major phase of reorganization. Both cER-mRNA and myoplasm domains localized in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight-cell stage the cER-mRNA domain compacts and gives rise to

  10. Action of tumor initiators and promoters in the Syrian hamster embryo cell transformation assay

    Energy Technology Data Exchange (ETDEWEB)

    Jones, C.A.; Huberman, E.

    1986-06-01

    The Syrian hamster embryo (SHE) cell transformation assay is unique among the rodent fibroblast transformation systems in that it uses normal, diploid cells. Alteration in the control of growth in carcinogen-treated cultures is used to indicate the onset of neoplastic development. An evaluation of the SHE assay for screening carcinogens is reported. Using coded chemicals, the degree of intra- and interlaboratory reproducibility with the system was evaluated. Overall, there was a good qualitative correlation between the carcinogenicity of the chemicals and their ability to induce morphological cell transformation. Unfortunately, the low level of response and lack of good dose-response relationships with certain chemical are still major constraints to the use of this system in routine testing. Further consideration needs to be given to developing procedures that select for, or amplify, expression of the transformed phenotype. 9 refs., 2 figs., 1 tab.

  11. Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells.

    Science.gov (United States)

    Tayeboon, Gh S; Ostad, S N; Nasri, S; Nili-Ahmadabadi, A; Tavakoli, F; Sabzevari, O

    2015-05-01

    CYP1A1, a P450 isoenzyme, is involved in the phase I xenobiotic metabolism including teratogen drugs. In the present study, the ability of teratogens to elevate the embryonic expression of CYP1A1 was examined. Micromass cell cultures prepared from day 13 rat embryo limb buds (LB). LB cells were cultivated and exposed for 5 days to retinoic acid (RA), hydrocortisone (HC), caffeine (CA) and quinine (QN). CYP1A1 protein expression and activity were measured using immunofluorescence staining and ethoxyresorufin O-deethylation (EROD) assay, respectively. The EROD activity increased significantly following LB cells exposure to RA and HC (pteratogens have potency to increase CYP1A1 activity.

  12. Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos.

    Science.gov (United States)

    Akshey, Y S; Malakar, D; De, A Kumar; Jena, M Kumar; Pawar, S Kumar; Dutta, R; Sahu, S

    2011-05-01

    The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups-contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods-one half by 2 μM Ca ionophore and another half by 2.31 kV/cm for 15 μSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. In vitro fertilization and stem cell harvesting from human embryos: the law and practice in the United States.

    Science.gov (United States)

    Hook, C Christopher

    2010-07-01

    The challenges before science and medicine are these: science must explore the natural world as thoroughly as possible, while still honoring, protecting, serving and preserving the subject of its investigations, and the human beings for whom it is a tool; medicine must confront disease and disability as effectively as possible, while also honoring, protecting, and preserving those beings for whom it serves - all of those beings, not just some, or even most, at the potential expense of others. These goals are challenged by embryo-destructive human embryonic stem cell research. The human embryo is a human being as clearly defined by embryology, and as such should be protected by the codes governing human subject research. However, because of the "potential" benefits offered by pluripotent stem cells, coupled with abortion politics and a very poorly regulated infertility industry, United States governmental advisory commissions and the scientific, medical, and political communities have attempted to define away the humanity of the human embryo, with a few notable exceptions. Because infertility treatments in the United States are poorly regulated, there are large numbers of supernumerary embryos in cryopreservation. However, only a tiny portion of these will ever be potentially available for research, and thus are not a realistic source of the cells necessary to provide treatments to the millions who might benefit from proposed stem cell based therapies. Cloning will not be the answer either, given the millions of women who must be exploited to provide sufficient numbers of eggs to generate the cloned cell lines. Moreover, the disposition decisions parents must make for their extra embryos are often agonizing, and not uncommonly change. The use of supernumerary embryos as a source for human embryonic stem cells is unethical, will never be a sufficient source for the medical treatments expected from stem cell research, and is often a source of great distress for the

  14. In vitro fertilization and stem cell harvesting from human embryos: the law and practice in the United States

    Directory of Open Access Journals (Sweden)

    C. Christopher Hook

    2010-07-01

    Full Text Available The challenges before science and medicine are these: science must explore the natural world as thoroughly as possible, while still honoring, protecting, serving and preserving the subject of its investigations, and the human beings for whom it is a tool; medicine must confront disease and disability as effectively as possible, while also honoring, protecting, and preserving those beings for whom it serves – all of those beings, not just some, or even most, at the potential expense of others. These goals are challenged by embryo-destructive human embryonic stem cell research. The human embryo is a human being as clearly defined by embryology, and as such should be protected by the codes governing human subject research. However, because of the “potential” benefits offered by pluripotent stem cells, coupled with abortion politics and a very poorly regulated infertility industry, United States governmental advisory commissions and the scientific, medical, and political communities have attempted to define away the humanity of the human embryo, witha few notable exceptions. Because infertility treatments in the United States are poorly regulated, there are large numbersof supernumerary embryos in cryopreservation. However, only a tiny portion of these will ever be potentially available for research, and thus are not a realistic source of the cells necessary to provide treatments to the millions who might benefit from proposed stem cell based therapies. Cloning willnot be the answer either, given the millions of women who must be exploited to provide sufficient numbers of eggs to generate the cloned cell lines. Moreover, the disposition decisions parents must make for their extra embryos are often agonizing, and not uncommonly change.The use of supernumerary embryos as a source for human embryonic stem cells is unethical, will never be a sufficient source for the medical treatments expected from stem cell research, and is often a source of

  15. In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

    Science.gov (United States)

    Dudczig, Stefanie; Currie, Peter D; Poggi, Lucia; Jusuf, Patricia R

    2017-03-22

    The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

  16. Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos.

    Science.gov (United States)

    Batista, M; Torres, A; Diniz, P; Mateus, L; Lopes-da-Costa, L

    2012-10-01

    The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days 2-8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for co-culture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P(4)) were evaluated. The production of P(4) was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P(4), PGF(2α), and PGE(2) compared to fresh cell cultures. This enables the use of pools of frozen-thawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P(4) quantified in the medium, but had no effect on PGF(2α) and PGE(2) quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed.

  17. High-sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus Embryo

    Directory of Open Access Journals (Sweden)

    Camille Lombard-Banek

    2016-10-01

    Full Text Available Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in blastomeres in the 16-cell embryo of the South African clawed frog (Xenopus laevis that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

  18. Effects of copper oxide nanoparticles and copper ions to zebrafish (Danio rerio) cells, embryos and fry

    DEFF Research Database (Denmark)

    Thit, Amalie; Skjolding, Lars Michael; Selck, Henriette

    2017-01-01

    with similar concentration-response relationships, whereas only Cu ions affected fry mortality (24h LC50≈30μM, ≈2mgCuL-1 for Cu ions and no significant mortality observed at up to 200μM, 12.7mgCuL-1 for CuO NP). Both Cu forms increased fry swimming activity during light cycles and decreased activity during......) and fry locomotion. In the ZFL tests, no significant cytotoxicity (cell death, decreased metabolic or cell membrane integrity) was detected for either treatment, though both significantly affected reactive oxygen species (ROS) production. Embryo mortality was affected by both Cu ions and CuO NPs...... dark cycles: Cu ions had significant impact at lower concentrations than CuO NPs. The implications are that Cu ions generally are more toxic than CuO NPs to embryos and fry but there is a marked difference in toxicity among the different zebrafish model systems. Metal NPs release into the environment...

  19. Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

    Directory of Open Access Journals (Sweden)

    Lin Yuan

    2014-11-01

    Full Text Available Cloned pigs generated by somatic cell nuclear transfer (SCNT show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP. q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.

  20. Forecasting science futures: legitimising hope and calming fears in the embryo stem cell debate.

    Science.gov (United States)

    Kitzinger, Jenny; Williams, Clare

    2005-08-01

    Controversies about biotechnologies often centre not so much on present scientific facts as on speculations about risks and benefits in the future. It is this key futuristic element in these arguments that is the focus of this article. We examine how competing visions of utopia or dystopia are defended through the use of diverse vocabularies, metaphors, associations and appeals to authority. Our case study explores how these rhetorical processes play out in the debate about embryo stem cell research in UK national press and TV news media. The findings show how predictions from those in favour of embryo stem cell research are supported by both hype and by anti-hype, by inconsistent appeals to the technologies' innovative status and by the selective deconstruction of concepts such as 'potential' and 'hope'. The debate also mobilises binary oppositions around reason versus emotion, science versus religion and fact versus fiction. This article highlights how traditional assertions of expertise are now combined with ideas about compassion and respect for democracy and diversity. It also highlights the fact that although news reporters are often responding to topical events the real focus is often on years, even decades ahead. Close attention to how images of the future are constructed, and the evolution of new strategies for legitimation are, we suggest, important areas of on-going research, particularly in discussions of scientific and medical developments and policy.

  1. Primordial germ cell proliferation is impaired in Fused Toes mutant embryos.

    Science.gov (United States)

    Kim, Bongki; Kim, Youngha; Sakuma, Rui; Hui, Chi-Chung; Rüther, Ulrich; Jorgensen, Joan S

    2011-01-15

    Over the first 4 days of their life, primordial germ cells invade the endoderm, migrate into and through the developing hindgut, and traverse to the genital ridge where they cluster and ultimately inhabit the nascent gonad. Specific signal-receptor combinations between primordial germ cells and their immediate environment establish successful migration and colonization. Here we demonstrate that disruption of a cluster of six genes on murine chromosome 8, as exemplified by the Fused Toes (Ft) mutant mouse model, results in severely decreased numbers of primordial germ cells within the early gonad. Primordial germ cell migration appeared normal within Ft mutant embryos; however, germ cell counts progressively decreased during this time. Although no difference in apoptosis was detected, we report a critical decrease in primordial germ cell proliferation by E12.5. The six genes within the Ft locus include the IrxB cluster (Irx3, -5, -6), Fts, Ftm, and Fto, of which only Ftm, Fto, and Fts are expressed in primordial germ cells of the early gonad. From these studies, we have discovered that the Ft locus on mouse chromosome 8 is associated with cell cycle deficits within the primordial germ cell population that initiates just before translocation into the genital ridge. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. A gene expression atlas of a bicoid-depleted Drosophila embryo reveals early canalization of cell fate.

    Science.gov (United States)

    Staller, Max V; Fowlkes, Charless C; Bragdon, Meghan D J; Wunderlich, Zeba; Estrada, Javier; DePace, Angela H

    2015-02-01

    In developing embryos, gene regulatory networks drive cells towards discrete terminal fates, a process called canalization. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos by depleting a key maternal input, bicoid (bcd), and measuring gene expression patterns of the network at cellular resolution. This method results in a gene expression atlas containing the levels of mRNA or protein expression of 13 core patterning genes over six time points for every cell of the blastoderm embryo. This is the first cellular resolution dataset of a genetically perturbed Drosophila embryo that captures all cells in 3D. We describe the technical developments required to build this atlas and how the method can be employed and extended by others. We also analyze this novel dataset to characterize the degree and timing of cell fate canalization in the segmentation network. We find that in two layers of this gene regulatory network, following depletion of bcd, individual cells rapidly canalize towards normal cell fates. This result supports the hypothesis that the segmentation network directly canalizes cell fate, rather than an alternative hypothesis whereby cells are initially mis-specified and later eliminated by apoptosis. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further computational modeling of canalization and gene regulation in this transcriptional network. © 2015. Published by The Company of Biologists Ltd.

  3. Co-localization of neural cell adhesion molecule and fibroblast growth factor receptor 2 in early embryo development.

    Science.gov (United States)

    Vesterlund, Liselotte; Töhönen, Virpi; Hovatta, Outi; Kere, Juha

    2011-01-01

    During development there is a multitude of signaling events governing the assembly of the developing organism. Receptors for signaling molecules such as fibroblast growth factor receptor 2 (FGFR2) enable the embryo to communicate with the surrounding environment and activate downstream pathways. The neural cell adhesion molecule (NCAM) was first characterized as a cell adhesion molecule highly expressed in the nervous system, but recent studies have shown that it is also a signaling receptor. Using a novel single oocyte adaptation of the proximity ligation assay, we here show a close association between NCAM and FGFR2 in mouse oocytes and 2-cell embryos. Real-time PCR analyses revealed the presence of messenger RNA encoding key proteins in downstream signaling pathways in oocytes and early mouse embryos. In summary these findings show a co-localization of NCAM and FGFR2 in early vertebrate development with intracellular signaling pathways present to enable a cellular response.

  4. Cryopreservation of Primordial Germ Cells by Rapid Cooling of Whole Zebrafish (Danio rerio) Embryos

    National Research Council Canada - National Science Library

    HIGAKI, Shogo; MOCHIZUKI, Kentaro; AKASHI, Yuichiro; YAMAHA, Etsuro; KATAGIRI, Seiji; TAKAHASHI, Yoshiyuki

    2010-01-01

    ...) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC...

  5. A novel supercapacitor-fuel cell hybrid cell

    Institute of Scientific and Technical Information of China (English)

    WANG Y; ZHENG Jim P

    2006-01-01

    A monolithic hybrid fuel cell (MHFC) with a novel configuration was proposed in an effort to improve the fuel cell performance during instantaneous power changes. A modified direct methanol fuel cell (DMFC) with a layer of hydrous ruthenium dioxide (RuO2·xH2O) sandwiched between the anode catalyst layer and membrane was used to demonstrate the principle of the MHFC. Experimental results indicate that the RuO2·xH2O layer is equivalent to a resistor-capacitor transmission line and functions similar to a capacitor in parallel with the anode electrode. The improvement in dynamic response of the MHFC was experimentally confirmed under step current change and square current pulse operating. The ionic conductivity of the RuO2·xH2O layer was also obtained.

  6. Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Briggs, Sharon F; Dominguez, Antonia A; Chavez, Shawn L; Reijo Pera, Renee A

    2015-06-01

    The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

  7. Neferine, an alkaloid from lotus seed embryo, inhibits human lung cancer cell growth by MAPK activation and cell cycle arrest.

    Science.gov (United States)

    Poornima, Paramasivan; Weng, Ching Feng; Padma, Viswanadha Vijaya

    2014-01-01

    Neferine is the major bisbenzylisoquinoline alkaloid isolated from the seed embryo of a traditional medicinal plant Nelumbo nucifera (Lotus). Epidemiological studies have revealed the therapeutic potential of lotus seed embryo. Although several mechanisms have been proposed, a clear anticancer action mechanism of neferine on lung cancer cells is still not known. Lung cancer is the most common cause of cancer death in the world, and the patients with advanced stage of nonsmall lung cancer require adjunct chemotherapy after surgical resection for the eradication of cancer cells. In this study, the effects of neferine were evaluated and characterized in A549 cells. Neferine induced apoptosis in a dose-dependent manner with the hypergeneration of reactive oxygen species, activation of MAPKs, lipid peroxidation, depletion of cellular antioxidant pool, loss of mitochondrial membrane potential, and intracellular calcium accumulation. Furthermore, neferine treatment leads to the inhibition of nuclear factor kappaB and Bcl2, upregulation of Bax and Bad, release of cytochrome C, activation of caspase cascade, and DNA fragmentation. In addition, neferine could induce p53 and its effector protein p21 and downregulation of cell cycle regulatory protein cyclin D1 thereby inducing G1 cell cycle arrest. These results suggest a novel function of neferine as an apoptosis inducer in lung cancer cells.

  8. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure

    Science.gov (United States)

    JEON, Yubyeol; NAM, Yeong-Hee; CHEONG, Seung-A; KWAK, Seong-Sung; LEE, Eunsong; HYUN, Sang-Hwan

    2016-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation. PMID:27064112

  9. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

    Science.gov (United States)

    Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

    2016-08-25

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

  10. Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo

    Directory of Open Access Journals (Sweden)

    McCarthy John J

    2005-08-01

    Full Text Available Abstract Background Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. Results We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. Conclusions Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.

  11. Study on Germination of Somatic Embryos of Liriodendrom Hybrids%杂交鹅掌楸体细胞胚萌发培养的研究

    Institute of Scientific and Technical Information of China (English)

    程强强; 戴小英; 宋晓琛; 江香梅; 肖复明

    2014-01-01

    The influences of development period of somatic embryos, major element and vitamin C, genotypes, different combinations with plant growth regulators and transplant medium on germination and transplanting survival for somatic embryos of Liriodendrom hybrids were studied. The results showed that the optimal developement phase of germination was torpedo-shaped embryos, and the base cultural medium of 3/4 MS and with the vitamin C used of keeping normal form from somatic embryos. The somatic embryo germination among different genotypes at the same cultural condition was different significantly, for example, the genotype 1×5002 was the highest, up to 82.46%, the genotype 1×4088 just was to 13.24%. The medium KT 0.1 mg/L+IBA 0.1mg/L can help to promote the germination of somatic embryogenesis. There were differences of seedling transplanting survival rate between the yellow subsoil and mixed peat soil, and the former was more suitable for transplanting than the later.%以杂交鹅掌楸体细胞胚为材料,研究体胚不同发育阶段、大量元素和维生素C、基因型、激素组合以及移栽基质对体胚苗萌发和移栽存活的影响。结果表明:鱼雷型胚萌发效果最好;3/4 MS培养基较适合体胚苗的萌发;Vc有利用保持体胚苗维持正常形态。不同基因型体胚苗萌发差异较大,基因型1×5002萌发效果最好,成苗率为84.26%;而1×4088仅有13.24%。 KT 0.1 mg/L+IBA 0.1 mg/L有利于促进体细胞胚的萌发。不同基因型体胚苗移栽存活率有差异,黄心土较混合泥炭土更适合体胚苗移栽。

  12. Presence and distribution of E-cadherin in the 4-cell golden hamster embryo. Effect of maternal age and parity.

    Science.gov (United States)

    Trejo, A; Ambriz, D; Navarro-Maldonado, M C; Mercado, E; Rosado, A

    2008-08-01

    Maternal age dependency of gestation time in hamster and in other mammals is a well demonstrated fact. We have recently shown that adult nulliparous and multiparous hamster females show significant asynchrony and retard on early embryo development (from two blastomeres to morula stages) when compared with nulliparous young females. The number of cell-cell adhesions between blastomeres in early embryo development has been reported to be a good indication of the ability of embryos to cleave and develop. In this work we studied, by indirect immunofluorescence, the presence and distribution of E-cadherin in 4-cell embryos obtained from nulliparous young (NYF), nulliparous adult (NAF) and multiparous adult (MAF) hamster females. Distribution and intensity of fluorescence was observed and registered using confocal microscopy. Staining intensities for E-cadherin were quantified by computed densitometry in the free membrane regions, in the cytoplasm region and in the cell-cell adhesion zones of each embryo. E-Cadherin in all the studied zones was significantly higher (p<0.01) in NYF. Cadherin concentration in the intercellular membranes was always statistically higher (p<0.05) than in the free membrane regions. An appreciable concentration of E-cadherin was found in the cytoplasm of the 4-cell embryos obtained from the three groups of females, but was significantly higher in NYF. No statistical differences were observed in any of the parameters studied between NAF and MAF. Our results seem to indicate that changes in the reproductive behavior related to age and/or multiparity may be correlated with changes in the processes related to intercellular adhesions during early cleavage.

  13. iPS cells-alternative pluripotent cells to embryo stem cells

    Institute of Scientific and Technical Information of China (English)

    PEI XueTao

    2010-01-01

    @@ Since the first murine and human embryonic stem cell lines were established by Drs.Evans and Kaufman [1] and Thomson et al.[2], respectively, great progress has been make in the field of stem cell research and regenerative medicine that gave promising futures for therapeutic interventions.However, ethical problems and complications from immune rejection have hindered the full development of ES cells into clinical practice for disease treatment.

  14. The migration of myogenic cells from the somites at the wing level in avian embryos.

    Science.gov (United States)

    Solursh, M; Drake, C; Meier, S

    1987-06-01

    This study is concerned with establishing a morphological basis for the initiation of migration of putative myogenic cells from the somites into the presumptive wing bud in avian embryos. At the 22 somite stage (stage 14) vasculogenesis is a prevalent activity. By use of a quail specific monoclonal antibody to vascular endothelial cells, vascular cells are recognized in the lateral plate, on the intermediate mesoderm, and on somite surfaces. Cells that are found between the lateral plate mesoderm and somites are shown to be vascular endothelial cells. The lateral body folds progressively bring the lateral plate mesoderm close to the lateral margin of the somites and vascular elements disappear from surface view. It is not until the 24 somite stage (stage 15) that some cells in the ventral lateral margin of somites at the wing level can be seen in scanning electron micrographs to extend basal cell processes toward adjacent vascular tubes. These results provide a morphological basis for the early migratory behavior of myogenic cells and demonstrate their close proximity to the prepatterned vascular network.

  15. Second heart field cardiac progenitor cells in the early mouse embryo.

    Science.gov (United States)

    Francou, Alexandre; Saint-Michel, Edouard; Mesbah, Karim; Théveniau-Ruissy, Magali; Rana, M Sameer; Christoffels, Vincent M; Kelly, Robert G

    2013-04-01

    At the end of the first week of mouse gestation, cardiomyocyte differentiation initiates in the cardiac crescent to give rise to the linear heart tube. The heart tube subsequently elongates by addition of cardiac progenitor cells from adjacent pharyngeal mesoderm to the growing arterial and venous poles. These progenitor cells, termed the second heart field, originate in splanchnic mesoderm medial to cells of the cardiac crescent and are patterned into anterior and posterior domains adjacent to the arterial and venous poles of the heart, respectively. Perturbation of second heart field cell deployment results in a spectrum of congenital heart anomalies including conotruncal and atrial septal defects seen in human patients. Here, we briefly review current knowledge of how the properties of second heart field cells are controlled by a network of transcriptional regulators and intercellular signaling pathways. Focus will be on 1) the regulation of cardiac progenitor cell proliferation in pharyngeal mesoderm, 2) the control of progressive progenitor cell differentiation and 3) the patterning of cardiac progenitor cells in the dorsal pericardial wall. Coordination of these three processes in the early embryo drives progressive heart tube elongation during cardiac morphogenesis. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

  16. Effects of melatonin on in vitro development of mouse two-cell embryos cultured in HTF medium.

    Science.gov (United States)

    Tian, Xiu-Zhi; Wen, Qing; Shi, Jian-Min; Liang-Wang; Zeng, Shen-Ming; Tian, Jian-Hui; Zhou, Guang-Bin; Zhu, Shi-En; Liu, Guo-Shi

    2010-01-01

    Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10-13 to 10-3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10-13 to 10-5 M and decreased by melatonin at 10-3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10-9 M. In comparison to control, 10-9 M melatonin increased blastocyst formation rate from 48.08 +/- 5.25% to 82.08 +/- 2.34% (p HTF medium.

  17. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    Science.gov (United States)

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  18. New hybrid model of proton exchange membrane fuel cell

    Institute of Scientific and Technical Information of China (English)

    WANG Rui-min; CAO Guang-yi; ZHU Xin-jian

    2007-01-01

    Model and simulation are good tools for design optimization of fuel cell systems. This paper proposes a new hybrid model of proton exchange membrane fuel cell (PEMFC). The hybrid model includes physical component and black-box component. The physical component represents the well-known part of PEMFC, while artificial neural network (ANN) component estimates the poorly known part of PEMFC. The ANN model can compensate the performance of the physical model. This hybrid model is implemented on Matlab/Simulink software. The hybrid model shows better accuracy than that of the physical model and ANN model. Simulation results suggest that the hybrid model can be used as a suitable and accurate model for PEMFC.

  19. A stamped PEDOT:PSS-silicon nanowire hybrid solar cell.

    Science.gov (United States)

    Moiz, Syed Abdul; Nahhas, Ahmed Muhammad; Um, Han-Don; Jee, Sang-Won; Cho, Hyung Koun; Kim, Sang-Woo; Lee, Jung-Ho

    2012-04-13

    A novel stamped hybrid solar cell was proposed using the stamping transfer technique by stamping an active PEDOT:PSS thin layer onto the top of silicon nanowires (SiNWs). Compared to a bulk-type counterpart that fully embeds SiNWs inside PEDOT:PSS, an increase in the photovoltaic efficiency was observed by a factor of ∼4.6, along with improvements in both electrical and optical responses for the stamped hybrid cell. Such improvements for hybrid cells was due to the formation of well-connected and linearly aligned active PEDOT:PSS channels at the top ends of the nanowires after the stamping process. These stamped channels facilitated not only to improve the charge transport, light absorption, but also to decrease the free carriers as well as exciton recombination losses for stamped hybrid solar cells.

  20. Developmental kinetics of the first cell cycles of bovine in vitro PRODUCED EMBRYOS IN RELATION TO THEIR IN VITRO VIABILITY AND SEX

    DEFF Research Database (Denmark)

    Holm, P; Shukri, N.N; Vajta, Gabor

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...

  1. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  2. Induction of apoptosis and inhibition of cell growth by tbx5 knockdown contribute to dysmorphogenesis in Zebrafish embryos

    Directory of Open Access Journals (Sweden)

    Tang Renbing

    2011-10-01

    Full Text Available Abstract Background The tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants. Methods Wild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope. Results Apoptosis-related genes (bad, bax, and bcl2, and cell cycle-related genes (cdk2, pcna, p27, and p57 showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos. Conclusion Our study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency

  3. Inhibition of MEK and GSK3 supports ES cell-like domed colony formation from avian and reptile embryos.

    Science.gov (United States)

    Nakanoh, Shota; Okazaki, Kenji; Agata, Kiyokazu

    2013-07-01

    As amniotes diversified, mammals may have modified mechanisms of cellular pluripotency along with the acquisition of a placenta. What then defined pluripotent states in the ancestral amniotes? To study the evolutionary background of pluripotency in amniotes, we tested the effects of extracellular effectors on primary culture cells from avian and reptile embryos in serum-free medium. When treated with a combination of a MEK inhibitor and a GSK3 inhibitor (2i condition), chicken early embryos formed domed colonies (DCs), which were morphologically indistinguishable from the colonies formed by mouse and rat naïve embryonic stem cells. However, no DCs formed when cells from further-developed embryos were cultured in the 2i condition, indicating that there is a clear boundary of DC-forming ability at around the stage of primitive streak elongation. Quail embryos at the blastoderm and cleavage stages also formed DCs in the 2i condition, which is consistent with the notion that the appearance of DCs corresponds with the presence of pluripotent cells in embryos. Gecko blastoderms also formed DCs in the 2i condition, but gastrulas did not. ERK activation by bFGF caused an effect opposite to that of the 2i condition, namely, it dispersed colonies of cells even from early embryos in all species examined. These results suggest that the regulation of pluripotency by FGF/ERK signaling may date back at least to the common ancestor of mammals, birds, and reptiles. However, gene expression analysis indicated the possibility that mammalian pluripotency transcription factors function differently in non-mammalian amniotes.

  4. Autonomous and non-autonomous roles of DNase II during cell death in C. elegans embryos.

    Science.gov (United States)

    Yu, Hsiang; Lai, Huey-Jen; Lin, Tai-Wei; Lo, Szecheng J

    2015-04-27

    Generation of DNA fragments is a hallmark of cell apoptosis and is executed within the dying cells (autonomous) or in the engulfing cells (non-autonomous). The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) method is used as an in situ assay of apoptosis by labelling DNA fragments generated by caspase-associated DNase (CAD), but not those by the downstream DNase II. In the present study, we report a method of ToLFP (topoisomerase ligation fluorescence probes) for directly visualizing DNA fragments generated by DNase II in Caenorhabditis elegans embryos. ToLFP analysis provided the first demonstration of a cell autonomous mode of DNase II activity in dying cells in ced-1 embryos, which are defective in engulfing apoptotic bodies. Compared with the number of ToLFP signals between ced-1 and wild-type (N2) embryos, a 30% increase in N2 embryos was found, suggesting that the ratio of non-autonomous and autonomous modes of DNase II was ~3-7. Among three DNase II mutant embryos (nuc-1, crn-6 and crn-7), nuc-1 embryos exhibited the least number of ToLFP. The ToLFP results confirmed the previous findings that NUC-1 is the major DNase II for degrading apoptotic DNA. To further elucidate NUC-1's mode of action, nuc-1-rescuing transgenic worms that ectopically express free or membrane-bound forms of NUC-1 fusion proteins were utilized. ToLFP analyses revealed that anteriorly expressed NUC-1 digests apoptotic DNA in posterior blastomeres in a non-autonomous and secretion-dependent manner. Collectively, we demonstrate that the ToLFP method can be used to differentiate the locations of blastomeres where DNase II acts autonomously or non-autonomously in degrading apoptotic DNA.

  5. Investigation of the photovoltaic cell/ thermoelectric element hybrid system performance

    Science.gov (United States)

    Cotfas, D. T.; Cotfas, P. A.; Machidon, O. M.; Ciobanu, D.

    2016-06-01

    The PV/TEG hybrid system, consisting of the photovoltaic cells and thermoelectric element, is presented in the paper. The dependence of the PV/TEG hybrid system parameters on the illumination levels and the temperature is analysed. The maxim power values of the photovoltaic cell, of the thermoelectric element and of the PV/TEG system are calculated and a comparison between them is presented and analysed. An economic analysis is also presented.

  6. Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryo.

    Science.gov (United States)

    Sritanaudomchai, Hathaitip; Pavasuthipaisit, Kanok; Kitiyanant, Yindee; Kupradinun, Piengchai; Mitalipov, Shoukhrat; Kusamran, Thanit

    2007-10-01

    Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, alpha-skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and alpha-fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes.

  7. 阿月浑子杂种幼胚培养技术的研究%Study on hybrid immature embryo culture technology of Pistacia vera L.

    Institute of Scientific and Technical Information of China (English)

    庞曼; 王洁; 董倩; 李旭新; 白志英; 路丙社

    2012-01-01

    以阿月浑子杂种果实和人工授粉后120 d的杂种幼胚为试材,对杂种果实生长发育规律及幼胚培养进行了研究,探讨了冷藏处理时间、不同光照条件和培养时间对幼胚萌发和成苗的影响.结果表明:阿月浑子人工授粉80 d后,单果鲜重和PF值仅为1.27 g和0.24;授粉后100 d,单果鲜重和PF值为1.57g和0.43;授粉后120 d,单果鲜重和PF值为2.81 g和0.80;3种冷藏处理中,以冷藏处理90 d时幼胚萌发及生长效果最好,幼胚萌率为91.67%,平均真叶数为6.09个,平均苗高为4.14 cm;在培养方式上,以黑暗处理后培养20 d效果最好,幼胚萌发率为86.67%,平均真叶数为5.38,平均苗高为4.23 cm.%In this experiment, hybrid fruits and immature embryos of Pistacia vera L.growing for 120 d after artificial pollination, were used as materials,The growth rule and immature embryo culture of pistachio's hybrid fruit were studied. The effects of cold treatment time, different light conditions and culture time on immature embryo development and normal seedling rate were also investigated, indicating that the single-fruit fresh weight and PF value were only 1. 27 g and 0. 24 when growing for 80 d after artificial pollination, reaching 1.57 g and 0. 43 in 100 d after, with the average fruit fresh weight and PF value being 2.81 g and 0. 80 respectively, in 120 d after. The suitable cold treatment time for immature embryo germination and growth is 90 d.the rate of embryo germination is 91. 67%, the average leaf number is 6. 09 and the average seedling height is 4. 14 cm. It was suggested that the best culture way is the dark treatment for 20 d, the rate of embryo germination is 86. 67%, the average leaf number is 5. 38 and the average seedling height is 4. 23 cm.

  8. Effects of donor cells on in vitro development of cloned bovine embryos

    Institute of Scientific and Technical Information of China (English)

    Jing Fu; Pengfei Guan; Leiwen Zhao; Hua Li; Shuzhen Huang; Fanyi Zeng; Yitao Zeng

    2008-01-01

    The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.

  9. PiggyBac Transposon Mediated Efficient eGFP Expression in Porcine Somatic Cells and Cloned Embryos

    Institute of Scientific and Technical Information of China (English)

    Luo Yi-bo; Zhang Li; Zhu Jiang; Wu Mei-ling; Huan Yan-jun; Yin Zhi; Mu Yan-shuang; Xia Ping; LiuZhong-hua

    2012-01-01

    PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein (eGFP) and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer (SCNT), the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.

  10. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

    Directory of Open Access Journals (Sweden)

    Yushi Ishibashi

    Full Text Available Reactive oxygen species (ROS promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA / abscisic acid (ABA metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI, an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells.

  11. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

    Science.gov (United States)

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells.

  12. Clinically failed eggs as a source of normal human embryo stem cells.

    Science.gov (United States)

    De Sousa, Paul A; Gardner, John; Sneddon, Sharon; Pells, Steve; Tye, Britt Jorgensen; Dand, Pawlina; Collins, Daniel M; Stewart, Karen; Shaw, Lisa; Przyborski, Stefan; Cooke, Michael; McLaughlin, K John; Kimber, Susan J; Lieberman, Brian A; Wilmut, Ian; Brison, Daniel R

    2009-05-01

    The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

  13. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

    Science.gov (United States)

    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

    Science.gov (United States)

    Landry, Joseph; Sharov, Alexei A.; Piao, Yulan; Sharova, Lioudmila V.; Xiao, Hua; Southon, Eileen; Matta, Jennifer; Tessarollo, Lino; Zhang, Ying E.; Ko, Minoru S. H.; Kuehn, Michael R.; Yamaguchi, Terry P.; Wu, Carl

    2008-01-01

    We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo. PMID:18974875

  15. Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Joseph Landry

    2008-10-01

    Full Text Available We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor, the largest subunit of NURF (Nucleosome Remodeling Factor in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/- embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

  16. HLA-matched embryos selected for siblings requiring haematopoietic stem cell transplantation: a psychological perspective.

    Science.gov (United States)

    Baetens, P; Van de Velde, H; Camus, M; Pennings, G; van Steirteghem, A; Devroey, P; Liebaers, I

    2005-02-01

    Allogeneic haematopoietic stem cell transplantation (HSCT) is a treatment for a number of acquired and congenital diseases. An important factor in the outcome of the treatment is the degree of human leukocyte antigen (HLA) compatibility between patient and donor. HLA identical siblings therefore provide the best chance for the recipient. Preimplantation genetic diagnosis (PGD) can be used to select HLA identical embryos if there is no HLA compatible sibling in the family. The Centre for Reproductive Medicine considered it morally justified to give medical assistance to couples in need of an HLA matched sibling. Two considerations played an important role in this respect: (i) the use as a donor should not be the only parents' motive for having the child and (ii) IVF and HLA typing on the embryos would be less of a burden for the parents than other alternatives. Since the first request in 2000, 12 couples have been referred for psychological counselling. The motivation of four couples will be discussed in depth. The validity of the arguments will be checked against the experience of the actual cases. The consequences of the treatment on the welfare of the future donor child will be discussed.

  17. Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.

    Science.gov (United States)

    Salvemini, Marco; D'Amato, Rocco; Petrella, Valeria; Ippolito, Domenica; Ventre, Giuseppe; Zhang, Ying; Saccone, Giuseppe

    2014-01-01

    The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) anddifferential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

  18. Day-3 Medium Changes can Affect Developmental Potential of Porcine Somatic Cell Nuclear Transfer and Parthenogenesis Embryos In Vitro

    Directory of Open Access Journals (Sweden)

    Dibyendu Biswas and Sang Hwan Hyun*

    2011-01-01

    Full Text Available The aim of the present study was to compare the developmental competence of porcine parthenotes and somatic cell nuclear transfer (SCNT embryos after day-3 medium change with fresh embryo culture medium to that of embryos that did not have a medium change (monoculture system. The parthenogenetic and SCNT blastocyst formation rates were significantly (P<0.05 higher in the no-medium-change group (43.3±2.3, 18.5±1.1%, respectively compared with the day-3 medium-change group (35.9±2.4, 7.9±0.9%, respectively. Total cell number in parthenotes and SCNT blastocysts was also significantly (P<0.05 higher in the no-media-change group (92.0±4.2, 66.9±7.7, respectively compared with the media-change group (81.5±3.1, 46.6±4.9, respectively. No significant difference in cleavage rate was found in either group for parthenotes or SCNT embryos. This result suggests that day-3 medium changes have negative effects on porcine parthenotes and SCNT embryos in vitro.

  19. Cathode-supported hybrid direct carbon fuel cells

    DEFF Research Database (Denmark)

    Gil, Vanesa; Gurauskis, Jonas; Deleebeeck, Lisa

    2017-01-01

    The direct conversion of coal to heat and electricity by a hybrid direct carbon fuel cell (HDCFC) is a highly efficient and cleaner technology than the conventional combustion power plants. HDCFC is defined as a combination of solid oxide fuel cell and molten carbonate fuel cell. This work...

  20. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research.

    Science.gov (United States)

    Manninen, Bertha Alvarez

    2008-01-31

    One argument used by detractors of human embryonic stem cell research (hESCR) invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event) as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical operation. Finally, I will end the

  1. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research

    Directory of Open Access Journals (Sweden)

    Manninen Bertha

    2008-01-01

    Full Text Available Abstract One argument used by detractors of human embryonic stem cell research (hESCR invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical

  2. Effect of synchronization of donor cells in early G1-phase using shake-off method on developmental potential of somatic cell nuclear transfer embryos in cattle.

    Science.gov (United States)

    Goto, Yuji; Hirayama, Muneyuki; Takeda, Kazuya; Tukamoto, Nobuyuki; Sakata, Osamu; Kaeriyama, Hiroshi; Geshi, Masaya

    2013-08-01

    In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0-phase (G0-SCNT group) or early G1-phase (eG1-SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0-phase and eG1-phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake-off method). The fusion rate in the G0-SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1-SCNT groups (39.2 ± 1.9%) (P cells in eG1-phase using the shake-off method improved the overall production efficiency of the clone offspring per transferred embryo.

  3. Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus Scrofa).

    Science.gov (United States)

    Zhang, Ting-Yu; Dai, Jian-Jun; Wu, Cai-Feng; Gu, Xiao-Long; Liu, Liang; Wu, Zhi-Qiang; Xie, Yi-Ni; Wu, Bin; Chen, Hui-Lan; Li, Yao; Chen, Xue-Jin; Zhang, De-Fu

    2012-02-01

    To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

  4. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris; Kiessling, Ann A

    2016-01-15

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  5. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

    2016-01-01

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  6. Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells.

    Science.gov (United States)

    Wei, Qinzhi; Huang, Haiyan; Yang, Linqing; Yuan, Jianhui; Yang, Xiaohua; Liu, Yungang; Zhuang, Zhixiong

    2014-04-01

    Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 μM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type. Copyright © 2012 Wiley Periodicals, Inc.

  7. Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development.

    Science.gov (United States)

    Park, Hyo-Young; Kim, Eun-Young; Lee, Seung-Eun; Choi, Hyun-Yong; Moon, Jeremiah Jiman; Park, Min-Jee; Son, Yeo-Jin; Lee, Jun-Beom; Jeong, Chang-Jin; Lee, Dong-Sun; Riu, Key-Jung; Park, Se-Pill

    2013-12-01

    Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT-MSC bioactive material (hAT-MSC-BM), and examined the effect of hAT-MSC-BM on porcine embryo development. hAT-MSC-BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT-MSC-BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT-MSC culture medium group, the best results were from the group cultured with 10% hAT-MSC-BM. Mitochondrial activity was also higher in the 10% hAT-MSC-BM-treated group. Moreover, the relative mRNA expression levels of development and anti-apoptosis genes were significantly higher in the 10% hAT-MSC-BM-treated group than in control, hAT-MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT-MSC-BM starting on Day 4 also improved the development rate and the total cell number of in vitro-fertilized embryos. This is the first report on the benefits of hAT-MSC-BM in a porcine embryo in vitro culture system. We conclude that hAT-MSC-BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro.

  8. X-chromosome inactivation in monkey embryos and pluripotent stem cells.

    Science.gov (United States)

    Tachibana, Masahito; Ma, Hong; Sparman, Michelle L; Lee, Hyo-Sang; Ramsey, Cathy M; Woodward, Joy S; Sritanaudomchai, Hathaitip; Masterson, Keith R; Wolff, Erin E; Jia, Yibing; Mitalipov, Shoukhrat M

    2012-11-15

    Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.

  9. A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans.

    Science.gov (United States)

    Lai, Yiling; Xiang, Meichun; Liu, Shuchun; Li, Erwei; Che, Yongsheng; Liu, Xingzhong

    2014-04-01

    Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500μg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.

  10. Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

    Science.gov (United States)

    Korpela, J; Kulomaa, M; Tuohimaa, P; Vaheri, A

    1983-01-01

    Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process. Images Fig. 2. Fig. 3. PMID:6315397

  11. Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

    Science.gov (United States)

    Korpela, J; Kulomaa, M; Tuohimaa, P; Vaheri, A

    1983-01-01

    Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process.

  12. β-Pix directs collective migration of anterior visceral endoderm cells in the early mouse embryo.

    Science.gov (United States)

    Omelchenko, Tatiana; Rabadan, M Angeles; Hernández-Martínez, Rocío; Grego-Bessa, Joaquim; Anderson, Kathryn V; Hall, Alan

    2014-12-15

    Collective epithelial migration is important throughout embryonic development. The underlying mechanisms are poorly understood but likely involve spatially localized activation of Rho GTPases. We previously reported that Rac1 is essential for generating the protrusive activity that drives the collective migration of anterior visceral endoderm (AVE) cells in the early mouse embryo. To identify potential regulators of Rac1, we first performed an RNAi screen of Rho family exchange factors (guanine nucleotide exchange factor [GEF]) in an in vitro collective epithelial migration assay and identified β-Pix. Genetic deletion of β-Pix in mice disrupts collective AVE migration, while high-resolution live imaging revealed that this is associated with randomly directed protrusive activity. We conclude that β-Pix controls the spatial localization of Rac1 activity to drive collective AVE migration at a critical stage in mouse development.

  13. Direct hydrogen fuel cell systems for hybrid vehicles

    Science.gov (United States)

    Ahluwalia, Rajesh K.; Wang, X.

    Hybridizing a fuel cell system with an energy storage system offers an opportunity to improve the fuel economy of the vehicle through regenerative braking and possibly to increase the specific power and decrease the cost of the combined energy conversion and storage systems. Even in a hybrid configuration it is advantageous to operate the fuel cell system in a load-following mode and use the power from the energy storage system when the fuel cell alone cannot meet the power demand. This paper discusses an approach for designing load-following fuel cell systems for hybrid vehicles and illustrates it by applying it to pressurized, direct hydrogen, polymer-electrolyte fuel cell (PEFC) systems for a mid-size family sedan. The vehicle level requirements relative to traction power, response time, start-up time and energy conversion efficiency are used to select the important parameters for the PEFC stack, air management system, heat rejection system and the water management system.

  14. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology

    Directory of Open Access Journals (Sweden)

    Qisheng Zuo

    2016-06-01

    Full Text Available The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus. Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA recombination assay, T7 endonuclease I (T7EI assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%. Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens.

  15. Relationship between Nucleus Swelling and Development Competence of Bovine Cloned Embryos Reconstructed by Enucleated Oocytes with Serum-starved or Serum-fed Fetal Somatic Cells

    Directory of Open Access Journals (Sweden)

    Mokhamad Fahrudin

    2015-10-01

    Full Text Available This study was conducted to examine the occurrence of nuclear remodeling (nucleus swelling and its effectson the subsequent in vitro development of bovine embryos reconstructed by serum-starved and serum-fed somaticcells. Results from this study demonstrated that all of the reconstructed embryos that received serum-starved andserum-fed somatic cells exhibited condensed-nuclei. More than 90% of the transferred nuclei exhibited nuclearenvelope breakdown and premature chromatin condensation which clearly distinct from an intact nucleus. Therewas no significant difference on the degree of nucleus swelling in SS-NT embryos or SF-NT embryos, indicatingthat either serum-starved or confluent somatic cell lines could be reprogrammed by the recipient cytoplasmenvironments in similar pattern. Although the fusion rate was not significantly different among the groups, theproportion of SS-NT embryos which developed to the 2- to 4-cell stage (89.7% and to the 8- to 16-cell stage (74.7%was significantly higher than that of SF-NT embryos. Whereas, the proportion of reconstructed embryos thatdeveloped to the morula and blastocyst stages were not significantly different among the groups. Results of thesestudies demonstrate that reconstructed embryos, which received either serum-starved or serum-fed confluentsomatic cells, showed similar developmental competence to the blastocyst stage.Keywords: nuclear transplantation technique-somatic cells-nucleus swelling

  16. [Influence of "zero" magnetic field on the growth of embryonic cells and primary embryos of mouse in vitro].

    Science.gov (United States)

    Osipenko, M A; Mezhevikina, L M; Krasts, I V; Iashin, V A; Novikov, V V; Fesenko, E E

    2008-01-01

    The present investigation reveals that a 250-fold screening of the geomagnetic field ("zero" geomagnetic fields, 200 nT) is a biologically active factor that adversely affects embryonic cells and the processes of early embryogenesis as a whole. In particular, the cultivation of primary embryonic fibroblasts in "zero" geomagnetic fields causes reduces the capacity for adhesion and proliferation, changes the monolayer morphology and increases cell death. In a more highly organized experimental model, two-celled mouse embryos, the exposure to the "zero" field results in an increase of plasma membrane permeability for dyes, a reorganization of the cytoskeleton because of alpha-actin redistribution, and the disturbance of the spatial orientation of blastomeres. As a result, the development of two-celled mouse embryos stops, and they do not reach the stage of blastocyst. These data show the significant role of geomagnetic fields in the normal growth of embryonic cells in vitro and the regulation of mammalian embryogenesis.

  17. mRNA translation during oocyte maturation plays a key role in development of primordial germ cells in Xenopus embryos

    Indian Academy of Sciences (India)

    Bahman Zeynali; Keith E Dixon

    2004-09-01

    It is believed that cytoplasmic localization in the egg is necessary for development of primordial germ cells (PGCs) in Xenopus embryos. In this study, we sought to determine if translation of maternal mRNA during oocyte maturation is involved in the development of PGCs. Donor oocytes were collected from both stimulated (those who receive gonadotropin) and unstimulated females, artificially matured and fertilized using a host transfer technique. Using chloramphenicol (50 M and 500 M RNA), RNA translation was inhibited during oocyte maturation. Our results showed that in unstimulated embryos treated with 50 M chloramphenicol, there was a significant reduction in the number of PGCs reaching genital ridges. In stimulated embryos, however, the number of PGCs was unchanged unless a higher concentration (500 M) of chloramphenicol was used. From these results it is suggested that maternal mRNA translation during oocyte maturation plays a key role in development of PGCs.

  18. Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos.

    Science.gov (United States)

    Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Sinha, Sunilima; Alexenko, Andrei P; Spate, Lee; Prather, Randall S; Roberts, R Michael

    2011-08-19

    The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.

  19. SINGLE CELL DEGENERATE OLIGONUCLEOTIDE PRIMER-PCR AND COMPARATIVE GENOMIC HYBRIDIZATION WITH MODIFIED CONTROL REFERENCE

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    For investigating the possibility of applying degenerate oligonucleotide primer PCR (DOP-PCR) and comparative genomic hybridization (CGH) technique to analyses of genomic genetics in a single cell, the whole genomic DNA of a single cell with XX, XY, XO, XXY, +13 or +21 was amplified by DOP-PCR. Single cell DOP-PCR CGHs with conventional and modified control references, the genomic DNA and a single cell DOP-PCR product from normal male, were carried out respectively. The results showed that the average profile of the fluorescence intensity ratio in CGH with the genomic DNA as reference fluctuates much and that the standard deviation in about 30% haploid is beyond the normal limits. False positive hyper-representation was found to exist in X chromosome while trisomy 13 and 21 were not detected. However, the distributions of the mean and the standard deviation of the ratio in the CGH with DOP-PCR product as reference were quite acceptable. The copy number changes of chromosome X,Y,13 and 21 were revealed. Those results suggested that there is unrandom unequal amplification in a single cell DOP-PCR. Using a single DOP-PCR product as reference can decrease its influence on CGH. Single cell DOP-PCR-CGH and its application in the genetic analyses of preimplantation embryo or fetal cell in maternal blood may be possible.

  20. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

    Science.gov (United States)

    Chinchar, V G; Floyd, A D; Chinchar, G D; Taylor, M W

    1979-02-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

  1. Neuroprotection and neuronal differentiation studies using substantia nigra dopaminergic cells derived from transgenic mouse embryos.

    Science.gov (United States)

    Son, J H; Chun, H S; Joh, T H; Cho, S; Conti, B; Lee, J W

    1999-01-01

    The major pathological lesion of Parkinson's disease (PD) is the selective cell death of dopaminergic (DA) neurons in substantia nigra (SN). Although the initial cause and subsequent molecular signaling mechanisms leading to DA cell death underlying the PD process remain elusive, brain-derived neurotrophic factor (BDNF) is thought to exert neuroprotective as well as neurotrophic roles for the survival and differentiation of DA neurons in SN. Addressing molecular mechanisms of BDNF action in both primary embryonic mesencephalic cultures and in vivo animal models has been technically difficult because DA neurons in SN are relatively rare and present with many heterogeneous cell populations in midbrain. We have developed and characterized a DA neuronal cell line of embryonic SN origin that is more accessible to molecular analysis and can be used as an in vitro model system for studying SN DA neurons. A clonal SN DA neuronal progenitor cell line SN4741, arrested at an early DA developmental stage, was established from transgenic mouse embryos containing the targeted expression of the thermolabile SV40Tag in SN DA neurons. The phenotypic and morphological differentiation of the SN4741 cells could be manipulated by environmental cues in vitro. Exogenous BDNF treatment produced significant neuroprotection against 1-methyl-4-phenylpyridinium, glutamate, and nitric oxide-induced neurotoxicity in the SN4741 cells. Simultaneous phosphorylation of receptor tyrosine kinase B accompanied the neuroprotection. This SN DA neuronal cell line provides a unique model system to circumvent the limitations associated with primary mesencephalic cultures for the elucidation of molecular mechanisms of BDNF action on DA neurons of the SN.

  2. Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation, colchicine treatment and gene transfection. Results are as follows: (Ⅰ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3% vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25μm) as donor nuclei was higher than that from large cells (25-33μm) and small cells (8-15μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS(11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05μmol/L colchicine could

  3. Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

    Institute of Scientific and Technical Information of China (English)

    WANG Hai; AO Hong; PAN QiuZhen; LI RongQi; ZHAO MengBin; LIAN ZhengXing; LI Ning; WU ChangXin

    2007-01-01

    To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation,colchicine treatment and gene transfection. Results are as follows: ( Ⅰ ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3%vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25 μm) as donor nuclei was higher than that from large cells (25-33 μm) and small cells (8-15 μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS( 11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05 μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05 μmol/L colchicine

  4. Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

    Science.gov (United States)

    Li, R; Wu, H; Zhuo, W W; Mao, Q F; Lan, H; Zhang, Y; Hua, S

    2015-10-01

    Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation.

  5. Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

    Science.gov (United States)

    Sun, Liangliang; Dubiak, Kyle M; Peuchen, Elizabeth H; Zhang, Zhenbin; Zhu, Guijie; Huber, Paul W; Dovichi, Norman J

    2016-07-05

    Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ∼0.8 μg (16-cell embryo) to ∼0.2 μg (50-cell embryo), the number of protein group identifications declined from 1 466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

  6. Exogenous gibberellins inhibit coffee (Coffea arabica cv. Rubi) seed germination and cause cell death in the embryo.

    Science.gov (United States)

    da Silva, E A Amaral; Toorop, Peter E; Nijsse, Jaap; Bewley, J Derek; Hilhorst, Henk W M

    2005-03-01

    The mechanism of inhibition of coffee (Coffea arabica cv. Rubi) seed germination by exogenous gibberellins (GAs) and the requirement of germination for endogenous GA were studied. Exogenous GA(4+7) inhibited coffee seed germination. The response to GA(4+7) showed two sensitivity thresholds: a lower one between 0 and 1 microM and a higher one between 10 and 100 microM. However, radicle protrusion in coffee seed depended on the de novo synthesis of GAs. Endogenous GAs were required for embryo cell elongation and endosperm cap weakening. Incubation of coffee seed in exogenous GA(4+7) led to loss of embryo viability and dead cells were observed by low temperature scanning microscopy only when the endosperm was surrounding the embryo. The results described here indicate that the inhibition of germination by exogenous GAs is caused by factors that are released from the endosperm during or after its weakening, causing cell death in the embryo and leading to inhibition of radicle protrusion.

  7. Detection of isoform-specific fibroblast growth factor receptors by whole-mount in situ hybridization in early chick embryos.

    Science.gov (United States)

    Nishita, Junko; Ohta, Sho; Bleyl, Steven B; Schoenwolf, Gary C

    2011-06-01

    We have developed "b" and "c" isoform-specific chicken fibroblast growth factor (FGF) receptor 1-3 probes for in situ hybridization. We rigorously demonstrate the specificity of these probes by using both dot blot hybridization and whole-mount in situ hybridization during neurulation and early postneurulation stages, and we compare expression patterns of each of the three isoform-specific probes to one another and to generic probes to each of the three (non-isoform-specific) FGF receptors. We show that the expression pattern of each receptor is represented by the collective expression of each of its two isoforms, with the expression of each FGF receptor being most similar to that of its "c" isoform at two of the three stages studied, and that tissue and stage differences exist in the patterns of expression of the six isoforms. We demonstrate the usefulness of these probes for defining the differential tissue expression of FGF receptor 1-3 isoforms.

  8. Misoprostol-induced radioprotection of Syrian hamster embryo cells in utero from cell death and oncogenic transformation

    Energy Technology Data Exchange (ETDEWEB)

    Miller, R.C.; LaNasa, P.; Hanson, W.R. [Loyola Univ., Maywood, IL (United States)

    1994-07-01

    Misoprostol, a PGE analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems. The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro. Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostal, and 2 h later the pregnant hamsters were exposed to graded doses of X rays. Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium. After a 2-week incubation period, clongenic cell survival and morphologically transformed foci were determined. Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone. In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone. These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events. 26 refs., 6 figs., 2 tabs.

  9. The impact of commercialisation on public perceptions of stem cell research: exploring differences across the use of induced pluripotent cells, human and animal embryos.

    Science.gov (United States)

    Critchley, Christine R; Bruce, Gordana; Farrugia, Matthew

    2013-10-01

    The development of pluripotent cells that enable stem cell research (SCR) without destroying human embryos is now a leading priority for science. Public and political controversies associated with human embryonic SCR experienced in the recent past should be alleviated if scientists no longer need to harvest cells from human embryos. This research suggests however additional issues needing attention in order to gain the public's trust and support: the use of mouse embryos and the commercialisation of research. Using a representative sample of 2,800 Australians, and an experimental telephone survey design, this research compared levels and predictors of public support for stem cell research across three cell source conditions: human embryo (HE), mouse embryo (ME) and induced pluripotent cells (iPSCs). The results revealed that the public were significantly more likely to support research using iPSCs than HE and ME cells and public compared to private research (regardless of the cell source). There was no significant difference in support for HE compared to ME research, but the former was viewed as more likely to lead to accessible health care benefits and to be associated with more trustworthy scientists. The results of a multimediation structural equation model showed that the primary reason support for SCR significantly dropped in a private compared to public context (i.e., the commercialisation effect) was because public scientists were trusted more than private scientists. This effect was consistent across all three SCR materials, suggesting that the use of mouse embryos or even iPSCs will not reduce the publics' concern with commercialised science. The implications these results have for public acceptance of stem cell and animal research are discussed in relation to possible solutions such as increasing public awareness of the regulation of animal research and benefit sharing.

  10. The effect of superovulation on the contributions of individual blastomeres from 2-cell stage CF1 mouse embryos to the blastocyst.

    Science.gov (United States)

    Katayama, Mika; Roberts, R Michael

    2010-01-01

    It remains controversial whether blastomeres of 2-cell stage mouse embryos show bias in their contribution to the blastocyst and whether there is any effect of superovulation. Two-cell stage embryos from CF1 mice were derived by either natural breeding (N) or superovulation (S) and cultured in vitro. At blastocyst, inner cell mass and trophectoderm were distinguished by Cdx2 and Oct4 immunostaining. A fluorescent dye (CM-Dil) was also used to tag individual blastomeres at the 2-cell stage, and the descendant cells identified by their red fluorescence. S and N embryos developed to blastocyst at the same rate and contained a similar number of cells. However, with S embryos, the descendants of the blastomere labeled with CM-DiI contributed predominantly to either the embryonic or abembryonic pole about 70% of the time, whereas most N embryos displayed random patterning, with no restriction to one or other of the poles. In S-embryos, but not N-embryos, the leading blastomere at second cleavage contributed preferentially to the embryonic pole of the blastocyst and the lagging blastomere to the abembryonic pole and hence mural trophectoderm. In addition, a tetrahedral rather than a flat morphology at the 4-cell stage of S-embryos was strongly biased to displaying the embryonic/abembryonic pattern at blastocyst. In contrast, S-embryos lacking a zona pellucida resembled N embryos in their patterning. In CF1 mice, superovulation has little effect on development to blastocyst, but enforces a greater degree of lineage restriction than natural breeding, most likely through constraints imposed by the zona pellucida.

  11. The Effects of Ethanol and Strontium on Growth and Development of Two-Cell Arrested Mouse Embryos

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Darabi

    2012-01-01

    Full Text Available Background: Arresting at a certain stage of development like the two-cell stage could be one of the causes of infertility. The aim of this study is to evaluate the effects of ethanol and strontium on growth and development of mice embryos arrested at the two-cell stage.Materials and Methods: In this experimental study, female mice were coupled with a male following superovulation. Positive vaginal plug mice were sacrificed 48 hours after human chorionic gonadotropin (hCG injection. Two-cell embryos were transferred to M16 medium and divided to four groups. The first control group was incubated without any exposure to low temperatures. Groups 2, 3 and 4 were exposed to 4°C for 24 hours. The second control group was incubated immediately, while the third and fourth groups were exposed to 10 mM strontium for five minutes and 0.1% ethanol for a further five minutes. Growth rate and developmental parameters of embryos were analyzed by one-way ANOVA. The significant difference between the groups was determined by Post Hoc.Results: The data shows that developmental rate is decreased significantly by 4°C exposure. The mean percentage of degenerated embryo was significantly different between groups but the mean cleavage rate was not significantly different. The mean percent of morula, blastocyst and hatched blastocyst formation were significantly different between groups during a 120 hours study post hCG injection.Conclusion: The effect of strontium and ethanol on arrested two-cell embryos had no significant effect on the mean percentage of morula, but ethanol treatment significantly increased the percentage of blastocyst and hatched blastocyst formation compared to strontium.

  12. Cell differentiation and germ-soma separation in Ediacaran animal embryo-like fossils

    Science.gov (United States)

    Chen, Lei; Xiao, Shuhai; Pang, Ke; Zhou, Chuanming; Yuan, Xunlai

    2014-12-01

    Phosphorites of the Ediacaran Doushantuo Formation (~600 million years old) yield spheroidal microfossils with a palintomic cell cleavage pattern. These fossils have been variously interpreted as sulphur-oxidizing bacteria, unicellular protists, mesomycetozoean-like holozoans, green algae akin to Volvox, and blastula embryos of early metazoans or bilaterian animals. However, their complete life cycle is unknown and it is uncertain whether they had a cellularly differentiated ontogenetic stage, making it difficult to test their various phylogenetic interpretations. Here we describe new spheroidal fossils from black phosphorites of the Doushantuo Formation that have been overlooked in previous studies. These fossils represent later developmental stages of previously published blastula-like fossils, and they show evidence for cell differentiation, germ-soma separation, and programmed cell death. Their complex multicellularity is inconsistent with a phylogenetic affinity with bacteria, unicellular protists, or mesomycetozoean-like holozoans. Available evidence also indicates that the Doushantuo fossils are unlikely crown-group animals or volvocine green algae. We conclude that an affinity with cellularly differentiated multicellular eukaryotes, including stem-group animals or algae, is likely but more data are needed to constrain further the exact phylogenetic affinity of the Doushantuo fossils.

  13. Cell differentiation and germ-soma separation in Ediacaran animal embryo-like fossils.

    Science.gov (United States)

    Chen, Lei; Xiao, Shuhai; Pang, Ke; Zhou, Chuanming; Yuan, Xunlai

    2014-12-11

    Phosphorites of the Ediacaran Doushantuo Formation (∼600 million years old) yield spheroidal microfossils with a palintomic cell cleavage pattern. These fossils have been variously interpreted as sulphur-oxidizing bacteria, unicellular protists, mesomycetozoean-like holozoans, green algae akin to Volvox, and blastula embryos of early metazoans or bilaterian animals. However, their complete life cycle is unknown and it is uncertain whether they had a cellularly differentiated ontogenetic stage, making it difficult to test their various phylogenetic interpretations. Here we describe new spheroidal fossils from black phosphorites of the Doushantuo Formation that have been overlooked in previous studies. These fossils represent later developmental stages of previously published blastula-like fossils, and they show evidence for cell differentiation, germ-soma separation, and programmed cell death. Their complex multicellularity is inconsistent with a phylogenetic affinity with bacteria, unicellular protists, or mesomycetozoean-like holozoans. Available evidence also indicates that the Doushantuo fossils are unlikely crown-group animals or volvocine green algae. We conclude that an affinity with cellularly differentiated multicellular eukaryotes, including stem-group animals or algae, is likely but more data are needed to constrain further the exact phylogenetic affinity of the Doushantuo fossils.

  14. Apical accumulation of MARCKS in neural plate cells during neurulation in the chick embryo

    Directory of Open Access Journals (Sweden)

    Arruti Cristina

    2001-04-01

    Full Text Available Abstract Background The neural tube is formed by morphogenetic movements largely dependent on cytoskeletal dynamics. Actin and many of its associated proteins have been proposed as important mediators of neurulation. For instance, mice deficient in MARCKS, an actin cross-linking membrane-associated protein that is regulated by PKC and other kinases, present severe developmental defects, including failure of cranial neural tube closure. Results To determine the distribution of MARCKS, and its possible relationships with actin during neurulation, chick embryos were transversely sectioned and double labeled with an anti-MARCKS polyclonal antibody and phalloidin. In the neural plate, MARCKS was found ubiquitously distributed at the periphery of the cells, being conspicuously accumulated in the apical cell region, in close proximity to the apical actin meshwork. This asymmetric distribution was particularly noticeable during the bending process. After the closure of the neural tube, the apically accumulated MARCKS disappeared, and this cell region became analogous to the other peripheral cell zones in its MARCKS content. Actin did not display analogous variations, remaining highly concentrated at the cell subapical territory. The transient apical accumulation of MARCKS was found throughout the neural tube axis. The analysis of another epithelial bending movement, during the formation of the lens vesicle, revealed an identical phenomenon. Conclusions MARCKS is transiently accumulated at the apical region of neural plate and lens placode cells during processes of bending. This asymmetric subcellular distribution of MARCKS starts before the onset of neural plate bending. These results suggest possible upstream regulatory actions of MARCKS on some functions of the actin subapical meshwork.

  15. Regulation of somatic embryo development in Norway spruce (Picea abies). A molecular approach to the characterization of specific developmental stages

    Energy Technology Data Exchange (ETDEWEB)

    Sabala, I. [Swedish Univ. of Agricultural Sciences, Uppsala (Sweden). Dept. of Forest Genetics

    1998-12-31

    Embryo development is a complex process involving a set of strictly regulated events. The regulation of these events is poorly understood especially during the early stages of embryo development. Somatic embryos go through the same developmental stages as zygotic embryos making them an ideal model system for studying the regulation of embryo development. We have used embryogenic cultures of Picea abies to study some aspects of the regulation of embryo development in gymnosperms. The bottle neck during somatic embryogenesis is the switch from the proliferation stage to the maturation stage. This switch is initiated by giving somatic embryos a maturation treatment i.e. the embryos are treated with abscisic acid (ABA). Somatic embryos which respond to ABA by forming mature somatic embryos were stimulated to secret a 70 kDa protein, AF70. The af70 gene was isolated and characterised. The expression of the af70 gene was constitutive in embryos but was highly ABA-induced in seedlings. Moreover, expression of this gene was stimulated during cold acclimation of Picea abies seedlings. A full length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins (LTPs), was isolated and characterised. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in nonembryogenic callus and seedlings. In situ hybridization showed that Pa18 gene is expressed in all embryonic cells of proliferating somatic embryos but the expression of the gene in mature somatic and zygotic embryos is restricted to the outer cell layer. Southern blot analysis at different stringencies was consistent with a single gene. An alteration in expression of Pa18 causes disturbance in the formation of the proper outer cell layer in the maturing somatic embryos. In addition to its influence on embryo development the Pa18 gene product also inhibits growth of Agrobacterium tumefaciens 195

  16. Solid Oxide Fuel Cell Hybrid System for Distributed Power Generation

    Energy Technology Data Exchange (ETDEWEB)

    Faress Rahman; Nguyen Minh

    2004-01-04

    This report summarizes the work performed by Hybrid Power Generation Systems, LLC (HPGS) during the July 2003 to December 2003 reporting period under Cooperative Agreement DE-FC26-01NT40779 for the U. S. Department of Energy, National Energy Technology Laboratory (DOE/NETL) entitled ''Solid Oxide Fuel Cell Hybrid System for Distributed Power Generation''. The main objective of this project is to develop and demonstrate the feasibility of a highly efficient hybrid system integrating a planar Solid Oxide Fuel Cell (SOFC) and a micro-turbine. In addition, an activity included in this program focuses on the development of an integrated coal gasification fuel cell system concept based on planar SOFC technology. Also, another activity included in this program focuses on the development of SOFC scale up strategies.

  17. Organic / IV, III-V Semiconductor Hybrid Solar Cells

    Directory of Open Access Journals (Sweden)

    Pang-Leen Ong

    2010-03-01

    Full Text Available We present a review of the emerging class of hybrid solar cells based on organic-semiconductor (Group IV, III-V, nanocomposites, which states separately from dye synthesized, polymer-metal oxides and organic-inorganic (Group II-VI nanocomposite photovoltaics. The structure of such hybrid cell comprises of an organic active material (p-type deposited by coating, printing or spraying technique on the surface of bulk or nanostructured semiconductor (n-type forming a heterojunction between the two materials. Organic components include various photosensitive monomers (e.g., phtalocyanines or porphyrines, conjugated polymers, and carbon nanotubes. Mechanisms of the charge separation at the interface and their transport are discussed. Also, perspectives on the future development of such hybrid cells and comparative analysis with other classes of photovoltaics of third generation are presented.

  18. Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo.

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    Elsa Abranches

    Full Text Available BACKGROUND: The in vitro generation of neurons from embryonic stem (ES cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor

  19. A study upon the influence of cyclophosphamide treatment on the red blood cells of the chicken embryo (Short Notes

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    Delia Anca HAS-LAZAU

    2006-05-01

    Full Text Available The aim of this study is to show the effect of cyclophosphamide on the eveloping red blood cells of the 3-4 days old chicken embryo, when the hematopoiesis is at its peack, located at the vitelline sack level.I have chosen to work with the chicken embryo red blood cells because they have an intense mitotic activity as well as a tumoural cell-like behaviour.It is vital to know the particularities of the cell cycle of the healthy and tumoural cells, keeping in mind that most of the cytostatics act upon the cell which are developing their cell cycle (Menkes B., Prelipceanu O., Checiu I., Căpălnăşan I. 1979.The cyclophosphamide is not stage-dependent, as it acts in all the stages of the cell cycle, its mutagen effect being accompanied also by a cell cycle stopping (Paşca C., Crăciun C., Ardelean A. 2000.Cyclophosphamide supply determines retrenchment of the cell division, transforming the normal cells into multinucleated cells, with normal ploydia. The cyclophosphamide is a cytostatic using for cancer therapy (Schiavoni G., Mattei F., Di Pucchio T., Santini S. M., Bracci L., Berardelli F., Proietti F. 2000.Reshearches have done lots of studies along the years both on mice and rats, concerning the effects of cyclophosphamide on: thymus and burse fabricio ( Giurgea R., Toma V., 1977, stromal cells of bone marrow (Anton E. 1997, pulmonary thrombocytopoiesis (Sulkowki S., Sulkowska M., Musiatowikz B. 1997.

  20. Comparative evaluation of cell culture-adapted and chicken embryo-adapted fowl pox vaccine strains.

    Science.gov (United States)

    Baxi, M K; Oberoi, M S

    1999-01-01

    Two types of vaccines, chicken embryo adapted (VacCE) and cell culture adapted (VacCC), were tested for their efficacy to elicite the immune response in birds vaccinated at 2 and 8 wk of age. The cell-mediated immune response studied by blastogenesis assay showed that birds vaccinated at the second week of age by both VacCE and VacCC vaccines had significant increase in T-lymphocyte count at 21 days postvaccination (PV) and 7 days postchallenge (PC), whereas in birds vaccinated at 8 wk of age, a significant increase was seen at 21 days PV and 7 days PC with the VacCC vaccine. The rise in passive hemagglutination titers was observed up to 21 days PV and 7 days PC in birds vaccinated at 2 wk of age. However, only the birds vaccinated with VacCC at 8 wk of age showed rise in titers at days 21 PV and 7 PC. Birds were challenged 90 days PV by scarification on the thigh region, and the birds vaccinated with VacCC showed 90% and 70% protection when vaccinated at 2 and 8 wk, respectively. The birds vaccinated with VacCE showed only 60% and 20% protection at the corresponding levels, respectively.

  1. Sustained centrosome-cortical contact ensures robust polarization of the one-cell C. elegans embryo.

    Science.gov (United States)

    Saturno, Dominique M; Castanzo, Dominic T; Williams, Margaret; Parikh, Devayu A; Jaeger, Eva C; Lyczak, Rebecca

    2017-02-15

    In C. elegans, the anterior-posterior axis is established at the one-cell stage when the embryo polarizes along its long axis. One model suggests that a cue from the centrosome triggers symmetry breaking and is then dispensable for further steps in the process. In the absence of the initial centrosome cue, a redundant mechanism, reliant on the centrosome's microtubules, can polarize the cell. Despite this model, data from multiple sources suggest that direct centrosome-contact with the cortex may play a role in ensuring robust polarization. Some of this past work includes analysis of pam-1 mutants, which lack a functional puromycin-sensitive aminopeptidase and have aberrant centrosome positioning and variable polarization defects. To better understand the role of centrosome dynamics in polarization, we looked in detail at centrosome behavior in relation to key polarity landmarks in pam-1 mutants as well as those lacking cortical flows. We provide evidence for a model in which sustained direct contact between the centrosome and the cortex acts to reinforce both the actomyosin and the microtubule-dependent pathways. This contact is necessary for polarization when flows are inhibited. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Effect of Mitochondrial Transplantation from Cumulus Granular Cells to the Early Embryos of Aged Mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To assess the role of mitochondria in the early embryonic development of ageing mice.Methods Mitochondria isolated from cumulus granular cells of aged mice were microinjected into oocytes or zygotes of aged mice. In the setting of oocyte injection, mitochondria were transferred via intracytoplasmic sperm injection (ICSI+MIT), and ICSI without mitochondrial transfer. In the setting of zygote injection, mitochondria were directly microinjected into fertilized oocytes (MIT), and those injected with buffer alone (mock injection) or not injected (uninjected) served as controls.Results Although the rates of oocyte cleavage between ICSI and ICSI+MIT groups were not statistically different (P>0.05), the rate of blastocyst in the ICSI+MIT group was significantly higher than that in ICSI group (P<0.05). Although both the cleavage and blastocyst rates of mock injection group were significantly lower than those of uninjected group (P<0.05), likely due to mechanical damages of the cells by microinjection, the decrease of these rates was prevented by mitochondrial transfer. After mitochondrial transfer, the rates of both cleavage and blastocyst were significantly improved over the mock-injection group (P<0.05).Conclusion Mitochondrial transplantation can improve the developmental potential of early embryos of aged mice.

  3. Patterned hybrid nanohole array surfaces for cell adhesion and migration.

    Science.gov (United States)

    Westcott, Nathan P; Lou, Yi; Muth, John F; Yousaf, Muhammad N

    2009-10-06

    We report the fabrication of hybrid nanohole array surfaces to study the role of the surface nanoevironment on cell adhesion and cell migration. We use polystyrene beads and reactive ion etching to control the size and the spacing between nanoholes on a tailored self-assembled monolayer inert gold surface. The arrays were characterized by scanning electron microscopy and brightfield microscopy. For cell adhesion studies, cells were seeded to these substrates to study the effect of ligand spacing on cell spreading, stress fiber formation, and focal adhesion structure and size. Finally, comparative cell migration rates were examined on the various nanohole array surfaces using time-lapse microscopy.

  4. Large scale in vivo risk assessment of bovine viral diarrhea virus (BVDV) transmission through transfer of bovine embryos produced via somatic cell nuclear transfer (SCNT).

    Science.gov (United States)

    Gregg, K; Gosch, G; Guerra, T; Chen, S H; Xiang, T; Broek, D; Bruner, B; Polejaeva, I

    2010-10-15

    The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission. Copyright © 2010 Elsevier Inc. All rights reserved.

  5. Pluripotent hybrid stem cells from transgenic Huntington's disease monkey.

    Science.gov (United States)

    Laowtammathron, Chuti; Chan, Anthony W S

    2013-01-01

    Huntington's disease (HD) is a devastating disease that currently has no cure. Transgenic HD monkeys have developed key neuropathological and cognitive behavioral impairments similar to HD patients. Thus, pluripotent stem cells derived from transgenic HD monkeys could be a useful comparative model for clarifying HD pathogenesis and developing novel therapeutic approaches, which could be validated in HD monkeys. In order to create personal pluripotent stem cells from HD monkeys, here we present a tetraploid technique for deriving pluripotent hybrid HD monkey stem cells.

  6. Lineage development of cell fusion hybrids upon somatic reprogramming

    OpenAIRE

    2011-01-01

    Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011 Somatic cell reprogramming has been extensively studied over the last years and opened new perspectives in the use of pluripotent cells for regenerative biomedical purposes. Spontaneous cell fusion has been suggested to be involved in regenerative processes in vivo. Strong evidences support the hypothesis that the reprogrammed hybrids resulting from the fusion between a pluripote...

  7. Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo.

    Science.gov (United States)

    Onjiko, Rosemary M; Morris, Sydney E; Moody, Sally A; Nemes, Peter

    2016-06-21

    Single-cell metabolic mass spectrometry enables the discovery (untargeted) analysis of small molecules in individual cells. Using single-cell capillary electrophoresis high-resolution mass spectrometry (CE-HRMS), we recently uncovered small-molecule differences between embryonic cells located along the animal-vegetal and dorsal-ventral axes of the 16-cell frog (Xenopus laevis) embryo, raising the question whether metabolic cell heterogeneity also exists along the left-right body axis. To address this question, we here advance single-cell CE-HRMS for identifying and quantifying metabolites in higher analytical sensitivity, and then use the methodology to compare metabolite production between left and right cells. Our strategy utilizes multiple solvents with complementary physicochemical properties to extract small molecules from single cells and improve electrophoretic separation, increasing metabolite ion signals for quantification and tandem HRMS. As a result, we were able to identify 55 different small molecules in D1 cells that were isolated from 8-cell embryos. To quantify metabolite production between left and right cells, we analyzed n = 24 different D1 cells in technical duplicate-triplicate measurements. Statistical and multivariate analysis based on 80 of the most repeatedly quantified compounds revealed 10 distinct metabolites that were significantly differentially accumulated in the left or right cells (p embryo.

  8. The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

    Directory of Open Access Journals (Sweden)

    Hitomi Suzuki

    Full Text Available BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

  9. Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  10. Dihydroartemisinin (DHA) treatment causes an arrest of cell division and apoptosis in rat embryonic erythroblasts in whole embryo culture.

    Science.gov (United States)

    Posobiec, Lorraine M; Clark, Robert L; Bushdid, Paul B; Laffan, Susan B; Wang, Kai-Fen; White, Tacey E K

    2013-12-01

    Within 24 hr after oral administration of the antimalarial artesunate to rats on Day 10 or 11 postcoitum (pc), there is depletion of embryonic erythroblasts (EEbs), leading to embryo malformation and death. The proximate agent is dihydroartemisinin (DHA), the primary metabolite. We investigated the causes of EEb depletion by evaluating effects of DHA on EEbs in whole embryo culture (WEC). Rat embryos cultured starting on Day 9 pc were treated with 1 or 7 μM DHA for 24 hr starting after 19 hr of culture (∼Day 10 pc) and for 2 to 12 hr starting after 43 hr of culture (∼Day 11 pc). DHA effects indicating the depletion of EEbs were paling of the visceral yolk sac and reductions in visible blood cells, H&E-stained normal (Type II or III) EEbs, and dividing (BrdU-stained) EEbs. DHA-induced abnormal cell division was indicated by increases in symmetric and asymmetric binuclear cells. DHA-induced apoptosis was indicated by increases in TUNEL- and Caspase-3-positive cells and EEbs with fragmented nuclei. In addition, although the overall number of EEbs was decreasing, DHA caused increases in the numbers of circulating early-stage (Type I or earlier) EEbs that could not be accounted for by cell division, suggesting the release of new, less sensitive erythroblasts from the yolk sac. In summary, treatment of Day 10 or 11 pc rat embryos with DHA in WEC resulted in defective and arrested cell division in EEbs followed by apoptosis, suggesting a mechanism for their depletion after artesunate treatment in vivo.

  11. Maternal hCG concentrations in early IVF pregnancies: associations with number of cells in the Day 2 embryo and oocytes retrieved.

    Science.gov (United States)

    Tanbo, T G; Eskild, A

    2015-12-01

    Do number of cells in the transferred cleavage stage embryo and number of oocytes retrieved for IVF influence maternal hCG concentrations in early pregnancies? Compared with transfer of a 2-cell embryo, transfer of a 4-cell embryo results in higher hCG concentrations on Day 12 after transfer, and more than 20 oocytes retrieved were associated with low hCG concentrations. Maternal hCG concentration in very early pregnancy varies considerably among women, but is likely to be an indicator of time since implantation of the embryo into the endometrium, in addition to number and function of trophoblast cells. We followed 1047 pregnancies after IVF/ICSI from oocyte retrieval until Day 12 after embryo transfer. Women were recruited in Norway during the years 2005-2013. Successful pregnancies after transfer of one single embryo that had been cultured for 2 days were included. Maternal hCG was quantified on Day 12 after embryo transfer by chemiluminescence immunoassay, which measures intact hCG and the free β-hCG chain. Information on a successful pregnancy, defined as birth after >16 weeks, was obtained by linkage to the Medical Birth Registry of Norway. Transfer of a 4-cell embryo resulted in higher maternal hCG concentrations compared with transfer of a 2-cell embryo (134.8 versus 87.8 IU/l, P 20) was associated with low hCG concentrations (P hCG concentrations in early pregnancy. Although embryo transfer was performed at the same time after fertilization, we do not know the exact time of implantation. A further limitation to our study is that the number of pregnancies after transfer of a 2-cell embryo was small (27 cases). Number of cells in the transferred embryo and number of oocytes retrieved may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations. Such knowledge may be important for interpretation of hCG concentrations in early pregnancy. © The Author 2015. Published by Oxford University

  12. Transcriptomic Analysis Of Purified Embryonic Neural Stem Cells From Zebrafish Embryos Reveals Signalling Pathways Involved In Glycine-dependent Neurogenesis

    Directory of Open Access Journals (Sweden)

    Eric eSAMARUT

    2016-03-01

    Full Text Available How is the initial set of neurons correctly established during the development of the vertebrate central nervous system? In the embryo, glycine and GABA are depolarizing due the immature chloride gradient, which is only reversed to become hyperpolarizing later in post-natal development. We previously showed that glycine regulates neurogenesis via paracrine signalling that promotes calcium transients in neural stem cells (NSCs and their differentiation into interneurons within the spinal cord of the zebrafish embryo. However, the subjacent molecular mechanisms are not yet understood. Our previous work suggests that early neuronal progenitors were not differentiating correctly in the developing spinal cord. As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis. Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting (FACS from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down. The strength of this approach is that it focused on the NSC population while tackling the biological issue in an in vivo context in whole zebrafish embryos. After sequencing the transcriptome by RNA-sequencing, we analyzed the genes whose expression was changed upon disruption of glycine signalling and we confirmed the differential expression by independent RTqPCR assay. While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signalling pathways (signalling by calcium, TGF-beta, sonic hedgehog, Wnt and p53-related apoptosis that are likely to mediate the promotion of neurogenesis by glycine.

  13. [First birth after preimplantation genetic diagnosis performed on thawed embryos].

    Science.gov (United States)

    Frydman, N; Ray, P; Romana, S; Fanchin, R; Lelorc'h, M; Kerbrat, V; Frydman, R; Tachdjian, G

    2003-06-01

    To report the birth of the first infant conceived after preimplantation genetic diagnosis (PGD) performed on frozen-thawed embryos in our PGD center. Three couples (C1, C2 and C3) who had frozen embryos from a previous in vitro fertilization attempt were enrolled in our PGD program. Embryos were thawed one day before the biopsy procedure for the couples C1 and C3 and the day of the biopsy for the couple C2. The single cell genetic analysis was performed by a multiplex PCR for the couple C1 and by fluorescent in situ hybridization for the couples C2 and C3. The embryos transfers were carried out on the third or fourth day. Out of ten thawed embryos, eight were biopsied and five were transferred during three embryos transfers. Two biochemical and one ongoing pregnancy were obtained yielded one birth. PGD may be offered to couples at risk of transmission of a serious and incurable genetic disease and having frozen embryos.

  14. Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

    DEFF Research Database (Denmark)

    Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe;

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI......, active caspase-3 and apoptotic nuclear morphology were observed in an untreated 8-cell stage, and TUNEL-labeling was observed from the 16-cell stage. Blastomeres concurrently displaying all apoptotic features were present in a few embryos at 16-cell and morula stages and in all blastocysts. All three...

  15. Response to: Dittrich et al.: Non-Embryo-Destructive Extraction of Pluripotent Embryonic Stem Cells - Overlooked Legal Prohibitions, Professional Legal Consequences and Inconsistencies in Patent Law.

    Science.gov (United States)

    Faltus, T; Storz, U

    2016-12-01

    The publication of "Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine" by Dittrich et al. in Geburtshilfe und Frauenheilkunde 2015; 75: 1239-1242 1 describes various possibilities which could result from the non-embryo-destructive extraction of embryonic stem cells from human blastocysts. But implementing this method is more problematic, both legally and ethically, than the authors have represented it to be and is illegal in Germany. German patent DE 10 2004 062 184 on the non-embryo-destructive extraction of embryonic stem cells referred to by Dittrich et al. contravenes the higher-ranking case-law of the European Court of Justice. Ultimately, the non-embryo-destructive harvesting of embryonic stem cells with the aim of storing these cells for use in potential therapies as proposed by Dittrich et al. is prohibited in Germany and could lead to criminal prosecution.

  16. Evaluation of cytotoxicity, genotoxicity and embryotoxicity of insecticide propoxur using flounder gill (FG) cells and zebrafish embryos.

    Science.gov (United States)

    Pandey, Manish Raj; Guo, Huarong

    2014-04-01

    Cytotoxicity, genotoxicity and embryotoxicity of carbamate insecticide propoxur were evaluated using flounder gill (FG) cells and zebrafish embryos. The cytotoxicity of propoxur in FG cells was analyzed by MTT, neutral red uptake (NRU), lactate dehydrogenase (LDH) release and Hoechst 33342 and propidium iodide double staining, and acute cytotoxic effects were observed in a concentration-dependent manner. The 24h-IC50 values of 89.96 ± 1.04, 103.4 ± 1.14 and 86.59 ± 1.13 μg/ml propoxur were obtained by MTT, NRU and LDH assays, respectively. The lethal effects were induced in FG cells mainly through necrosis but not apoptosis as evidenced by double fluorescence staining. Comet assay showed weak genotoxic effects and statistically significant DNA damages were recorded in the cells exposed to highest tested concentration of 75 μg/ml propoxur (p<0.05). Propoxur exerted obvious acute toxic effects on the survival, spontaneous movement, hatching and heart rate, and development (yolk and pericardial sac edema) of zebrafish embryos in both time- and concentration-dependent manner only at ⩾ 100 μg/ml. The corresponding 24h-, 48 h- and 96 h-LC50 values of propoxur in zebrafish embryos were 166.4 ± 1.06, 146.3 ± 1.07 and 134.8 ± 1.06 μg/ml, respectively. The above data obtained suggest a low acute toxicity of propoxur to the in vitro cultured FG cells and zebrafish embryos.

  17. Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers.

    Science.gov (United States)

    Bergem, M; Norberg, K; Roseth, A; Meuwissen, T; Lien, S; Aamodt, R H

    2006-04-01

    The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

  18. Identification of proliferating cells in chicken embryos using 5-bromo- 2'-deoxyuridine immunohistochemical detection

    Directory of Open Access Journals (Sweden)

    Mário Lúcio Lopes

    2000-03-01

    Full Text Available Chicken embryos were incubated with BrdU, embedded in plastic resin, sectioned and screened immunohistochemically to identify proliferating cells in the neural tube and somites. Fixation in 4% paraformaldehyde for 1 h was essential for detecting specific colorimetric signals of BrdU incorporation into cells during the S phase of the cell cycle. Transverse sections of the neural tube showed that the nuclei of proliferating cells (BrdU positive had a uniform and centralized distribution, whereas unstained nuclei were found only along the extremities of the neural tube. Transverse sections of differentiated somites showed proliferating cells in the scleratome and dermatome. However, no incorporation of BrdU was observed in myotomic cells, which give rise to axial skeletal muscle. In spite of their proximity, the dermatome and myotome showed marked differences in cell proliferation. The excellent preservation of morphological characteristics in the embryonic tissues facilitated identification of variations in BrdU incorporation.Embriões de frango foram incubados na presença de BrdU e montados em resina plástica. A detecção de células em proliferação nos somitos e tubo neural foi feita através de anticorpos contra BrdU. Um ponto essencial para a otimização do método foi a fixação dos embriões por apenas uma hora em paraformaldeído a 4%. Análise de cortes transversais revelou que no tubo neural os núcleos marcados se posicionavam na região central. Cortes transversais em somitos diferenciados revelaram a presença de células em proliferação no dermátomo e esclerótomo, no entanto não foi observado nenhum sinal no miótomo. A metodologia aqui apresentada permitiu identificar com clareza e boa resolução as células em proliferação presentes em tecidos embrionários.

  19. CXCR4(+) dendritic cells promote angiogenesis during embryo implantation in mice.

    Science.gov (United States)

    Barrientos, Gabriela; Tirado-González, Irene; Freitag, Nancy; Kobelt, Peter; Moschansky, Petra; Klapp, Burghard F; Thijssen, Victor L J L; Blois, Sandra M

    2013-04-01

    Early pregnancy is characterized by decidual adaption to the developing embryo involving angiogenesis and vascular growth. Failure of decidual vascular expansion is linked to diseases of pregnancy. Dendritic cells (DC) have been associated with vascular growth during early gestation, though it is unknown whether their capacity to modulate angiogenesis is ubiquitous to all DC subsets. Here, we show that DC normally found associated with the decidual vasculature co-express the C-X-C chemokine receptor type 4 (CXCR4). In addition, we demonstrate that impaired homing of CXCR4(+)DC during early gestation provoked a disorganized decidual vasculature with impaired spiral artery remodeling later in gestation. In contrast, adoptive transfer experiments provided evidence that CXCR4(+)DC are able to rescue early pregnancy by normalizing decidual vascular growth and delivery of pro-angiogenic factors, which results in adequate remodeling of the spiral arteries during placental development. Taken together, our results indicate an important role of CXCR4(+)DC in the regulation of decidual angiogenesis and highlight the importance of the CXCL12/CXCR4 pathway during this process, suggesting that this may represent a key pathway to evaluate during pregnancy pathologies associated with impaired vascular expansion.

  20. Rationality and religion in the public debate on embryo stem cell research and prenatal diagnostics.

    Science.gov (United States)

    Myskja, Bjørn K

    2009-06-01

    Jürgen Habermas has argued that religious views form a legitimate background for contributions to an open public debate, and that religion plays a particular role in formulating moral intuitions. Translating religious arguments into "generally accessible language" (Habermas, Eur J Philos 14(1):1-25, 2006) to enable them to play a role in political decisions is a common task for religious and non-religious citizens. The article discusses Habermas' view, questioning the particular role of religion, but accepting the significance of including such counter-voices to the predominant views. Furthermore it is pointed out that not only religious but also numerous secular views stand in need of translation to be able to bear on policy matters. Accepting Habermas' general framework, I raise the question whether experts (such as clinicians working in relevant specialised areas of care) participating in political debates on biomedical issues have a duty to state their religious worldview, and to what extent the American government decision to restrict embryo stem cell research is an illegitimate transgression of the State-Church divide.

  1. Maize rough endosperm3 encodes an RNA splicing factor required for endosperm cell differentiation and has a nonautonomous effect on embryo development.

    Science.gov (United States)

    Fouquet, Romain; Martin, Federico; Fajardo, Diego S; Gault, Christine M; Gómez, Elisa; Tseung, Chi-Wah; Policht, Tyler; Hueros, Gregorio; Settles, A Mark

    2011-12-01

    Endosperm and embryo development are coordinated via epigenetic regulation and signaling between these tissues. In maize (Zea mays), the endosperm-embryo signals are not known, but endosperm cellularization is a key event for embryos to form shoots and roots. We screened seed mutants for nonautonomous functions in endosperm and embryo development with genetically nonconcordant seeds and identified the recessive mutant rough endosperm3 (rgh3). The wild-type Rgh3 allele is required in the endosperm for embryos to develop and has an autonomous role in embryo and seedling development. Endosperm cell differentiation is defective in rgh3. Results from endosperm cell culture indicate that rgh3 mutants remain in a proliferative state through mid-seed development. Rgh3 encodes the maize U2AF(35) Related Protein (URP), an RNA splicing factor involved in both U2 and U12 splicing. The Rgh3 allele produces at least 19 alternative splice variants with only one isoform encoding a full-length ortholog to URP. The full-length RGH3α isoform localizes to the nucleolus and displays a speckled pattern within the nucleoplasm, and RGH3α colocalizes with U2AF(65). A survey of alternatively spliced transcripts found that, in the rgh3 mutant, a fraction of noncanonical splicing events are altered. Our findings suggest that differentiation of maize endosperm cell types is necessary for embryos to develop. The molecular cloning of Rgh3 suggests that alternative RNA splicing is needed for cell differentiation, development, and plant viability.

  2. ‘奥迪亚’葡萄杂交后代胚挽救及无核性状分子鉴定%Embryo rescue and seedlessness molecular identification of hybrid off- springs of ‘Otilia' grape

    Institute of Scientific and Technical Information of China (English)

    程和禾; 吴雅琴; 吴永杰; 李玉生; 陈龙; 周锡明; 赵艳华

    2012-01-01

    【目的】为了研究影响杂交后代胚挽救萌发率的主要因素,并对其无核性状进行早期鉴定,【方法】以无核、抗病葡萄品种‘奥迪亚’为母本,‘玫瑰香’、‘摩尔多瓦’和‘红地球’3个主栽品种为父本进行杂交,通过L9(3^3)正交试验,建立简单高效的胚挽救体系,并用分子标记对杂交苗无核性状进行鉴定。【结果】结果表明,影响胚挽救萌发率的最关键因素为接种时期,其次为培养基种类和NAA浓度;以‘奥迪亚’为母本的杂交胚其适宜接种程序为授粉后49~56d剥取胚珠,接种在B5或NN69培养基上充分发育70d,再转入1/2B5培养基上萌芽,‘奥迪亚’ב摩尔多瓦’杂交胚通过此程序萌发率达到了50.48%;经分子标记鉴定,各组合后代的无核株系均超过50%。【结论】利用胚挽救方法以‘奥迪亚’为杂交母本可高效创新葡萄无核种质。%[Objective]What was the main factor which influenced the embryo germination rate and whether the hybrid off-springs were seedless or not were studied in this test. [Method ]Three hybrid combi- nations were set up which were ‘Otilia' בMuscat Hamburg', ‘ Otilia' בMoldova' and ‘Otilia' × ‘Red Globe', and the female parent ‘Otilia' was seedless and disease-resistant. L9 (33) orthogonal test were designed to establish simple and efficient embryo rescue progress, and using molecular marker to identify whether the hybrid off-springs were seedless or not. [Result] The results were as follows: the most important factor that influenced the embryo germination rate was inoculation period, which is followed by the medium type and NAA concentration; the suitable embryo rescue progress for hybrid embryos, whose female parents was Otilia, was that the embryos were first inoculated at 49-56 DAF (days after fertiliza- tion), and after developed on the B5 or NN69 medium for 70 d

  3. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination

    OpenAIRE

    Yushi Ishibashi; Shinsuke Kasa; Masatsugu Sakamoto; Nozomi Aoki; Kyohei Kai; Takashi Yuasa; Atsushi Hanada; Shinjiro Yamaguchi; Mari Iwaya-Inoue

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidas...

  4. Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

    Directory of Open Access Journals (Sweden)

    M Imron

    2007-06-01

    Full Text Available In vivo embryo production was limited by number of donor, wide variability respond due to superovulation program and also immunoactifity of superovulation hormone (FSH. Splitting technology could be an alternative to increase the number of transferrable embryos into recipien cows. Splitting is done with cutting embryo becoming two equal pieces (called demi embrio base on ICM orientation. The objective of this research was to determine the viability of demi embryo obtained from embryo splitting of fresh and frozen thawed embryo. The results showed that demi embryos which performed blastocoel reexpansion 3 hours after embryo splitting using fresh and frozen thawed embryos were 76.9 and 76.2% respectively. Base on existention of inner cell mass (ICM, the number of demi embryos developed with ICM from fresh and frozen thawed embryos were not significantly different (90.6 and 85.7% respectively. The cell number of demi embryo from fresh embryos splitting was not different compared with those from frozen thawed embryos (36.1 and 35.9 respectively. These finding indicated that embryo splitting can be applied to frozen thawed embryos with certain condition as well as fresh embryos.

  5. miRNA-17 members that target Bmpr2 influence signaling mechanisms important for embryonic stem cell differentiation in vitro and gastrulation in embryos.

    Science.gov (United States)

    Larabee, Shannon M; Coia, Heidi; Jones, Shiloh; Cheung, Eric; Gallicano, G Ian

    2015-02-01

    Body axes and germ layers evolve at gastrulation, and in mammals are driven by many genes; however, what orchestrates the genetic pathways during gastrulation remains elusive. Previously, we presented evidence that microRNA-17 (miRNA-17) family members, miR-17-5p, miR-20a, miR-93, and miR-106a were differentially expressed in mouse embryos and functioned to control differentiation of the stem cell population. Here, we identify function(s) that these miRNAs have during gastrulation. Fluorescent in situ hybridization miRNA probes reveal that these miRNAs are localized at the mid/posterior primitive streak (ps) in distinct populations of primitive ectoderm, mesendoderm, and mesoderm. Seven different miRNA prediction algorithms are identified in silico bone morphogenic protein receptor 2 (Bmpr2) as a target of these miRNAs. Bmpr2 is a member of the TGFβ pathway and invokes stage-specific changes during gastrulation. Recently, Bmpr2 was shown regulating cytoskeletal dynamics, cell movement, and invasion. Our previous and current data led to a hypothesis by which members of the miR-17 family influence gastrulation by suppressing Bmpr2 expression at the primitive streak. This suppression influences fate decisions of cells by affecting genes downstream of BMPR2 as well as mesoderm invasion through regulation of actin dynamics.

  6. Teratogen-induced apoptotic cell death: does the apoptotic machinery act as a protector of embryos exposed to teratogens?

    Science.gov (United States)

    Torchinsky, Arkady; Fein, Amos; Toder, Vladimir

    2005-12-01

    Considerable evidence has been collected demonstrating that many teratogens induce apoptotic cell death in embryonic structures that turn out to be malformed in fetuses and newborns. Apoptosis is a genetically regulated process that is realized by the activation of death and pro-survival signaling cascades, and the interplay between these cascades determines whether the cell exposed to apoptotic stimuli dies or survives. Therefore, there is intense interest in understanding how the apoptotic machinery functions in embryos exposed to teratogens. However, the interpretation of the results obtained remains problematic. The main problem is that excessive embryonic cell death, regardless of its nature, if uncompensated for, ultimately leads to maldevelopment or embryonic death. Therefore, we can easily interpret results when the intensity of teratogen-induced cell death and the severity or incidence of teratogen-induced anomalies directly correlate with each other. However, when teratogen-induced cell death is not followed by the formation of anomalies, a usual explanation is that teratogen-induced apoptotic cell death contributes to the renewal of teratogen-targeted cell populations by promoting the removal of injured cells. It is clear that such an explanation leaves vague the role of the anti-apoptotic signaling mechanism (and, hence, the apoptotic machinery as a whole) with respect to protecting the embryo against teratogenic stress. In this review, we summarize the data from studies addressing the function of the apoptotic machinery in embryos exposed to teratogens, and then we discuss approaches to interpreting the results of these studies. We hypothesize that activation of a proapoptotic signaling in teratogen-targeted cell populations is a necessary condition for an anti-apoptotic signaling that counteracts the process of maldevelopment to be activated. If such a scenario is true, we need to modify our approaches to choosing molecular targets for studies

  7. Aneuploidy analysis of non-pronuclear embryos from IVF with use of array CGH: a case report.

    Science.gov (United States)

    Lixin, Deng; Zhifeng, Xiang; Cong, He; Jinzhou, Zhang; Hongbin, Xie

    2014-06-01

    By using array comparative genomic hybridization (array CGH), to analyze the aneuploidy of the single blastomeres from non-pronuclear embryos on cleavage-stage in IVF cycle. Four non-pronuclear embryos were got from an IVF cycle, and the each single cell was biopsied from the four cleavage-stage embryos on the third day after the insemination which was investigated by using array CGH. After the biopsy, all the embryos continued to cleave, and lately entered the morula stage on the fifth day, just one embryo 3 was developed to early blastocyst stage on the sixth day. The four blastomere 24 chromosomes showed one X monomer and three normal XY diploids; the autosome chromosomes of blastomeres were abnormally gained or lost at different chromosome from four embryos, such as Embryo 1 : 49,X (-1, -5, -11, -19, -20, -21, -Y, +3, +6, +7, +8, +10, +13, +14, +16, +17, +18); Embryo 2 : 44,XY (-12, -15); Embryo 3: 47,XY (-3, -8, -9, -21, +7, +17, +18, +19, +20); Embryo 4 : 54,XY (+4, +7, +10, +12, +13, +16, +17, +22). With the use of the array CGH, the aneuploidy analysis could review the abnormal chromosomes of single blastomere from the non-pronuclear embryos, which can harbor the risk of abnormal sex chromosome and autosome chromosomes.

  8. Effect of L-carnitine on in vitro developmental rate, the zona pellucida and hatching of blastocysts and their cell numbers in mouse embryos

    OpenAIRE

    Khanmohammadi, Nasrin; Movahedin, Mansoureh; Safari, Manouchehr; Sameni, Hamid Reza; Yousefi, Behpour; Jafari, Behnaz; Zarbakhsh, Sam

    2016-01-01

    L-carnitine (LC) is an antioxidant with the ability to promote the growth in vitro embryo. Objective: The goal was to evaluate the effect of LC on some indicators of embryo development and blastocyst quality including zona pellucid (ZP) thickness, the hatching of blastocysts and their cell numbers. Materials and Methods: Mouse embryos were randomly divided into five groups and incubated with different concentrations of LC (I; 0, II; 0.5, III; 1, IV; 2 and V; 4 mg/ml) from 2-cell to hatched bl...

  9. Gadolinium induced apoptosis of human embryo liver L02 cell line by ROS-mediated AIF pathway

    Institute of Scientific and Technical Information of China (English)

    YE Lihua; SHI Zhe; LIU Huixue; YANG Xiaoda; WANG Kui

    2011-01-01

    Gd3+ complexes have a variety of medical applications. In order to shed light on the mechanism of hepatotoxicity of Gd3+ compounds, we investigated the effects of GdCl3 on human embryo liver cell strand (L02 cells). The experimental results showed that long-time exposure to GdC13 resulted in L02 cell apoptosis. The incubation of L02 cells with GdCl3 first induced increase in cellular reactive oxygen species (ROS) and decrease in mitochondrial inner membrane potential (△Ψm). It later resulted in the activation of poly (ADP-ribose) polymerase (PARP) and the release of mitochondrial apoptosis-inducing factor (AIF). The activation of caspase 3, however, was not observed.Antioxidants could significantly reduce GdCl3-induced decrease of △Ψm, release of AIF, and cell apoptosis. Although GdCl3 caused a significant increase in cell membrane permeability in L02, the change of cell membrane permeability was unlikely to be involved in GdCl3-induced cell apoptosis. Overall, our experimental results suggested that GdCl3 induced apoptosis of human embryo liver L02 cell line by ROS-mediated AIF pathway.

  10. Microarray analysis of embryo-derived bovine pluripotent cells: The vulnerable state of bovine embryonic stem cells

    Science.gov (United States)

    Kim, Daehwan; Jung, Yeon-Gil

    2017-01-01

    Although there are many studies about pluripotent stem cells, little is known about pluripotent pathways and the difficulties of maintaining the pluripotency of bovine cells in vitro. Here, we investigated differently expressed genes (DEG) in bovine embryo-derived stem-like cells (eSLCs) from various origins to validate their distinct characteristics of pluripotency and differentiation. We identified core pluripotency markers and additional markers which were not determined as pluripotency markers yet in bovine eSLCs. Using the KEGG database, TGFβ, WNT, and LIF signaling were related to the maintenance of pluripotency. In contrast, some DEGs related to the LIF pathway were down-regulated, suggesting that reactivation of the pathway may be required for the establishment of true bovine embryonic stem cells (ESCs). Interestingly, oncogenes were co-down-regulated, while tumor suppressor genes were co-up-regulated in eSLCs, implying that this pattern may induce abnormal teratomas. These data analyses of signaling pathways provide essential information on authentic ESCs in addition to providing evidence for pluripotency in bovine eSLCs. PMID:28257460

  11. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    Science.gov (United States)

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.

  12. Beyond the 'embryo question': human embryonic stem cell ethics in the context of biomaterial donation in the UK.

    Science.gov (United States)

    Bahadur, G; Morrison, M; Machin, L

    2010-12-01

    Discussion about the ethics of human embryonic stem cell (ESC) research in the UK tends to be dominated by the divisive and potentially intractable issue of the moral status of the embryo. This can have the effect of silencing or marginalizing other concerns, especially in the context of public engagement with science in this field. One such area of potential public concern is the donation of oocytes and embryos to stem cell research. Contemporary research on the views of donors and potential donors about a wide range of biomaterials, from solid organs to gametes and bone marrow, is reviewed and used to illustrate the range and types of ethical concerns articulated by this important group of stakeholders. Attitudes to donation are found to vary according to the type of tissue being donated or collected, the purpose for which donation is being sought and the nature of the recipient of the donation. Pertinently, attitudes towards donating oocytes are found to differ in some respects from donation of embryos or fetal tissue. The implications of these findings for ensuring ethically robust informed consent and publicly acceptable sourcing of human biomaterials for stem cell research are then considered.

  13. Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with digitonin and Xenopus egg extract

    DEFF Research Database (Denmark)

    Liu, Ying; Østrup, Olga; Li, Juan

    2011-01-01

    Pre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from...... Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells...... cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor...

  14. HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

    Directory of Open Access Journals (Sweden)

    Parisa Imanirad

    2014-01-01

    Full Text Available Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α, a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver, and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages.

  15. File list: Oth.ALL.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.10.DNA-RNA_hybrids.AllCell.bed ...

  16. File list: Oth.ALL.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.05.DNA-RNA_hybrids.AllCell.bed ...

  17. File list: Oth.ALL.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.20.DNA-RNA_hybrids.AllCell.bed ...

  18. File list: Oth.ALL.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.50.DNA-RNA_hybrids.AllCell.bed ...

  19. Hybrid multiscale modeling and prediction of cancer cell behavior.

    Science.gov (United States)

    Zangooei, Mohammad Hossein; Habibi, Jafar

    2017-01-01

    Understanding cancer development crossing several spatial-temporal scales is of great practical significance to better understand and treat cancers. It is difficult to tackle this challenge with pure biological means. Moreover, hybrid modeling techniques have been proposed that combine the advantages of the continuum and the discrete methods to model multiscale problems. In light of these problems, we have proposed a new hybrid vascular model to facilitate the multiscale modeling and simulation of cancer development with respect to the agent-based, cellular automata and machine learning methods. The purpose of this simulation is to create a dataset that can be used for prediction of cell phenotypes. By using a proposed Q-learning based on SVR-NSGA-II method, the cells have the capability to predict their phenotypes autonomously that is, to act on its own without external direction in response to situations it encounters. Computational simulations of the model were performed in order to analyze its performance. The most striking feature of our results is that each cell can select its phenotype at each time step according to its condition. We provide evidence that the prediction of cell phenotypes is reliable. Our proposed model, which we term a hybrid multiscale modeling of cancer cell behavior, has the potential to combine the best features of both continuum and discrete models. The in silico results indicate that the 3D model can represent key features of cancer growth, angiogenesis, and its related micro-environment and show that the findings are in good agreement with biological tumor behavior. To the best of our knowledge, this paper is the first hybrid vascular multiscale modeling of cancer cell behavior that has the capability to predict cell phenotypes individually by a self-generated dataset.

  20. Comparative studies in Rous sarcoma with virus, tumor cells, and chick embryo cells transformed in vitro by virus. II. Response of normal and immunized chicks.

    Science.gov (United States)

    DOUGHERTY, R M; MORGAN, H R

    1962-01-01

    Chick embryo fibroblasts infected in vitro with Rous sarcoma virus have properties similar to tumor cells when injected into virus-immune chickens. When such virus-transformed fibroblasts are injected into normal chickens, they apparently participate in the production of tumors independent of their release of virus and are thus apparently malignant in vivo.

  1. 小鼠胚胎干细胞与四倍体胚胎的嵌合%Chimera of mouse ES cells and tetraploid embryos

    Institute of Scientific and Technical Information of China (English)

    李相运; 窦忠英; 李松

    2003-01-01

    The oviducts of superovulated Kunming white females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1074 late two-cell-stage embryos.The embryos were placed twenty at a time between two platinum electrodes laid 1 mm apart in 0.3M mannitol in the electrode chamber.The blastomeres were fused by a short electric pulse(80V for 50μsec) applied by a pulse generator.Fusion of blastomeres was usually completed in 20-60minutes.After 25 hours of culture,most of the tetraploid embryos developed to the four-cell stage.Zonae pellucidae of 387 four-cell-stage tetraploid embryos were removed by treatment with acid Tyrode's buffer.The embryos were plated on an ES cell layer,After 40 hours of coculture,248 embryos aggregated with ES cells were collected and transferred into the uteri of twenty four 2.5-day pseudopregnant recipinets.Ten recipients were pregnant.but no live fetuses were born.Three pregnant recipients were routinely subject to a Caesarean section on day 18 of pregnancy and seven abnormal fetuses were obtained.The results demonstrate that ES cells derived from C57BL/6 mice are pluripotential to a certain extent.

  2. Self-correction in human embryos%胚胎自我修复

    Institute of Scientific and Technical Information of China (English)

    徐艳文

    2013-01-01

    Reanalysis of aneuploid embryos diagnosed by preimplantation genetic screening (PGS) using fluorescence in situ hybridization(FISH) showed that part or all cells in some human cleavage-stage embryos may undergo self-correction during preimplantation development. Putative embryo self-correction mechanisms include embryonic mosaicism, preferential segregation of chromosomal abnormalities to the trophectoderm and extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy. However, embryo self-correction has not been proved in the study using a single nucleotide polymorphism (SNP)microarray-based 24 chromosome aneuploidy screening technology. Neither preferential segregation of aneuploidy to trophectoderm nor uniparental disomy was found. Further study to improve the accuracy of karyotyping on cleavage-stage embryos is definitely needed.

  3. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi

    2014-01-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...

  4. Graphene-based transparent electrodes for hybrid solar cells

    Directory of Open Access Journals (Sweden)

    Pengfei eLi

    2014-11-01

    Full Text Available The graphene-based transparent and conductive films were demonstrated to be cost-effective electrodes working in organic-inorganic hybrid Schottky solar cells. Large area graphene films were produced by chemical vapor deposition (CVD on copper foils and transferred onto glass as transparent electrodes. The hybrid solar cell devices consist of solution processed poly (3, 4-ethlenedioxythiophene: poly (styrenesulfonate (PEDOT: PSS which is sandwiched between silicon wafer and graphene electrode. The solar cells based on graphene electrodes, especially those doped with HNO3, has comparable performance to the reference devices using commercial indium tin oxide (ITO. Our work suggests that graphene-based transparent electrode is a promising candidate to replace ITO.

  5. Pluripotency of embryo-derived stem cells from rodents, lagomorphs, and primates: Slippery slope, terrace and cliff.

    Science.gov (United States)

    Savatier, Pierre; Osteil, Pierre; Tam, Patrick P L

    2017-03-01

    The diverse cell states and in vitro conditions for the derivation and maintenance of the mammalian embryo-derived pluripotent stem cells raise the questions of whether there are multiple states of pluripotency of the stem cells of each species, and if there are innate species-specific variations in the pluripotency state. We will address these questions by taking a snapshot of our knowledge of the properties of the pluripotent stem cells, focusing on the maintenance of pluripotency and inter-conversion of the different types of pluripotent stem cells from rodents, lagomorphs and primates. We conceptualize pluripotent stem cells acquiring a series of cellular states represented as terraces on a slope of descending gradient of pluripotency. We propose that reprogramming pluripotent stem cells from a primed to a naive state is akin to moving upstream over a steep cliff to a higher terrace. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols.

    Science.gov (United States)

    Mei, Xin; Wu, Yuan-Yuan; Mao, Xiao; Tu, You-Ying

    2011-01-01

    Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene.

  7. Solid Oxide Fuel Cell Hybrid System for Distributed Power Generation

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen Minh

    2005-12-01

    This report summarizes the work performed by Hybrid Power Generation Systems, LLC (HPGS) under Cooperative Agreement DE-FC2601NT40779 for the US Department of Energy, National Energy Technology Laboratory (DoE/NETL) entitled ''Solid Oxide Fuel Cell Hybrid System for Distributed Power Generation''. The main objective of this project is to develop and demonstrate the feasibility of a highly efficient hybrid system integrating a planar Solid Oxide Fuel Cell (SOFC) and a gas turbine. A conceptual hybrid system design was selected for analysis and evaluation. The selected system is estimated to have over 65% system efficiency, a first cost of approximately $650/kW, and a cost of electricity of 8.4 cents/kW-hr. A control strategy and conceptual control design have been developed for the system. A number of SOFC module tests have been completed to evaluate the pressure impact to performance stability. The results show that the operating pressure accelerates the performance degradation. Several experiments were conducted to explore the effects of pressure on carbon formation. Experimental observations on a functioning cell have verified that carbon deposition does not occur in the cell at steam-to-carbon ratios lower than the steady-state design point for hybrid systems. Heat exchanger design, fabrication and performance testing as well as oxidation testing to support heat exchanger life analysis were also conducted. Performance tests of the prototype heat exchanger yielded heat transfer and pressure drop characteristics consistent with the heat exchanger specification. Multicell stacks have been tested and performance maps were obtained under hybrid operating conditions. Successful and repeatable fabrication of large (>12-inch diameter) planar SOFC cells was demonstrated using the tape calendering process. A number of large area cells and stacks were successfully performance tested at ambient and pressurized conditions. A 25 MW plant configuration was

  8. Effect of Laryngeal Squamous Cell Carcinoma Tissue Implantation on the Chick Embryo Chorioallantoic Membrane: Morphometric Measurements and Vascularity

    Directory of Open Access Journals (Sweden)

    Virgilijus Uloza

    2015-01-01

    Full Text Available Background. The aim of this study was to develop chick embryo chorioallantoic membrane (CAM model of laryngeal squamous cell carcinoma (LSCC and to evaluate the morphological and morphometric characteristics and angiogenic features of it. Methods. Fresh LSCC tissue samples obtained from 6 patients were implanted onto 15 chick embryo CAMs. Morphological, morphometric, and angiogenic changes in the CAM and chorionic epithelium were evaluated up to 4 days after the tumor implantation. Immunohistochemical analysis (34βE12, CD31, and Ki67 staining was performed to detect cytokeratins and tumor endothelial cells and to evaluate the proliferative capacity of the tumor before and after implantation on the CAM. Results. The implanted LSCC tissue samples survived on the CAM in all the experiments and retained the essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM (103–417%, p=0.0001 and the chorionic epithelium (70–140%, p=0.0001 and increase in number of blood vessels (75–148%, p=0.0001 in the CAM. Conclusions. This study clarifies that chick embryo CAM is a relevant assay for implanting LSCC tissue and provides the first morphological and morphometric characterization of the LSCC CAM model that opens new perspectives to study this disease.

  9. Instructing cells with programmable peptide DNA hybrids

    Science.gov (United States)

    Freeman, Ronit; Stephanopoulos, Nicholas; Álvarez, Zaida; Lewis, Jacob A.; Sur, Shantanu; Serrano, Chris M.; Boekhoven, Job; Lee, Sungsoo S.; Stupp, Samuel I.

    2017-07-01

    The native extracellular matrix is a space in which signals can be displayed dynamically and reversibly, positioned with nanoscale precision, and combined synergistically to control cell function. Here we describe a molecular system that can be programmed to control these three characteristics. In this approach we immobilize peptide-DNA (P-DNA) molecules on a surface through complementary DNA tethers directing cells to adhere and spread reversibly over multiple cycles. The DNA can also serve as a molecular ruler to control the distance-dependent synergy between two peptides. Finally, we use two orthogonal DNA handles to regulate two different bioactive signals, with the ability to independently up- or downregulate each over time. This enabled us to discover that neural stem cells, derived from the murine spinal cord and organized as neurospheres, can be triggered to migrate out in response to an exogenous signal but then regroup into a neurosphere as the signal is removed.

  10. DON抑制小麦种胚细胞周期启动的研究%Inhibition Effect of DON on Cell Cycle Activation in Wheat Embryo Cells

    Institute of Scientific and Technical Information of China (English)

    刘晓; 杜昱光; 白雪芳

    2000-01-01

    小麦种子在0.5~4.0 mg/L的脱氧雪腐镰刀菌烯醇(DON)溶液中萌发,用流式细胞仪检测胚细胞的周期时相并观察黄化苗的生长。结果表明,对照组胚细胞在萌发12 h开始启动细胞周期,此后S期细胞比率迅速增加并在20 h达到最大值,较萌发10 h的6.5%增加1.78倍,约14%的胚细胞在萌发的22~26 h间完成第一次分裂;DON抑制G1期胚细胞的启动,小麦胚中S期细胞比率随DON浓度增加呈指数下降,3.5与2.0 mg/L的DON可分别将小麦胚细胞阻滞在G1期和S期;小麦黄化苗的苗高与主胚根长均随DON浓度升高呈线性下降,但DON刺激小麦初生根数目增加,刺激作用以1.5 mg/L最强。以上结果说明DON因阻止了胚细胞周期的启动而抑制小麦种子萌发。%The activation of the cell cycle in embryo cells of wheat (Triticum aestivum L., cv. Lu-Mai No. 22) seeds imbibing in 0.5~4.0 mg/L deoxynivalenol (DON) was studied by flow cytometric analyses. The results indicated that the majority of the embryo cells in wheat seeds were arrested at the G1 phase of the cell cycle. The cell cycle was activated after the seeds imbibing in distilled water for 12 h, and then the relative percentage of S-phase cells in wheat embryos were observed to increase steadily until a peak was reached on the 20 h imbibition. The number of S-phase cells in wheat embryos imbibing in water for 20 h was 1.78 fold more than that imbibing for 10 h. When imbibing for 22 to 26 h, about 14% embryo cells in wheat seeds had finished their mitosis. The entry of the embryo cells at G1 phase into S phase was strongly inhibited by imbibing the seeds in DON solution. The percentage of wheat embryo cells at S phase decreased exponentially with increasing concentration of DON treatment up to 4.0 mg/L. Embryo cells in wheat seeds could be blocked completely at G1 phase and S phase by 3.5 and 2.0 mg/L DON treatment respectively. The seedling height and radicle

  11. A hybrid sequential deposition fabrication technique for micro fuel cells

    Science.gov (United States)

    Stanley, Kevin G.; Czyzewska, Eva K.; Vanderhoek, Tom P. K.; Fan, Lilian L. Y.; Abel, Keith A.; Wu, Q. M. Jonathan; Parameswaran, M. Ash

    2005-10-01

    Micro fuel cell systems have elicited significant interest due to their promise for instantly rechargeable, longer duration and portable power. Most micro fuel cell systems are either built as miniaturized plate-and-frame or silicon-based microelectromechanical systems (MEMS). Plate-and-frame systems are difficult to fabricate smaller than 20 cm3. Existing micro fuel cell designs cannot meet the cost, scale and power requirements of some portable power markets. Traditional MEMS scaling advantages do not apply to fuel cells because the minimum area for the fuel cell is fixed by the catalyst area required for a given power output, and minimum volume set by mass transport limitations. We have developed a new hybrid technique that borrows from both micro and macro machining techniques to create fuel cells in the 1-20 cm3 range, suitable for cell phones, PDAs and smaller devices.

  12. Fuel-Cell-Powered Vehicle with Hybrid Power Management

    Science.gov (United States)

    Eichenberg, Dennis J.

    2010-01-01

    Figure 1 depicts a hybrid electric utility vehicle that is powered by hydrogenburning proton-exchange-membrane (PEM) fuel cells operating in conjunction with a metal hydride hydrogen-storage unit. Unlike conventional hybrid electric vehicles, this vehicle utilizes ultracapacitors, rather than batteries, for storing electric energy. This vehicle is a product of continuing efforts to develop the technological discipline known as hybrid power management (HPM), which is oriented toward integration of diverse electric energy-generating, energy-storing, and energy- consuming devices in optimal configurations. Instances of HPM were reported in five prior NASA Tech Briefs articles, though not explicitly labeled as HPM in the first three articles: "Ultracapacitors Store Energy in a Hybrid Electric Vehicle" (LEW-16876), Vol. 24, No. 4 (April 2000), page 63; "Photovoltaic Power Station With Ultracapacitors for Storage" (LEW- 17177), Vol. 27, No. 8 (August 2003), page 38; "Flasher Powered by Photovoltaic Cells and Ultracapacitors" (LEW-17246), Vol. 27, No. 10 (October 2003), page 37; "Hybrid Power Management" (LEW-17520), Vol. 29, No. 12 (December 2005), page 35; and "Ultracapacitor-Powered Cordless Drill" (LEW-18116-1), Vol. 31, No. 8 (August 2007), page 34. To recapitulate from the cited prior articles: The use of ultracapacitors as energy- storage devices lies at the heart of HPM. An ultracapacitor is an electrochemical energy-storage device, but unlike in a conventional rechargeable electrochemical cell or battery, chemical reactions do not take place during operation. Instead, energy is stored electrostatically at an electrode/electrolyte interface. The capacitance per unit volume of an ultracapacitor is much greater than that of a conventional capacitor because its electrodes have much greater surface area per unit volume and the separation between the electrodes is much smaller.

  13. Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Cortes, J L; Sánchez, L; Catalina, P; Cobo, F; Bueno, C; Martínez-Ramirez, A; Barroso, A; Cabrera, C; Ligero, G; Montes, R; Rubio, R; Nieto, A; Menendez, P

    2008-04-01

    The optimization of human embryonic stem (hES) cell line derivation methods is challenging because many worldwide laboratories have neither access to spare human embryos nor ethical approval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological material. In this study, we developed a new strategy based on the combination of whole-blastocyst culture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of inner cell mass (ICM) isolation and ES cell derivation using murine embryos. Embryos were divided into good- and poor-quality embryos. We demonstrate that the efficiency of both ICM isolation and ES cell derivation using this strategy is significantly superior to whole-blastocyst culture or laser drilling technology itself. Regardless of the ICM isolation method, the ES cell establishment depends on a feeder cell growth surface. Importantly, this combined methodology can be successfully applied to poor-quality blastocysts that otherwise would not be suitable for laser drilling itself nor immunosurgery in an attempt to derive ES cell lines due to the inability to distinguish the ICM. The ES cell lines derived by this combined method were characterized and shown to maintain a typical morphology, undifferentiated phenotype, and in vitro and in vivo three germ layer differentiation potential. Finally, all ES cell lines established using either technology acquired an aneuploid karyotype after extended culture periods, suggesting that the method used for ES cell derivation does not seem to influence the karyotype of the ES cells after extended culture. This methodology may open up new avenues for further improvements for the derivation of hES cells, the majority of which are derived from frozen, poor-quality human embryos.

  14. Effect of epigallocatechin-3-gallate on the in vitro developmental potential of porcine oocytes and embryos obtained parthenogenetically and by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Yunsheng Li

    2014-03-01

    Full Text Available The present study aimed to investigate the effects of epigallocatechin-3-gallate (EGCG on the in vitro development of porcine oocytes, parthenogenetic activation embryos (PA, and somatic cell nuclear transfer (SCNT embryos. In Experiment 1, 0 (control, 10, 30, and 50 μg/mL EGCG were added to in vitro maturation (IVM medium to explore the effect of EGCG on IVM of pig oocytes. The matured oocytes were then used to produce PA and SCNT embryos. Either for nuclear maturation of oocytes or for the rates of cleavage and blastocyst of PA and SCNT embryos, no significant difference was found among all groups. However, the total cell number per cloned blastocyst was significantly lower in blastocysts derived from oocytes matured in 50 μg/mL EGCG (P<0.05 as compared with the other groups. In Experiment 2, we cultured pig SCNT and PA embryos in medium containing various concentrations of EGCG to examine the effect of EGCG on preimplantation development. The cleavage and blastocyst rates and the total cell number per blastocyst did not significantly differ between PA and SCNT embryos among all groups. However, the reactive oxygen species level was significantly lower in the PA embryos cultured in 10 μg/mL EGCG than the other groups (P<0.05. Our results suggest that high doses of EGCG in IVM are harmful to the oocytes as evidenced by the decreased quality of SCNT embryos, and EGCG has no beneficial effects on in vitro development of pig cloned embryos.

  15. Embryo genome profiling by single-cell sequencing for preimplantation genetic diagnosis in a β-thalassemia family

    DEFF Research Database (Denmark)

    Xu, Yanwen; Chen, Shengpei; Yin, Xuyang

    2015-01-01

    for a β-thalassemia-carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome...... leukocyte antigen matching tests. CONCLUSIONS: This retrospective study in a β-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single...

  16. Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.

  17. Review of Polymer, Dye-Sensitized, and Hybrid Solar Cells

    Directory of Open Access Journals (Sweden)

    S. N. F. Mohd-Nasir

    2014-01-01

    Full Text Available The combination of inorganic nanoparticles semiconductor, conjugated polymer, and dye-sensitized in a layer of solar cell is now recognized as potential application in developing flexible, large area, and low cost photovoltaic devices. Several conjugated low bandgap polymers, dyes, and underlayer materials based on the previous studies are quoted in this paper, which can provide guidelines in designing low cost photovoltaic solar cells. All of these materials are designed to help harvest more sunlight in a wider range of the solar spectrum besides enhancing the rate of charge transfer in a device structure. This review focuses on developing solid-state dye-synthesized, polymer, and hybrid solar cells.

  18. Solid Oxide Fuel Cell Hybrid System for Distributed Power Generation

    Energy Technology Data Exchange (ETDEWEB)

    David Deangelis; Rich Depuy; Debashis Dey; Georgia Karvountzi; Nguyen Minh; Max Peter; Faress Rahman; Pavel Sokolov; Deliang Yang

    2004-09-30

    This report summarizes the work performed by Hybrid Power Generation Systems, LLC (HPGS) during the April to October 2004 reporting period in Task 2.3 (SOFC Scaleup for Hybrid and Fuel Cell Systems) under Cooperative Agreement DE-FC26-01NT40779 for the U. S. Department of Energy, National Energy Technology Laboratory (DOE/NETL), entitled ''Solid Oxide Fuel Cell Hybrid System for Distributed Power Generation''. This study analyzes the performance and economics of power generation systems for central power generation application based on Solid Oxide Fuel Cell (SOFC) technology and fueled by natural gas. The main objective of this task is to develop credible scale up strategies for large solid oxide fuel cell-gas turbine systems. System concepts that integrate a SOFC with a gas turbine were developed and analyzed for plant sizes in excess of 20 MW. A 25 MW plant configuration was selected with projected system efficiency of over 65% and a factory cost of under $400/kW. The plant design is modular and can be scaled to both higher and lower plant power ratings. Technology gaps and required engineering development efforts were identified and evaluated.

  19. Hybrid window layer for photovoltaic cells

    Science.gov (United States)

    Deng, Xunming

    2010-02-23

    A novel photovoltaic solar cell and method of making the same are disclosed. The solar cell includes: at least one absorber layer which could either be a lightly doped layer or an undoped layer, and at least a doped window-layers which comprise at least two sub-window-layers. The first sub-window-layer, which is next to the absorber-layer, is deposited to form desirable junction with the absorber-layer. The second sub-window-layer, which is next to the first sub-window-layer, but not in direct contact with the absorber-layer, is deposited in order to have transmission higher than the first-sub-window-layer.

  20. Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo.

    Directory of Open Access Journals (Sweden)

    Ioannis Kokkinopoulos

    Full Text Available In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.

  1. Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo.

    Science.gov (United States)

    Kokkinopoulos, Ioannis; Ishida, Hidekazu; Saba, Rie; Ruchaya, Prashant; Cabrera, Claudia; Struebig, Monika; Barnes, Michael; Terry, Anna; Kaneko, Masahiro; Shintani, Yasunori; Coppen, Steven; Shiratori, Hidetaka; Ameen, Torath; Mein, Charles; Hamada, Hiroshi; Suzuki, Ken; Yashiro, Kenta

    2015-01-01

    In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.

  2. Fuel cell hybrid taxi life cycle analysis

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, Patricia, E-mail: patricia.baptista@ist.utl.pt [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Ribau, Joao; Bravo, Joao; Silva, Carla [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Adcock, Paul; Kells, Ashley [Intelligent Energy, Charnwood Building, HolywellPark, Ashby Road, Loughborough, LE11 3GR (United Kingdom)

    2011-09-15

    A small fleet of classic London Taxis (Black cabs) equipped with hydrogen fuel cell power systems is being prepared for demonstration during the 2012 London Olympics. This paper presents a Life Cycle Analysis for these vehicles in terms of energy consumption and CO{sub 2} emissions, focusing on the impacts of alternative vehicle technologies for the Taxi, combining the fuel life cycle (Tank-to-Wheel and Well-to-Tank) and vehicle materials Cradle-to-Grave. An internal combustion engine diesel taxi was used as the reference vehicle for the currently available technology. This is compared to battery and fuel cell vehicle configurations. Accordingly, the following energy pathways are compared: diesel, electricity and hydrogen (derived from natural gas steam reforming). Full Life Cycle Analysis, using the PCO-CENEX drive cycle, (derived from actual London Taxi drive cycles) shows that the fuel cell powered vehicle configurations have lower energy consumption (4.34 MJ/km) and CO{sub 2} emissions (235 g/km) than both the ICE Diesel (9.54 MJ/km and 738 g/km) and the battery electric vehicle (5.81 MJ/km and 269 g/km). - Highlights: > A Life Cycle Analysis of alternative vehicle technologies for the London Taxi was performed. > The hydrogen powered vehicles have the lowest energy consumption and CO{sub 2} emissions results. > A hydrogen powered solution can be a sustainable alternative in a full life cycle framework.

  3. Whole mount in situ hybridization of E8.5 to E12.5 rat embryos%E8.5-E12.5大鼠胚胎整体原位杂交技术

    Institute of Scientific and Technical Information of China (English)

    张吉霞; 蔡玉瑾; 谢耀丽; 李海荣; 乔从进; 崔慧林

    2013-01-01

    目的:通过优化实验条件,建立大鼠胚胎整体原位杂交方法.方法:E8.5-E10.5大鼠胚胎4%多聚甲醛固定6h,不经蛋白酶K消化;E11.5-E12.5胚胎4%多聚甲醛固定过夜,蛋白酶K系列稀释实验确定消化浓度和时间.E8.5-E12.5胚胎预杂交2h,地高辛标记的甘油二酯激酶ζ(DGKζ)寡核苷酸探针杂交24h,山羊血清封闭2.5~3h,地高辛抗体4℃过夜,冷TBST和NTMT漂洗1~2h,NBT/BCIP避光显色.应用免疫组织化学方法观察DGKζ在E12.5大鼠胚胎组织的表达.结果:DGKζ在不同胚龄大鼠胚胎均有清晰的杂交信号,主要集中在神经系统;E12.5大鼠胚胎脑泡、背根神经节有较强表达,与免疫组化结果一致.结论:本研究建立了简便、可行、重复性高的E8.5-E12.5大鼠胚胎整体原位杂交方法.%Objective:To establish a method for whole mount in situ hybridization of rat embryos by optimizing experimental conditions.Methods:E8.5-E10.5 rat embryos were fixed in 4% paraformaldehyde for 6 hours,and had no proteinase K digestion.E11.5-E12.5 rat embryos were fixed in 4% paraformaldehyde overnight.The concentration and time of proteinase K digestion were analyzed by serial dilution.E8.5-E12.5 embryos were pre-hybridized for 2 hours and hybridized with digoxigenin-labeled diacylglycerol kinase ζ (DGKζ) oligonucleotide probe for 24 hours,blocked by goat serum for 2.5-3 hours,and incubated anti-digoxigenin antibody overnight at 4℃.Then the embryos were rinsed in cool TBST and NTMT for 1-2 hours and developed with NBT/BCIP in dark.The expression of DGKζ protein in E12.5 embryos was observed by using immunohistochemistry.Results:DGKζ positive signals mainly located in nervous system of rat embryos at different ages and were viewed obviously.DGKζ positive signals were observed strongly in cephalic vesicles,dorsal root ganglions of E12.5 rat embryos,which was consistent with the immunohistochemical results.Conclusion:The present study has established

  4. Generation of mouse chimeras with high contribution of tetraploid embryonic stem cells and embryonic stem cell-fibroblast hybrid cells.

    Science.gov (United States)

    Matveeva, Natalia M; Kizilova, Elena A; Serov, Oleg L

    2015-01-01

    The in vitro long-term cultivation of embryonic stem (ES) cells derived from pre-implantation embryos offers the unique possibility of combining ES cells with pre-implantation embryos to generate chimeras, thus facilitating the creation of a bridge between in vitro and in vivo investigations. Genomic manipulation using ES cells and homologous recombination is one of the most outstanding scientific achievements, resulting in the generation of animals with desirable genome modifications. As such, the generation of ES cells with different ploidy via cell fusion also deserves much attention because this approach allows for the production of chimeras that contain somatic cells with various ploidy. Therefore, this is a powerful tool that can be used to study the role of polyploidy in the normal development of mammals.

  5. Nanocomposite-Based Bulk Heterojunction Hybrid Solar Cells

    Directory of Open Access Journals (Sweden)

    Bich Phuong Nguyen

    2014-01-01

    Full Text Available Photovoltaic devices based on nanocomposites composed of conjugated polymers and inorganic nanocrystals show promise for the fabrication of low-cost third-generation thin film photovoltaics. In theory, hybrid solar cells can combine the advantages of the two classes of materials to potentially provide high power conversion efficiencies of up to 10%; however, certain limitations on the current within a hybrid solar cell must be overcome. Current limitations arise from incompatibilities among the various intradevice interfaces and the uncontrolled aggregation of nanocrystals during the step in which the nanocrystals are mixed into the polymer matrix. Both effects can lead to charge transfer and transport inefficiencies. This paper highlights potential strategies for resolving these obstacles and presents an outlook on the future directions of this field.

  6. Ethical consideration of experimentation using living human embryos: the Catholic Church's position on human embryonic stem cell research and human cloning.

    Science.gov (United States)

    Ohara, N

    2003-01-01

    Although the potential applications of human embryonic stem cells and therapeutic cloning hold promise for the alleged medical benefits, these technologies have posed profound ethical issues because they necessitate the destruction of human embryos. A fundamental point in the issues is the concept of the moral status of human embryos. The Catholic Church has held that human life begins at the moment of conception and therefore, has defended the dignity, inviolable right to life and integrity of human embryos. The Catholic Church has opposed human embryonic stem cell research and any kind of human cloning because they are contrary to the dignity of procreation, of conjugal union and of human embryos. Moreover, these techniques have the risk of creating a sub-category of human beings that are destined basically for the convenience of others. In conclusion, science and technology can never be independent of the criterion of morality, since technology exists for man and must respect his finality.

  7. THE EFFECT OF RECOMBINANT HUMAN LEUKEMIA INHIBITORY FACTOR (rhLIF ON IN VITRO DEVELOPMENT OF MOUSE 2-CELL EMBRYOS AND THEIR ISOLATED BLASTOMERES

    Directory of Open Access Journals (Sweden)

    MOHAMMAD AKBARI

    2004-09-01

    Full Text Available In this study effect of recombinant human leukemia inhibitory factor on invitro development of 2 cells embryos and isolated blastomeres derived from mouse 2 cell embryos were investigated. Female ICR mice that were between 8 to 10 weeks old received intraperitoneal injection of 7.5 IU of PMSG for super ovulation followed by intraperitoneal administration of 7.5 IU of HCG 48 hours later. The mice were then mated to mature ICR male mice and were checked for vaginal plugs after 13-14 hours. Mice were killed 46-48 hours after HCG injection by cervical dislocation, their oviducts were removed and flushing 2 cell embryos were collected. The zona pellucida of 2 cell embryos were removed by Acid Tyrod solution and blastomeres separated with oocyte preparation pipette and then all embryos and blastomeres were cultured in Potassium Simplex Optimized Medium (KSOM +Aminoacid (AA different amounts of rhLIF (500IU/ml, 1000IU/ml and 1500IU/ml. Some embryos and individual blastomere also were cultured without rhLIF as control group. All samples were cultured in an incubator at 370C with 0.05 CO2 for 120 hours. The rate of embryo and individual blastomeres which reached to 2 cell, 4 cell, 8 cell and 9-16 cell were the same in all groups. However in further developmental stages, morula and blastocyst between experimental and control groups were significantly different. Therefore it may be concluded that: cultivation of isolated blastomers up to the blastocyst stage with rhLIF has stimulatory effect on the preimplantation stage (morula and blastocyst but it has no stimulatory and inhibitory effects when was added to culture media at the early cleavage stage.

  8. Stable organic-inorganic hybrid multilayered photoelectrochemical cells

    Science.gov (United States)

    Park, Sun-Young; Kim, Min-gyeong; Jung, Jaehoon; Heo, Jinhee; Hong, Eun Mi; Choi, Sung Mook; Lee, Joo-Yul; Cho, Shinuk; Hong, Kihyon; Lim, Dong Chan

    2017-02-01

    The production of hydrogen from water via solar energy conversion has attracted immense attention as a potential solution for addressing energy supply issues. We demonstrated a stable and efficient organic-inorganic hybrid photoelectrochemical (H-PEC) cell. Modifying the surface energy and structure of the organic photoactive layer using multi-functional nanomaterials including -OH-modified NiO nanoparticles and reduced graphene oxide (RGO) led to a 2.8-fold enhancement of the water splitting performance in a single junction H-PEC cell. The enhanced performance was attributed to the i) improved water-wettability, ii) enhanced charge extraction property by band-edge alignment, and iii) the catalytic effect of the introduced NiO-OH nanoparticles. In addition, because of the effects of the RGO layer preventing water penetration and photo-corrosion during the oxidation of water, a distinguishable long-term stability was achieved from the H-PEC cell with an RGO capping layer. The best performance was obtained from the organic-inorganic hybrid multi-junction PEC cells consisting of the WO3 photo-anode (activated under UV irradiation) and the H-PEC cell (activated under visible light irradiation). The H-PEC cell with a WO3 photo-anode exhibited significantly enhanced stability and performance by a factor of 11.6 higher than photocurrent of the single H-PEC cell.

  9. Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

    Science.gov (United States)

    Brazolot, C L; Petitte, J N; Etches, R J; Verrinder Gibbins, A M

    1991-12-01

    Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Convergent extension: using collective cell migration and cell intercalation to shape embryos.

    Science.gov (United States)

    Tada, Masazumi; Heisenberg, Carl-Philipp

    2012-11-01

    Body axis elongation represents a common and fundamental morphogenetic process in development. A key mechanism triggering body axis elongation without additional growth is convergent extension (CE), whereby a tissue undergoes simultaneous narrowing and extension. Both collective cell migration and cell intercalation are thought to drive CE and are used to different degrees in various species as they elongate their body axis. Here, we provide an overview of CE as a general strategy for body axis elongation and discuss conserved and divergent mechanisms underlying CE among different species.

  11. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

    Science.gov (United States)

    Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments.

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