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Sample records for mouse embryo cells

  1. 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

  2. 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.

  3. 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

  4. 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)

  5. 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

  6. 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...

  7. 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.

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

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    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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  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. 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.

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. [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.

  14. 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.

  15. 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.

  16. 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.

  17. ROCK inhibition prevents early mouse embryo development.

    Science.gov (United States)

    Duan, Xing; Chen, Kun-Lin; Zhang, Yu; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-08-01

    ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.

  18. Mouse embryos' fusion for the tetraploid complementation assay.

    Science.gov (United States)

    Gertsenstein, Marina

    2015-01-01

    Production of the germline-competent chimeras using genetically modified ES cell lines is an essential step in the establishment of novel mouse models. In addition chimeras provide a powerful tool to study the cell lineage and to analyze complex phenotypes of mutant mice. Mouse chimeras with tetraploid embryos are used to rescue extraembryonic defects, to analyze an impact of gene function on specific lineage, to study the interaction between embryonic and extraembryonic tissues, and to produce mutant embryos and mice for the phenotype analysis. Tetraploid embryos are generated by the fusion of two blastomeres of the mouse embryo. The applications of tetraploid complementation assay and the protocol are described below.

  19. 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

  20. 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.

  1. 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

  2. Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells.

    Science.gov (United States)

    Fresa, K L; Karjalainen, H E; Tevethia, S S

    1987-02-15

    The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.

  3. In amnio MRI of mouse embryos.

    Directory of Open Access Journals (Sweden)

    Thomas A Roberts

    Full Text Available Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px. To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community.

  4. Wagging the dogma; tissue-specific cell cycle control in the mouse embryo.

    Science.gov (United States)

    Pagano, Michele; Jackson, Peter K

    2004-09-03

    The family of cyclin-dependent kinases (Cdks) lies at the core of the machinery that drives the cell division cycle. Studies in cultured mammalian cells have provided insight into the cellular functions of many Cdks. Recent Cdk and cyclin knockouts in the mouse show that the functions of G1 cell cycle regulatory genes are often essential only in specific cell types, pointing to our limited understanding of tissue-specific expression, redundancy, and compensating mechanisms in the Cdk network.

  5. 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...

  6. Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.

    Science.gov (United States)

    Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

    2014-07-16

    Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

  7. 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.

  8. Expression of TRAIL in Mouse Uterine Endometrium during Embryo Implantation

    Institute of Scientific and Technical Information of China (English)

    Dong-mei TAN; Ming-zhong HE; Qi CHEN; Guo-qi LAI; Li-zhi WANG; Yi TAN

    2006-01-01

    Objective To investigate the expression of TRAIL in mouse uterine endometrium during embryo implantation and its role in the apoptosis of decidual cells.Methods Expression of TRAIL in uterine endometrium of pregnant mouse from d 1 to d 8 was detected with RT-PCR and immunohistochemistry.Results The expressed level of TRAIL mRNA in uterine endometrium of pregnant mouse from d 1 to d 8 was higher during embryo implantation than that prior to embryo implantation (P<0. 05). No expression of TRAIL protein in mouse utrine endometrium was detected through d 1 to d 3. However, TRAIL protein was found in the luminal epithelial cells to which embryos attached on d 4. Moreover, TRAIL was expressed solely in decidual cells around invadting embryos through d 5 to d 6 while in trophoblastic cells adjacent to decidua through d 7 to d 8.Conclusion Apoptosis of luminal epithelial cells of endometrium induced by TRAIL could be one of mechanisms with which embryos penertrated the epithelial barrier,and apoptosis of both decidual cells and trophoblastic cells induced by TRAIL may play an important role during accruate invasion of trophoblastic cells.

  9. Effect of 935-MHz phone-simulating electromagnetic radiation on endometrial glandular cells during mouse embryo implantation.

    Science.gov (United States)

    Liu, Wenhui; Zheng, Xinmin; Qu, Zaiqing; Zhang, Ming; Zhou, Chun; Ma, Ling; Zhang, Yuanzhen

    2012-10-01

    This study examined the impact of 935MHz phone-simulating electromagnetic radiation on embryo implantation of pregnant mice. Each 7-week-old Kunming (KM) female white mouse was set up with a KM male mouse in a single cage for mating overnight after induction of ovulation. In the first three days of pregnancy, the pregnant mice was exposed to electromagnetic radiation at low-intensity (150 μW/cm(2), ranging from 130 to 200 μW/cm(2), for 2- or 4-h exposure every day), mid-intensity (570 μW/cm(2), ranging from 400 to 700 μW/cm(2), for 2- or 4-h exposure every day) or high-intensity (1400 μW/cm(2), ranging from 1200 to 1500 μW/cm(2), for 2- or 4-h exposure every day), respectively. On the day 4 after gestation (known as the window of murine embryo implantation), the endometrium was collected and the suspension of endometrial glandular cells was made. Laser scanning microscopy was employed to detect the mitochondrial membrane potential and intracellular calcium ion concentration. In high-intensity, 2- and 4-h groups, mitochondrial membrane potential of endometrial glandular cells was significantly lower than that in the normal control group (Pelectromagnetic radiation and longer length of the radiation are required to inflict a remarkable functional and structural damage to mitochondrial membrane. Our data demonstrated that electromagnetic radiation with a 935-MHz phone for 4 h conspicuously decreased mitochondrial membrane potential and lowered the calcium ion concentration of endometrial glandular cells. It is suggested that high-intensity electromagnetic radiation is very likely to induce the death of embryonic cells and decrease the chance of their implantation, thereby posing a high risk to pregnancy.

  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. New patterns of bulk DNA repair in ultraviolet irradiated mouse embryo carcinoma cells following differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Rasko, I.; Georgieva, M.; Farkas, G.; Santhan, M.; Burg, K. (Genetics Institute, Szeged (Hungary)); Coates, J.; Johnson, R.T. (Univ. of Cambridge (United Kingdom)); Mitchell, D.L. (M.D. Anderson Cancer Center, Smithville, TX (United States))

    1993-05-01

    Mouse embryocarcinoma stem cells differentiate in culture, given the appropriate induction. The authors examined whether these cells could provide information about the regulation of nucleotide excision repair in relation to differentiation by measuring the rate-limiting incision step, the removal of cyclobutane dimers and (6-4) photoproducts from the genome as a whole and the effect of the bacteriophage T4 endonuclease (denV) gene on repair in differentiated cells. It was found that differentiation is accompanied by a marked decline in the early incision ability after UV irradiation (sixfold for P19, fourfold for PCC7 and twofold for F9), and the authors measured, in parallel, the loss of two common UV photoproducts [cyclobutane dimers and (6-4) photoproducts] from P19 cells. After differentiation, the excellent overall cyclobutane dimer repair capacity of proliferating cells (84% removal in 24 h) is lost (no removal in 24 h), while removal of (6-4) photoproducts, although normal at 24 h (94%), is much slower than in undifferentiated P19 at 3 h (no removal versus 64%). The presence of the denV gene greatly stimulates the repair of cyclobutane dimers in undifferentiated P19 cells (94% removal at 3 h vs. no removal) and also in differentiated cells (50% removal at 24 h vs. no removal). The denV gene also stimulates the early repair of (6-4) photoproducts in both differentiated and undifferentiated cells.

  12. Enhancement of NMRI Mouse Embryo Development In vitro

    Directory of Open Access Journals (Sweden)

    Abedini, F.

    2013-12-01

    Full Text Available Most of the systematic studies used in the development of human embryo culture media have been done first on mouse embryos. The general use of NMRI outbred mice is a model for toxicology, teratology and pharmacology. NMRI mouse embryo exhibit the two-cell block in vitro. The objective of this study was to evaluate and compare the effects of four kinds of culture media on the development of zygotes (NMRI after embryo vitrification. One-cell mouse embryos were obtained from NMRI mice after superovulation and mating with adult male NMRI mice. And then randomly divided into 4 groups for culture in four different cultures media including: M16 (A, DMEM/Ham, F-12 (B, DMEM/Ham's F-12 co-culture with Vero cells(C and DMEM/Ham's F-12 co-culture with MEF cells (D. Afterward all of the embryos were vitrified in EFS40 solution and collected. Results of our study revealed, more blastocysts significantly were developed with co-culture with MEF cells in DMEM/Ham's F-12 medium. More research needed to understand the effect of other components of culture medium, and co-culture on NMRI embryo development.

  13. Calciumreleasing activity induced by nuclei of mouse fertilized early embryos

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    At fertilization, repetitive transient rises of intracellular calcium concentration occur in all mammals studied so far. It has been shown that calcium rises could be induced when mouse fertilized 1-, 2-cell nuclei were transplanted into unfertilized eggs and that the reconstituted embryo could be activated. However, whether the capability of inducing calcium rises occurs in all stages of mammalian embryos remains unknown. In this study, by using the nuclear transplantation technique and measurement of intracellular calcium rises in living cells, we showed that only the nuclei from mouse fertilized 1-cell and 2-cell embryos, neither the nuclei from 4-, 8-cell and ethanol activated parthenogenetic embryos nor 2 or 3 nuclei of electrofused 4-cell stage syncytium, have calcium-releasing activity when they were transferred into unfertilized mature oocytes. Our results indicate that the calcium-releasing activity in nuclei of 1-, 2-cell embryos is produced during fertilization and exists at the special stage of fertilized early embryos. These suggested that the capacity of inducing calcium release activity in fertilized early embryos is important for normal embryonic development.

  14. The perfect host: a mouse host embryo facilitating more efficient germ line transmission of genetically modified embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Robert A Taft

    Full Text Available There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs. We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium. ESC germline transmission was observed in 9/11 (82% lines using PH blastocysts, compared to 6/11 (55% when conventional host blastocysts were used. Furthermore, less than 35% (83/240 of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137 of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the

  15. Aberrant DNA methylation patterns in cultured mouse embryos

    Institute of Scientific and Technical Information of China (English)

    HOU Jian; CUI Xiuhong; LEI Tinghua; LIU Lei; AN Xiaorong; CHEN Yongfu

    2005-01-01

    Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofiuorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.

  16. Fundamental cryobiology of mouse ova and embryos

    Energy Technology Data Exchange (ETDEWEB)

    Leibo, S. P.

    1977-01-01

    An increasing fraction of mouse ova and embryos are killed as the rate at which they are cooled to -196/sup 0/C is increased. The survival of these cells depends not only on cooling rate, but also on the minimum subzero temperature to which the cells are cooled. Low temperature microscopy demonstrates that lethal cooling rates are coincident with those that produce intracellular ice formation, and that the lethal temperature appears to be that at which intracellular ice forms. Furthermore, the microscopy shows that ova do not dehydrate when cooled at rates that produce intracellular ice and cell death, but undergo substantial shrinkage when cooled at rates that produce little intracellular ice and high survival. Measurements of the water permeability of mouse ova and the temperature coefficient of that permeability can be used to test a mathematical model formulated to describe the kinetics of water loss at subzero temperatures from a hypothetical cell. The observed dehydration of ova cooled to subzero temperatures at given rates is approximately predicted by the mathematical model, although there is some quantitative discrepancy between the observed and calculated responses.

  17. Epiblast ground state is controlled by canonical Wnt/β-catenin signaling in the postimplantation mouse embryo and epiblast stem cells.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Sumi

    Full Text Available Epiblast stem cells (EpiSCs are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells.

  18. 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.

  19. In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

    Science.gov (United States)

    Kelley, Rebecca L; Gardner, David K

    2017-02-15

    Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture.

  20. Zygotic G2/M cell cycle arrest induced by ATM/Chk1 activation and DNA repair in mouse embryos fertilized with hydrogen peroxide-treated epididymal mouse sperm.

    Directory of Open Access Journals (Sweden)

    Bin Wang

    Full Text Available Human sperm cryopreservation for assisted reproduction is compromised by ROS-induced sperm cryodamage. Our previous model study in which mouse sperm were treated with H₂O₂ to simulate sperm DNA-damage caused by cryopreservation-induced ROS have discovered that mouse embryos fertilized with treated sperm showed a delay in cleavage that might be associated with cell cycle arrest. The DNA-damage checkpoint pathway underlying the delay remained elusive. Moreover, our previous study have also indicated that γH2AX, the DNA-damage repair marker, was functional in mouse embryos similarly fertilized, but the completeness and correctness are unknown and warrant more studies because insufficiency of completeness and correctness of DNA repair would otherwise trigger apoptosis. Based on the aforementioned model, we used embryo culture, inverted microscope, BrdU incorporation and immunofluorescence to explore the cell cycle phase that arrest occurred and the underlying DNA-damage checkpoint pathway in mouse zygotes fertilized with H₂O₂-treated sperm. We also adopted Tunel to investigate the apoptosis of mouse embryos similarly fertilized at different developmental stages to testify the completeness and correctness of sperm-derived DNA-damage repair. We found G2/M cell cycle arrest in zygotes fertilized with H₂O₂-treated sperm. ATM (pSer-1981 and Chk1 (pSer-345 activations, rather than ATR (pSer-428 and Chk2 (pThr-68, were detected in zygotes of the treated group. The apoptosis of embryos of different developmental stages of the treated group weren't different from those of the untreated group. In conclusions, ATM (pSer-1981-Chk1 (pSer-345 cascade might have mediated G2/M cell cycle arrest and allowed time to facilitate sperm-derived DNA-damage repair in mouse zygotes fertilized with oxygen-stressed sperm, and the DNA-damage repair might be effective.

  1. Zygotic G2/M Cell Cycle Arrest Induced by ATM/Chk1 Activation and DNA Repair in Mouse Embryos Fertilized with Hydrogen Peroxide-Treated Epididymal Mouse Sperm

    Science.gov (United States)

    Chen, Man; Xiao, Jianfeng; Wu, Xiaoyan; Xiao, Wanfen; Song, Yu; Wang, Xiaoyan

    2013-01-01

    Human sperm cryopreservation for assisted reproduction is compromised by ROS-induced sperm cryodamage. Our previous model study in which mouse sperm were treated with H2O2 to simulate sperm DNA-damage caused by cryopreservation-induced ROS have discovered that mouse embryos fertilized with treated sperm showed a delay in cleavage that might be associated with cell cycle arrest. The DNA-damage checkpoint pathway underlying the delay remained elusive. Moreover, our previous study have also indicated that γH2AX, the DNA-damage repair marker, was functional in mouse embryos similarly fertilized, but the completeness and correctness are unknown and warrant more studies because insufficiency of completeness and correctness of DNA repair would otherwise trigger apoptosis. Based on the aforementioned model, we used embryo culture, inverted microscope, BrdU incorporation and immunofluorescence to explore the cell cycle phase that arrest occurred and the underlying DNA-damage checkpoint pathway in mouse zygotes fertilized with H2O2-treated sperm. We also adopted Tunel to investigate the apoptosis of mouse embryos similarly fertilized at different developmental stages to testify the completeness and correctness of sperm-derived DNA-damage repair. We found G2/M cell cycle arrest in zygotes fertilized with H2O2-treated sperm. ATM (pSer-1981) and Chk1 (pSer-345) activations, rather than ATR (pSer-428) and Chk2 (pThr-68), were detected in zygotes of the treated group. The apoptosis of embryos of different developmental stages of the treated group weren’t different from those of the untreated group. In conclusions, ATM (pSer-1981)-Chk1 (pSer-345) cascade might have mediated G2/M cell cycle arrest and allowed time to facilitate sperm-derived DNA-damage repair in mouse zygotes fertilized with oxygen-stressed sperm, and the DNA-damage repair might be effective. PMID:24040138

  2. Cumulus Cell Role on Mouse Germinal Vesicle Oocyte Maturation, Fertilization, and Subsequent Embryo Development to Blastocyst Stage In Vitro

    Directory of Open Access Journals (Sweden)

    Reza Mahmodi

    2009-01-01

    Full Text Available Objective: The purpose of this study is to investigate the effect of cumulus cells on maturation,fertilization and subsequent development of mouse germinal vesicle oocytes.Materials and Methods: A total of 470 germinal vesicle (GV oocytes were obtained from26 ovaries of 3- 4 week old ICR female mice 48 hours after injection of 5 IU pregnant mareserum gonadotropin (PMSG. Collected oocytes were divided into two groups; group I: GVoocytes without cumulus cells (denuded oocyte, group II: GV oocytes with cumulus cells(cumulus-oocyte complex. The oocytes in both groups were cultured in TCM-199 mediumsupplemented with 10% fetal bovine serum (FBS for 22- 24 hours in a humidified atmosphereof 5% CO2 in air at 37°C. Oocyte maturation was scored under inverted microscope.To do in vitro fertilization, matured oocytes from each group were placed in T6 mediumand capacitated spermatozoa were added. Then the fertilized oocytes were cultured andassessed for cleavage to the 2-cell stage 24 hours and production of blastocyst 120 hoursafter fertilization. Data was analyzed by chi-square test and differences in the values wereconsiderable significant when p<0.05.Results: Maturation, fertilization, cleavage and blastocyst rates in denuded oocytes were:76.32%, 57.49%, 51.15% and 19.14% respectively. In the cumulus-oocyte complex rateswere: 89.41%, 80.76%, 75.58% and 45.62% respectively; all in the cumulus-oocyte complexwere significantly higher than those of denuded oocytes (p<0.05.Conclusion: The present study indicates that cumulus cells have important role duringmaturation, fertilization and subsequent embryo development to the blastocyst stage.

  3. Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate the effects of different kinds of nuclear recipients from Kunming (KM) mouse on developmental potential of somatic nuclear transfer em- bryos, the enucleated MⅡ oocytes, enucleated zygotes and 2-cell blastomere were used to produce cloned mouse embryos. Using fibroblast deriving from C57/BL6 ear tissue as nuclear donor, we produced cloned embryos by transferring the fibroblast nuclei into enucleated KM mouse oocytes (single nuclear transfer, SNT), transferring pronuclei from the SNT embryos into enucleated KM zygotes (nuclear into zygote, NZ), and 2-cell blastomere nuclei from SNT embryos into enucleated KM mouse oocytes (nuclear into oocytes, NO); tetraploid embryos (tetraploid embryos, TE) were obtained by fusing two blastomeres, one is from the SNT cloned embryos, and the other from normal 2-cell KM mouse embryos. In group SNT, the cloned embryos could not develop beyond 8-cell stage and the rate of 8-cell stage is only 0.3%; in group NO, the reconstructed embryos could develop to morula stage, the rate of 8-cell stage was significantly greater than that of SNT group (P < 0.05); in group NZ, the development rate was further improved, and the reconstructed embryos could develop into blastocyst stage, the rate of blastocyst was 1.9%; in group TE, as high as 62.3% of the reconstructed embryos could develop into blastocyst. Results suggested that different nuclear recipients could significantly affect the developmental potential of cloned mouse embryos; KM MⅡ oocyte cytoplasm was not so effective as zygotes to reprogram the mouse somatic cell nuclei; serial nuclear transfer could improve the developmental potential of cloned mouse embryos.

  4. Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells

    DEFF Research Database (Denmark)

    Schneider, H R; Reichert, G H; Issinger, O G

    1986-01-01

    , increased phosphorylation of a 110-kDa protein is observed. Treatment of the embryo extracts with heparin, a highly specific inhibitor of CKII activity, results in a drastic reduction of the 110-kDa protein phosphorylation indicating that the protein might be a CKII-specific substrate. Rapidly proliferating...... mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme...

  5. Cell degeneration and mitosis in the buccopharyngeal and branchial membranes in the mouse embryo.

    Science.gov (United States)

    Poelmann, R E; Dubois, S V; Hermsen, C; Smits-van Prooije, A E; Vermeij-Keers, C

    1985-01-01

    The frequencies of cell degeneration and mitosis were investigated in the rupturing buccopharyngeal membrane (BPM) and in the persistent first branchial membrane (BM). In the BPM, cell degeneration starts many hours before rupture is visible, but mitotic figures are absent. In the BM this situation is reversed: mitotic figures are regularly observed, but a degenerating cell only occasionally. It is concluded that the ratio between the numbers of degenerating and dividing cells regulates the fate of both the BPM and the BM.

  6. 小鼠孤雌胚2-细胞阻滞的研究%Studies on mouse parthenogenetic embryos 2-cell block

    Institute of Scientific and Technical Information of China (English)

    何志全; 李三华; 陈伟; 陆祥

    2011-01-01

    In order to study the mechanism of mouse parthenogenetic embryos 2-cell block,we selected oviduct epithelial cells and pituitary cells for feeder layer to culture mouse parthenogenetic embryos. The mouse oocytes which were activated after dealing with 5 min in 70mL/L alcohol and 3 h in 2 mmol/L 6- DMAP and 5 g/mL CB, were cultured in KSOM medium containing different feeder layers. We observed and compared mouse parthenogenetic embryos developmental situation which were in various culture conditions. In result,the embryos had no significant difference in each group after 24 h culture,and all of them had high cleavage rate(P〉0. 05). After being cultured to 48 h, the parthenogenetic embryos cultured in KSOM medium with two types of feeder cells had developmental effect of 4 to 8-cell,it was significantly different(P〈0.05) compared with single feeder layer cell and cotrol group(P〈0.01). The ratio of embryos which can develop to Morula/blastocyst was 49.4%(42/85)(P〈0.01). We inferred that KSOM medium containing oviduct epithelial cells and pituitary cells can effectively promote the development of mouse parthenogenetic embryos which can breakthrough 2-cell block(block rate to 74.1%), and further improve embryos development(morula/blastocyst rate to 49.4 %).%为探讨小鼠孤雌胚2-细胞阻滞的机制,本试验以昆明小鼠为研究对象,通过输卵管上皮细胞和垂体细胞作为饲养层培养小鼠孤雌胚胎,分析其作用机理。小鼠卵母细胞通过70mL/L乙醇激活5min,再用2mmol/L 6-DMAP、5μg/mL CB激活3h后,分别在不同饲养层的KSOM培养液中进行培养,观察并比较各培养条件下孤雌胚胎的发育情况。结果,培养至第24小时,各组无显著差异(P〉0.05),都有较高卵裂率。培养至第48小时,用两种饲养层细胞培养的孤雌胚胎4-细胞~8-细胞发育效果好,与用单独一种饲养层细胞培养相比,差异显著(P〈0.05),与对照

  7. Mouse one-cell embryos undergoing a radiation-induced G2 arrest may re-enter S-phase in the absence of cytokinesis

    Energy Technology Data Exchange (ETDEWEB)

    Jacquet, P.; Buset, J. [Lab. of Radiobiology, Dept. of Radioprotection, CEN/SCK, Boeretang, Mol (Belgium); Vankerkom, J. [Div. of Environmental Research, VITO, Mol (Belgium); Baatout, S.; De Saint-Georges, L.; Schoonjans, W.; Desaintes, C. [Lab. of Radiobiology, Dept. of Radioprotection, CEN/SCK, Boeretang, Mol (Belgium)

    2002-07-01

    PCC (premature chromosome condensation) can be used for visualizing and scoring damage induced by radiation in the chromatin of cells undergoing a G1 or G2 arrest. A method involving the fusion of irradiated single embryonic cells with single MI oocytes was used to induce PCC in mouse zygotes of the BALB/c strain, which suffer a drastic G2 arrest after X-irradiation (dose used 2.5 Gy). Other G2-arrested embryos were exposed in vitro to the phosphatase inhibitor calyculin A. Both methods furnished excellent chromosome preparations of the G2-arrested embryos. The mean number of chromosome fragments did not change significantly during G2 arrest, suggesting that zygotes of this strain are unable to repair DNA damage leading to such aberrations. Forty to fifty percent of the irradiated embryos were unable to cleave after G2 arrest and remained blocked at the one-cell stage for a few days before dying. PCC preparations obtained from such embryos suggested that about 30% of them had undergone a late mitosis not followed by cytokinesis and had entered a new DNA synthesis. These results are discussed in the light of recent observations in irradiated human cells deficient in the p53/14-3-3{sigma} pathway. (author)

  8. Mouse embryos and chimera cloned from neural cells in the postnatal cerebral cortex.

    Science.gov (United States)

    Makino, Hatsune; Yamazaki, Yukiko; Hirabayashi, Takahiro; Kaneko, Ryosuke; Hamada, Shun; Kawamura, Yoshimi; Osada, Tomoharu; Yanagimachi, Ryuzo; Yagi, Takeshi

    2005-01-01

    Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.

  9. Generation of targeted mouse mutants by embryo microinjection of TALENs.

    Science.gov (United States)

    Wefers, Benedikt; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

    2014-08-15

    Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.

  10. 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.

  11. 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.

  12. 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.

  13. Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To investigate the effect of clenbuterol hydrochloride on the in vitro devel-opment of both 1-cell and 2-cell mouse embryos.Methods The cultural systems of both 1-cell and 2-cell mouse embryo were used todetermine the effect of clenbuterol hydrochloride at doses of 1 ng/mL, 3 ng/mL, and10 ng/mL on developmental rates of mouse embryos.Results When 1-cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride,developmental rates from the 4-cell stage to blastocyst stage were significantly lowerthan those in the control group (P< 0. 05), but on dosages of 3 ng/mL and 10ng/mL,the inhibiting effects on embryo development were significantly increased (P< 0. 01).When 2-cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, obvious dif-ferences in developmental rates were not found between the 2-cell embryo group and thecontrol (P> 0. 05). However, at levels of 3 ng/mL and 10 ng/mL, significant de-crease of developmental rates in 2-cell embryos was observed from the 4-cell and fromthe 8-cell stage, respectively (P< 0. 05). Embryos cultured with clenbuterol hydrochlo-ride appeared to have more granules, fragments and degeneration than those in thecontrol.Conclusion Clenbuterol hydrochloride has a toxic effect on the mouse embryos, and theeffect is in a dose-dependent. 1-cell mouse embryos cultured with clenbuterolhydrochloride could be easily inhibited at 2-cell stage, but the effect of clenbuterolhydrochloride on development of the late 2-cell embryos would be reduced.

  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. Thymocyte clones from 14-day mouse embryos. I. State of T cell receptor genes, surface markers, and growth requirements

    OpenAIRE

    1987-01-01

    We have established in culture 13 clones from the thymus of a 14-d B10.BR mouse embryo and characterized 8 of them. All eight FT clones have the TCR-gamma and -beta genes in germline configuration. They express mRNA for the gamma, but not for the beta nor the alpha genes. All eight FT clones are Thy-1+, Ly-1+, LFA-1+, Pgp-1+, H-2K+, and T3-. Three phenotypes could be distinguished on the basis of Lyt-2, L3T4, and IL-2-R expression: Lyt-2+, L3T4-, IL-2-R+ (I); Lyt-2+, L3T4-, IL-2- R- (II); and...

  16. Photobiomodulation of early mouse embryo development

    Science.gov (United States)

    Sviridova-Chailakhyan, T. A.; Fakhranurova, L. I.; Simonova, N. B.; Khramov, R. N.; Manokhin, A. A.; Paskevich, S. I.; Chailakhyan, L. M.

    2008-04-01

    The effect of artificial sunlight (AS) from a xenon source and of converted AS with an additional orange-red luminescent (λ MAX=626 nm) component (AS+L) on the development of mouse zygotes was investigated. A plastic screen with a photoluminophore layer was used for production of this orange-red luminescent (L) component. A single short-term (15 min) exposure produced a long-term stable positive effect on early embryo development of mice, which persisted during several days. After exposure to AS+L, a stimulating influence on preimplantation development was observed, in comparison with the control group without AS exposure. The positive effects were as follows: increase in percent of embryos (P <= 0.05) developed to the blastocyst stage (96.2 %) with hatching from the zona pellucida (80.8 %) within 82-96 hours in vitro compared to the control (67.1 % and 28.8 %, respectively).

  17. A Rapid and Efficient 2D/3D Nuclear Segmentation Method for Analysis of Early Mouse Embryo and Stem Cell Image Data

    Directory of Open Access Journals (Sweden)

    Xinghua Lou

    2014-03-01

    Full Text Available Segmentation is a fundamental problem that dominates the success of microscopic image analysis. In almost 25 years of cell detection software development, there is still no single piece of commercial software that works well in practice when applied to early mouse embryo or stem cell image data. To address this need, we developed MINS (modular interactive nuclear segmentation as a MATLAB/C++-based segmentation tool tailored for counting cells and fluorescent intensity measurements of 2D and 3D image data. Our aim was to develop a tool that is accurate and efficient yet straightforward and user friendly. The MINS pipeline comprises three major cascaded modules: detection, segmentation, and cell position classification. An extensive evaluation of MINS on both 2D and 3D images, and comparison to related tools, reveals improvements in segmentation accuracy and usability. Thus, its accuracy and ease of use will allow MINS to be implemented for routine single-cell-level image analyses.

  18. Resveratrol prevents nicotine-induced teratogenesis in cultured mouse embryos

    NARCIS (Netherlands)

    Lin, C.; Yon, J.M.; Jung, A.Y.; Lee, J.G.; Jung, K.Y.; Kang, J.K.; Lee, B.J.; Yun, Y.W.; Nam, S.Y.

    2012-01-01

    Nicotine, a major toxic component in tobacco smoke, leads to severe embryonic damage during organogenesis in embryos. We investigated whether resveratrol would positively influence nicotine-induced teratogenesis in mouse embryos (embryonic day 8.5) cultured for 48 h using a whole embryo culture syst

  19. Effects of Placental Isoferritin on the Mouse Embryo Development in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHU Ying; WU Chaoying; SUN Yongyu

    2007-01-01

    To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell,8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more em-bryos development to the blastocyst and hatching blastocyst (P0.05). It was concluded that PLF at the concentration of 10--100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantion blastocysts.

  20. The synthetic retinoid AGN 193109 but not retinoic acid elevates CYP1A1 levels in mouse embryos and Hepa-1c1c7 cells.

    Science.gov (United States)

    Soprano, D R; Gambone, C J; Sheikh, S N; Gabriel, J L; Chandraratna, R A; Soprano, K J; Kochhar, D M

    2001-07-15

    The synthetic retinoid AGN 193109 is a potent pan retinoic acid receptor (RAR) antagonist. Treatment of pregnant mice with a single oral 1 mg/kg dose of this antagonist on day 8 postcoitum results in severe craniofacial (median cleft face or frontonasal deficiency) and eye malformations in virtually all exposed fetuses. Using differential display analysis, we have determined that CYP1A1 mRNA levels are elevated in mouse embryos 6 h following treatment with AGN 193109. Similarly, an elevation in CYP1A1 mRNA levels, protein levels, and aryl hydrocarbon hydoxylase activity occurs in Hepa-1c1c7 cells, with the maximal elevation observed when the cells were treated with 10(-5) M AGN 193109 for 4 to 8 h. Elevation in CYP1A1 mRNA levels in mouse embryos and Hepa-1c1c7 cells does not occur upon treatment with the natural retinoid, all-trans-retinoic acid. Finally, elevation in CYP1A1 mRNA levels was not observed when mutant Hepa-1c1c7 cells, which are defective in either the aryl hydrocarbon receptor (AhR) or aryl hydrocarbon receptor nuclear translocator (ARNT), were treated with AGN 193109. This suggests that the AhR/ARNT pathway and not the RAR/RXR pathway is mediating the elevation of CYP1A1 mRNA levels by AGN 193109, at least in the Hepa-1c1c7 cells. This is the first example of a retinoid that displays the abililty to regulate both the RAR/RXR and AhR/ARNT transcriptional regulatory pathways.

  1. TGF-β/Smad2/3 signaling directly regulates several miRNAs in mouse ES cells and early embryos.

    Directory of Open Access Journals (Sweden)

    Nicholas Redshaw

    Full Text Available The Transforming Growth Factor-β (TGF-β signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-β signaling in mouse embryonic stem (ES cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-β activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341∼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-β inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341∼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-β/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-β/Smad2/3 pathway underlie tumor

  2. Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

    Energy Technology Data Exchange (ETDEWEB)

    Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A [N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow (Russian Federation); Ryabova, A V [A M Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation)

    2015-05-31

    Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated. (extreme light fields and their applications)

  3. In vitro study of cell differentiation by two type mouse embryo stem cells on mono- and multilayer nanocarbon tubes

    Science.gov (United States)

    Imai, Koichi; Akasaka, Tsukasa; Watari, Fumio; Tanoue, Akito; Nakamura, Kazuaki; Suese, Kazuhiko; Takashima, Hiromasa; Nishikawa, Tetsunari; Tanaka, Akio; Takeda, Shoji

    2012-09-01

    The effects of nanomaterials on human reproduction and development remain unknown. The risks of nanomaterials for future generations should be elucidated. Thus, it is important to establish an experimental method to accurately examine embryotoxicity. We previously investigated the myocardial cell differentiation of ES-D3 cells using monolayer (SWCNTs) and multilayer (MWCNTs) nanocarbon tubes. As a result, in spite of having the same carbon composition, the effects on the cell differentiation levels differed between the tubes. We investigated their cell differentiation and cytotoxic effects on EL M3 and ES-R1-EGFP B2/EGFP cells, which require feeder cells. As a result, myocardial pulse rates differed between the presence of SWCNTs and MWCNTs even when feeder cells existed between the samples and cells. The different surface structures of SWCNTs and MWCNTs may have influenced ES cell differentiation.

  4. Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

    Science.gov (United States)

    Yan, Zheng; Liang, Hongxing; Deng, Li; Long, Hui; Chen, Hong; Chai, Weiran; Suo, Lun; Xu, Chen; Kuang, Yanping; Wu, Lingqian; Lu, Shengsheng; Lyu, Qifeng

    2015-01-01

    Increased risk of monozygotic twinning (MZT) has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM) splitting in human '8'-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i) in vivo developed blastocysts and (ii-iii) in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that '8'-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P split distribution of ICM cells was observed around the small zona opening of '8'-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in '8'-shaped hatching and hatched blastocysts than in 'U'-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

  5. Gene expression profiles in mouse embryo fibroblasts lacking stathmin, a microtubule regulatory protein, reveal changes in the expression of genes contributing to cell motility

    Directory of Open Access Journals (Sweden)

    Cassimeris Lynne

    2009-07-01

    Full Text Available Abstract Background Stathmin (STMN1 protein functions to regulate assembly of the microtubule cytoskeleton by destabilizing microtubule polymers. Stathmin over-expression has been correlated with cancer stage progression, while stathmin depletion leads to death of some cancer cell lines in culture. In contrast, stathmin-null mice are viable with minor axonopathies and loss of innate fear response. Several stathmin binding partners, in addition to tubulin, have been shown to affect cell motility in culture. To expand our understanding of stathmin function in normal cells, we compared gene expression profiles, measured by microarray and qRT-PCR, of mouse embryo fibroblasts isolated from STMN1+/+ and STMN1-/- mice to determine the transcriptome level changes present in the genetic knock-out of stathmin. Results Microarray analysis of STMN1 loss at a fold change threshold of ≥ 2.0 revealed expression changes for 437 genes, of which 269 were up-regulated and 168 were down-regulated. Microarray data and qRT-PCR analysis of mRNA expression demonstrated changes in the message levels for STMN4, encoding RB3, a protein related to stathmin, and in alterations to many tubulin isotype mRNAs. KEGG Pathway analysis of the microarray data indicated changes to cell motility-related genes, and qRT-PCR plates specific for focal adhesion and ECM proteins generally confirmed the microarray data. Several microtubule assembly regulators and motors were also differentially regulated in STMN1-/- cells, but these changes should not compensate for loss of stathmin. Conclusion Approximately 50% of genes up or down regulated (at a fold change of ≥ 2 in STMN1-/- mouse embryo fibroblasts function broadly in cell adhesion and motility. These results support models indicating a role for stathmin in regulating cell locomotion, but also suggest that this functional activity may involve changes to the cohort of proteins expressed in the cell, rather than as a direct

  6. MicroRNA-450a-3p represses cell proliferation and regulates embryo development by regulating Bub1 expression in mouse.

    Directory of Open Access Journals (Sweden)

    Min Luo

    Full Text Available Bub1 is a critical component of the spindle assembly checkpoint (SAC and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3'-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.

  7. The ACE2/Ang-(1-7)/Mas Axis Regulates the Development of Pancreatic Endocrine Cells in Mouse Embryos.

    Science.gov (United States)

    Wang, Lin; Liang, Juan; Leung, Po Sing

    2015-01-01

    Angiotensin-converting enzyme 2 (ACE2), its product Angiotensin-(1-7) [Ang-(1-7)], and Ang-(1-7) receptor Mas, have been shown to regulate organogenesis during embryonic development in various species. However, it is not known whether a local ACE2/Ang-(1-7)/Mas axis is present in the fetal pancreas. It is hypothesized that there is a local ACE2/Ang-(1-7)/Mas axis in the embryonic pancreas in mice that is involved in regulating islet cell development. To address this issue, the endogenous expression profile of axis constituents in embryonic mouse pancreata was examined. Involvement of the ACE2 axis in the regulation of pancreatic development was also examined. The present experiments showed in an in vivo animal model that endogenous expression levels of ACE2 and the Mas receptor were upregulated in mouse pancreata in late embryogenesis, peaking on embryonic day E16.5, when it reached 3 folds compared to that seen at E12.5. Consistently, endogenous expression of Ang-(1-7) also peaked at E16.5. Treatment with the ACE2 inhibitor DX600 did not alter islet development. However, prenatal treatment with A779, a Mas receptor antagonist, reduced the β-cell to α-cell ratio in neonatal islets, impaired islet insulin secretory function, and impaired the pups' glucose tolerance. In ex vivo pancreas explant cultures, A779 again decreased the β-cell to α-cell ratio, apparently through its effects on β-cell proliferation (reduced proliferation shown with Ki67 staining), and also decreased Insulin and Ngn3 mRNA expression. Furthermore, treatment of explant cultures with Ang-(1-7) increased mRNA levels of Insulin and pancreatic progenitor marker Ngn3, as well as Nox4, the ROS generation enzyme; these stimulatory effects were attenuated by co-treatment with A779, suggesting that Ang-(1-7), via Mas receptor signaling, may promote differentiation of pancreatic progenitors into insulin-producing cells via modulation of reactive oxygen species. These data together suggest that a Mas

  8. A quantification model for apoptosis in mouse embryos in the early stage of fetation

    Institute of Scientific and Technical Information of China (English)

    WANG PengFei; FU JianHua; MA WanYun; CHEN DieYan; Lü DanYu; BAI WenJia

    2009-01-01

    Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development. Using a two-photon laser scanning microscopy (TPLSM) system, we obtained 3-dimensional (3D) fluorescent images of preimplantation mouse embryos. A model for quantification was established. The statistical results for the spatial location of apoptosis bodies in embryos was obtained following image processing, as well as investigation of the kinetics of apoptosis. It was found that most (70%) apoptosis occurred in the trophectoderm, and the departure between the centroid and geometric center of embryos had a step transition when embryos developed into the 32-cell stage,which was consistent with the theoretical prediction that the blastocele would induce a symmetry break of the distribution of cells in embryos.

  9. A quantification model for apoptosis in mouse embryos in the early stage of fetation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development. Using a two-photon laser scanning microscopy (TPLSM) system, we obtained 3-dimensional (3D) fluorescent images of preimplantation mouse embryos. A model for quantification was established. The statistical results for the spatial location of apoptosis bodies in embryos was obtained following image processing, as well as investigation of the kinetics of apoptosis. It was found that most (70%) apoptosis occurred in the trophectoderm, and the departure between the centroid and geometric center of embryos had a step transition when embryos developed into the 32-cell stage, which was consistent with the theoretical prediction that the blastocele would induce a symmetry break of the distribution of cells in embryos.

  10. Role of glucose in cloned mouse embryo development

    National Research Council Canada - National Science Library

    Zhiming Han; Rita Vassena; Maggie M. Y. Chi; Santhi Potireddy; Miriam Sutovsky; Kelle H. Moley; Peter Sutovsky; Keith E. Latham

    2008-01-01

    Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early...

  11. DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Wyrobek, Andrew J.

    2009-01-18

    Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

  12. Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

    Directory of Open Access Journals (Sweden)

    Zheng Yan

    Full Text Available Increased risk of monozygotic twinning (MZT has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM splitting in human '8'-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i in vivo developed blastocysts and (ii-iii in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that '8'-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01. Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of '8'-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in '8'-shaped hatching and hatched blastocysts than in 'U'-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01. Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of '8'-shaped hatching, and '8'-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

  13. Endodcytic labelling of visceral endoderm of mouse perigastrulation embryos

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Yoh Wada, Minako Aoyama, Ge-Hong Sun-Wada, Nobuyuki Kawamura & Hiroyuki Tabata ### Abstract In this protocol we describe methods for observation endocytic activity in the mouse embryos. The methods are optimised for mouse embryos at E5.5~E7.2 pregastrulation/gastrulation stages. We optimise three different experimental schemes for tracing the embryonic endocytosis. In utero labelling scheme, an endocytic tracer is introduced into circulation of a pregnant mother to follow...

  14. Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos.

    Directory of Open Access Journals (Sweden)

    Helen M Phillips

    Full Text Available Neural crest cells (NCC give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN form of Rho kinase (Rock in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.

  15. 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.

  16. Identification of CD146 Expression in Human and Mouse Preimplantation Embryo

    Institute of Scientific and Technical Information of China (English)

    Hong-bo WANG; Xuan DU; Ya-hui XU; Ze-hua WANG

    2008-01-01

    Objective To investigate whether CD146, a cell adhesion molecule, is expressed in mouse and human preimplantation blastocysts and to localize CD146 in the layer of trophectoderm(TE) and/or inner cell mass(ICM). Methods Human and mouse embryos were collected. Using reverse transcription polymerase chain reaction(RT-PCR), the expression of CD146 mRNA in blastocyst was evaluated in human and mouse embryos. Single embryo immunohistochemical staining was applicated in the examination of the expression of CD146 in protein level. The statistical significance of the data was analyzed using t-test. Results CD146 transcript was detected in all human and mouse preimplantation morula and blastocyst. The expression of CD146 was found to localize in human and mouse compacted morula stage embryos and the TE and ICM of the expanded blastocysts. Conclusion mRNA and protein of CD146 was expressed in preimplantation embryos,which may have a profound influence on early preimplantation development for the differentiation of the trophectoderm and the morphogenesis of the blastocyst.Furthermore, the expression of CD146 in blastocyst stage may be implicated in the assistance of embryo implantation.

  17. IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line

    Directory of Open Access Journals (Sweden)

    Hsu Teng-Tsao

    2003-09-01

    Full Text Available Abstract Background Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1 plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1 may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line. Results In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%. IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively. IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively. Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27

  18. AVE protein expression and visceral endoderm cell behavior during anterior-posterior axis formation in mouse embryos: Asymmetry in OTX2 and DKK1 expression.

    Science.gov (United States)

    Hoshino, Hideharu; Shioi, Go; Aizawa, Shinichi

    2015-06-15

    The initial landmark of anterior-posterior (A-P) axis formation in mouse embryos is the distal visceral endoderm, DVE, which expresses a series of anterior genes at embryonic day 5.5 (E5.5). Subsequently, DVE cells move to the future anterior region, generating anterior visceral endoderm (AVE). Questions remain regarding how the DVE is formed and how the direction of the movement is determined. This study compares the detailed expression patterns of OTX2, HHEX, CER1, LEFTY1 and DKK1 by immunohistology and live imaging at E4.5-E6.5. At E6.5, the AVE is subdivided into four domains: most anterior (OTX2, HHEX, CER1-low/DKK1-high), anterior (OTX2, HHEX, CER1-high/DKK1-low), main (OTX2, HHEX, CER1, LEFTY1-high) and antero-lateral and posterior (OTX2, HHEX-low). The study demonstrates how this pattern is established. AVE protein expression in the DVE occurs de novo at E5.25-E5.5. Neither HHEX, LEFTY1 nor CER1 expression is asymmetric. In contrast, OTX2 expression is tilted on the future posterior side with the DKK1 expression at its proximal domain; the DVE cells move in the opposite direction of the tilt.

  19. 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.

  20. Silicon-nanowire based attachment of silicon chips for mouse embryo labelling.

    Science.gov (United States)

    Durán, S; Novo, S; Duch, M; Gómez-Martínez, R; Fernández-Regúlez, M; San Paulo, A; Nogués, C; Esteve, J; Ibañez, E; Plaza, J A

    2015-03-21

    The adhesion of small silicon chips to cells has many potential applications as direct interconnection of the cells to the external world can be accomplished. Hence, although some typical applications of silicon nanowires integrated into microsystems are focused on achieving a cell-on-a-chip strategy, we are interested in obtaining chip-on-a-cell systems. This paper reports the design, technological development and characterization of polysilicon barcodes featuring silicon nanowires as nanoscale attachment to identify and track living mouse embryos during their in vitro development. The chips are attached to the outer surface of the Zona Pellucida, the cover that surrounds oocytes and embryos, to avoid the direct contact between the chip and the embryo cell membrane. Two attachment methodologies, rolling and pushpin, which allow two entirely different levels of applied forces to attach the chips to living embryos, are evaluated. The former consists of rolling the mouse embryos over one barcode with the silicon nanowires facing upwards, while in the latter, the barcode is pushed against the embryo with a micropipette. The effect on in vitro embryo development and the retention rate related to the calculated applied forces are stated. Field emission scanning electron microscopy inspection, which allowed high-resolution imaging, also confirms the physical attachment of the nanowires with some of them piercing or wrapped by the Zona Pellucida and revealed extraordinary bent silicon nanowires.

  1. Obesity does not aggravate vitrification injury in mouse embryos: a prospective study

    Directory of Open Access Journals (Sweden)

    Ma Wenhong

    2012-08-01

    Full Text Available Abstract Background Obesity is associated with poor reproductive outcomes, but few reports have examined thawed embryo transfer in obese women. Many studies have shown that increased lipid accumulation aggravates vitrification injury in porcine and bovine embryos, but oocytes of these species have high lipid contents (63 ng and 161 ng, respectively. Almost nothing is known about lipids in human oocytes except that these cells are anecdotally known to be relatively lipid poor. In this regard, human oocytes are considered to be similar to those of the mouse, which contain approximately 4 ng total lipids/oocyte. To date, no available data show the impact of obesity on vitrification in mouse embryos. The aim of this study was to establish a murine model of maternal diet-induced obesity and to characterize the effect of obesity on vitrification by investigating the survival rate and embryo developmental competence after thawing. Methods Prospective comparisons were performed between six–eight-cell embryos from obese and normal-weight mice and between fresh and vitrified embryos. Female C57BL/6 mice were fed standard rodent chow (normal-weight group or a high-fat diet (obese group for 6 weeks. The mice were mated, zygotes were collected from oviducts and cultured for 3 days, and six–eight-cell embryos were then selected to assess lipid content in fresh embryos and to evaluate differences in apoptosis, survival, and development rates in response to vitrification. Results In fresh embryos from obese mice, the lipid content (0.044 vs 0.030, Pvs.9.3%, Pvs. 93.1%, P Conclusions This study demonstrated that differences in survival and developmental rates between embryos from obese and normal-weight mice were eliminated after vitrification. Thus, maternal obesity does not aggravate vitrification injury, but obesity alone greatly impairs pre-implantation embryo survival and development.

  2. A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

    Directory of Open Access Journals (Sweden)

    Mikiko Tokoro

    Full Text Available Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

  3. A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

    Science.gov (United States)

    Tokoro, Mikiko; Fukunaga, Noritaka; Yamanaka, Kaori; Itoi, Fumiaki; Terashita, Yukari; Kamada, Yuko; Wakayama, Sayaka; Asada, Yoshimasa; Wakayama, Teruhiko

    2015-01-01

    Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

  4. Gene expression profiling of mouse embryos with microarrays

    Science.gov (United States)

    Sharov, Alexei A.; Piao, Yulan; Ko, Minoru S. H.

    2011-01-01

    Global expression profiling by DNA microarrays provides a snapshot of cell and tissue status and becomes an essential tool in biological and medical sciences. Typical questions that can be addressed by microarray analysis in developmental biology include: (1) to find a set of genes expressed in a specific cell type; (2) to identify genes expressed commonly in multiple cell types; (3) to follow the time-course changes of gene expression patterns; (4) to demonstrate cell’s identity by showing similarities or differences among two or multiple cell types; (5) to find regulatory pathways and/or networks affected by gene manipulations, such as overexpression or repression of gene expression; (6) to find downstream target genes of transcription factors; (7) to find downstream target genes of cell signaling; (8) to examine the effects of environmental manipulation of cells on gene expression patterns; and (9) to find the effects of genetic manipulation in embryos and adults. Here we describe strategies for executing these experiments and monitoring changes of cell state with gene expression microarrays in application to mouse embryology. Both statistical assessment and interpretation of data are discussed. We also present a protocol for performing microarray analysis on a small amount of embryonic materials. PMID:20699157

  5. Antiteratogenic Effects of beta-Carotene in Cultured Mouse Embryos Exposed to Nicotine

    OpenAIRE

    C. Lin; Yon, J. M.; Jung, A.Y.; Lee, J. G.; Jung, K. Y.; Lee, B.J.; Yun, Y.W.; Nam, S Y

    2013-01-01

    After maternal intake, nicotine crosses the placental barrier and causes severe embryonic disorders and fetal death. In this study, we investigated whether beta -carotene has a beneficial effect against nicotine-induced teratogenesis in mouse embryos (embryonic day 8.5) cultured for 48 h in a whole embryo culture system. Embryos exposed to nicotine (1 mM) exhibited severe morphological anomalies and apoptotic cell death, as well as increased levels of TNF- alpha , IL-1 beta , and caspase 3 mR...

  6. Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

    Science.gov (United States)

    Majidi Gharenaz, Nasrin; Movahedin, Mansoureh; Mazaheri, Zohreh; Pour beiranvand, Shahram

    2016-01-01

    Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage. PMID:27679826

  7. Efficient delivery of DNA and morpholinos into mouse preimplantation embryos by electroporation.

    Directory of Open Access Journals (Sweden)

    Hui Peng

    Full Text Available Mouse preimplantation development is characterized by three major transitions and two lineage segregations. Each transition or lineage segregation entails pronounced changes in the pattern of gene expression. Thus, research into the function of genes with obvious changes in expression pattern will shed light on the molecular basis of preimplantation development. We have described a simplified and effective method--electroporation--of introducing plasmid DNA and morpholinos into mouse preimplantation embryos and verified effectiveness of this approach by testing the procedure on the endogenous gene Oct4. Before electroporation, the zona pellucida was weakened by the treatment of acid Tyrode's solution. Then we optimized the parameters such as voltage, pulse duration, number of pulses and repeats, and applied these parameters to subsequent experiments. Compared with the control groups, the number of apoptotic cells and the expression and localization of OCT3/4 or CDX2 was not significantly changed in blastocysts developed from 1-cell embryos, which were electroporated with pIRES2-AcGFP1-Nuc eukaryotic expression vector or mismatched morpholino oligonucleotides. Furthermore, electroporated plasmid DNA and morpholinos targeting the endogenous gene Oct4 were able to sharply down regulate expression of OCT4 protein and actually cause expected phenotypes in mouse preimplantation embryos. In conclusion, plasmid DNA and morpholinos could be efficient delivered into mouse preimplantation embryos by electroporation and exert their functions, and normal development of preimplantation embryos was not affected.

  8. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos

    Science.gov (United States)

    Yokomizo, Tomomasa; Dzierzak, Elaine

    2010-01-01

    Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Despite their importance, hematopoietic clusters have not been systematically quantitated or mapped because of technical limitations posed by the opaqueness of whole mouse embryos. Here, we combine an approach to make whole mouse embryos transparent, with multicolor marking, to allow observation of hematopoietic clusters using high-resolution 3-dimensional confocal microscopy. Our method provides the first complete map and temporal quantitation of all hematopoietic clusters in the mouse embryonic vasculature. We show that clusters peak in number at embryonic day 10.5, localize to specific vascular subregions and are heterogeneous, indicating a basal endothelial to non-basal (outer cluster) hematopoietic cell transition. Clusters enriched with the c-Kit+CD31+SSEA1– cell population contain functional hematopoietic progenitors and stem cells. Thus, three-dimensional cartography of transparent mouse embryos provides novel insight into the vascular subregions instrumental in hematopoietic progenitor/stem cell development, and represents an important technological advancement for comprehensive in situ hematopoietic cluster analysis. PMID:20876651

  9. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos.

    Science.gov (United States)

    Yokomizo, Tomomasa; Dzierzak, Elaine

    2010-11-01

    Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Despite their importance, hematopoietic clusters have not been systematically quantitated or mapped because of technical limitations posed by the opaqueness of whole mouse embryos. Here, we combine an approach to make whole mouse embryos transparent, with multicolor marking, to allow observation of hematopoietic clusters using high-resolution 3-dimensional confocal microscopy. Our method provides the first complete map and temporal quantitation of all hematopoietic clusters in the mouse embryonic vasculature. We show that clusters peak in number at embryonic day 10.5, localize to specific vascular subregions and are heterogeneous, indicating a basal endothelial to non-basal (outer cluster) hematopoietic cell transition. Clusters enriched with the c-Kit(+)CD31(+)SSEA1(-) cell population contain functional hematopoietic progenitors and stem cells. Thus, three-dimensional cartography of transparent mouse embryos provides novel insight into the vascular subregions instrumental in hematopoietic progenitor/stem cell development, and represents an important technological advancement for comprehensive in situ hematopoietic cluster analysis.

  10. Mouse embryo manipulations with OCT guidance

    Science.gov (United States)

    Garcia, Monica D.; Syed, Saba H.; Coughlin, Andrew J.; Wang, Shang; West, Jennifer L.; Larin, Kirill V.; Larina, Irina V.

    2014-03-01

    Optical coherence tomography (OCT) is a three-dimensional, non-invasive optical imaging technique that relies on low-coherence interferometry. OCT has the capability of imaging 2 - 3 mm into tissue, which enables imaging of deeper structures within the embryo with a relatively high spatial resolution (2 - 15μm). Within the past decade, OCT has been increasingly used as a live imaging tool for embryonic cardiovascular research in several animal models. Research in our lab has recently shown that OCT can be used in combination with embryo culture for the visualization of early mammalian cardiovascular development (E7.5 - E10.0). Here, we demonstrate that OCT can be used for the guided microinjection of gold-silica nanoshell suspension into the cardiovascular system in live embryos without deleterious effect. This approach shows a promising application for the OCT guided delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, signaling molecules or dyes to specific organ systems or tissues in live embryos and demonstrates a great potential for gold-silica nanoshells as a contrast agent in embryonic studies.

  11. 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

  12. Near-infrared laser irradiation improves the development of mouse pre-implantation embryos.

    Science.gov (United States)

    Yokoo, Masaki; Mori, Miho

    2017-05-27

    The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing of blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Role of aVb3 integrin in embryo implantation in the mouse

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Integrin, a heterodimeric adhesive molecule composed of a and b subunits, can regulate cell adhesion and trafficking. Recent data have documented that, at the "implantation window" stage, aVb3 integrin participates in the maternal-fetal interaction and becomes a potential marker of uterine receptivity. Furthermore, it can affect invasiveness of embryo. This work made a further study about its action mechanism. Results of indirect immunofluorescence and laser scanning confocal microscopy showed that aVb3 integrin was clearly expressed in the mouse blastocyst. Injection of aVb3 integrin antiserum into a uterine horn of a pregnant mouse on day 3 markedly decreased the number of embryos implanted (P < 0.001). In a co-culture model, aVb3 integrin antisera at 1:100 and 1:200 dilutions significantly depressed the attachment and outgrowth reactions of blastocysts on monolayer of uterine epithelial cells. Analysis of correlation manifested that the inhibitory effect of aVb3 integrin antiserum was dosage/dilution-dependent. Thus, aVb3 integrin is an essential factor in the uterine endometrium for embryo implantation in the mouse. This integrin distinctly expressed in the mouse blastocyst at "implantation" stage affected the process of embryo implantation by route of mediating both the attachment and the outgrowth processes of blastocyst on uterine epithelial cells.

  14. HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

    NARCIS (Netherlands)

    P. Imanirad (Parisa); P. Solaimani Kartalaei (Parham); M. Crisan (Mihaela); C.S. Vink (Chris); T. Yamada-Inagawa (Tomoko); E. de Pater (Emma); D. Kurek (Dorota); P. Kaimakis (Polynikis); R. van der Linden (Reinier); N.A. Speck (Nancy); E.A. Dzierzak (Elaine)

    2014-01-01

    textabstractHypoxia 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 maj

  15. Effects of cryopreservation on cytoskeleton of mouse 2 -4 cell embryos%冷冻对小鼠2-4细胞胚胎细胞骨架结构的影响

    Institute of Scientific and Technical Information of China (English)

    罗孟军; 刘伟信; 王颖佳

    2014-01-01

    目的:探讨胚胎冷冻技术对小鼠2-4细胞胚胎细胞骨架结构的影响。方法用抗骨架蛋白 actin 的单克隆抗体进行免疫染色,激光共聚焦显微镜观察经过慢速冷冻小鼠2-细胞、4-细胞胚胎的细胞骨架结构的状况。结果以丙二醇为冷冻保护剂,采用慢速冷冻-快速解冻的方式冷冻小鼠2-细胞、4-细胞胚胎对胚胎细胞骨架结构的分布特点没有明显影响,荧光强度定量分析表明冷冻胚胎荧光强度与未冷冻胚胎的荧光强度相比,差异无统计学意义(P >0.05)。结论冷冻对小鼠2-细胞和4-细胞胚胎的细胞骨架结构没有明显影响。%Objective TO investigate the effects Of cryOPreservatiOn On the cytOskeletOn Of 2 - 4cell mOuse embryOs. Methods ImmunOhistOchemical staining was used tO assess the status Of skeletOn PrOtein actin Of mOuse embryOs. The cytOskeletOn Of mOuse cryOPreserved embryOs was Observed by laser - cOnfOcal micrOscOPe. Results The slOwing - freezing and fast - thawing PrOtOcOl with PROH as cryOPrOtectant had little effect On the distributing characteristic Of cytOskeletOn Of 2 - cell,4 - cell mOuse embryOs,and had little effect On the fluOrescent intensity by fluOrescence quantitative analysis,with nO significant differences(P > 0. 05). Conclusion CryOPreservatiOn had little effect On the cytOskeletOn Of 2 - 4 cell mOuse embryOs.

  16. Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

    Directory of Open Access Journals (Sweden)

    Xinyu Liu

    Full Text Available Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF failure due to poor embryo quality.

  17. Low cost labeling with highlighter ink efficiently visualizes developing blood vessels in avian and mouse embryos.

    Science.gov (United States)

    Takase, Yuta; Tadokoro, Ryosuke; Takahashi, Yoshiko

    2013-12-01

    To understand how blood vessels form to establish the intricate network during vertebrate development, it is helpful if one can visualize the vasculature in embryos. We here describe a novel labeling method using highlighter ink, easily obtained in stationery stores with a low cost, to visualize embryo-wide vasculatures in avian and mice. We tested 50 different highlighters for fluorescent microscopy with filter sets equipped in a standard fluorescent microscope. The yellow and violet inks yielded fluorescent signals specifically detected by the filters used for green fluorescent protein (GFP) and red fluorescent protein (RFP) detections, respectively. When the ink solution was infused into chicken/quail and mouse embryos, vasculatures including large vessels and capillaries were labeled both in living and fixed embryos. Ink-infused embryos were further subjected to histological sections, and double stained with antibodies including QH-1 (quail), α smooth muscle actin (αSMA), and PECAM-1 (mouse), revealing that the endothelial cells were specifically labeled by the infused highlighter ink. Highlighter-labeled signals were detected with a resolution comparable to or higher than signals of fluorescein isothiocyanate (FITC)-lectin and Rhodamine-dextran, conventionally used for angiography. Furthermore, macroconfocal microscopic analyses with ink-infused embryos visualized fine vascular structures of both embryo proper and extra-embryonic plexus in a Z-stack image of 2400 μm thick with a markedly high resolution. Together, the low cost highlighter ink serves as an alternative reagent useful for visualization of blood vessels in developing avian and mouse embryos and possibly in other animals.

  18. Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro.

    Science.gov (United States)

    Kelley, Rebecca L; Gardner, David K

    2016-11-01

    Embryos are routinely cultured individually, although this can reduce blastocyst development. Culture in atmospheric (20%) oxygen is also common, despite multiple detrimental effects on embryos. Although frequently occurring together, the consequences of this combination are unknown. Mouse embryos were cultured individually or grouped, under physiological (5%) or atmospheric (20%) oxygen. Embryos were assessed by time-lapse and blastocyst cell allocation. Compared with the control group (5% oxygen group culture), 5-cell cleavage (t5) was delayed in 5% oxygen individual culture and 20% oxygen group culture (59.91 ± 0.23, 60.70 ± 0.29, 63.06 ± 0.32 h post-HCG respectively, P culture were delayed earlier (3-cell cleavage), and at t5 cleaved later than embryos in other treatments (66.01 ± 0.40 h, P culture and 20% oxygen group culture (134.1 ± 3.4, 104.5 ± 3.2, 73.4 ± 2.2 cells, P culture (57.0 ± 2.8 cells, P culture and 20% oxygen is detrimental to embryo development.

  19. Mouse endometrial stromal cells produce basement-membrane components

    DEFF Research Database (Denmark)

    Wewer, U M; Damjanov, A; Weiss, J;

    1986-01-01

    During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The dec...

  20. Efficient blastomere biopsy for mouse embryo splitting for future applications in human assisted reproduction.

    Science.gov (United States)

    Illmensee, K; Kaskar, K; Zavos, P M

    2005-12-01

    The objective of the current study was to establish a safe, efficient biopsy procedure for embryo splitting using the mouse model for future applications in human assisted reproduction. From mouse embryos at the 2-, 4-, 6- and 8-cell stage, half the number of blastomeres were microsurgically biopsied and transferred into empty mouse zonae pellucidae. Twin embryonic development was monitored during in-vitro culture. Blastocyst developmental rate using 2-, 4-, 6-, and 8-cell splitting was 74.4, 75.0, 66.7 and 38.4 respectively, with corresponding hatching rates of 94.9, 97.5, 92.7 and 83.8%. Blastocysts from 2-, 4-, and 6-cell splitting resulted in elevated hatching rates compared with non-operated blastocysts (87.5%), due to the Tyrode-assisted hatching effect. Blastocyst morphology was superior from 2- and 4-cell splitting when compared with 6- and 8-cell splitting. Furthermore, outgrowth of twin blastocysts from 2- and 4-cell splitting showed well-developed colonies with trophoblast cells and clusters of ICM cells, whereas those obtained from 6- and 8-cell splitting frequently formed small-sized colonies. Due to the high twinning success rate obtained under the experimental conditions employed in this study, it appears that with further modifications and proper safeguards, such embryo splitting efforts could have potential applications in humans.

  1. 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.

  2. 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.

  3. Effect of washing mineral oil on development of mouse embryos in vitro and in vivo after embryo transfer

    Institute of Scientific and Technical Information of China (English)

    Li Hui; Zhang Li-xuan; Zhong Yu; Zhu Kai; Zhang Tian; Wang Min-kang

    2008-01-01

    Objective:To establish a simple and effective washing procedure for both used and purchased mineral oil,that can be used for embryo culture.Methods:A complete test system has been used for this purpose.There are 3 steps in our new washing proto-col.First,the oil was mixed with 95% ethanol at 1:1,the bottle being shaken by hand for 10 minutes,then sepa-rated.Second,the oil was heated to boiling point with 0.31 mol/L NaCl for 30 minutes.Third,anhydrous Na:SO4 was put into the oil for further treatment.1-cell stage embryos of a KM strain mouse have been collected surgically and cultured.Cleavage and blastocyst stage development were recorded and some embryos were transferred into re-cipients.Results:The results show that recycled oil can promote the development from 2-cell to blastocyst stage(23.3%)when compared with that of control(16.9%).Offspring have been obtained at 44 %(7/16),16 %(3/19)from washed recycled oil and control oil respectively.Conclusion:This washing procedure is safe and effective for the used treatment and for other sources of mineral oil used for embryo culture.

  4. [Production and characterization of some mouse embryo cellular substrates in vitro].

    Science.gov (United States)

    Voiculescu, C; Stoian, I; Nachtigal, S; Gaicu, N; Duldurescu, D; Palade, V

    1976-01-01

    Cell cultures with a different multiplication potential in vitro, depending upon the strain source used, were obtained from mouse embryos belonging to the CVA, C57 Black A2G and Swiss strains. Only the Swiss 12 culture underwent spontaneous transformation and was carried through more than 50 passages in vitro. The Swiss-12 substrate proved not to be contaminated either by viruses or micoplasma. It is less sensitive than other elective cell substrates to infection with attenuated polioviruses, cytopathogenic Coxsackie A9 virus and vaccinia virus, but its sensitivity to infection with Herpes simplex type 1 virus is similar to that of human embryo fibroblasts. After a high number of passages the Swiss-12 substrate permits, in comparison to other cell substrates (human heteroploid Hep-2 line, human embryo fibroblasts), a highly efficient qualitative differentiation between the growth media and calf serum.

  5. Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching.

    Science.gov (United States)

    Erbach, Gregory T; Biggers, John D; Manning, Peter C; Nowak, Romana A

    2013-08-01

    To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development. Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy. PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos' outer surface. PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.

  6. Relevance of LIF and EGF on Mouse Preimplantation Embryo Development

    Directory of Open Access Journals (Sweden)

    Iraj Amiri

    2008-01-01

    Full Text Available Objective: Recent evidence suggests that Leukemia Inhibitory Factor (LIF, a member ofinterleukin-6 family, has biological actions on preimplantation embryo development. Alsoit is established that Epidermal Growth Factor (EGF, a strong mitosis-promoting agent,improves the preimplantation embryo development by increasing the cell metabolism andproliferation. The purpose of the present study is to investigate the effects of these factors,alone and in combination together, on preimplantation and development of the embryo.Materials and Methods: Six to eight weeks old NMRI mice were super ovulated by injectionof 10IU PMSG and 10IU hCG, then the mated mice were killed 46 hours later. Theiroviducts were flushed, two-cell embryos collected and divided randomly to the four groupsas following: Control, treatment 1 (LIF, treatment 2 (EGF, treatment 3 (LIF+EGF. In eachgroup, the embryos were cultured in an incubator at 37°C with 5% CO2 and 90% humidityfor 72hrs. The state of embryo development was evaluated in 24,36,48,60 and 72hrsfollowing the embryos cultures. By the end of the cultures, cell apoptosis was studiedby the terminal deoxynucleotidyl transferas-mediated dUTP nick end-labeling (TUNELtechnique.Results: Significant difference was detected in the rate of hatching in the LIF and LIF+EGFgroups. This difference was also seen in the rate of blastocyst formation after 36hrs(p<0.05 and in the average of the total cell number (p<0.05 after 72hrs. In comparison tothe apoptotic index, there was no significant difference between the control and treatmentgroups.Conclusion: The findings in this study show a beneficial effect of LIF and EGF on theblastocyst formation, hatching and its total cell numbers in vitro.

  7. Rapid and simple method for in vivo ex utero development of mouse embryo explants.

    Science.gov (United States)

    Gonçalves, André B; Thorsteinsdóttir, Sólveig; Deries, Marianne

    2016-01-01

    The in utero development of mammals drastically reduces the accessibility of the mammalian embryo and therefore limits the range of experimental manipulation that can be done to study functions of genes or signaling pathways during embryo development. Over the past decades, tissue and organ-like culture methods have been developed with the intention of reproducing in vivo situations. Developing accessible and simple techniques to study and manipulate embryos is an everlasting challenge. Herein, we describe a reliable and quick technique to culture mid-gestation explanted mouse embryos on top of a floating membrane filter in a defined medium. Viability of the cultured tissues was assessed by apoptosis and proliferation analysis showing that cell proliferation is normal and there is only a slight increase in apoptosis after 12h of culture compared to embryos developing in utero. Moreover, differentiation and morphogenesis proceed normally as assessed by 3D imaging of the transformation of the myotome into deep back muscles. Not only does muscle cell differentiation occur as expected, but so do extracellular matrix organization and the characteristic splitting of the myotome into the three epaxial muscle groups. Our culture method allows for the culture and manipulation of mammalian embryo explants in a very efficient way, and it permits the manipulation of in vivo developmental events in a controlled environment. Explants grown under these ex utero conditions simulate real developmental events that occur in utero.

  8. The expression and function of VEGF at embryo implanta- tion "window" in the mouse

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays a critical role in angiogenesis. Recent reports indicated that VEGF was closely involved in embryo implantation and embryonic vasculogenesis. However, very little information is available about the detailed expression and function of VEGF at implantation "window". In this work, VEGFs were primarily present on uterine epithelial cell monolayer and blastocysts including the outgrew trophoblasts at implantation window. VEGF antibodies decreased the number of mice embryos implanted and the percentage of blastocysts with attachment and outgrowth in a co-culture model in a dose-dependant manner. These findings demonstrate that VEGF is one of the essential cytokines for embryo implantation in mouse. VEGF may act as a local mediator to regulate the maternal-fetal interaction, and facilitate blastocyst implantation.

  9. Antiteratogenic Effects of β-Carotene in Cultured Mouse Embryos Exposed to Nicotine

    Directory of Open Access Journals (Sweden)

    Chunmei Lin

    2013-01-01

    Full Text Available After maternal intake, nicotine crosses the placental barrier and causes severe embryonic disorders and fetal death. In this study, we investigated whether β-carotene has a beneficial effect against nicotine-induced teratogenesis in mouse embryos (embryonic day 8.5 cultured for 48 h in a whole embryo culture system. Embryos exposed to nicotine (1 mM exhibited severe morphological anomalies and apoptotic cell death, as well as increased levels of TNF-α, IL-1β, and caspase 3 mRNAs, and lipid peroxidation. The levels of cytoplasmic superoxide dismutase (SOD, mitochondrial manganese-dependent SOD, cytosolic glutathione peroxidase (GPx, phospholipid hydroperoxide GPx, hypoxia inducible factor 1α, and Bcl-xL mRNAs decreased, and SOD activity was reduced compared to the control group. However, when β-carotene (1×10−7 or 5×10−7μM was present in cultures of embryos exposed to nicotine, these parameters improved significantly. These findings indicate that β-carotene effectively protects against nicotine-induced teratogenesis in mouse embryos through its antioxidative, antiapoptotic, and anti-inflammatory activities.

  10. Primary cilia are not required for normal canonical Wnt signaling in the mouse embryo.

    Directory of Open Access Journals (Sweden)

    Polloneal Jymmiel R Ocbina

    Full Text Available Sonic hedgehog (Shh signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a or IFT complex B proteins (Ift172 or Ift88. We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1. The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.

  11. Development of mouse embryos cryopreserved by an ultra-rapid method of freezing.

    Science.gov (United States)

    Wilson, L; Quinn, P

    1989-01-01

    High concentrations of cryoprotectant combined with sucrose were utilized in an ultra-rapid freezing protocol for mouse preimplantation embryos. Dimethylsulphoxide (DMSO, 1.5 or 3.5 M) or propanediol (PROH, 1.5 or 3.0 M) combined with 0.25 M sucrose were used as freezing solutions. One-, 2- or 8-cell embryos were placed directly into these solutions at room temperature, loaded into straws and plunged into liquid nitrogen within 2-3 min. The straws were rapidly thawed and the embryos expelled into the solution in which they were frozen for 10 min. The cryoprotectants were then removed by single- or multi-step dilution. Survival and development of the embryos in vitro and in vivo were assessed. DMSO (1.5 M) and both concentrations of PROH were totally inadequate as a cryoprotectant in this freezing protocol. A concentration of 3.5 M DMSO gave high survival and development rates when a multi-step dilution procedure was used, but not with a single-step dilution. One-cell embryos gave 71% survival, 35% in-vitro development and 10% in-vivo viability; 2-cell embryos showed 87% survival, 77% in-vitro development and 66% in-vivo viability; and 8-cell embryos showed 97% survival, 87% in-vitro development and 62% in-vivo viability. The results for the 2- and 8-cell stages compared favourably with non-frozen controls, which had 71% in-vivo viability. This method of cryopreservation is therefore fast and viable.

  12. Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

    National Research Council Canada - National Science Library

    Chung, Young Gie; Mann, Mellissa R W; Bartolomei, Marisa S; Latham, Keith E

    2002-01-01

    .... Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined...

  13. Nuclear-Cytoplasmic “Tug of War” During Cloning: Effects of Somatic Cell Nuclei on Culture Medium Preferences of Preimplantation Cloned Mouse Embryos1

    National Research Council Canada - National Science Library

    Young Gie Chung; Mellissa R. W. Mann; Marisa S. Bartolomei; Keith E. Latham

    2002-01-01

    .... Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined...

  14. 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...

  15. 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.

  16. Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro

    Directory of Open Access Journals (Sweden)

    O'Neill Chris

    2010-06-01

    Full Text Available Abstract This study investigates the suitability of surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF and electrospray ionization (ESI mass spectrometry for analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10 protein chips detected a protein peak at m/z ~8570 released by both C57BL6 and hybrid embryos. No other peaks unique to the presence of the embryo were identified with this method. ESI mass spectrometry of tryptic digests of embryo-conditioned media identified a total of 20 proteins released during development from the zygote to blastocyst stage. Four proteins were expressed in at least 7 out of 8 cultures tested, one of these (lactate dehydrogenase B was in all cultures. A further five proteins were in at least half of the cultures and 11 more proteins were in at least one culture. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 and peptidyl arginine deiminase, type VI. A further four proteins detected have roles in redox regulation of cells, and three others are capable of inducing post-translational modifications of proteins. This study shows the feasibility of ESI mass spectrometry for identifying the proteins secreted by the preimplantation embryo in vitro. This analysis identifies a range of targets that now require detailed functional analysis to assess whether their release by the embryo is an important property of early embryo development.

  17. The effect of peritoneal fluid from patients with endometriosis on mitochondrial function and development of early mouse embryos.

    Directory of Open Access Journals (Sweden)

    Jing Shu

    Full Text Available BACKGROUND: Peritoneal fluid (PF from patients with endometriosis can inhibit early embryo development via probable functional changes of embryo mitochondria in the early stage of embryo development. The purpose of this study was to determine the effect of PF from patients with endometriosis on mitochondrial function and development of early mouse embryos. METHODOLOGY/PRINCIPAL FINDINGS: PF was collected from patients with infertility and endometriosis, infertility due to tubal factors, and normal control subjects, and the level of NO was measured. Early murine embryos were then cultured with PF from normal control subjects, those with endometriosis, and with human tubal fluid (HTF, respectively. Cleavage and blastulation rates, mitochondrial DNA (mtDNA copy numbers, adenosine triphosphate (ATP level, and mitochondrial membrane potential (ΔΨm of the different groups were compared. The NO level in the PF of patients with endometriosis was significantly greater than in those without endometriosis and control patients. The embryos cultures with PF from patients with endometriosis had a lower cleavage rate and blastulation rate, and higher ATP and ΔΨm level at the 2- and 4-cell stages. No significant difference was found in mtDNA copies among the 3 groups. CONCLUSIONS/SIGNIFICANCE: PF from patients with endometriosis can inhibit early embryo development via probable functional changes of embryo mitochondria in the early stage of embryo development. Understanding the effects of PF on embryo development may assist in developing new methods of treatment for infertility.

  18. Alterations of intraembryonic metabolites in preimplantation mouse embryos exposed to elevated concentrations of glucose: a metabolic explanation for the developmental retardation seen in preimplantation embryos from diabetic animals.

    Science.gov (United States)

    Moley, K H; Chi, M M; Manchester, J K; McDougal, D B; Lowry, O H

    1996-06-01

    Preimplantation mouse embryos exposed to hyperglycemia, whether in vivo or in vitro, experience delayed development from the 2-cell to blastocyst stage. By comparing metabolites from embryos exposed to high vs. normal glucose conditions, a metabolic explanation for the delayed growth pattern was sought. Fertilized 1-cell embryos obtained from superovulated B5 x CBA F1 mice were cultured for 96 h in medium containing 2.8 mM glucose (C) or in medium with added glucose to give 10 mM, 30 mM, or 52 mM glucose (HG). After incubation, each embryo was quick-frozen and freeze-dried. Metabolites were assayed by the ultramicrofluorometric technique and enzymatic cycling to obtain measurable levels in single embryos. Embryos cultured in HG exhibited 7-fold higher intracellular glucose levels than those cultured in C (C: 2.25 +/- 0.6 vs. HG: 16.61 +/- 2.4 mmol/kg wet weight; p Krebs cycle metabolites are elevated and correspond to the degree of developmental delay. These findings suggest that a metabolic abnormality may be responsible for retarded development experienced by embryos exposed to high glucose.

  19. A microfluidic system supports single mouse embryo culture leading to full-term development

    NARCIS (Netherlands)

    Esteves, Telma Cristina; Rossem, van Fleur; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele; Le Gac, Séverine

    2013-01-01

    The present study demonstrates the feasibility of application of a microfluidic system for in vitro culture of pre-implantation mouse embryos, with subsequent development to full-term upon embryo transfer. Specifically, embryos cultured in groups in nL volume chambers achieve pre-implantation develo

  20. Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos

    Science.gov (United States)

    Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mous...

  1. Effect of Intracytoplasmic Sperm Injection (ICSI on Mouse Embryos Preimplantational Development

    Directory of Open Access Journals (Sweden)

    Claudia Cârstea

    2012-05-01

    Full Text Available It is known that the in vitro culture (IVC of preimplantation embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared the development of mouse blastocysts produced by intracytoplasmic sperm injection (ICSI versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC. At the end of cultivation (96 hrs for blastocyst stage embryos, expanded blastocysts of each group were randomly selected, and ICM and total cells number were differentially stained. The total cell number of blastocysts was estimated by counting the total number of nuclei using DAPI staining. Cell number for inner cell mass (ICM was estimated by counting the OCT4 (POU5FL positive cells. Digitally recombined, composite images were analyzed using the Zeiss Axion Vision software and Zeiss Apotome. All 5–10 optical sections were divided using a standard grid over each layer to count all. Comparing the total cells and the ICM cells number, it appears that each method of fertilization has a unique pattern development. The developmental rate and the total cell number of the blastocyst were significantly lower in ICSI versus in vivo fertilized embryos which affect the embryonic developmental rate and the total cell number of blastocysts.

  2. 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

  3. The expression profile of microRNAs in mouse embryos.

    Science.gov (United States)

    Mineno, Junichi; Okamoto, Sachiko; Ando, Tatsuya; Sato, Masahiro; Chono, Hideto; Izu, Hiroyuki; Takayama, Masanori; Asada, Kiyozo; Mirochnitchenko, Oleg; Inouye, Masayori; Kato, Ikunoshin

    2006-01-01

    MicroRNAs (miRNAs), which are non-coding RNAs 18-25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering approximately 80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

  4. 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)

  5. Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ping [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Wang, Ningling [Department of Assisted Reproduction, Shanghai Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Lin, Xianhua; Jin, Li [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Xu, Hong, E-mail: xuhong1168@126.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Li, Rong [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Huang, Hefeng, E-mail: huanghefg@hotmail.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China)

    2016-02-26

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.

  6. The effects of MRI on mouse embryos during fetal stage

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Takashi; Sakazaki, Takahiko; Itokawa, Yuka [Suzuka University of Medical Science, Koriyama (Japan)] (and others)

    2006-06-15

    The effects of Magnetic Resonance Imaging (MRI) on mouse embryos at the early stage of organogenesis were investigated. Pregnant ICR mice were exposed on day 8 of gestation to MRI at 0.5 T for 0.5 hour to 3 hours. The mortality rates of embryos or fetuses, the incidence of external malformations, fetal body weight and sex ratio were observed at day 18 of gestation. A significant increase in embryonic mortality was observed after exposure to either 0.5 T MRI for 0.5 hour or 2 hours. However, the exposure to MRI for 1 hour or 3 hours did not induce any significant increase in embryonic mortality when compared with control. External malformations such as exencephaly, cleft palate and anomalies of tail were observed in all experimental groups exposed to each MRI. A statistically significant increase of external malformations was observed in all groups treated with 0.5 T MRI for 0.5 hour and 3 hours. The incidence of external malformations in the mice group exposed to 0.5 T MRI for 0.5-hour was found to be higher than those of mice group exposed to 0.5 T MRI for 2 hours. The effects of MRI on the external malformations might not to be dose-dependent. There was no statistically significant difference in fetal body weight and sex ratio among each MRI exposure groups.

  7. Post-Transcriptional Control of Gene Expression in Mouse Early Embryo Development: A View from the Tip of the Iceberg

    Directory of Open Access Journals (Sweden)

    Claudio Sette

    2011-04-01

    Full Text Available Fertilization is a very complex biological process that requires the perfect cooperation between two highly specialized cells: the male and female gametes. The oocyte provides the physical space where this process takes place, most of the energetic need, and half of the genetic contribution. The spermatozoon mostly contributes the other half of the chromosomes and it is specialized to reach and to penetrate the oocyte. Notably, the mouse oocyte and early embryo are transcriptionally inactive. Hence, they fully depend on the maternal mRNAs and proteins stored during oocyte maturation to drive the onset of development. The new embryo develops autonomously around the four-cell stage, when maternal supplies are exhausted and the zygotic genome is activated in mice. This oocyte-to-embryo transition needs an efficient and tightly regulated translation of the maternally-inherited mRNAs, which likely contributes to embryonic genome activation. Full understanding of post-transcriptional regulation of gene expression in early embryos is crucial to understand the reprogramming of the embryonic genome, it might help driving reprogramming of stem cells in vitro and will likely improve in vitro culturing of mammalian embryos for assisted reproduction. Nevertheless, the knowledge of the mechanism(s underlying this fundamental step in embryogenesis is still scarce, especially if compared to other model organisms. We will review here the current knowledge on the post-transcriptional control of gene expression in mouse early embryos and discuss some of the unanswered questions concerning this fascinating field of biology.

  8. Expression of chemokine receptors CCR3,CCR5 and CXCR3 on CD4+ T cells in CBA/JxDBA/2 mouse model,selectively induced by IL-4 and IL-10,regulates the embryo resorption rate

    Institute of Scientific and Technical Information of China (English)

    JIANG Pei-juan; ZHAO Ai-min; BAO Shi-min; XIAO Shi-jin; XIONG Miao

    2009-01-01

    Background Chemokines and their receptors have been a research focus in transplantation immunology.Chemokines and their receptors play a role in lymphocyte recruitment and differentiation process.This study aimed to observe whether IL-4 and IL-10 may regulate the expression of chemokine receptors CCR3,CCR5 and CXCR3 on CD4+ T cells in CBA/JxDBA/2 mouse model and to explore the role of CCR3,CCR5,CXCR3 in immune tolerance in pregnancy.Methods The mouse model of spontaneous abortion (CBA/JxDBA/2) and the normal pregnant mouse model (CBA/JxBALB/c) were used.CBA/JxDBA/2 mice were injected with IL-4 (CBA/JxDBA/2-1L-4),IL-4 and IL-10 (CBA/JxDBA/2-1L-4+IL-10),or normal saline (CBA/JxDBA/2-NS) as a control.The expression of CCR3,CCR5 and CXCR3 on CD4+ T cells from mouse peripheral blood was measured by the double-labelled FCM method,and the embryo resorption rate was also examined.Results The embryo resorption rate in the CBA/JxDBA/2 group without any treatment was significantly higher than that in the CBA/JxBALB/c group (17.9% vs 3.7%,P<0.01).The embryo resorption rate in the CBA/JxDBA/2 group immunized with IL-4 or IL-4 together with IL-10 was significantly decreased,compared with that in the control and NS groups respectively.CCR3 expression on CD4+ T cells in the CBA/JxDBA/2 group without any treatment was significantly lower than that in the CBA/JxBALB/c group (0.3738±0.3575 vs 1.2190±0.2772,P<0.01 );both CCR5 (3.0900±1.5603 vs 1.2390±0.6361,P <0.01)and CXCR3 (2.4715±0.9074 vs 0.9200±0.5585,P <0.01 ) expressions on CD4+ T cells of the CBA/JxDBA/2 group without any treatment were significantly higher than those of the CBA/JxBALB/c group.Significant up-regulation of CCR3 and down-regulation of CXCR3 were found in the CBA/JxDBA/2 group treated with IL-4 (CCR3:2.0360±0.6944,CXCR3:1.3510±0.5263,P <0.01) or IL-4 and IL-10 (CCR3:1.8160±1.0947,CXCR3:1.0940±0.7168,P<0.01).Because of the CCR5,IL-4 and IL-10 (1.9400±0.8504 vs 3.0900±1.5603,P <0.05),but

  9. Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro.

    Science.gov (United States)

    Ishiwata, Isamu; Tamagawa, Tomoharu; Tokieda, Yuko; Iguchi, Megumi; Sato, Kahei; Ishikawa, Hiroshi

    2003-03-01

    Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.

  10. Lin28a is dormant, functional, and dispensable during mouse oocyte-to-embryo transition.

    Science.gov (United States)

    Flemr, Matyas; Moravec, Martin; Libova, Veronika; Sedlacek, Radislav; Svoboda, Petr

    2014-06-01

    The oocyte-to-embryo transition (OET) denotes transformation of a highly differentiated oocyte into totipotent blastomeres of the early mammalian embryo. OET depends exclusively on maternal RNAs and proteins accumulated during oocyte growth, which implies importance of post-transcriptional control of gene expression. OET includes replacement of abundant maternal microRNAs (miRNAs), enriched also in differentiated cells and exemplified by the Let-7 family, with embryonic miRNAs common in pluripotent stem cells (the miR-290 family in the mouse). Lin28a and its homolog Lin28b encode RNA-binding proteins, which interfere with Let-7 maturation and facilitate reprogramming of induced pluripotent stem cells. Both Lin28a and Lin28b transcripts are abundant in mouse oocytes. To test the role of maternal expression of Lin28a and Lin28b during oocyte-to-zygote reprogramming, we generated mice with oocyte-specific knockdown of both genes by using transgenic RNA interference. Lin28a and Lin28b are dispensable during oocyte growth because their knockdown has no effect on Let-7a levels in fully grown germinal vesicle (GV)-intact oocytes. Furthermore, transgenic females were fertile and produced healthy offspring, and their overall breeding performance was comparable to that of wild-type mice. At the same time, 2-cell embryos derived from transgenic females showed up-regulation of mature Let-7, suggesting that maternally provided LIN28A and LIN28B function during zygotic genome activation. Consistent with this conclusion is increased translation of Lin28a transcripts upon resumption of meiosis. Our data imply dual repression of Let-7 during OET in the mouse model, the selective suppression of Let-7 biogenesis by Lin28 homologs superimposed on previously reported global suppression of miRNA activity. © 2014 by the Society for the Study of Reproduction, Inc.

  11. 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.

  12. Micronucleus formation causes perpetual unilateral chromosome inheritance in mouse embryos.

    Science.gov (United States)

    Vázquez-Diez, Cayetana; Yamagata, Kazuo; Trivedi, Shardul; Haverfield, Jenna; FitzHarris, Greg

    2016-01-19

    Chromosome segregation defects in cancer cells lead to encapsulation of chromosomes in micronuclei (MN), small nucleus-like structures within which dangerous DNA rearrangements termed chromothripsis can occur. Here we uncover a strikingly different consequence of MN formation in preimplantation development. We find that chromosomes from within MN become damaged and fail to support a functional kinetochore. MN are therefore not segregated, but are instead inherited by one of the two daughter cells. We find that the same MN can be inherited several times without rejoining the principal nucleus and without altering the kinetics of cell divisions. MN motion is passive, resulting in an even distribution of MN across the first two cell lineages. We propose that perpetual unilateral MN inheritance constitutes an unexpected mode of chromosome missegregation, which could contribute to the high frequency of aneuploid cells in mammalian embryos, but simultaneously may serve to insulate the early embryonic genome from chromothripsis.

  13. Deferoxamine immobilized poly(D,L-lactide) membrane via polydopamine adhesive coating: The influence on mouse embryo osteoblast precursor cells and human umbilical vein endothelial cells.

    Science.gov (United States)

    Li, Huihua; Luo, Binghong; Wen, Wei; Zhou, Changren; Tian, Lingling; Ramakrishna, Seeram

    2017-01-01

    Osteogenesis and angiogenesis play the prominent role in the bone regeneration. In this study, deferoxamine (DFO), an induced agent for osteogenesis and angiogenesis, was modified onto the surface of poly(D,L-lactide) (PDLLA) membrane via a facile and convenient approach based on the self-polymerization of dopamine (DOPA). The surface composition, morphology, hydrophilicity and surface energy of the original and modified PDLLA membranes were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electronic microscopy (SEM), atomic force microscopy (AFM) and contact angle measurement. The surface roughness and hydrophilicity of the PDLLA membrane were obviously increased by introducing either the single polydopamine (PDOPA) or the dual layers of PDOPA and DFO. In vitro cells culture experiments indicated that both the PDLLA/PDOPA and PDLLA/PDOPA-DFO composite membranes were more beneficial to the attachment, proliferation and spreading of MC3T3-E1 cells and HUVECs compared to the original PDLLA membrane. The PDLLA/PDOPA-DFO membrane was supportive for the proliferation of both MC3T3-E1 cells and HUVECs, and especially for HUVECs. The results suggested that the as-prepared PDLLA/PDOPA-DFO composite can be expected to be used as a promising bone regenerative material with promoted angiogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides.

    Science.gov (United States)

    Wefers, Benedikt; Meyer, Melanie; Ortiz, Oskar; Hrabé de Angelis, Martin; Hansen, Jens; Wurst, Wolfgang; Kühn, Ralf

    2013-03-05

    The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.

  15. Virtual histology of transgenic mouse embryos for high-throughput phenotyping.

    Directory of Open Access Journals (Sweden)

    John T Johnson

    2006-04-01

    Full Text Available A bold new effort to disrupt every gene in the mouse genome necessitates systematic, interdisciplinary approaches to analyzing patterning defects in the mouse embryo. We present a novel, rapid, and inexpensive method for obtaining high-resolution virtual histology for phenotypic assessment of mouse embryos. Using osmium tetroxide to differentially stain tissues followed by volumetric X-ray computed tomography to image whole embryos, isometric resolutions of 27 mum or 8 mum were achieved with scan times of 2 h or 12 h, respectively, using mid-gestation E9.5-E12.5 embryos. The datasets generated by this method are immediately amenable to state-of-the-art computational methods of organ patterning analysis. This technique to assess embryo anatomy represents a significant improvement in resolution, time, and expense for the quantitative, three-dimensional analysis of developmental patterning defects attributed to genetically engineered mutations and chemically induced embryotoxicity.

  16. Mouse Leydig Tumor Cells

    Directory of Open Access Journals (Sweden)

    Bo-Syong Pan

    2011-01-01

    Full Text Available Cordycepin is a natural pure compound extracted from Cordyceps sinensis (CS. We have demonstrated that CS stimulates steroidogenesis in primary mouse Leydig cell and activates apoptosis in MA-10 mouse Leydig tumor cells. It is highly possible that cordycepin is the main component in CS modulating Leydig cell functions. Thus, our aim was to investigate the steroidogenic and apoptotic effects with potential mechanism of cordycepin on MA-10 mouse Leydig tumor cells. Results showed that cordycepin significantly stimulated progesterone production in dose- and time-dependent manners. Adenosine receptor (AR subtype agonists were further used to treat MA-10 cells, showing that A1, A 2A , A 2B , and A3, AR agonists could stimulate progesterone production. However, StAR promoter activity and protein expression remained of no difference among all cordycepin treatments, suggesting that cordycepin might activate AR, but not stimulated StAR protein to regulate MA-10 cell steroidogenesis. Meanwhile, cordycepin could also induce apoptotic cell death in MA-10 cells. Moreover, four AR subtype agonists induced cell death in a dose-dependent manner, and four AR subtype antagonists could all rescue cell death under cordycepin treatment in MA-10 cells. In conclusion, cordycepin could activate adenosine subtype receptors and simultaneously induce steroidogenesis and apoptosis in MA-10 mouse Leydig tumor cells.

  17. Culture and identification of neural stem cell derived from auditory cortex of embryo mouse%胚胎小鼠听皮层区域神经干细胞的分离培养及鉴定

    Institute of Scientific and Technical Information of China (English)

    任红苗; 王宜南; 陈继川; 吴晓平; 张波; 刘媛; 张世昌

    2011-01-01

    目的 通过剖腹手术从C57BL/6胎鼠(El4左右)中获得听皮层区域组织,进行神经干细胞体外培养和分化鉴定,探索新的自体NSC移植治疗来源.方法 选取孕14天左右的C57BL/6小鼠,剖腹取出胎鼠,分离听皮层区域组织,在体外无血清培养得到NSC,用免疫荧光细胞染色法进行NSC特异性标记物(Nestin)、增殖能力(BrdU)及多向分化潜能的鉴定.结果 听皮层区域组织在体外通过无血清培养能够得到大量细胞球,经Nestin及Brdu鉴定为NSC球且具有较高增殖能力.NSC球在体外分化后产生β微管蛋白(β-TubulinⅢ)阳性的神经元以及胶质纤维酸性蛋白(GFAP)阳性的星形胶质细胞.结论 胎鼠听皮层区域在体外无血清培养可获得大量具有增殖能力和多向分化潜能NSCs,是自体NSCs移植治疗新的种子来源.%Objective To culture and identify the neural stem cells (NSCs) derived from auditory cortex in C57BI7 6 embryo mouse (E14 days) by laparotomy and search for a new source of NSCs for self-transplantation therapy. Methods Auditory cortex was obtained from embryo mouse acquired from C57BL/6 pregnancy mouse (E14days) by laparotomy. NSCs were isolated from auditory cortex and cultured in serum-free medium in vitro. Fluorescence immunocytochemis-try was carried out to examine the expression of NSCs marker (nestin), self-proliferation (Brdu), and the multipotential of differentiation. Results A large number of NSCs examined by Nestin could be harvested from Al in vitro in serum-free media. They expressed Brdu, showing their potential of self-proliferation. They were also able to differentiate into β-Tubu-lin Ⅲ positive neurons and GFAP positive astrocytes. Conclusion NSCs can be obtained from Al of embryo mouse by in vitro serum-free culture, with potentials of self-proliferation and multipotential of differentiation. This can be applied to self-transplantation therapy as a new source of NSCs.

  18. ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses

    NARCIS (Netherlands)

    Schwarzer, Caroline; Esteves, Telma Cristina; Arau´zo-Bravo, Marcos J.; Le Gac, Séverine; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele

    2012-01-01

    Do different human ART culture protocols prepare embryos differently for post-implantation development? ... Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the

  19. Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development.

    Science.gov (United States)

    Bulusu, Vinay; Prior, Nicole; Snaebjornsson, Marteinn T; Kuehne, Andreas; Sonnen, Katharina F; Kress, Jana; Stein, Frank; Schultz, Carsten; Sauer, Uwe; Aulehla, Alexander

    2017-02-27

    How metabolism is rewired during embryonic development is still largely unknown, as it remains a major technical challenge to resolve metabolic activities or metabolite levels with spatiotemporal resolution. Here, we investigated metabolic changes during development of organogenesis-stage mouse embryos, focusing on the presomitic mesoderm (PSM). We measured glycolytic labeling kinetics from (13)C-glucose tracing experiments and detected elevated glycolysis in the posterior, more undifferentiated PSM. We found evidence that the spatial metabolic differences are functionally relevant during PSM development. To enable real-time quantification of a glycolytic metabolite with spatiotemporal resolution, we generated a pyruvate FRET-sensor reporter mouse line. We revealed dynamic changes in cytosolic pyruvate levels as cells transit toward a more anterior PSM state. Combined, our approach identifies a gradient of glycolytic activity across the PSM, and we provide evidence that these spatiotemporal metabolic changes are intrinsically linked to PSM development and differentiation.

  20. EDS-40玻璃化液冻存小鼠4细胞胚胎的研究%Study on the cryopreservation of mouses 4 cell embryo by the EDS-40 vitrification

    Institute of Scientific and Technical Information of China (English)

    鲁栋梁; 崔曙; 陈在贤

    2012-01-01

    Objective To study the cryoprotective effect of the solution of EDS-40 on mouse 4 cell embryo . Methods First test the verifying effect of EDS-40 and EFS-40 solutions, Then random chose 4 cell embryos of female mice to vitrify in EFS-40 or EDS-40 solutions at 25℃ and immerse into liquid nitrogen. Control group embryos were placed in program freezing e-quipment. All samples were cryopreserved 3 months. The embryos were rewarmed rapidly in 25℃ water,and agitated gently ,then embryos were transferred into cultural medium and cultured. Then watch the survival rates and the merogenic rates. Results Both EDS-40 and EFS-40 solutions had vitrifying effects. The survival rates of EFS-40. EDS-40 and control group were 87. 8%、 93. 47% and 80. 77% respectively;the merogenic rates of EFS-4O、EDS-4O and control groups were79. 83% 、 86.53% and 69. 23% respectively. Conclusion The effect of EDS-40 solution is better than that of EFS-40.%目的 研究玻璃化液EDS-40对冻贮小鼠4细胞胚胎的效果.方法 ①EFS-40和EDS-40玻璃化溶液的玻璃化测试.②随机将4细胞小鼠胚胎,在25℃室温下分别加入EDS-40和EFS-40玻璃化溶液,玻璃化后装管并迅速投入液氮中.对照组置于程序冷冻仪中进行程序化冷冻.各组均冻存3个月后,在25℃的水浴中复温后,用培养液反复洗涤后移入培养孔板培养,观察胚胎存活率及卵裂率.结果 两种玻璃化溶液能达到玻璃化效果;EFS-40、EDS-40及对照组冷冻复温后的存活率分别87.8%、93.47%、80.77%;EFS-40、EDS-40及对照组冻胚的卵裂率分别:79.83%、86.53%、69.23%.结论 EDS-40玻璃化液冻贮小鼠4细胞胚胎的效果明显优于EFS-40,是一种理想的玻璃化液.

  1. Expression of LIF, VEGF,CD57 and CD68 after the transfer of rat embryos to mouse uteri

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The high failure rate of interspecific pregnancy is a major obstacle to the successful interspecific cloning of mammals. To investigate the reasons for the failure of interspecfic pregnancy between rats and mice, we transferred rat blastocysts into mouse uteri on the third day of pseudopregnancy (D3). Our previous study showed that intact rat embryos could still be observed in mouse uteri on D9. In the present study, we found that expression of CD57 and CD68 increased significantly at the maternal-fetal interface following the transfer of rat embryos. Similarly, Leukaemia inhibitory factor (LIF) expression increased, but vascular endothelial growth factor (VEGF) expession decreased. In a co-culture system, the percentage of rat ectoplacental cones (EPCs) with adhesion and outgrowth and outgrowth area on mouse uterine decidual cells were less than that of mouse EPCs. These results indicate that an increase in the immunological rejection response and a decrease in the invasiveness of rat embryos may be important reasons for the failure of interspecific pregnancy between rat and mouse.

  2. Genetic analysis reveals an unexpected role of BMP7 in initiation of ureteric bud outgrowth in mouse embryos.

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    Alexandre Gonçalves

    Full Text Available BACKGROUND: Genetic analysis in the mouse revealed that GREMLIN1 (GREM1-mediated antagonism of BMP4 is essential for ureteric epithelial branching as the disruption of ureteric bud outgrowth and renal agenesis in Grem1-deficient embryos is restored by additional inactivation of one Bmp4 allele. Another BMP ligand, BMP7, was shown to control the proliferative expansion of nephrogenic progenitors and its requirement for nephrogenesis can be genetically substituted by Bmp4. Therefore, we investigated whether BMP7 in turn also participates in inhibiting ureteric bud outgrowth during the initiation of metanephric kidney development. METHODOLOGY/PRINCIPAL FINDINGS: Genetic inactivation of one Bmp7 allele in Grem1-deficient mouse embryos does not alleviate the bilateral renal agenesis, while complete inactivation of Bmp7 restores ureteric bud outgrowth and branching. In mouse embryos lacking both Grem1 and Bmp7, GDNF/WNT11 feedback signaling and the expression of the Etv4 target gene, which regulates formation of the invading ureteric bud tip, are restored. In contrast to the restoration of ureteric bud outgrowth and branching, nephrogenesis remains aberrant as revealed by the premature loss of Six2 expressing nephrogenic progenitor cells. Therefore, very few nephrons develop in kidneys lacking both Grem1 and Bmp7 and the resulting dysplastic phenotype is indistinguishable from the one of Bmp7-deficient mouse embryos. CONCLUSIONS/SIGNIFICANCE: Our study reveals an unexpected inhibitory role of BMP7 during the onset of ureteric bud outgrowth. As BMP4, BMP7 and GREM1 are expressed in distinct mesenchymal and epithelial domains, the localized antagonistic interactions of GREM1 with BMPs could restrict and guide ureteric bud outgrowth and branching. The robustness and likely significant redundancy of the underlying signaling system is evidenced by the fact that global reduction of Bmp4 or inactivation of Bmp7 are both able to restore ureteric bud outgrowth

  3. Role of reactive oxygen species in diabetes-induced embryotoxicity: studies on pre-implantation mouse embryos cultured in serum from diabetic pregnant women.

    Science.gov (United States)

    Ornoy, A; Kimyagarov, D; Yaffee, P; Abir, R; Raz, I; Kohen, R

    1996-11-01

    Sera from diabetic patients or sera with high levels of diabetic metabolic products, were found to affect mouse and rat blastocysts. In the present study we examined the earliest developmental stages at which human diabetic serum will be lethal to mouse pre-implantation embryos, and whether reactive oxygen species (ROS) are involved in these diabetes-induced injuries. We cultured 2-4 cell-stage embryos and blastocysts in a medium containing 30 or 50% serum obtained from pregnant women with diabetes Type I, Type II and gestational diabetes (GDM) for 72 h. The development of the 2-4 cell-stage embryos was delayed when cultured in 30% diabetic serum, but the viability was impaired to a lesser extent. Viability was reduced in blastocysts cultured in 50% diabetic serum, but the development of the living embryos was not delayed. Cyclic voltametry measures the oxidation potential of the tissue and the concentration of antioxidants, thus reflecting the total antioxidative activity of the embryos. Pre-implantation embryos cultured in diabetic serum had a lower concentration of antioxidants than embryos cultured in non-diabetic serum. It seems, therefore, that diabetic metabolic factors may induce embryotoxicity in pre-implantation embryos through derangement of the antioxidant defense mechanism. A similar mechanism is suggested for the diabetes-induced teratogenicity in post-implantation embryos.

  4. Microdrop preparation factors influence culture-media osmolality, which can impair mouse embryo preimplantation development.

    Science.gov (United States)

    Swain, J E; Cabrera, L; Xu, X; Smith, G D

    2012-02-01

    Because media osmolality can impact embryo development, the effect of conditions during microdrop preparation on osmolality was examined. Various sizes of microdrops were prepared under different laboratory conditions. Drops were pipetted directly onto a dish and covered by oil (standard method) or pipetted on the dish, overlaid with oil before removing the underlying media and replaced with fresh media (wash-drop method). Drops were made at 23°C or on a heated stage (37°C) and with or without airflow. Osmolality was assessed at 5 min and 24h. The biological impact of osmolality change was demonstrated by culturing 1-cell mouse embryos in media with varying osmolality. Reduced drop volume, increased temperature and standard method were associated with a significant increase in osmolality at both 5 min and 24h (P-values media with elevated osmolality (>310mOsm/kg; P<0.05). Procedures in the IVF laboratory can alter osmolality and impact embryo development.

  5. Mouse immature oocytes irradiated in vivo at 14-days of age and evaluated for transmitted effects using the aggregation embryo chimera assay

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    Straume, T. [University of California Lawrence Livermore National Laboratory, Livermore, CA (United States); Raabe, O.G.; Walsh, K.J.; Wiley, L.M. [Institute of Toxicology and Environmental Health, Davis, CA (United States)

    1996-09-23

    A previous study using the mouse-preimplantation-embryo-chimera assay demonstrated a reproducible transmitted effect (proliferation disadvantage observed in early embryos) from females irradiated as 49-day-old adults using 0.15 Gy of gamma rays and then mated seven weeks later, i.e., embryos were from oocytes that were immature at time of irradiation. Because mouse immature oocytes are known to be much more radiosensitive to cell killing in juveniles than in adults, a follow-on study was performed here using 14-day-old juvenile mice. In contrast to adults, the exposure of juveniles to 0.15 Gy of gamma rays did not result in a detectable transmitted proliferation disadvantage when animals were mated 7 or 12 weeks later. This observation is discussed in light of previous studies on mouse immature oocytes and embryo chimeras.

  6. Offspring from mouse embryos developed using a simple incubator-free culture system with a deoxidizing agent.

    Science.gov (United States)

    Itoi, Fumiaki; Tokoro, Mikiko; Terashita, Yukari; Yamagata, Kazuo; Fukunaga, Noritaka; Asada, Yoshimasa; Wakayama, Teruhiko

    2012-01-01

    To culture preimplantation embryos in vitro, water-jacketed CO(2) incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O(2) was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.

  7. Offspring from mouse embryos developed using a simple incubator-free culture system with a deoxidizing agent.

    Directory of Open Access Journals (Sweden)

    Fumiaki Itoi

    Full Text Available To culture preimplantation embryos in vitro, water-jacketed CO(2 incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O(2 was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.

  8. Potential role of miR-29b in modulation of Dnmt3a and Dnmt3b expression in primordial germ cells of female mouse embryos

    NARCIS (Netherlands)

    Takada, S.; Berezikov, E.; Choi, Y.L.; Yamashita, Y.; Mano, H.

    2009-01-01

    MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs and are implicated in an increasing number of biological processes. To examine whether miRNAs might contribute to sexual differentiation, we performed expression profiling of miRNAs in mouse embryonic gonads with the use of a

  9. Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos.

    Science.gov (United States)

    Biechele, Steffen; Cox, Brian J; Rossant, Janet

    2011-07-15

    Wnt signaling plays important roles in development and disease. The X-chromosomal Porcupine homolog gene (Porcn) encodes an evolutionary conserved member of the membrane bound O-acyl transferase (MBOAT) superfamily that has been shown to be required for the palmitoylation and secretion of Wnt3a, a mechanism that has been suggested to be conserved for all mammalian Wnt ligands. PORCN mutations in humans cause Focal Dermal Hypoplasia (FDH), a disorder causing developmental defects in heterozygous females and embryonic lethality in hemizygous males. In this study, Porcn mutant mouse embryonic stem (ES) cells were used to analyze the role of Porcn in mammalian embryonic development. In vitro, we show an exclusive requirement for Porcn in Wnt secreting cells and further, that any of the four Porcn isoforms is sufficient to allow for the secretion of functional Wnt3a. Embryos generated by aggregation of Porcn mutant ES cells with wildtype embryos fail to complete gastrulation in vivo, but remain in an epiblast-like state, similar to Wnt3 and Gpr177/Wls mutants. Consistent with this phenotype, in vitro differentiated mutant ES cells fail to generate endoderm and mesoderm derivatives. Taken together, these data confirm the importance of Porcn for Wnt secretion and gastrulation and suggest that disruption of early development underlies the male lethality of human PORCN mutants.

  10. 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

  11. 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.

  12. 4D atlas of the mouse embryo for precise morphological staging.

    Science.gov (United States)

    Wong, Michael D; van Eede, Matthijs C; Spring, Shoshana; Jevtic, Stefan; Boughner, Julia C; Lerch, Jason P; Henkelman, R Mark

    2015-10-15

    After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development.

  13. 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

  14. Comparative Culturing of 3T3 Swiss J2 Mouse Embryo Fibroblasts on Modified Chitosan Matrices.

    Science.gov (United States)

    Alekhin, A I; Gaenko, G P

    2016-07-01

    Comparative culturing of mouse embryo fibroblasts on chitosan matrices and culture plastic was carried out. During the first 2 h of culturing (lag phase), cell adhesion to chitosan and chitosan-gelatin matrices was 20-30% higher than adhesion to culture plastic (control). During the stationary phase, 80% cells adhered to chitosan matrices (vs. 60% in the control). Cell culturing on chitosan matrices was carried out without medium replacement with fresh portions. The cells remained viable within 5 days of culturing. Cell death phase was observed on day 6 of culturing on chitosan matrices: cell adhesion dropped to 50%. Culturing on culture plastic was carried out with daily medium replacement with a fresh portion. Cell death phase (50% decrease in the number of adherent cell) under these condition was observed on day 5. It seems that the observed effect was a result of contact interactions of cell integrins and chitosan ligands, modulation of transmembrane signal, eventually modifying the expression of cell genes. This effect can be required in regenerative medicine for production of primary cell culture.

  15. Sox17-dependent gene expression and early heart and gut development in Sox17-deficient mouse embryos.

    Science.gov (United States)

    Pfister, Sabine; Jones, Vanessa J; Power, Melinda; Truisi, Germaine L; Khoo, Poh-Lynn; Steiner, Kirsten A; Kanai-Azuma, Masami; Kanai, Yoshiakira; Tam, Patrick P L; Loebel, David A F

    2011-01-01

    Sox17 is a transcription factor that is required for maintenance of the definitive endoderm in mouse embryos. By expression profiling of wild-type and mutant embryos and Sox17-overexpressing hepatoma cells, we identified genes with Sox17-dependent expression. Among the genes that were up-regulated in Sox17-null embryos and down-regulated by Sox17 expressing HepG2 cells is a set of genes that are expressed in the developing liver, suggesting that one function of Sox17 is the repression of liver gene expression, which is compatible with a role for Sox17 in maintaining the definitive endoderm in a progenitor state. Consistent with these findings, Sox17(-/-) cells display a diminished capacity to contribute to the definitive endoderm when transplanted into wild-type hosts. Analysis of gene ontology further revealed that many genes related to heart development were downregulated in Sox17-null embryos. This is associated with the defective development of the heart in the mutant embryos, which is accompanied by localised loss of Myocd-expressing cardiogenic progenitors and the malformation of the anterior intestinal portal.

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

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    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.

  17. Imaging and differentiation of mouse embryo tissues by ToF-SIMS

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    Wu, L; Lu, X; Kulp, K; Knize, M; Berman, E; Nelson, E; Felton, J; Wu, K J

    2006-06-16

    Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) equipped with a gold ion gun was used to image mouse embryos and differentiate tissue types (brain, spinal cord, skull, rib, heart and liver). Embryos were paraffin-embedded and then de-paraffinized. The robustness and repeatability of the method was determined by analyzing nine tissue slices from three different embryos over a period of several weeks. Using Principal Component Analysis (PCA) to reduce the spectral data generated by ToF-SIMS, histopathologically identified tissue types of the mouse embryos can be differentiated based on the characteristic differences in their mass spectra. These results demonstrate the ability of ToF-SIMS to determine subtle chemical differences even in fixed histological specimens.

  18. Supravital fluorometric apoptosis detection in a single mouse embryo using lab-on-a-chip.

    Science.gov (United States)

    Walczak, Rafał; Śniadek, Patrycja; Dziuban, Jan A; Kluger, Joanna; Soyta, Anna Chełmońska

    2011-10-07

    Detection of apoptosis is one of the main criteria of preimplantation embryo growth potential assessment. Recent developments in lab-on-a-chip techniques has led to apoptosis detection and monitoring on a single cell or embryo level. However, single embryo apoptosis detection without a change in embryo developmental competence and post-examination "recovery" still remains a challenge. In this paper we present a lab-on-a-chip, co-working with miniaturized optical instrumentation, which allows supravital examination of single embryos for the presence of apoptotic blastomers with full after lab-on-a-chip study "recovery" and maintenance of their further developmental capacity.

  19. Phosphorylated ERK5/BMK1 transiently accumulates within division spindles in mouse oocytes and preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Maria A. Ciemerych

    2011-10-01

    Full Text Available MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells, and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular, vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 528–534

  20. Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles.

    Science.gov (United States)

    de Jong, Esther; Barenys, Marta; Hermsen, Sanne A B; Verhoef, Aart; Ossendorp, Bernadette C; Bessems, Jos G M; Piersma, Aldert H

    2011-06-01

    The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds,(1) flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. [Embryotoxicity of Senecionis Scandentis Hebra on in vitro cultured mouse embryos].

    Science.gov (United States)

    Han, Jia-Yin; Yi, Yan; Liang, Ai-Hua; Zhang, Yu-Shi; Li, Chun-Ying; Zhao, Yong; Cui, Hong-Yu; Lu, Yu-Ting

    2014-09-01

    The purpose of this study is to evaluate the embryotoxicity of alkaloids in Senecionis Scandentis Hebra on in vitro cultured mouse embryos. Mouse whole embryo culture (WEC) was applied in this study. Post-implantation (8.5 d) mouse embryos were isolated from their mothers, and cultured in medium of immediately centrifuged serum (ICS) with different concentrations of seneciphylline (target concentrations were 100, 50, 25 and 12.5 μg x mL(-1)) or senkirkine (target concentrations were 50, 25 and 12.5 μg x mL(-1)) for 48 h. After culturing completed, the development and organic morphodifferentiation of the cultured embryos were evaluated microscopically. Treatment with seneciphylline and senkirkine had adverse effects on the development and organic morphodifferentiation of embryos. The effect also had clear dose-response. Alkaloidals in Senecionis Scandentis Hebra had embryotoxicity on cultured embryos, which indicated that pregnant people exposed to Senecionis Scandentis Hebra may get potential risk on fetus.

  2. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes

    Science.gov (United States)

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S.; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming. PMID:27472658

  3. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

    Science.gov (United States)

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

  4. The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo.

    Science.gov (United States)

    Nowotschin, Sonja; Costello, Ita; Piliszek, Anna; Kwon, Gloria S; Mao, Chai-an; Klein, William H; Robertson, Elizabeth J; Hadjantonakis, Anna-Katerina

    2013-05-01

    Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

  5. The effect of sevoflurane on developing A/J strain mouse embryos using a whole-embryo culture system--the incidence of cleft lip in culture embryos.

    Science.gov (United States)

    Yamada, Morimasa; Yamamoto, Naoki; Ohgami, Saori; Kanazawa, Mayuko; Harada, Jun; Ohno, Norikazu; Natsume, Nagato

    2014-03-01

    A/J strain mice have a high spontaneous incidence of cleft lip (ICL) and/or palate. The primary palate-related effects of sevoflurane on developing A/J strain mouse embryos (embryos) were studied using a whole-embryo culture (WEC) system. This system could separate the direct effects of sevoflurane from those that are maternally mediated. A total of 205 10.5-d embryos were cultured for 24 h in either a control group (control gas: 95% O2 and 5% CO2) or sevoflurane-administered groups (1/4, 1/2, and 1 minimum alveolar concentration (MAC) with control gas) for 8 h. After 16 h, 11.5-d culture embryos were examined in terms of crown-rump length, number of somites, and protein content. Crown-rump length in the 1 MAC was significantly shorter than in the control group (p < 0.05). Protein content in the 1/2 MAC (p < 0.05) and 1 MAC (p < 0.001) was significantly lower than in the control group. The ICL showed no significant differences between each group. (The ICL rose with an increase in the sevoflurane concentration, but this was not significant). The positive findings in this study indicate that a WEC system is useful for studying the mechanisms of ICL (teratogenicity) associated with sevoflurane.

  6. Phenotyping structural abnormalities in mouse embryos using high-resolution episcopic microscopy

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    Wolfgang J. Weninger

    2014-10-01

    Full Text Available The arrival of simple and reliable methods for 3D imaging of mouse embryos has opened the possibility of analysing normal and abnormal development in a far more systematic and comprehensive manner than has hitherto been possible. This will not only help to extend our understanding of normal tissue and organ development but, by applying the same approach to embryos from genetically modified mouse lines, such imaging studies could also transform our knowledge of gene function in embryogenesis and the aetiology of developmental disorders. The International Mouse Phenotyping Consortium is coordinating efforts to phenotype single gene knockouts covering the entire mouse genome, including characterising developmental defects for those knockout lines that prove to be embryonic lethal. Here, we present a pilot study of 34 such lines, utilising high-resolution episcopic microscopy (HREM for comprehensive 2D and 3D imaging of homozygous null embryos and their wild-type littermates. We present a simple phenotyping protocol that has been developed to take advantage of the high-resolution images obtained by HREM and that can be used to score tissue and organ abnormalities in a reliable manner. Using this approach with embryos at embryonic day 14.5, we show the wide range of structural abnormalities that are likely to be detected in such studies and the variability in phenotypes between sibling homozygous null embryos.

  7. Gradual meiosis-to-mitosis transition in the early mouse embryo.

    Science.gov (United States)

    Courtois, Aurélien; Hiiragi, Takashi

    2012-01-01

    The transition from meiosis to mitosis is a fundamental process to guarantee the successful development of the embryo. In the mouse, the transition includes extensive reorganisation of the division machinery, centrosome establishment and changes in spindle proprieties and characteristic. Recent findings indicate that this transition is gradual and lasts until the late blastocyst stage. In-depth knowledge of the mechanisms underlying the transition would provide new insight into de novo centrosome formation and regulation of spindle size and proprieties. Here, we review recent advances in the understanding of acentrosomal spindle formation, centriole establishment and the meiosis-to-mitosis transition in the mouse pre-implantation embryo.

  8. Preliminar toxicological assesement of Ruta graveolens, Origanum vulgare and Persea americana on the preimplantational mouse embryos

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    V. Benavides

    2014-06-01

    Full Text Available The growing interest in natural medicine makes it necessary to study plant properties as well as their possible secondary effects. In recent years the toxic effects of many medicinal plants on the preimplantational mouse embryo development have been studied. Many of them produce malformations and alterations in the embryonic development. Ruta graveolens "ruda", Origanum vulgare "oregano" and Persea americana "palta" are used in rural areas to menstrual colic and to provoke abortion (estrella, 1995. This study is aimed at assessing "in vivd'the effect of extracts of "oregano", "ruda" and "palta" to 20% on the morphology and growth of preimplantational mouse embryos.

  9. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    Institute of Scientific and Technical Information of China (English)

    Amjad Riaz; Xiaoyang Zhao; Xiangpeng Dai; Wei Li; Lei Liu; Haifeng Wan; Yang Yu; Liu Wang; Qi Zhou

    2011-01-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem(ES)cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved.Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  10. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    Science.gov (United States)

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  11. Amnion formation in the mouse embryo: the single amniochorionic fold model.

    Science.gov (United States)

    Pereira, Paulo N G; Dobreva, Mariya P; Graham, Liz; Huylebroeck, Danny; Lawson, Kirstie A; Zwijsen, A N

    2011-08-01

    Despite the detailed knowledge obtained over the last decade on the molecular regulation of gastrulation in amniotes, the process of amnion development has been poorly described and illustrated in mice, and conflicting descriptions exist. Understanding the morphogenesis and development not only of the early mouse embryo, but also of its extraembryonic tissues, is crucial for correctly interpreting fate-mapping data and mouse mutants with gastrulation defects. Moreover, the recent isolation from amnion of cells with stem cell features further argues for a better understanding of the process of amnion formation. Here, we revisit the highly dynamic process of amnion formation in the mouse. Amnion development starts early during gastrulation and is intimately related to the formation of the exocoelom and the expansion of the amniotic fold. The authoritative description involves the fusion of two amniotic folds, a big posterior and a smaller anterior fold. We challenged this 'two amniotic folds' model by performing detailed histomorphological analyses of dissected, staged embryos and 3D reconstructions using historical sections. A posterior fold of extraembryonic ectoderm and associated epiblast is formed early during gastrulation by accumulation of extraembryonic mesoderm posterior to the primitive streak. Previously called the "posterior amniotic fold", we rename it the "amniochorionic fold" (ACF) because it forms both amnion and chorion. Exocoelom formation within the ACF seems not to involve apoptosis within the mesoderm. The ACF and exocoelom expand without disrupting the anterior junction of epiblast, extraembryonic ectoderm and visceral endoderm. No separate anterior fold is formed; its absence was confirmed in 3D reconstructions. Amnion and chorion closure is eccentric, close to the anterior margin of the egg cylinder: we name it the "anterior separation point". Here, we reconcile previous descriptions of amnion formation and provide new nomenclature, as well as

  12. Amnion formation in the mouse embryo: the single amniochorionic fold model

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    Huylebroeck Danny

    2011-08-01

    Full Text Available Abstract Background Despite the detailed knowledge obtained over the last decade on the molecular regulation of gastrulation in amniotes, the process of amnion development has been poorly described and illustrated in mice, and conflicting descriptions exist. Understanding the morphogenesis and development not only of the early mouse embryo, but also of its extraembryonic tissues, is crucial for correctly interpreting fate-mapping data and mouse mutants with gastrulation defects. Moreover, the recent isolation from amnion of cells with stem cell features further argues for a better understanding of the process of amnion formation. Here, we revisit the highly dynamic process of amnion formation in the mouse. Amnion development starts early during gastrulation and is intimately related to the formation of the exocoelom and the expansion of the amniotic fold. The authoritative description involves the fusion of two amniotic folds, a big posterior and a smaller anterior fold. We challenged this 'two amniotic folds' model by performing detailed histomorphological analyses of dissected, staged embryos and 3D reconstructions using historical sections. Results A posterior fold of extraembryonic ectoderm and associated epiblast is formed early during gastrulation by accumulation of extraembryonic mesoderm posterior to the primitive streak. Previously called the "posterior amniotic fold", we rename it the "amniochorionic fold" (ACF because it forms both amnion and chorion. Exocoelom formation within the ACF seems not to involve apoptosis within the mesoderm. The ACF and exocoelom expand without disrupting the anterior junction of epiblast, extraembryonic ectoderm and visceral endoderm. No separate anterior fold is formed; its absence was confirmed in 3D reconstructions. Amnion and chorion closure is eccentric, close to the anterior margin of the egg cylinder: we name it the "anterior separation point". Conclusions Here, we reconcile previous descriptions

  13. [Site of fidget gene action and its interaction with the ocular retardation gene in cultured mouse embryo retinas].

    Science.gov (United States)

    Koniukhov, B V; Ugol'kova, T S

    1978-01-01

    The eye rudiments of 10 and 11 days old mouse embryos of the genotypes +/+ +/+, fi/fi +/+, +/+ or/or, fi/fi or/or and 11 days old embryos of the genotype fi/+ or/+ were cultivated in vitro during 24, 48 and 72 hrs. The expression of the fi gene was shown in the cells of the cultivated fi/fi +/+ retina: its proliferative activity was inhibited. The fi gene was not active in the cells of the developing lens and the anomalies of the latter in homozygotes arose secondarily, due to the inhibition of growth of the retinal rudiment. The fi and or genes interacted synergically in the cultivated fi/fi or/or retina, thus resulting in the marked inhibition of its mitotic activity. This suggests that both the genes act in the retinal cells and, apparently, affect different links of the biochemical chain of events in the preparation of DNA replication.

  14. TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

    Science.gov (United States)

    Carvacho, Ingrid; Ardestani, Goli; Lee, Hoi Chang; McGarvey, Kaitlyn; Fissore, Rafael A.; Lykke-Hartmann, Karin

    2016-01-01

    The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development. PMID:27681336

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Effect of an Improved Mechanical Method for Assisted Hatching on the in vitro Development of Mouse Embryos

    Institute of Scientific and Technical Information of China (English)

    Ai-jun ZHANG; Yun FENG; Xiao-yan HUANG; Lan XIA; Yi-juan SUN; Yan LI

    2006-01-01

    Objective To evaluate the safety and efficiency of an improved shape of opening for mechanical assisted hatching (AH) on the in virto development of mouse embryos.Methods A total of 622 KM BAI mouse embryos in 2-cell-4-cell stage were randomly divided into group A, group B and control group. A new mechanical AH method by improving the shape of opening in the ZP was used in group A, and a "-/ "-shaped opening was created. A "+" -shaped opening was made in group B, while no opening was made in control group. Comparisons have been made among the three groups with regard to the duration of AH, the blastocyst formation and complete hatching rate, etc.Results The duration of AH in group A (43.25 ± 3.46 s) was significantly shorter than that in group B (52.81 ±4.32 s, P <0.05). The blastocyst formation rate on d 5was not significantly different among the three groups (92.27%, 93.66% and 94.92%respectively, P >0.05). The complete hatching rate of blastocysts on d 6 between group A and group B was no statistical difference (94.09% vs 92.71%, P >0.05), but significantly higher than that in control group (43.32%, P <0.001). No significant difference in the percentage of grade 1 blastocysts was found among the three groups on d 5 (85.22%, 82.81% and 86.63% respectively, P >0.05).Conclusion It could enhance the process of embryo hatching and facilitate the hatching rate of blastocysts by using the improved mechanical AH method, which is of safety and efficiency to mouse embryo in the in vitro development.

  19. Protective effect of [6]-gingerol on the ethanol-induced teratogenesis of cultured mouse embryos.

    Science.gov (United States)

    Yon, Jung-Min; Baek, In-Jeoung; Lee, Se-Ra; Kim, Mi-Ra; Hong, Jin Tae; Yong, Hwanyul; Lee, Beom Jun; Yun, Young Won; Nam, Sang-Yoon

    2012-01-01

    Excessive ethanol consumption during pregnancy causes fetal alcohol syndrome. We investigated the effect of [6]-gingerol on ethanol-induced embryotoxicity using a whole embryo culture system. The morphological changes of embryos and the gene expression patterns of the antioxidant enzymes cytosolic glutathione peroxidase (cGPx), cytoplasmic Cu/Zn superoxide dismutase (SOD1), and Mn-SOD (SOD2), and SOD activity were examined in the cultured mouse embryos exposed to ethanol (5 μL/3 mL) and/or [6]-gingerol (1×10(-8) or 1×10(-7) μg/mL) for 2 days. In ethanol-exposed embryos, the standard morphological score of embryos was significantly decreased compared with those of the control (vehicle) group. However, cotreatment of embryos with [6]-gingerol and ethanol significantly improved all of the developmental parameters except crownrump length and head length, compared with those of the ethanol alone group. The mRNA expression levels of cGPx and SOD2, not SOD1, were decreased consistently, SOD activity were significantly decreased compared with the control group. However, the decreases in mRNA levels of antioxidant enzymes and SOD activity were significantly restored to the control levels by [6]-gingerol supplement. These results indicate that [6]-gingerol has a protective effect against ethanol-induced teratogenicity during mouse embryogenesis.

  20. Antiteratogenic Effects of beta-Carotene in Cultured Mouse Embryos Exposed to Nicotine

    NARCIS (Netherlands)

    Lin, C.; Yon, J.M.; Jung, A.Y.; Lee, J.G.; Jung, K.Y.; Lee, B.J.; Yun, Y.W.; Nam, S.Y.

    2013-01-01

    After maternal intake, nicotine crosses the placental barrier and causes severe embryonic disorders and fetal death. In this study, we investigated whether beta -carotene has a beneficial effect against nicotine-induced teratogenesis in mouse embryos (embryonic day 8.5) cultured for 48 h in a whole

  1. COMPARATIVE PATHOGENESIS OF HALOACETIC ACID AND PROTEIN KINASE INHIBITOR EMBRYOTOXICITY IN MOUSE WHOLE EMBRYO CULTURE

    Science.gov (United States)

    Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.Ward KW, Rogers EH, Hunter ES 3rd.Curriculum in Toxicology, University of North Carolina at Chapel Hill, 27599-7270, USA.Haloacetic acids ...

  2. Evaluation of OPEN zinc finger nucleases for direct gene targeting of the ROSA26 locus in mouse embryos.

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    Mario Hermann

    Full Text Available Zinc finger nucleases (ZFNs enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.

  3. Study of Elaeagnus angustifolia Fruit Aqueous Extract on the Histomorphometrical Changes of Retina in Mouse Embryo

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    Jamshid Arum

    2016-09-01

    Full Text Available Background Modern societies tend to use herbs or traditional medicine because of fewer side effects than synthetic drugs. Elaeagnus angustifolia is a plant with many therapeutic effects. Objectives This study aimed to investigate the effect of aqueous extract of Elaeagnus angustifolia on fetal mice retina in Balb/C by evaluating of histomophometrical and immunohistochemical parameters in the eye mouse embryos Methods In this experimental study, thirty pregnant mice were randomly divided into two groups. Control group received food and drinking water, and the experimental group received food and the aqueous extract at a dose of 500 mg/kg in the form of water solution from the day zero of pregnancy up to the 18th day. Pregnant mice were killed and their fetuses were taken, fixed and stained with hematoxylin-eosin. Histomorphometrical and immunohistochemical changes of retin examined. Results The mean of Crown-Rump length and weight of fetuses significantly increased in experimental group compared to control group. In experimental group, a significant decrease was shown in the mean of diameters and weight of placenta compared to control group. Retinal thickness in posterior, superior and inferior part significantly reduced in experimental group compared to control group. however, retinal thickness in anterior part in experimental group was not different when compared to control group. The number of Ki-67-positive cells showed that the retinal cells proliferation in experimental group decreased compared to control group. Conclusions The use of aqueous extract of Elaeagnus angustifolia at dose of 500 mg/kg to pregnant mice caused the growth and histological changes on fetal mouse and in the retinal mouse development.

  4. A novel regulatory element for Shh expression in the lung and gut of mouse embryos.

    Science.gov (United States)

    Tsukiji, Nagaharu; Amano, Takanori; Shiroishi, Toshihiko

    2014-02-01

    Hedgehog (Hh) signaling plays pivotal roles in morphogenesis of several embryonic tissues, including the primitive gut. In the mouse embryo, Sonic hedgehog (Shh) is expressed in endodermal epithelia from the oral cavity to the intestine, and contributes to cell proliferation in the underlying mesenchyme and subsequent differentiation into the gastrointestinal smooth muscle. Three evolutionary conserved non-coding sequences in the region upstream of the Shh coding sequence contain endoderm-specific enhancers for Shh expression. Although Shh expression in the endodermal epithelial lining is mostly attributed to these three enhancers, none of them regulates gene expression in the gastroesophageal epithelium. Here, we found that a 1.7Kb fragment located 100Kb upstream of the Shh coding sequence contains a functional element for Shh expression in endodermal organs, including the esophagus and stomach. Compared with the three known endodermal enhancers, this novel enhancer shows less evolutionary conservation, even among rodents. In mouse embryonic endodermal tissues, the seamless expression of Shh is achieved by a patchwork of multiple enhancers with different rates of evolution.

  5. Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines.

    Science.gov (United States)

    Wilson, Robert; McGuire, Christina; Mohun, Timothy

    2016-01-01

    The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate.

  6. MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro.

    Science.gov (United States)

    Feng, Ruizhi; Sang, Qing; Zhu, Yan; Fu, Wei; Liu, Miao; Xu, Yan; Shi, Huijuan; Xu, Yao; Qu, Ronggui; Chai, Renjie; Shao, Ruijin; Jin, Li; He, Lin; Sun, Xiaoxi; Wang, Lei

    2015-03-03

    Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.

  7. 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...

  8. Paternal Benzo[a]pyrene Exposure Modulates MicroRNA Expression Patterns in the Developing Mouse Embryo

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    Asgeir Brevik

    2012-01-01

    Full Text Available Little attention has been given to how microRNA expression is affected by environmental contaminants exposure. We investigate the effects of paternal exposure to benzo[a]pyrene (B[a]P on miRNA expression in the developing mouse embryo. Male mice were exposed to B[a]P (150 mg/kg i.p., and their sperm was used four days later in in-vitro fertilization experiments. Twenty embryos each from 2-, 8-cell and the blastocyst stage were used for genome-wide miRNA expression profiling. Paternal exposure to B[a]P affected the expression of several miRNAs, and the target genes for some of the dysregulated miRNAs were enriched in many different pathways that are likely to be relevant for the developing mouse embryo. By linking the miRNA target genes to publicly available databases, we identified some miRNA target genes that may serve as global markers of B[a]P-mediated genotoxic stress. The dysregulated miRNAs may provide valuable knowledge about potential transgenerational effects of sublethal exposure to chemicals.

  9. Localization of HstI transcripts to the apical ectodermal ridge in the mouse embryo.

    Science.gov (United States)

    Suzuki, H R; Sakamoto, H; Yoshida, T; Sugimura, T; Terada, M; Solursh, M

    1992-03-01

    The HstI gene is a transforming gene, coding for a protein of the fibroblast growth factor family (Sakamoto et al., 1986). Previous RNA hybridization studies with the mouse homolog demonstrated the presence of a 3.0-kb transcript in Day 11 and 14 mouse embryos. Here we detect a 3.0-kb transcript in the limb and body of the dissected Day 11 mouse embryo. PCR amplification using HstI-specific primers also showed comparable results. In order to localize the HstI transcripts during development, corresponding HstI cDNA was isolated, and an HstI-specific region was used as a probe for in situ hybridization analysis. Serial sections of embryos from Day 8 (early-somite stages) through Days 9, 10, 11, and 12 of gestation were examined. With the antisense probe, a signal was detected in the Day 11 and 12 embryo, where it was localized to the apical ectodermal ridge (AER) of the limb bud. This structure is well known for its role in promoting the distal outgrowth of the developing limb bud. Signal was detected in both fore- and hindlimbs during the period of rapid distal growth. This restricted localization suggests a role for HstI in normal embryogenesis, including outgrowth of the limb bud.

  10. Early Onset of Heat-Shock Response in Mouse Embryos Revealed by Quantification of Stress-Inducible hsp70i RNA

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    Lawrence J. Wangh

    2007-01-01

    Full Text Available Heat shock response is fully established in mouse embryos at the blastocyst stage, but it is unclear when this response first arises during development. To shed light on this question, we used a single-tube method to quantify mRNA levels of the heat shock protein genes hsp70.1 and hsp70.3 (hsp70i in individual cleavage-stage embryos that had or had not been heat-shocked. While untreated, healthy embryos contained very low copy numbers of hsp70i RNA, heat shock rapidly induced the synthesis of hundreds of hsp70i transcripts per blastomere at both the 4-cell and the 8-cell stages. In addition, we performed hsp70i measurements in embryos that had not been heat-shocked but had been very slow in developing.Quantification of hsp70i RNA and genomic DNA copy numbers in these slow-growing embryos demonstrated the presence of two distinct populations. Some of the embryos contained considerable levels of hsp70i RNA, a finding consistent with the hypothesis of endogenous metabolic stress accompanied by cell cycle arrest and delayed development. Other slow-growing embryos contained no hsp70i RNA and fewer than expected hsp70i gene copies, suggesting the possibility of ongoing apoptosis. In conclusion, this study demonstrates that mouse embryos can activate hsp70i expression in response to sub-lethal levels of stress as early as at the 4-cell stage. Our results also indicate that quantification of hsp70i DNA and RNA copy numbers may provide a diagnostic tool for embryonic health.

  11. 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.

  12. 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.

  13. Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

    Science.gov (United States)

    Morange, M; Diu, A; Bensaude, O; Babinet, C

    1984-04-01

    In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.

  14. Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos.

    Science.gov (United States)

    Hirasawa, Ryutaro; Sasaki, Hiroyuki

    2009-01-01

    The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.

  15. Dynamic changes in leptin distribution in the progression from ovum to blastocyst of the pre-implantation mouse embryo

    Science.gov (United States)

    Schulz, Laura C.; Roberts, R. Michael

    2011-01-01

    The hormone leptin, which is primarily produced by adipose tissue, is a critical permissive factor for multiple reproductive events in the mouse, including implantation. In the CD1 strain, maternally-derived leptin from the oocyte becomes differentially distributed among blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased towards a particular fate as early as the 2-cell stage. Here, we have confirmed that embryonic leptin is of maternal origin and re-examined leptin distribution in two distinct strains in which embryos were derived after either normal ovulation or superovulation. A polarized pattern of leptin distribution was found in the majority of both CD1 and CF1 embryos (79.1 % and 76.9 %, respectively) collected following superovulation, but was reduced, particularly in CF1 embryos (29.8 %; p < 0.0001), after natural ovulation. The difference in leptin asymmetries in the CF1 strain arose between ovulation and the first cleavage division, and was not affected by removal of the zona pellucida. Presence or absence of leptin polarization was not linked to differences in ability of embryos to develop normally to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm but upon blastocoel expansion it was lost from cells. Throughout development leptin co-localized with LRP2, a multi-ligand transport protein, and its patterning resembled that noted for the maternal-effect proteins OOEP, NLRP5, and PADI6, suggesting that it is a component of the subcortical maternal complex with as yet unknown significance in pre-implantation development. PMID:21444625

  16. Differential distribution of cubilin and megalin expression in the mouse embryo.

    Science.gov (United States)

    Drake, Christopher J; Fleming, Paul A; Larue, Amanda C; Barth, Jeremy L; Chintalapudi, Mastan R; Argraves, W Scott

    2004-03-01

    Cubilin and megalin are cell surface proteins that work cooperatively in many absorptive epithelia to mediate endocytosis of lipoproteins, vitamin carriers, and other proteins. Here we have investigated the coordinate expression of these receptors during mouse development. Our findings indicate that while there are sites where the receptors are co-expressed, there are other tissues where expression is not overlapping. Apical cubilin expression is pronounced in the extraembryonic visceral endoderm (VE) of 6-9.5 days postcoitum (dpc) embryos. By contrast, little megalin expression is evident in the VE at 6 dpc. However, megalin expression in the VE increases as development progresses (7.5-9.5 dpc), although it is not as uniformly distributed as cubilin. Punctate expression of megalin is also apparent in the region of the ectoplacental cone associated with decidual cells, whereas cubilin expression is not seen in association with the ectoplacenta. Strong expression of megalin is observed in the neural ectoderm, neural plate and neural tube (6-8.5 dpc), but cubilin expression is not apparent in any of these tissues. At 8.5 dpc, megalin is expressed in the developing endothelial cells of blood islands, whereas cubilin is absent from these cells. Finally, cubilin, but not megalin, is expressed by a subpopulation of cells dispersed within the 7.5 dpc embryonic endoderm and having a migratory morphology. In summary, the co-expression of cubilin and megalin in the VE is consistent with the two proteins functioning jointly in this tissue. However, the differential distribution pattern indicates that the proteins also function independent of one another. Furthermore, the finding of megalin expression in blood island endothelial cells and cubilin expression in embryonic endoderm highlight potential new developmental roles for these proteins.

  17. 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.

  18. Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening.

    Science.gov (United States)

    Adams, David; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J; Dickinson, Mary; Greene, Nicholas D E; Henkelman, Mark; Justice, Monica; Mohun, Timothy; Murray, Stephen A; Pauws, Erwin; Raess, Michael; Rossant, Janet; Weaver, Tom; West, David

    2013-05-01

    Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data.

  19. Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening

    Directory of Open Access Journals (Sweden)

    David Adams

    2013-05-01

    Full Text Available Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC, supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1 the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2 a common framework to harmonise international efforts within this context; (3 the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4 the importance of centralising data in a standardised form to facilitate data mining; and (5 the development of online tools to allow open access to and dissemination of the phenotyping data.

  20. Relationship between gelatin concentrations in silk fibroin-based composite scaffolds and adhesion and proliferation of mouse embryo fibroblasts.

    Science.gov (United States)

    Orlova, A A; Kotlyarova, M S; Lavrenov, V S; Volkova, S V; Arkhipova, A Yu

    2014-11-01

    Porous scaffolds of silk fibroin and composite porous scaffolds with 10, 20, 30, 40, and 50% gelatin were made by the freezing-thawing method. The relationship between adhesion and proliferation rate mouse embryo fibroblast and the scaffold composition was studied by laser confocal scanning microscopy. Addition of gelatin to the scaffold structure stimulated adhesion and proliferation of mouse embryo fibroblasts; the optimal content of gelatin was 30%.

  1. 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.

  2. Role of the first mitosis in the remodeling of the parental genomes in mouse embryos

    Institute of Scientific and Technical Information of China (English)

    Hong Lin LIU; Kentaro T.HARA; Fugaku AOKI

    2005-01-01

    Although male and female pronuclei reside in the same zygotic cytoplasm, they differ in many respects, such as volume and transcriptional activity. The aim of this study is to investigate whether these differences are lost during the first mitosis. For this purpose, a new method was developed to inhibit the mixing of two parental chromosomes during mitosis, thus to induce the formation of two nuclei after they exit from the mitotic phase. In this method, one-cell embryos are arrested at metaphase by treatment with nocodazole, and whn exitting from the mitotic phase, two nuclei were formed in a single karyocyte following treatment with 6-dimethylaminopurine (6-DMAP). These embryos were designated as post-mitotic embryos (PM-embryos), in which the two nuclei were derived from the male and female genomes. We found that in the control one-cell embryos that had not been treated with the reagents, the volume of the male pronucleus was about 1.65-fold greater than that of the female pronucleus, whereas the volumes of the two nuclei in the PM-embryos were similar (volume ratio of 1.01). Although a two-fold difference in transcriptional activity was detected between the male and female pronuclei in the control embryos, no difference in transcriptional activity was detected between the two nuclei of PM-embryos. The ratio of transcriptional activity in the nucleus derived from the paternal genome to that from the maternal genome was 1.02, for which no significant difference was detected by the x2fitness test. Therefore, the volumes and transcriptional activities of the male and female nuclei were approximately equal in PM-embryos, which suggests that the asymmetries of pronuclear volume and transcriptional activity between male and female genomes are somehow losted during the first mitosis.

  3. 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.

  4. 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.

  5. Gene expression profiling of mouse embryos with microarrays

    OpenAIRE

    Sharov, Alexei A; Piao, Yulan; Minoru S.H. Ko

    2010-01-01

    Global expression profiling by DNA microarrays provides a snapshot of cell and tissue status and becomes an essential tool in biological and medical sciences. Typical questions that can be addressed by microarray analysis in developmental biology include: (1) to find a set of genes expressed in a specific cell type; (2) to identify genes expressed commonly in multiple cell types; (3) to follow the time-course changes of gene expression patterns; (4) to demonstrate cell’s identity by showing s...

  6. 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

  7. Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos.

    Science.gov (United States)

    Jin, Yong-Xun; Zheng, Zhong; Yu, Xian-Feng; Zhang, Jia-Bao; Namgoong, Suk; Cui, Xiang-Shun; Hyun, Sang-Hwan; Kim, Nam-Hyung

    2016-02-01

    The mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI).

  8. Conservation of DNA Methylation Programming Between Mouse and Human Gametes and Preimplantation Embryos.

    Science.gov (United States)

    White, Carlee R; MacDonald, William A; Mann, Mellissa R W

    2016-09-01

    In mice, assisted reproductive technologies (ARTs) applied during gametogenesis and preimplantation development can result in disruption of genomic imprinting. In humans, these technologies and/or subfertility have been linked to perturbations in genomic imprinting. To understand how ARTs and infertility affect DNA methylation, it is important to understand DNA methylation dynamics and the role of regulatory factors at these critical stages. Recent genome studies performed using mouse and human gametes and preimplantation embryos have shed light onto these processes. Here, we comprehensively review the current state of knowledge regarding global and imprinted DNA methylation programming in the mouse and human. Available data highlight striking similarities in mouse and human DNA methylation dynamics during gamete and preimplantation development. Just as fascinating, these studies have revealed sex-, gene-, and allele-specific differences in DNA methylation programming, warranting future investigation to untangle the complex regulation of DNA methylation dynamics during gamete and preimplantation development.

  9. Automated pipeline for anatomical phenotyping of mouse embryos using micro-CT.

    Science.gov (United States)

    Wong, Michael D; Maezawa, Yoshiro; Lerch, Jason P; Henkelman, R Mark

    2014-06-01

    The International Mouse Phenotyping Consortium (IMPC) plans to phenotype 20,000 single-gene knockout mice to gain an insight into gene function. Approximately 30% of these knockout mouse lines will be embryonic or perinatal lethal. The IMPC has selected three-dimensional (3D) imaging to phenotype these mouse lines at relevant stages of embryonic development in an attempt to discover the cause of lethality using detailed anatomical information. Rate of throughput is paramount as IMPC production centers have been given the ambitious task of completing this phenotyping project by 2021. Sifting through the wealth of data within high-resolution 3D mouse embryo data sets by trained human experts is infeasible at this scale. Here, we present a phenotyping pipeline that identifies statistically significant anatomical differences in the knockout, in comparison with the wild type, through a computer-automated image registration algorithm. This phenotyping pipeline consists of three analyses (intensity, deformation, and atlas based) that can detect missing anatomical structures and differences in volume of whole organs as well as on the voxel level. This phenotyping pipeline was applied to micro-CT images of two perinatal lethal mouse lines: a hypomorphic mutation of the Tcf21 gene (Tcf21-hypo) and a knockout of the Satb2 gene. With the proposed pipeline we were able to identify the majority of morphological phenotypes previously published for both the Tcf21-hypo and Satb2 mutant mouse embryos in addition to novel phenotypes. This phenotyping pipeline is an unbiased, automated method that highlights only those structural abnormalities that survive statistical scrutiny and illustrates them in a straightforward fashion. © 2014. Published by The Company of Biologists Ltd.

  10. Response of Mouse Zygotes Treated with Mild Hydrogen Peroxide as a Model to Reveal Novel Mechanisms of Oxidative Stress-Induced Injury in Early Embryos

    Science.gov (United States)

    2016-01-01

    Our study aimed to develop embryo models to evaluate the impact of oxidative stress on embryo development. Mouse zygotes, which stayed at G1 phase, were treated with prepared culture medium (containing 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, or 0.1 mM hydrogen peroxide (H2O2)) for 30 min in experiment 1. The dose-effects of H2O2 on embryo development were investigated via comparisons of the formation rate at each stage (2- and 4-cell embryos and blastocysts). Experiment 2 was carried out to compare behaviors of embryos in a mild oxidative-stressed status (0.03 mM H2O2) with those in a control (0 mM H2O2). Reactive oxygen species (ROS) levels, variation of mitochondrial membrane potential (MMP), expression of γH2AX, and cell apoptosis rate of blastocyst were detected. We observed a dose-dependent decrease on cleavage and blastocyst rates. Besides, higher level of ROS, rapid reduction of MMP, and the appearance of γH2AX revealed that embryos are injured early in mild oxidative stress. Additionally, γH2AX may involve during DNA damage response in early embryos. And the apoptotic rate of blastocyst may significantly increase when DNA damage repair is inadequate. Most importantly, our research provides embryo models to study cell cycle regulation and DNA damage response under condition of different levels of oxidative stress.

  11. 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.

  12. 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.

  13. Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

    Science.gov (United States)

    Wong, Christopher Yee; Mills, James K

    2017-03-01

    Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures.

  14. Folic acid supplementation changes the fate of neural progenitors in mouse embryos of hyperglycemic and diabetic pregnancy.

    Science.gov (United States)

    Yuan, Qiuhuan; Zhao, Shidou; Liu, Shangming; Zhang, Yanmin; Fu, Jie; Wang, Fuwu; Liu, Qian; Ling, Eng-Ang; Hao, Aijun

    2013-07-01

    Folic acid has been shown to decrease the incidence of neural tube defects (NTDs) in normal and hyperglycemic conditions, but the influence of folic acid on the development of central nervous system is not fully understood. Here, we aimed to explore the effects of folic acid, especially high dose of folic acid, on the characteristics of neural progenitors in embryos of hyperglycemic and diabetic mouse. Hyperglycemic and diabetic pregnant mice were given 3 mg/kg or 15 mg/kg folic acid from embryonic day 0.5 (E0.5) and were euthanased on E11.5, E13.5 or E18.5. The incidence of NTDs at E13.5 was counted. The proliferation, apoptosis and differentiation of neural progenitors and neuronal migration were determined using BrdU incorporation assay, TUNEL assay, immunofluorescence, Western blot and real-time reverse transcriptase polymerase chain reaction. Both normal and high doses of folic acid decreased the incidence of NTDs, promoted proliferation and reduced apoptosis of neuroepithelial cells in embryos of hyperglycemic and diabetic mice. Importantly, folic acid, especially at high dose, might affect the premature differentiation of neural progenitors in embryos of hyperglycemic and diabetic pregnancy. This may be attributed to changes of messenger RNA expression levels of some basic-helix-loop-helix transcription factors. In addition, folic acid might be involved in regulating neuronal migration in embryos of hyperglycemic and diabetic pregnancy. These findings suggest that periconceptional supplementation of folic acid, especially at high dose, may be a double-edged sword because it may result in undesirable outcomes affecting both the neuronal and glial differentiation in hyperglycemic and diabetic pregnancy.

  15. Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

    Directory of Open Access Journals (Sweden)

    Bell Graham

    2005-12-01

    Full Text Available Abstract Background Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. Results We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Conclusion Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

  16. 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.

  17. Novel gene expression patterns along the proximo-distal axis of the mouse embryo before gastrulation

    Directory of Open Access Journals (Sweden)

    Greenfield Andy

    2007-02-01

    Full Text Available Abstract Background To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region. Results To contribute to elucidating the mechanisms underlying the events involved in early patterning of the mouse embryo, we have carried out a microarray screen to identify novel genes that are differentially expressed between the distal and proximal parts of the E5.5 embryo. Secondary screening of resulting candidates by in situ hybridisation at E5.5 and E6.5 revealed novel expression patterns for known and previously uncharacterised genes, including Peg10, Ctsz1, Cubilin, Jarid1b, Ndrg1, Sfmbt2, Gjb5, Talia and Plet1. The previously undescribed gene Talia and recently identified Plet1 are expressed specifically in the distal-most part of the extraembryonic ectoderm, adjacent to the epiblast, and are therefore potential candidates for regulating early patterning events. Talia and the previously described gene XE7 define a gene family highly conserved among metazoans and with a predicted protein structure suggestive of a post-transcriptional regulative function, whilst Plet1 appears to be mammal-specific and of unknown function. Conclusion Our approach has allowed us to compare expression between dissected parts of the egg cylinder and has identified multiple genes with novel expression patterns at this developmental stage. These genes are potential candidates for regulating tissue interactions following implantation.

  18. 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.

  19. Researchers Put Embryo Development 'On Hold' in Mice

    Science.gov (United States)

    ... medlineplus.gov/news/fullstory_162187.html Researchers Put Embryo Development 'On Hold' in Mice Putting stem cells ... were able to halt development of early mouse embryos for up to a month in the lab ...

  20. 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.

  1. [Distribution of foreign mitochondrial DNA during the first splittings of the transmitochondrial mouse embryos].

    Science.gov (United States)

    Kustova, M E; Sokolova, V A; Bass, M G; Zakharova, F M; Sorokin, A V; Vasil'ev, V B

    2008-01-01

    Distribution of human mitochondrial DNA (mtDNA) among separate murine blastomeres was analyzed during the splitting of embryos in which the suspension of human mitochondria had been injected at the one- or two-cell stage. Human mtDNA was detected by PCR with species specific primers. The total amount of the two- and four-cell murine embryos analyzed in the study was 339. In all embryos examined the copies of human mitochondrial genome were revealed along with murine mtDNA, which indicated the phenomenon of an artificially modeled heteroplasmy. The foreign mtDNA was not ubiquitous among the blastomeres of transmitochondrial embryos. Mathematical analysis of the results showed that in the period between the injection of human mitochondria and the subsequent splitting no equal distribution of the human mtDNA occurred in the cytoplasm. These results also point at the presence of more than 2-3 segregation units of mtDNA in the entire pool of mitochondria (about 5 x 10(2)) introduced into an embryo by microinjection.

  2. 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.

  3. Dose-response relationship of cadmium or radiation-induced embryotoxicity in mouse whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Kiyohito; Kawamata, Akitoshi; Matsuoka, Masato; Wakisaka, Takashi; Fujiki, Yoshishige (Asahi University School of Dentistry, Gifu (Japan))

    1988-12-01

    Mouse embryos of B6C3F/sub 1/ strain were exposed in vitro to 1.2 to 2.2 {mu}M cadmium chloride (Cd) or to 100 to 320 R x-rays, and the effects of the exposure on development were examined after 39 h of culture. Development of embryos was assessed from lethality, formation of the neural tube defect, diameter and protein of yolk sac, crown-rump and head lengths, embryonic protein, and number of somites. Incidence of the neural tube defect increased from 3.4 to 100% by 1.2 to 2.0 {mu}M Cd, while embryo deaths increased from 13.8 to 93.3% by 2.0 to 2.2 {mu}M Cd. Embryonic protein was significantly reduced at the teratogenic range, but the number of somites was only affected by 1.6 to 2.0 {mu}M Cd. X-irradiation at 100 to 320 R induced the neural tube defect in 2.9 to 72.7% of the embryos. An embryolethal effect was observed only at the 320 R dose. Crown-rump and head lengths and embryonic protein were significantly affected at the teratogenic range, but the diameter and protein of yolk sac and number of somites were hardly affected. Cadmium- or radiation-induced response data of both teratogenicity and endpoints indicating inhibition of embryonic development were acceptably fitted to a linear log-probit regression. These regressions suggest that as an estimation of interference in development of embryos, embryonic protein and head length are sensitive endpoints while the number of somites is an insensitive criterion. (author).

  4. Disruption of overlapping transcripts in the ROSA βgeo 26 gene trap strain leads to widespread expression of β-galactosidase in mouse embryos and hematopoietic cells

    Science.gov (United States)

    Zambrowicz, Brian P.; Imamoto, Akira; Fiering, Steve; Herzenberg, Leonard A.; Kerr, William G.; Soriano, Philippe

    1997-01-01

    The ROSAβgeo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral βgeo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5′ ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans. PMID:9108056

  5. 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.

  6. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos

    NARCIS (Netherlands)

    T. Yokomizo (Tomomasa); E.A. Dzierzak (Elaine)

    2010-01-01

    textabstractHematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Despite their importance, hematopoietic clusters have not been systematically quantitated or mapped because of technical limitations posed by the opaqueness of w

  7. Detecting cardiac contractile activity in the early mouse embryo using multiple modalities

    Directory of Open Access Journals (Sweden)

    Chiann-mun eChen

    2015-01-01

    Full Text Available The heart is one of the first organs to develop during mammalian embryogenesis. In the mouse, it starts to form shortly after gastrulation, and is derived primarily from embryonic mesoderm. The embryonic heart is unique in having to perform a mechanical contractile function while undergoing complex morphogenetic remodelling. Approaches to imaging the morphogenesis and contractile activity of the developing heart are important in understanding not only how this remodelling is controlled but also the origin of congenital heart defects. Here, we describe approaches for visualising contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients. We describe an algorithm for enhancing this image data and quantifying contractile activity from it. Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.

  8. Oxidative stress in mouse sperm impairs embryo development, fetal growth and alters adiposity and glucose regulation in female offspring.

    Directory of Open Access Journals (Sweden)

    Michelle Lane

    Full Text Available Paternal health cues are able to program the health of the next generation however the mechanism for this transmission is unknown. Reactive oxygen species (ROS are increased in many paternal pathologies, some of which program offspring health, and are known to induce DNA damage and alter the methylation pattern of chromatin. We therefore investigated whether a chemically induced increase of ROS in sperm impairs embryo, pregnancy and offspring health. Mouse sperm was exposed to 1500 µM of hydrogen peroxide (H2O2, which induced oxidative damage, however did not affect sperm motility or the ability to bind and fertilize an oocyte. Sperm treated with H2O2 delayed on-time development of subsequent embryos, decreased the ratio of inner cell mass cells (ICM in the resulting blastocyst and reduced implantation rates. Crown-rump length at day 18 of gestation was also reduced in offspring produced by H2O2 treated sperm. Female offspring from H2O2 treated sperm were smaller, became glucose intolerant and accumulated increased levels of adipose tissue compared to control female offspring. Interestingly male offspring phenotype was less severe with increases in fat depots only seen at 4 weeks of age, which was restored to that of control offspring later in life, demonstrating sex-specific impacts on offspring. This study implicates elevated sperm ROS concentrations, which are common to many paternal health pathologies, as a mediator of programming offspring for metabolic syndrome and obesity.

  9. Regulation of the expression of proto-oncogenes by autocrine embryotropins in the early mouse embryo.

    Science.gov (United States)

    Jin, Xing Liang; O'Neill, C

    2011-06-01

    Autocrine embryotropins act as survival signals for the preimplantation embryo. In this study we examined the role of Paf in the transcription of the key proto-oncogenes Bcl2 and Fos. Transcripts were detected in oocytes and some cohorts of zygotes but not in cohorts of 2-cell, 8-cell, and blastocyst stage embryos. Immunolocalization of BCL2 and FOS showed little staining in oocytes and zygotes but increased staining in the embryo from the 2-cell to blastocyst stage. Paf (37 nM) treatment of 2-cell embryos caused an alpha-amanitin (26 μM)-sensitive increase in Bcl2 and Fos transcripts 20 min after treatment that subsided by 40 min. This increase was blocked by inhibition of calcium (by BAPTA-AM) or phosphatidylinositol-3-kinase signaling (by LY294002). Paf challenge also caused increased staining of BCL2 and FOS. Increased staining of FOS required new protein synthesis that had a half-life of 2-4 h after Paf challenge. Only a small proportion (∼12%) of individual 2-cell embryos collected from the reproductive tract had detectable Bcl2 and Fos. This dichotomous pattern of transcript expression is consistent with the known periodic actions of Paf (which has a periodicity of ∼90 min) and the relatively short half-life of the resulting transcripts. A BCL2 antagonist (HA14-1) caused a dose-dependent decrease in the capacity of cultured zygotes to develop to morphological blastocysts, which was partially reversed by the simultaneous addition of Paf to medium. The results show that Paf induces periodic transient transcriptions of key proto-oncogenes that result in the persistent presence of the resulting proteins in the preimplantation phase of development.

  10. 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.

  11. 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.

  12. 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.

  13. Intracellular mediators of transforming growth factor β superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo

    Directory of Open Access Journals (Sweden)

    Ishii Shunsuke

    2007-06-01

    Full Text Available Abstract Background Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Results Proteins that are downstream of the transforming growth factor-β superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFβ superfamily for their normal development. Phosphorylated Smad1 (pSmad1, pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Conclusion Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-β superfamily to endosomes is important for the regulation of growth factor signaling.

  14. A Preliminary Observation on the Development of Mouse Embryos Co-cultured with Human Oviductal Tissue or Conditioned Medium in Vitro

    Institute of Scientific and Technical Information of China (English)

    钟瑜; 张春雪; 潘善培

    1994-01-01

    The Present investigation has been carried out to examine the effect of human oviductal tissue co-culture system on the development of mouse embryos in vitro.Two-cell embryos collected from superovulated mouse were co-cultured with human oviductal tissue suspended in Ham'd F10+10%Fetal Calf Serum(F10 FCS),or in oviductal tissue conditioned medium and F10FCS as control.The results showed that the proportion developed into blastocyst,proportion of hatched and the velocity of cmbryo development were higher in both tissue co-culture and conditioned medium as compared with F10 FCS control.Furthermore,the velocity and percentage of embryomic devetopmem were higher in co-culture with ampullary tissue or its conditioned medium than that of isthmus,the effects of co-culture and conditioned medium on embryo development had no significant difference.All the embryos obtained from two co-culture systems could cleave normally,This experimental observation indicated that human oviductal epithelium might secrete some factors to promote the embryonic development in vitro.

  15. Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos

    Energy Technology Data Exchange (ETDEWEB)

    Yoneda, Akihiro, E-mail: ayoneda@sci.hokudai.ac.jp [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan); Division of Molecular Therapeutics, Center for Food & Medical Innovation, Hokkaido University (Japan); Watanabe, Tomomasa [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan)

    2015-05-01

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca{sup 2+}) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca{sup 2+} oscillatory pattern, whether PLCζ-induced Ca{sup 2+} oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca{sup 2+} oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca{sup 2+} oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca{sup 2+} oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca{sup 2+} oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca{sup 2+} oscillations continued after pronuclear formation. • The Ca{sup 2+} oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts.

  16. Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

    Science.gov (United States)

    Matson, P L; Graefling, J; Junk, S M; Yovich, J L; Edirisinghe, W R

    1997-07-01

    Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha-chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.

  17. Aberrant behavior of mouse embryo development after blastomere biopsy as observed through time-lapse cinematography.

    Science.gov (United States)

    Ugajin, Tomohisa; Terada, Yukihiro; Hasegawa, Hisataka; Velayo, Clarissa L; Nabeshima, Hiroshi; Yaegashi, Nobuo

    2010-05-15

    To analyze whether blastomere biopsy affects early embryonal growth as observed through time-lapse cinematography. Comparative prospective study between embryos in which a blastomere was removed and embryos in which a blastomere was not removed. An experimental laboratory of the university. We calculated the time between blastocele formation and the end of hatching, the time between the start and end of hatching, the number of contractions and expansions between blastocyst formation and the end of hatching, and the maximum diameter of the expanded blastocyst. In blastomere removal embryos, compaction began at the six-cell stage instead of at the eight-cell stage. We also found that hatching was delayed in these embryos as compared with matched controls. Moreover, the frequency of contraction and expansion movements after blastocyst formation was significantly higher in the blastomere removal group as compared with the control group. Finally, the maximum diameter of the expanded blastocyst just before hatching was not significantly different between both groups. These findings suggested that blastomere removal has an adverse effect on embryonic development around the time of hatching. Thus, future developments in preimplantation genetic diagnosis and screening should involve further consideration and caution in light of the influence of blastomere biopsy on embryonal growth. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  18. 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.

  19. 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.

  20. Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos

    Directory of Open Access Journals (Sweden)

    B Cisterna

    2009-08-01

    Full Text Available In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in premRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

  1. Transgenerational developmental effects and genomic instability after X-irradiation of preimplantation embryos: Studies on two mouse strains

    Energy Technology Data Exchange (ETDEWEB)

    Jacquet, P., E-mail: pjacquet@sckcen.be [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium); Buset, J.; Neefs, M. [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium); Vankerkom, J. [Division of Environmental Research, VITO, Boeretang 200, B-2400 Mol (Belgium); Benotmane, M.A.; Derradji, H. [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium); Hildebrandt, G. [Department of Radiotherapy and Radiation Oncology, University of Leipzig, Stephanstrasse 9a, D-04103 Leipzig (Germany); Department of Radiotherapy, University of Rostock, Suedring 75, D-18059 Rostock (Germany); Baatout, S. [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium)

    2010-05-01

    Recent results have shown that irradiation of a single cell, the zygote or 1-cell embryo of various mouse strains, could lead to congenital anomalies in the fetuses. In the Heiligenberger strain, a link between the radiation-induced congenital anomalies and the development of a genomic instability was also suggested. Moreover, further studies showed that in that strain, both congenital anomalies and genomic instability could be transmitted to the next generation. The aim of the experiments described in this paper was to investigate whether such non-targeted transgenerational effects could also be observed in two other radiosensitive mouse strains (CF1 and ICR), using lower radiation doses. Irradiation of the CF1 and ICR female zygotes with 0.2 or 0.4 Gy did not result in a decrease of their fertility after birth, when they had reached sexual maturity. Moreover, females of both strains that had been X-irradiated with 0.2 Gy exhibited higher rates of pregnancy, less resorptions and more living fetuses. Additionally, the mean weight of living fetuses in these groups had significantly increased. Exencephaly and dwarfism were observed in CF1 fetuses issued from control and X-irradiated females. In the control group of that strain, polydactyly and limb deformity were also found. The yields of abnormal fetuses did not differ significantly between the control and X-irradiated groups. Polydactyly, exencephaly and dwarfism were observed in fetuses issued from ICR control females. In addition to these anomalies, gastroschisis, curly tail and open eye were observed at low frequencies in ICR fetuses issued from X-irradiated females. Again, the frequencies of abnormal fetuses found in the different groups did not differ significantly. In both CF1 and ICR mouse strains, irradiation of female zygotes did not result in the development of a genomic instability in the next generation embryos. Overall, our results suggest that, at the moderate doses used, developmental defects

  2. Live dynamic OCT imaging of cardiac structure and function in mouse embryos with 43 Hz direct volumetric data acquisition

    Science.gov (United States)

    Wang, Shang; Singh, Manmohan; Lopez, Andrew L.; Wu, Chen; Raghunathan, Raksha; Schill, Alexander; Li, Jiasong; Larin, Kirill V.; Larina, Irina V.

    2016-03-01

    Efficient phenotyping of cardiac dynamics in live mouse embryos has significant implications on understanding of early mammalian heart development and congenital cardiac defects. Recent studies established optical coherence tomography (OCT) as a powerful tool for live embryonic heart imaging in various animal models. However, current four-dimensional (4D) OCT imaging of the beating embryonic heart largely relies on gated data acquisition or postacquisition synchronization, which brings errors when cardiac cycles lack perfect periodicity and is time consuming and computationally expensive. Here, we report direct 4D OCT imaging of the structure and function of cardiac dynamics in live mouse embryos achieved by employing a Fourier domain mode-locking swept laser source that enables ~1.5 MHz A-line rate. Through utilizing both forward and backward scans of a resonant mirror, we obtained a ~6.4 kHz frame rate, which allows for a direct volumetric data acquisition speed of ~43 Hz, around 20 times of the early-stage mouse embryonic heart rate. Our experiments were performed on mouse embryos at embryonic day 9.5. Time-resolved 3D cardiodynamics clearly shows the heart structure in motion. We present analysis of cardiac wall movement and its velocity from the primitive atrium and ventricle. Our results suggest that the combination of ultrahigh-speed OCT imaging with live embryo culture could be a useful embryonic heart phenotyping approach for mouse mutants modeling human congenital heart diseases.

  3. Electroactivation of Oocyte and Electrofusion of Two-cell Embryos of Mouse%小鼠卵母细胞的电激活及胚胎电融合

    Institute of Scientific and Technical Information of China (English)

    李莉; 赵浩斌; 魏庆信

    2001-01-01

    The electric impulse was applied in oocyte electroactivation and two-cell embryonic electrofusion of mouse. The results showed that the difference of the rate of oocyte activation was obvious(P<0.01) as a result of different electric field strength,while using 30μs ,two pulses and 0.1 kV/cm pulse could get the best result(76.9%). The difference existed among different electric field strength,pulse number and pulse duration,for embryonic fusion too.The best fusion result(84.6%) could be obtained by using 0.1kV/cm, 30μs and two pulses.

  4. 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.

  5. Transglutaminase 3 expression in C57BL/6J mouse embryo epidermis and the correlation with its differentiation

    Institute of Scientific and Technical Information of China (English)

    Jian ZHANG; Hui Ying ZHI; Fang DING; Ai Ping LUO; Zhi Hua LIU

    2005-01-01

    Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornified envelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then we determined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. We found a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cells from E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed in the granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin development provided several morphological evidences for the epidermal differentiation. The above findings suggest that the expression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.

  6. Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

    Science.gov (United States)

    Wang, Lu; Lu, Angeleem; Zhou, Hong-Xia; Sun, Ran; Zhao, Jie; Zhou, Cheng-Jie; Shen, Jiang-Peng; Wu, Sha-Na; Liang, Cheng-Guang

    2013-01-01

    Casein kinase I alpha (CK1α) is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1) extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP), an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

  7. Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available Casein kinase I alpha (CK1α is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1 extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP, an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

  8. 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.

  9. A mutant of SWAP-70, a phosphatidylinositoltrisphosphate binding protein, transforms mouse embryo fibroblasts, which is inhibited by sanguinarine.

    Directory of Open Access Journals (Sweden)

    Yasuhisa Fukui

    Full Text Available SWAP-70, a phosphatidylinositol trisphosphate (PtdIns(3,4,5P(3 binding protein, has been suggested to be involved in transformation of mouse embryo fibroblasts (MEFs as well as membrane ruffling after growth factor stimulation of the cells. A mutant, SWAP-70-374, was found to be able to bind to F-actin in vitro, whereas wild-type SWAP-70 failed to do so. This mutant was present at the plasma membrane without any stimulation while the wild-type protein was present only in the cytosol unless cells were stimulated with EGF. Expression of this mutant in MEFs resulted in morphologic transformation, fast growth, and loss of contact inhibition, suggesting that SWAP-70 with this mutation can transform the cells. ERK1/2 was activated in SWAP-70-374-transformed cells. Use of MEK inhibitors revealed that the ERK1/2 pathway does not affect the cell growth of MEFs but is responsible for loss of contact inhibition. To investigate the function of SWAP-70 further, drugs that can inhibit SWAP-70-dependent cell responses were screened. Among various drugs, sanguinarine was found to inhibit transformation of MEFs by SWAP-70-374. This drug was able to inhibit SWAP-70-mediated membrane ruffling as well, suggesting that its effect was closely related to the SWAP-70 signaling pathway. These results suggest that SWAP-70-374 can activate some signaling pathways, including the ERK1/2 pathway, to transform MEFs.

  10. Sexing of Mouse Preimplantation Embryos Using Polymerase Chain Reaction%运用PCR对小鼠植入前胚胎进行性别诊断

    Institute of Scientific and Technical Information of China (English)

    李汶; 陆长富; 卢光琇

    2001-01-01

    In order to determine the sex of mouse embryo, 1 or 2 blastomeres were biopsied from Kun-ming-white mouse preimplantation embryo at 4-8 cells stage. The gDNA of the single-blastomere was abstracted. According to the base sequence of 145C5, a repititive sequence of Y chromosome of mouse C57BL6, a pair of primer were asigned and synthesized. The gDNA was amplified using these primers. 108 mouse preimplantation embryos were sexed via this technique. 46 male embryos and 62 female embryos were transfered into five pseudopreganant mothers respectively. 4 male litters and 9 female litters were obtained. The diagnosis positive rate was 100%(4/4) and 70% (9/13)respectively. The result of PCR indecated that there was no difference between the repititive sequence of Y chromosome in mouse C57BL6 and Kun-ming-white mouse. The technique developed in this study might be further used for preimplantation genetic diagnosis of single-gene defects.%根据C57BL6小鼠Y染色体重复序列145C5的碱基顺序, 设计并合成一对引物, 运用PCR扩增昆明白小鼠植入前胚胎卵裂球DNA, 以确定其性别。共对108枚活检胚胎的相应卵裂球进行了性别诊断, 获雄性胚46枚, 雌性胚62枚, 移植后分别获雄性仔鼠4只, 准确率100%(4/4), 雌性仔鼠9只, 准确率70%(9/13)。本研究结果表明小鼠Y染色体重复序列145C5的碱基顺序在C57BL6小鼠和昆明白小鼠中基本一致;为农牧业动物进行性别选择和运用PCR进行单基因病植入前遗传学诊断提供了方法学基础。

  11. 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

  12. Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

    Science.gov (United States)

    Ruane, Peter T; Berneau, Stéphane C; Koeck, Rebekka; Watts, Jessica; Kimber, Susan J; Brison, Daniel R; Westwood, Melissa; Aplin, John D

    2017-09-01

    How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model? Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium. In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models. This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells. Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to

  13. Mouse newborn cells allow highly productive mouse cytomegalovirus replication, constituting a novel convenient primary cell culture system.

    Science.gov (United States)

    Le-Trilling, Vu Thuy Khanh; Trilling, Mirko

    2017-01-01

    Mammalian cell culture is indispensable for most aspects of current biomedical research. Immortalized cell lines are very convenient, but transforming principles (e.g. oncogenic viruses or their oncogenes) can heavily influence the experimental outcome. Primary cells do not share this apparent disadvantage but are more laborious to generate. Certain viruses (e.g. mouse cytomegalovirus) do not replicate efficiently in most transformed cell lines. In the past, such viruses have been routinely propagated on primary mouse embryonic fibroblasts (MEF) established around day 17 (d17) of gestation. According to new regulations of the European Union, experiments using gravid mammals and/or their embryos in the last trimester (>d14 in the case of mice) of gestation do require explicit permission of the local authorities responsible for animal care and use. Applying for such permission is time-consuming and often inflexible. Embryonic fibroblasts could also be produced at earlier time points of pregnancy from younger and smaller embryos. Obviously, this approach consumes more pregnant mice and embryos. Newborn mice are larger thus yielding more cells per sacrificed animal and the new Directive (2010/63/EU) excludes the killing of animals solely for the use of their organs or tissues. We established a convenient protocol to generate adherent mouse newborn cells (MNC). A direct comparison of MNC with MEF revealed that MNC fully recapitulate all tested aspects of a broad panel of virological parameters (plaque size, final titers, viral replication kinetics, viral gene expression, drug and interferon susceptibility as well as species specificity). The herein described approach allows researchers the legal use of primary cells and contributes to the 3R (replace, reduce, refine) guiding principles-especially the 'reduce' aspect-for the use of animals in scientific research. Additionally, it offers the option to directly compare in vitro and in vivo experiments when MNC are

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

    Science.gov (United States)

    van den Brink, Susanne C; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A; Martinez Arias, Alfonso

    2014-11-01

    Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. Trim43a, Trim43b, and Trim43c: Novel mouse genes expressed specifically in mouse preimplantation embryos.

    Science.gov (United States)

    Stanghellini, Ilaria; Falco, Geppino; Lee, Sung-Lim; Monti, Manuela; Ko, Minoru S H

    2009-12-01

    We describe the identification and characterization of Trim43a, Trim43b, and Trim43c genes, whose expression are restricted to preimplantation stages and peak at the 8-cell to morula stage. We identified a 5kb DNA fragment that covers upstream region of Trim43a as a putative promoter, which can drive the expression of mStrawberry fluorescent protein in a manner similar to endogenous Trim43 genes. Trim43 genes will be useful stage-specific markers for the study of preimplantation embryos.

  5. Black ginseng inhibits ethanol-induced teratogenesis in cultured mouse embryos through its effects on antioxidant activity.

    Science.gov (United States)

    Lee, Se-Ra; Kim, Mi-Ra; Yon, Jung-Min; Baek, In-Jeoung; Park, Chun Gui; Lee, Beom Jun; Yun, Young Won; Nam, Sang-Yoon

    2009-02-01

    Fetal alcohol syndrome is caused by excessive ethanol consumption during pregnancy. We investigated the effect of black ginseng (red ginseng that is subjected to 9 cycles of 95-100 degrees C for 2-3h) on ethanol-induced teratogenesis using an in vitro whole embryo culture system. Postimplantational mouse embryos at embryonic day 8.5 were exposed to ethanol (1 microl/ml) in the presence or absence of black ginseng (1, 10, and 100 microg/ml) for 2 days, and then morphological scoring and real-time PCR analysis were carried out. In ethanol-treated embryos, the total morphological score and individual scores for flexion, heart, fore-, mid-, and hindbrains, otic, optic, and olfactory systems, branchial bars, maxillary and mandibular processes, caudal neural tube, and somites were significantly lower than the control group (pteratogenesis through the augmentation of antioxidative activity in embryos.

  6. Biological response to ionizing radiation in mouse embryo fibroblasts with a targeted disruption of the DNA polymerase beta gene.

    Science.gov (United States)

    Miura, M; Watanabe, H; Okochi, K; Sasaki, T; Shibuya, H

    2000-06-01

    Base excision repair (BER) is carried out by two distinct pathways in mammalian cells, one dependent on DNA polymerase beta (Polb) and the other on proliferating cell nuclear antigen (Pcna). We studied whether the Polb-dependent pathway plays an important role in BER in vivo after exposure to ionizing radiation. For this purpose, we used mouse embryo fibroblasts derived from wild-type and Polb gene knockout littermates. Both cell lines had essentially the same clonogenic cell survival and low levels of apoptosis as determined by a colony formation assay and by a change in mitochondrial membrane potential, respectively. No significant cleavage of protein kinase C delta (Pkcd) in vivo, which is a substrate for caspase 3, was detected, and intact Pkcd was retained in both cell lines for at least 72 h after irradiation. Similar significant increases in caspase 3-like activities as measured by Asp-Glu-Val-Asp (DEVD) cleaving activity in vitro were observed in both cell lines after irradiation. Radiation induced cell cycle arrest in the form of a G(2)-phase block, and G(2)/M-phase fractions reached a peak approximately 10 h after irradiation and decreased thereafter with a similar time course in both cell lines. Similar levels of chromatin-bound Pcna were observed immediately after irradiation in non-S-phase cells of both cell lines and disappeared by 4 h after irradiation. We conclude that the deficiency in Polb does not have a significant influence on the radiation responses of these cells. Together with evidence accumulated in vitro, these results strongly support the idea that the Pcna-dependent pathway predominantly acts in BER of radiation-induced DNA damage in vivo.

  7. The combined effects of MRI and x-rays on ICR mouse embryos during organogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Yeunhwa; Hasegawa, Takeo; Yamamoto, Youichi [Suzuka Univ. of Medical Science, Mie (Japan); Kai, Michiaki; Kusama, Tomoko

    2001-09-01

    The combined effects of X-rays and magnetic resonance imaging (MRI) on mouse embryos at an early stage of organogenesis were investigated. Pregnant ICR mice were irradiated on day 8 of gestation with X-rays at a dose of 1 Gy and/or MRI at 0.5 T for 1 hour. The mortality rates of the embryos or fetuses, the incidence of external malformations, the fetal body weight and the sex ratio were observed at day 18 of gestation. A significant increase in embryonic mortality was observed after exposure to either 1 Gy of X-radiation or 0.5 T MRI. However, the combined X-rays and MRI did not show a statistically significant increase in embryonic mortality compared with the control. External malformations, such as exencephaly, a cleft palate and anophthalmia, were observed in mice irradiated with X-rays and/or MRI. The incidence of each malformation in all treated groups increased with statistical significance compared with the control mice. The incidence in mice irradiated with both X-rays and MRI was lower than in mice irradiated with only X-rays. The combined effects of the combination of radiation and MRI on the external malformations might be antagonistic. There were no statistically significant differences in fetal death, fetal body weight and sex ratio among all experimental groups. (author)

  8. The effect of hepatocyte growth factor on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

    Directory of Open Access Journals (Sweden)

    Mohammad H. Bahadori

    2011-05-01

    Full Text Available Background: Oocyte invitro maturation is an enormously promising technology for the treatment of infertility, yet its clinical application remains limited owing to poor success rates. Therefore, this study was devised to evaluate the effect of hepatocyte growth factor (HGF on in vitro maturation of immature mouse oocytes and resulting embryos development. Materials and Method: Cumulus – oocyte complex and germinal vesicle were obtained from eighteen 6-8 weeks-old female NMRI mice 46-48 hours after administration of an injection of 5 IU PMSG (Pregnant Mares’ Serum Gonadotrophin. Oocytes were culture in TCM199 (Tissue culture medium-199 supplemented with dosages of 0, 10, 20, 50 and 100 ng/ml of HGF. After 24 hours, metaphase ІІ oocytes were co-incubated with sperms for 4-6 hours in T6 medium. Following isolation of two pronucleus embryos, cleavage of embryos was assessed in the same medium till blastocyst stage. The number of oocytes and embryos was recorded under an invert microscope and the rate of oocyte maturation, fertilization and embryos cleavage until blastocyst stage compared using of student χ2 test. Results: In all compared groups, oocytes growth and embryos development rate in the 20 ng/ml of HGF treatment group was significantly higher (p<0.05 than the control group (p<0.05.Conclusion: 20 ng/ml of HGF improved the nuclear maturation and embryo development up to blastocyst stage during culture condition

  9. Inhibition of fumonisin B1 cytotoxicity by nanosilicate platelets during mouse embryo development.

    Directory of Open Access Journals (Sweden)

    Yu-Jing Liao

    Full Text Available Nanosilicate platelets (NSP, the form of natural silicate clay that was exfoliated from montmorillonite (MMT, is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1, and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD. In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1 in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.

  10. 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

  11. Fertilization-induced autophagy in mouse embryos is independent of mTORC1.

    Science.gov (United States)

    Yamamoto, Atsushi; Mizushima, Noboru; Tsukamoto, Satoshi

    2014-07-01

    Autophagy is a dynamically regulated intracellular degradation system that is important for cellular processes such as amino acid production during starvation and intracellular quality control. Previously, we reported that autophagy is suppressed in oocytes but is rapidly up-regulated after fertilization. During this period, autophagy is thought to be important for the generation of amino acids from the bulk degradation of maternal proteins that have accumulated during oogenesis. However, the mechanism of autophagy induction after fertilization is presently unknown. In most cell types, autophagy is negatively controlled by mammalian target of rapamycin complex 1 (mTORC1), which is typically regulated by amino acids and insulin or related growth factors. In this study, we determined the role of mTORC1 in fertilization-induced autophagy. On the basis of the phosphorylation status of mTORC1 substrates, we found that mTORC1 activity was relatively high in metaphase II (MII) oocytes but was rapidly decreased within 3 h of fertilization. However, chemical inhibition of mTORC1 by Torin1 or PP242 in MII oocytes or fertilized embryos did not induce autophagy. In addition, activation of mTORC1 by cycloheximide did not inhibit fertilization-induced autophagy in fertilized embryos. By contrast, the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 effectively suppressed autophagy in these embryos. These data suggest that, even though autophagy induction and postfertilization mTORC1 activity are inversely correlated with each other, as observed in other cell types, mTORC1 suppression is neither essential nor sufficient for fertilization-induced autophagy, highlighting a unique feature of the regulation mechanism of autophagy-mediated intracellular turnover in early embryos. © 2014 by the Society for the Study of Reproduction, Inc.

  12. Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea

    Directory of Open Access Journals (Sweden)

    Edward R. Hills

    2010-04-01

    Full Text Available Women are advised not to attempt pregnancy while on hydroxyurea (HU due to the teratogenic effects of this agent, based on results obtained from animal studies. Several case reports suggest that HU may have minimal or no teratogenic effects on the developing human fetus. Fourteen cases of HU therapy in pregnant patients diagnosed with acute or chronic myelogenous leukemia, primary thrombocythemia, or sickle cell disease (SCD have been reported. Three pregnancies were terminated by elective abortion; 1 woman developed eclampsia and delivered a phenotypically normal stillborn infant. All other patients delivered live, healthy infants without congenital anomalies. We contend that case studies such as these have too few patients and cannot effectively address the adverse effect of HU on preimplantation embryo or fetuses. The objective of this study was to assess the risks associated with a clinically relevant dose of HU used for the treatment of SCD, on ovulation rate and embryo development, using adult C57BL/6J female mice as a model. In Experiment 1, adult female mice were randomly assigned to a treatment or a control group (N = 20/group. Treatment consisted of oral HU (30 mg/kg for 28 days; while control mice received saline (HU vehicle. Five days to the cessation of HU dosing, all mice were subjected to folliculogenesis induction with pregnant mare serum gonadotropin (PMSG. Five mice/group were anesthetized at 48 hours post PMSG to facilitate blood collection via cardiac puncture for estradiol-17β (E2 measurement by RIA. Ovulation was induced in the remaining mice at 48 hours post PMSG with human chorionic gonadotropin (hCG and immediately caged with adult males for mating. Five plugged female mice/group were sacrificed for the determination of ovulation rate. The remaining mated mice were sacrificed about 26 hours post hCG, ovaries excised and weighed and embryos harvested and cultured in Whitten’s medium (WM supplemented with CZBt. In

  13. Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos

    DEFF Research Database (Denmark)

    Matzen, Sara Maj Hyldig; Østrup, Olga; Vejlsted, Morten

    2011-01-01

    -positive primordial germ cells (PGCs) compared with neighboring somatic cells in porcine embryos at Embryonic Day 15 (E15), E17, E20, E21, and E28. We show that, in agreement with the mouse model, a significantly lower level of DNA methylation was observed in the early migrating PGCs. This level...

  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. Effect of Partial Incision of the Zona Pellucida by Piezo-Micromanipulator for In Vitro Fertilization Using Frozen-Thawed Mouse Spermatozoa on the Developmental Rate of Embryos Transferred at the 2-Cell Stage

    National Research Council Canada - National Science Library

    Yosuke Kawase; Takamitsu Iwata; Otoya Ueda; Nobuo Kamada; Takanori Tachibe; Yukari Aoki; Kou-ichi Jishage; Hiroshi Suzuki

    2002-01-01

    .... The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared...

  16. 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.

  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. A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

    DEFF Research Database (Denmark)

    Klinck, Rasmus; Füchtbauer, Ernst-Martin; Ahnfelt-Rønne, Jonas;

    2011-01-01

    of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)(1Hri), to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates...

  19. 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

  20. Mmu-miR-193 Is Involved in Embryo Implantation in Mouse Uterus by Regulating GRB7 Gene Expression

    Science.gov (United States)

    Li, Rong; He, Junlin; Chen, Xuemei; Ding, YuBin; Wang, Yingxiong; Long, ChunLan; Shen, Lianju

    2014-01-01

    Embryo implantation is a complicated process involving a series of endometrial changes that depend on differential gene expression. MicroRNAs (miRNAs) are important for regulation of gene expression. Previous studies have shown that miRNAs may participate in the regulation of gene expression during embryo implantation. To explore the role of endometrial miRNAs in early murine pregnancy, we used microarrays to investigate whether miRNAs were differentially expressed in the mouse endometrium on pregnancy day 4 (D4) and day 6 (D6). The results demonstrated that 17 miRNAs were upregulated and 18 were downregulated (>2-fold) in D6 endometria compared to D4. We identified that mmu-miR-193 exhibited the highest upregulation on D6, and the upregulation of mmu-miR-193 before embryo implantation could reduce the embryo implantation rate. Further, we demonstrated that mmu-miR-193 influenced embryo implantation by regulating growth factor receptor-bound protein 7 expression. In summary, our study suggests that mmu-miR-193 plays an important role in embryo implantation. PMID:24336674

  1. 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.

  2. 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.

  3. Nucleus downscaling in mouse embryos is regulated by cooperative developmental and geometric programs

    OpenAIRE

    Elina Tsichlaki; Greg FitzHarris

    2016-01-01

    Maintaining appropriate nucleus size is important for cell health, but the mechanisms by which this is achieved are poorly understood. Controlling nucleus size is a particular challenge in early development, where the nucleus must downscale in size with progressive reductive cell divisions. Here we use live and fixed imaging, micromanipulation approaches, and small molecule analyses during preimplantation mouse development to probe the mechanisms by which nucleus size is determined. We find a...

  4. Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion.

    Science.gov (United States)

    Kidder, Benjamin L; Oseth, Leann; Miller, Shanna; Hirsch, Betsy; Verfaillie, Catherine; Coucouvanis, Electra

    2008-06-01

    Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.

  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. Protective Effect of Quercetin on the Development of Preimplantation Mouse Embryos against Hydrogen Peroxide-Induced Oxidative Injury

    Science.gov (United States)

    Zhang, Qin-hua; Yan, Zhi-guang; Liang, Hong-xing; Chai, Wei-ran; Yan, Zheng; Kuang, Yan-ping; Qi, Cong

    2014-01-01

    Quercetin, a plant-derived flavonoid in Chinese herbs, fruits and wine, displays antioxidant properties in many pathological processes associated with oxidative stress. However, the effect of quercetin on the development of preimplantation embryos under oxidative stress is unclear. The present study sought to determine the protective effect and underlying mechanism of action of quercetin against hydrogen peroxide (H2O2)-induced oxidative injury in mouse zygotes. H2O2 treatment impaired the development of mouse zygotes in vitro, decreasing the rates of blastocyst formation and hatched, and increasing the fragmentation, apoptosis and retardation in blastocysts. Quercetin strongly protected zygotes from H2O2-induced oxidative injury by decreasing the reactive oxygen species level, maintaining mitochondrial function and modulating total antioxidant capability, the activity of the enzymatic antioxidants, including glutathione peroxidase and catalase activity to keep the cellular redox environment. Additionally, quercetin had no effect on the level of glutathione, the main non-enzymatic antioxidant in embryos. PMID:24586844

  7. RESEARCHES REGARDING THE INFLUENCE OF RECOVERY MEDIA ON THE IN VITRO DEVELOPMENT CAPACITY OF THE PREIMPLANTATIONAL MOUSE EMBRYO

    Directory of Open Access Journals (Sweden)

    ADA CEAN

    2009-05-01

    Full Text Available Phosphate Bufered Saline with 0.4% BSA and M2 medium are one of the most common media used in embryorecovery. The aim of our paper was to investigate if the recovery media used for the recovery of the mouseembryo is influencing in vitro developmental capacity. As biological material we used 10 used were mousefemales, age 2 months superovulated with 5UI PMSG (Pregnant Mare Serum Gonadotropine and 5 UI hCG(human Corionic Gonadotropine. The embryos used were recovered, by oviduct flushing, at 24 hours from theidentification of the vaginal plug. The majority of the embryos (78.3% were in two cells stage. A total of 123, 2cells embryos were cultivated in M16 medium. The evolution of the embryos was examined at 24, 48 and 72hours interval. The proportion of hatched blastocyst was higher at the embryos recovered with M2 (53.7%compared with the embryos recovered with PBS 0.4% BSA. The difference is statistically very significant(p<0.001. Embryos recovered in M2 media have a higher in vitro developmental capacity compared with theembryos recovered in PBS media supplemented with 0,4% BSA, possibly because of the sodium bicarbonate andlactate used in M2 media for pH regulation.

  8. Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation

    Directory of Open Access Journals (Sweden)

    Watson Andrew J

    2007-01-01

    Full Text Available Abstract Background Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2. The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments. Results Our results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4 are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels. Conclusion These results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK.

  9. Deprenyl enhances the teratogenicity of hydroxyurea in organogenesis stage mouse embryos.

    Science.gov (United States)

    Schlisser, Ava E; Hales, Barbara F

    2013-08-01

    Hydroxyurea, an antineoplastic drug, is a model teratogen. The administration of hydroxyurea to CD1 mice on gestation day 9 induces oxidative stress, increasing the formation of 4-hydroxy-2-nonenal adducts to redox-sensitive proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the caudal region of the embryo. GAPDH catalytic activity is reduced, and its translocation into the nucleus is increased. Because the nuclear translocation of GAPDH is associated with oxidative stress-induced cell death, we hypothesized that this translocation plays a role in mediating the teratogenicity of hydroxyurea. Deprenyl (also known as selegiline), a drug used as a neuroprotectant in Parkinson's disease, inhibits the nuclear translocation of GAPDH. Hence, timed pregnant CD1 mice were treated with deprenyl (10mg/kg) on gestation day 9 followed by the administration of hydroxyurea (400 or 600mg/kg). Deprenyl treatment significantly decreased the hydroxyurea-induced nuclear translocation of GAPDH in the caudal lumbosacral somites. Deprenyl enhanced hydroxyurea-mediated caudal malformations, inducing specifically limb reduction, digit anomalies, tail defects, and lumbosacral vertebral abnormalities. Deprenyl did not augment the hydroxyurea-induced inhibition of glycolysis or alter the ratio of oxidized to reduced glutathione. However, it did dramatically increase cleaved caspase-3 in embryos. These data suggest that nuclear GAPDH plays an important, region-specific, role in teratogen-exposed embryos. Deprenyl exacerbated the developmental outcome of hydroxyurea exposure by a mechanism that is independent of oxidative stress. Although the administration of deprenyl alone did not affect pregnancy outcome, this drug may have adverse consequences when combined with exposures that increase the risk of malformations.

  10. [The surface glycolipid antigen specific for the internal cell mass of the mouse blastocyst and of the stem cells of murine teratocarcinoma F9].

    Science.gov (United States)

    Anfimova, M L; Bannikov, G A; Troianovskiĭ, S M

    1989-01-01

    A new monoclonal antibody that recognizes a new antigen on the surface of mouse teratocarcinoma F9 stem cells has been described. This antigen is a glycolipid as demonstrated by inhibition of immunofluorescence by different monosaccharides, glycoproteins and glycolipid fraction of F9 cells as well as by chemical analysis. Immunofluorescent staining of in vitro cultivated preimplantation mouse embryos has demonstrated that this antigen is specific only of internal cell mass cells of late blastocyst.

  11. The effects of MRI and X -radiation on ICR mouse embryos during organogenesis

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    Gu, Yeunhwa; Hasegawa, Takeo; Muto, Hiroe; Santokuya, Takumi; Suzuki, Sachiyo [Suzuka University, Mie (Japan); Kusama, Tomoko [Oita University, Oita (Japan)

    1999-07-01

    The combined effects of X -radiation and magnetic resonance imaging (MRI) on mouse embryos during organogenesis were investigated. Pregnant ICR mice were irradiated with whole-body X -rays at 1 Gy with a dose rate of 0.2 Gy/min and/or a single whole-body MRI at 0.5 T for 1 hour. Embryonic and fetal mortalities, the incidence of external gross malformations, fetal body weight and sex ratios of mice treated on day 8 of gestation were determined at day 18 of gestation. There were no significant differences in the frequency of prenatal death, fetal body weight and sex ratios between control and 0.5 T irradiated mice. The frequencies of embryonic deaths in mice irradiated with X -rays increased significantly. The frequencies of external malformations such as exencephaly and anophthalmia in mice irradiated with X -rays or MRI increased significantly. However the frequencies of external malformation and prenatal death in the mice irradiated with both X -rays and MRI decreased more than in mice irradiated with X -rays alone. The combined effects of radiation and MRI on the external malformations and embryonic death might be antagonistic.

  12. 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.

  13. Establishment of in vitro Co-culture to Mouse Parthenogenetic Embryos%小鼠孤雌胚胎体外共培养体系的建立

    Institute of Scientific and Technical Information of China (English)

    何志全; 陈伟; 李三华; 刘淑娟

    2011-01-01

    [目的]在输卵管上皮细胞和/或垂体细胞饲养层下培养小鼠孤雌胚胎,探讨促进小鼠孤雌胚发育突破2-cell阻滞的机制,建立完善的培养体系.[方法]复式激活小鼠卵母细胞后分别在不同饲养层的CZB培养液中进行培养,并观察、比较各培养条件下孤雌胚胎的发育情况.[结果]培养至24 h,各组无显著差异,都有较高卵裂率.培养至48 h,2类饲养层细胞下的孤雌胚胎4~8-cell发育效果好,与单独一类饲养层细胞培养差异显著,与对照组比较差异极显著.培养至96 h,2类饲养层细胞下有49.4%(42/85)孤雌胚胎发育为桑囊胚,与其他各组比较差异极显著.[结论]含有输卵管上皮细胞+垂体细胞的CZB培养液可有效促进小鼠孤雌胚胎发育突破2-ceU阻滞,并进一步提高其发育率.%[ Objective ] In order to study the mechanism of 2-cell block in mouse parthenogenetic embryos development,mouse parthenogenetic embryos were cultured based on oviduct epithelial cells and/or pituitary cells for feeder layer. [ Method ] After compound activation,the mouse oocytes were cultured in CZB medium which containing different feeder layers, then observed and compared parthenogenetic embryos situation under various culture conditions. [ Result ] Cultured for 24 h,no significant differences in each group,all have a higher cleavage rate. Cultured for 48 h,has developmental effect of 4 -8-cell with two types of feeder cells,compared with single feeder layer cell,it has significant difference,compared with control group,it has extremely significant difference. Cultured for 96 h,49.4% (42/85) parthenogenetic embryos developed to morula/blastocyst under two types of feeder cells,and other groups significant difference. [ Conclusion] CZB medium which containing oviduct epithelial cells + pituitary cells could effectively promote the development of mouse parthenogenetic embryos and breakthroughs the 2-cell block,further improve the development

  14. ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo.

    Science.gov (United States)

    De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M

    2015-05-15

    A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition.

  15. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  16. High osmolality vitrification: a new method for the simple and temperature-permissive cryopreservation of mouse embryos.

    Directory of Open Access Journals (Sweden)

    Keiji Mochida

    Full Text Available Procedures for cryopreserving embryos vary considerably, each having its specific advantages and disadvantages in terms of technical feasibility, embryo survival yield, temperature permissibility and species- or strain-dependent applicability. Here we report a high osmolality vitrification (HOV method that is advantageous in these respects. Cryopreservation by vitrification is generally very simple, but, unlike slow freezing, embryos should be kept at a supercooling temperature (below -130°C to avoid cryodamage. We overcame this problem by using an HOV solution containing 42.5% (v/v ethylene glycol, 17.3% (w/v Ficoll and 1.0 M sucrose. This solution is more viscous than other cryopreservation solutions, but easy handling of embryos was assured by employing a less viscous equilibration solution before vitrification. Most (>80% embryos cryopreserved in this solution survived at -80°C for at least 30 days. Normal mice were recovered even after intercontinental transportation in a conventional dry-ice package for 2-3 days, indicating that special containers such as dry shippers with liquid nitrogen vapor are unnecessary. The HOV solution could also be employed for long-term storage in liquid nitrogen, as with other conventional cryoprotectants. Finally, we confirmed that this new vitrification method could be applied successfully to embryos of all six strains of mice we have tested so far. Thus, our HOV method provides an efficient and reliable strategy for the routine cryopreservation of mouse embryos in animal facilities and biomedical laboratories, and for easy and cheap transportation.

  17. Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos.

    Science.gov (United States)

    Yao, Li-Juan; Zhong, Zhi-Sheng; Zhang, Li-Sheng; Chen, Da-Yuan; Schatten, Heide; Sun, Qing-Yuan

    2004-05-01

    Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.

  18. 胚胎时期小鼠小肠的组织发生%Histogenesis of mouse small intestine in embryo

    Institute of Scientific and Technical Information of China (English)

    郎尉雅; 贾琴; 张敏; 崔梦卿; 李良平; 张琳

    2013-01-01

    Objective To observe the morphogenesis of mouse small intestine tissues and distribution of caliciform cells in embryo at different stages in order to provide morphological data for the development of mouse small intestine.Methods HE and PAS stain were used in the paraffin section of mouse embryo aged from embryo 13.5 day (E13.5 d) to postnatal 1 (P1) day.Rseults (1) At E13.5d,the small intestine of mouse was differentiated,the mucosa,submucosa,muscularis,serosa appeared.(2) At E15.5 d,the epithelium of intestine started differentiating into intestinal villus,and caliciform cell gradually formed at the day E16.5 embryo,at P1day embryo small intestinal gland builded up.At this time of embryo,the basic structure of small intestine came into being.(3)The number of the caliciform cells increased as mice age,that caliciform cells were most abundant at P1 day (P<0.05).Caliciform cells were distributed mainly over the epithelial villus,i predominate in the terminal ileum,and deminishing from ileum,jejunum to duodenum (P<0.05).Conclusion The differentiation of small intestine is fastest at E15.5~E17.5 d,the basic structure of the small intestine with certain digestive and absorptive function is formed during the embryonic period.%目的 观察小鼠胚胎各个时期小肠组织的形态结构及杯状细胞在小肠内的分布规律,为小鼠小肠的组织发生提供形态学依据.方法 采用HE染色和PAS染色,对小鼠胚胎第13.5天(E13.5d)至出生后第1天(Pld)胚胎的石蜡切片染色并行光学显微镜观察.结果 (1)小鼠肠壁于E13.5 d已分化出现黏膜层、黏膜下层、肌层及浆膜.(2)肠绒毛于E15.5 d分化形成,杯状细胞于E16.5 d逐渐发育分化出现,肠腺于E18.5~P1 d发育分化形成,此时小肠基本结构形成.(3)杯状细胞主要分布于小肠绒毛上皮,其中以回肠末端最多,回肠、空肠、十二指肠顺次递减(P<0.05).杯状细胞数量随胎龄逐渐增加而逐渐增多,于P1 d

  19. 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.

  20. Utero-tubal embryo transfer and vasectomy in the mouse model.

    Science.gov (United States)

    Bermejo-Alvarez, Pablo; Park, Ki-Eun; Telugu, Bhanu P

    2014-02-28

    The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

  1. 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.

  2. Restraint stress alters immune parameters and induces oxidative stress in the mouse uterus during embryo implantation.

    Science.gov (United States)

    Liu, Guanhui; Dong, Yulan; Wang, Zixu; Cao, Jing; Chen, Yaoxing

    2014-12-01

    The influence of stress on embryo implantation is not well understood. Prior studies have focused on later gestational stages and the long-term impact of stress on immune function. The objective of this study is to investigate the effects of restraint stress on the immune parameters and the oxidative states of the uterus during implantation. In this study, pregnant CD1 mice were subjected to restraint stress (4 h/d) on embryonic day 1 (E1) and sacrificed on E3, E5, and E7. Maternal plasma corticosterone (CORT) secretion and implantation sites in the uterus were examined. The uterine (excluding embryos) homogenate and uterine lymphocytes were collected to examine oxidative stress states and associated immune parameters. The results demonstrated that restraint stress increased maternal plasma CORT secretion and reduced the number of implantation sites by 15.3% on E5 and by 26.1% on E7. Moreover, restraint stress decreased the density of uterine natural killer (uNK) cells in the endometrium by 22.1-47.9% and increased the density of mast cells in the myometrium by 55.6-76.9%. Restraint stress remarkably decreased the CD3(+)CD4(+) T/CD3(+)CD8(+) T cell ratio (by 26.2-28.9%) and attenuated uterine lymphocyte proliferation and secretion of cytokines. In addition, restraint stress threatened the intracellular equilibrium between oxidants and antioxidants, resulting in decreased glutathione peroxidase (GSH-PX) (32.2% and 45.7%), superoxide dismutase (SOD) (15.5% and 26.1%), and total antioxidant capacity (T-AOC) (18.4% and 18.2%) activities and increased malondialdehyde (MDA) (34.4% and 43.0%) contents on E5 and E7. In conclusion, these findings demonstrate that restraint stress causes abnormal implantation and negatively impacts immune parameters in association with oxidative stress in mice.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Effects of Catalytic Subunitαof Protein Phosphatase 2A on Cell Migration of Mouse Embryo Fibroblasts%蛋白磷酸酶2A 催化亚基α在小鼠胚胎成纤维细胞迁移中的作用研究

    Institute of Scientific and Technical Information of China (English)

    王庆华; 王生存; 李斌; 刘春; 吴刘成; 王旭; 邵义祥

    2016-01-01

    利用体外基因敲除技术检测小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)周期和迁移能力的变化,探究蛋白磷酸酶2A 的 Cα亚基在 MEFs 细胞迁移过程中的作用。用 Cαfl/fl的纯合子雄鼠与雌鼠1∶2配对,取 E12.5 d 的胚胎制作小鼠胚胎成纤维细胞。利用表达 Cre 重组酶(Ad-Cre-EGFP)以及GFP 荧光蛋白(Ad-EGFP)的腺病毒载体,对 P3代 MEFs 进行感染,分别用 PCR,RT-PCR 和 Western blot方法进行基因型鉴定。同时利用流式分选技术检测感染后 MEFs 的细胞周期的变化,利用细胞划痕试验检测了其细胞迁移能力的变化。结果显示,胰酶消化的方法成功获得了小鼠胚胎成纤维细胞,DNA,RNA 和蛋白水平的鉴定获得了3对3的野生型和基因敲除 MEFs。流式分选发现 Ad-Cre 处理的 MEFs 与 Ad-EG-FP 处理的 MEFs 相比,处于 G2期的细胞比例为30.8%±2.57%,高于 Ad-EGFP MEFs 的23.9%±2.46%,并且细胞碎片的比例也远高于后者。划痕试验表明,Cα亚基缺失后细胞迁移能力下降,12 h 就显著低于对照组。结果表明,腺病毒携带的 Cre 重组酶能够在体外有效地敲除掉 MEFs 中的蛋白磷酸酶2A 的Cα亚基,PP2A Cα亚基的缺失会导致 MEFs 细胞周期倾向于阻滞在 G2期,并会降低细胞迁移的能力。%To investigate the effects of catalytic subunit α of protein phosphatase 2A (PP2A)on cell cycle and cell migration in mouse embryo fibroblasts(MEFs),the mouse embryos were got at E12.5 by mating the heterozygotes of Cαsubunit conditional knockout male and female homozygous mice at the ratio of 1∶2.MEFs were prepared by trypsin digestion and genotyped by PCR,RT-PCR and Western blot to identify the genetic basis of each embryo.Adenovirus associated Cre recombinase and GFP immunofluorescence protein were constructed and delivered to the P3 MEFs to knockout the Cαgene,GFP was used as control

  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. Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    TsungHsiaochien; christine,L.Mummery

    1990-01-01

    In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice,the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF).Compared with the feeder layer of MEF cells,medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyotypic normality of ES cells only in short term cell propagation.Besides,ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras.Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more converient and efficient than the conventional microdrop method.

  10. 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.

  11. 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...

  12. Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2 regulates axon integrity in the mouse embryo.

    Directory of Open Access Journals (Sweden)

    Amy N Hicks

    Full Text Available Using transposon-mediated gene-trap mutagenesis, we have generated a novel mouse mutant termed Blad (Bloated Bladder. Homozygous mutant mice die perinatally showing a greatly distended bladder, underdeveloped diaphragm and a reduction in total skeletal muscle mass. Wild type and heterozygote mice appear normal. Using PCR, we identified a transposon insertion site in the first intron of Nmnat2 (Nicotinamide mononucleotide adenyltransferase 2. Nmnat2 is expressed predominantly in the brain and nervous system and has been linked to the survival of axons. Expression of this gene is undetectable in Nmnat2(blad/blad mutants. Examination of the brains of E18.5 Nmnat2(blad/blad mutant embryos did not reveal any obvious morphological changes. In contrast, E18.5 Nmnat2(blad/blad homozygotes showed an approximate 60% reduction of spinal motoneurons in the lumbar region and a more than 80% reduction in the sensory neurons of the dorsal root ganglion (DRG. In addition, facial motoneuron numbers were severely reduced, and there was virtually a complete absence of axons in the hind limb. Our observations suggest that during embryogenesis, Nmnat2 plays an important role in axonal growth or maintenance. It appears that in the absence of Nmnat2, major target organs and tissues (e.g., muscle are not functionally innervated resulting in perinatal lethality. In addition, neither Nmnat1 nor 3 can compensate for the loss of Nmnat2. Whilst there have been recent suggestions that Nmnat2 may be an endogenous modulator of axon integrity, this work represents the first in vivo study demonstrating that Nmnat2 is involved in axon development or survival in a mammal.

  13. Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

    Science.gov (United States)

    Gadue, Paul; Gouon-Evans, Valerie; Cheng, Xin; Wandzioch, Ewa; Zaret, Kenneth S; Grompe, Markus; Streeter, Philip R; Keller, Gordon M

    2009-09-01

    The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

  14. 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.

  15. 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.

  16. Analysis of the sex-chromosome constitution of digynic triploid mouse embryos.

    Science.gov (United States)

    Speirs, S; Kaufman, M H

    1989-01-01

    LT/Sv strain mice regularly ovulate up to 50% of their eggs as primary oocytes, which are fertilisable and give rise to digynic triploid embryos. A similar number of eggs are ovulated as secondary oocytes and, following fertilisation, give rise to normal diploid embryos. Pregnant LT/Sv females were autopsied at about midday on day 10 of gestation, when normal diploid embryos would be expected to possess between 25 and 30 pairs of somites. While a few of the triploid embryos either consisted of disorganised embryonic masses or were resorbing, most were at readily recognisable embryonic stages. Just over half of the embryos recovered were "unturned," while the remainder had "turned" and possessed between 15 and 25 pairs of somites. The triploids were usually readily recognised, owing to their small size and because they often displayed neural tube and cardiac defects. All of the embryos recovered were analysed cytogenetically by G-banding to establish their ploidy and sex-chromosome constitution. The XY:XX sex ratio of the 105 diploid embryos recovered, all of which had "turned," was 1.06:1, while the overall XXY:XXX sex ratio of the 120 triploids was 1:1. Analysis of only the developmentally most advanced triploid embryos (i.e., the 49 that had "turned") revealed that the XXY:XXX sex ratio in this group was 1.13:1, which was not significantly different from the expected ratio of 1:1. The crown-rump lengths of the XY and XX "turned" embryos were almost identical, as were those of the XXY and XXX "turned" embryos, although the triploids were significantly smaller than the diploids. No obvious effect of sex-chromosome constitution on developmental potential was therefore observed in this study in relation to either the digynic triploid or the control diploid embryos.

  17. Endogenously EGFP-Labeled Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Zhang, Junli; Rao, Rammohan V; Spilman, Patricia; Mangada, Julie; Xie, Lin; Vitelli, Cathy; Gorostiza, Olivia F; Madden, David T; Zeng, Xianmin; Jin, Kunlin; Hart, Matthew J; Bredesen, Dale E; Galvan, Veronica

    2011-02-01

    Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping β-actin promoter The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759-769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies.

  18. 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.

  19. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

    Science.gov (United States)

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella

    2016-09-02

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.

  20. 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.

  1. Isolation and Characterization of Node/Notochord-like Cells from Mouse Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Winzi, Maria Karin; Maddox-Hyttel, Poul; Dale, J Kim

    2011-01-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However......, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed...... the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells...

  2. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

    Science.gov (United States)

    Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

    2011-11-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

  3. Interaction of Mouse Pem Protein and Cell Division Cycle 37 Homolog

    Institute of Scientific and Technical Information of China (English)

    Fen GUO; Yue-Qin LI; Shi-Qian LI; Zhi-Wen LUO; Xin ZHANG; Dong-Sheng TANG; Tian-Hong ZHOU

    2005-01-01

    Mouse Pem, a homeobox gene, encodes a protein consisting of 210 amino acid residues. To study the function of mouse Pem protein, we used the yeast two-hybrid system to screen the library of 7-day mouse embryo with full-length mouse Pem eDNA. Fifty-two colonies were obtained after 1.57×108 colonies were screened by nutrition limitation and β-galactosidase assay. Seven individual insert fragments were obtained from the library, and three of them were identified, one of which was confirmed to be the cell division cycle 37 (Cdc37) homolog gene by sequencing. The interaction between mouse Pem and Cdc37homolog was then confirmed by glutathione S-transferase pull-down assay, and the possible interaction model was suggested.

  4. Embryonic death, dwarfism and fetal malformations after irradiation of embryos at the zygote stage. Studies on two mouse strains

    Energy Technology Data Exchange (ETDEWEB)

    Jacquet, P.; Saint-Georges, L. de; Baugnet-Mahieu, L. [Laboratory of Radiobiology, Department of Radioprotection, CEN/SCK, Mol (Belgium); Vankerkom, J. [Division of Environmental Research, VITO, Mol (Belgium)

    1995-11-01

    Female mice of the BALB/c and CF1 strains were mated and irradiated with various doses of X-rays 7 h after presumed fertilization. 18 days later, females were killed and their uteri examined for prenatal mortality at the different stages of development. Living fetuses were weighed and examined for the presence of external malformations. A number of them were also examined for skeletal anomalies. Radiation induced mainly a dose-dependent increase of the preimplantation loss in the BALB/c strain and of the early postimplantation loss in the CF1 strain. Embryos of the BALB/c strain were refractory to the induction of teratogenic effects after such preimplantation irradiation. In CF1 mice, the frequency of malformed fetuses increased regularly after irradiation, the difference with controls being significant for the doses of 10, 50 and 100 cGy. Dwarfism occurrence also appeared to be increased by irradiation in this strain, although the importance of this effect varied depending on the criterion chosen for the assessment of dwarfs. With the definition proposed in the present paper, the increase in the frequency of dwarfs paralleled that of malformed fetuses, being significant after doses of 50 and 100 cGy. Irradiation did not increase the frequency of skeletal anomalies. A careful examination of the various data obtained to date led us to conclude that radiation may possibly be teratogenic in several mouse strains, when administered as early as during the one-cell stage and, to a lesser extent, during the following preimplantation stages. However, early prenatal mortality will remain by far the greatest risk associated with an exposure to radiation during this period. Moreover, the relativity of the risk of abnormality due to such irradiation should be considered in the context of the high prevalence of developmental defects spontaneously occurring during human pregnancy.

  5. Isolation and culture of neural stem cells from Kunming mouse embryo%昆明种小鼠胚胎神经干细胞的分离与培养

    Institute of Scientific and Technical Information of China (English)

    宫晓洁; 孙莉; 黎靖宇; 袁路; 刘琦; 陈维平

    2009-01-01

    背景:对于神经干细胞的分离培养,目前多采用胰蛋白酶对组织细胞予以消化,但消化时间较难把握.目的:采用胰酶消化与机械分离法相结合的方式对昆明种小鼠胚胎脑神经干细胞进行分离、培养,并进行初步的免疫组织化学检测.设计、时间及地点:细胞学体外观察,于2006-10/2007-09在广西医科大学基础医学实验室完成.材料:孕14~16d的昆明种小鼠由广西医科大学实验动物中心提供.方法:分离昆明种小鼠胎鼠的脑组织,经胰酶消化加机械吹打后,在加入碱性成纤维细胞生长因子和表皮细胞生长因子的B27无血清DMEM/F12培养基中培养.主要观察指标:用免疫细胞化学方法鉴定分离的神经干细胞.结果:培养24 h后,细胞以悬浮方式生长,聚集成团;48 h后形成由数十个细胞组成的细胞球,形态规则,体积大小不等,细胞无突起,形成典型的神经球,可传代扩增.免疫细胞化学染色结果示细胞巢蛋白呈阳性表达.结论:在含有表皮细胞生长因子、碱性成纤维细胞生长因子的无血清B27培养基条件下,有利于小鼠胚胎神经干细胞的体外培养和传代增殖.%BACKGROUND: During the process of isolating and culturing neural stem cells, trypsin is used to digest tissues and cells, but it is difficult to control the digestion time.OBJECTIVE: To isolate and cultivate neural stem cells from fetal brain of Kunming mice by trypsinization combined with mechanical isolation and to identify by the immunohistochemical method.DESIGN, TIME AND SETTING: In vitro observation of cytology. The experiment was performed at Basic Medical Laboratory of Guangxi Medical University from October 2006 to September 2007.MATERIALS: Kunming mice of pregnant 14 to 16 days were provided by the Laboratory Animal Center of Guangxi Medical University.METHODS: Brain tissues were isolated from the fetal brain of Kunming mice, pipettad mechanically after trypsinization, and

  6. 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.

  7. The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

    Science.gov (United States)

    Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. © 2016. Published by The Company of Biologists Ltd.

  8. 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.

  9. Mouse cell culture: methods and protocols

    OpenAIRE

    Elvira M. Guerra Shinohara

    2010-01-01

    The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases), starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward ...

  10. 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...

  11. 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).

  12. Non-destructive monitoring of mouse embryo development and its qualitative evaluation at the molecular level using Raman spectroscopy

    Science.gov (United States)

    Ishigaki, Mika; Hashimoto, Kosuke; Sato, Hidetoshi; Ozaki, Yukihiro

    2017-03-01

    Current research focuses on embryonic development and quality not only by considering fundamental biology, but also by aiming to improve assisted reproduction technologies, such as in vitro fertilization. In this study, we explored the development of mouse embryo and its quality based on molecular information, obtained nondestructively using Raman spectroscopy. The detailed analysis of Raman spectra measured in situ during embryonic development revealed a temporary increase in protein content after fertilization. Proteins with a β-sheet structure—present in the early stages of embryonic development—are derived from maternal oocytes, while α-helical proteins are additionally generated by switching on a gene after fertilization. The transition from maternal to embryonic control during development can be non-destructively profiled, thus facilitating the in situ assessment of structural changes and component variation in proteins generated by metabolic activity. Furthermore, it was indicated that embryos with low-grade morphology had high concentrations of lipids and hydroxyapatite. This technique could be used for embryo quality testing in the future.

  13. Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Dinnyes Andras

    2007-03-01

    Full Text Available Abstract Background Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data. Results Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly. Conclusion Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb

  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. A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Ahuja, Akshay K.; Jodkowska, Karolina; Teloni, Federico

    2016-01-01

    Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX...

  16. Fv-1 locus restriction of mouse retroviruses in glucocorticoid-treated cells

    Energy Technology Data Exchange (ETDEWEB)

    Tennant, R.W.; Jones, S.C.; Otten, J.A.; Yang, W.K.; Brown, A.

    1978-08-01

    Treatment of mouse embryo cells with hydrocortisone (10/sup -6/M) or dexamethasone (10/sup -4/ to 10/sup -6/M) increases virus synthesis whether the cells are permissive or restrictive at the Fv-1 locus. However, the number of cells infected was not increased in either permissive or restrictive cells by treatment with either glucocorticoid, and the two-hit titration pattern in restrictive cells remained unaltered. Therefore, the enhancement of virus replication by the glucocorticoids is independent of Fv-1 restriction and appears to occur after the Fv-1 locus-sensitive step in virus synthesis.

  17. Isolation of Mouse salivary gland stem cells

    NARCIS (Netherlands)

    Pringle, Sarah; Nanduri, Lalitha; van der Zwaag, Marianne; van Os, Ronald; Coppes, Rob

    2011-01-01

    Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent

  18. 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...

  19. 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.

  20. The Establishment of Method on Early Mouse Embryo Transplantation%小鼠早期胚胎不同移植方法的建立

    Institute of Scientific and Technical Information of China (English)

    邹悦; 张守纯

    2013-01-01

    Objective Establish mouse superovulation and embryo transfer technology,in order to further use the technology to control their offspring,and gradually will these technologies applied to other animals,so as to speed up its breed improvement,improve the utilization rate of good varieties and individual.Method Of 4-week-old C57BL/6 female mouse by intraperitoneal injection 7.5 IU PMSG (pregnant mare serum gonadotropin) 48 hours after intraperitoneal injection of superovulation 7.5 IU HCG (human chorionic gonadotropin) and C57BL/6 male rats mated,see bolt 0.5 days,1.5 days and 3.5 days,remove the corresponding embryo,the resulting embryo screening,elect better quality embryos and embryonic stages (single cells,2 cells capsule fallopian tube or uterus KM pseudopregnant mice mouse embryo) into estrus synchronization see bolt after KM ligation male rats mating stimulus be in production pregnancy after transplantation in mouse.Result Superovulation 88 C57BL/6 mice,see bolt 62,a total of 510 single-cell embryos,400 2-cell stage embryos,blastocysts 35,the ministry of tubal transplantation,tubal notches,uterine transplant transplant pseudopregnant female mice 43,pregnancy producing 39 farrowing 361.Conclusion Tubal fimbria transplantation compared with tubal Jiankou transplant was no significant difference tubal transplantation hurt small animals,but for patients who require a higher; fallopian tube the Jiankou transplant operation is relatively quick,but the larger animal injury; uterine transplant the pregnancy best effect at this stage of embryo easily implantation.%目的 建立小鼠胚胎移植技术,探索小鼠胚胎输卵管伞部移植、输卵管剪口移植和子宫移植技术要点,为进一步将该项技术应用到生物净化等领域提供参考数据.方法 对4周龄C57BL/6雌性小鼠采用腹腔注射7.5 IUPMSG(孕马血清促性腺激素),48 h后腹腔注射7.5 IU HCG(人绒毛膜促性腺激素)进行超数排卵处理,并与C57BL/6

  1. Retinoic acid controls the bilateral symmetry of somite formation in the mouse embryo.

    Science.gov (United States)

    Vermot, Julien; Gallego Llamas, Jabier; Fraulob, Valérie; Niederreither, Karen; Chambon, Pierre; Dollé, Pascal

    2005-04-22

    A striking characteristic of vertebrate embryos is their bilaterally symmetric body plan, which is particularly obvious at the level of the somites and their derivatives such as the vertebral column. Segmentation of the presomitic mesoderm must therefore be tightly coordinated along the left and right embryonic sides. We show that mutant mice defective for retinoic acid synthesis exhibit delayed somite formation on the right side. Asymmetric somite formation correlates with a left-right desynchronization of the segmentation clock oscillations. These data implicate retinoic acid as an endogenous signal that maintains the bilateral synchrony of mesoderm segmentation, and therefore controls bilateral symmetry, in vertebrate embryos.

  2. Estrogen-induced transcription factor EGR1 regulates c-Kit transcription in the mouse uterus to maintain uterine receptivity for embryo implantation.

    Science.gov (United States)

    Park, Mira; Kim, Hye-Ryun; Kim, Yeon Sun; Yang, Seung Chel; Yoon, Jung Ah; Lyu, Sang Woo; Lim, Hyunjung Jade; Hong, Seok-Ho; Song, Haengseok

    2017-09-28

    Early growth response 1 (Egr1) is a key transcription factor that mediates the action of estrogen (E2) to establish uterine receptivity for embryo implantation. However, few direct target genes of EGR1 have been identified in the uterus. Here, we demonstrated that E2 induced EGR1-regulated transcription of c-Kit, which plays a crucial role in cell fate decisions. Spatiotemporal expression of c-Kit followed that of EGR1 in uteri of ovariectomized mice at various time points after E2 treatment. E2 activated ERK1/2 and p38 to induce EGR1, which then activated c-Kit expression in the uterus. EGR1 transfection produced rapid and transient induction of c-KIT in a time- and dose-dependent manner. Furthermore, luciferase assays to measure c-Kit promoter activity confirmed that a functional EGR1 binding site(s) (EBS) was located within -1 kb of the c-Kit promoter. Site-directed mutagenesis and chromatin immunoprecipitation-PCR for three putative EBS within -1 kb demonstrated that the EBS at -818/-805 was critical for EGR1-dependent c-Kit transcription. c-Kit expression was significantly increased in the uterus on day 4 and administration of Masitinib, a c-Kit inhibitor, effectively interfered with embryo implantation. Collectively, our results showed that estrogen induces transcription factor EGR1 to regulate c-Kit transcription for uterine receptivity for embryo implantation in the mouse uterus. Copyright © 2017. Published by Elsevier B.V.

  3. 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

  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

    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

  5. 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.

  6. Pinoresinol-4,4'-di-O-beta-D-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts.

    Science.gov (United States)

    Do, Kee Hun; Choi, Young Whan; Kim, Eun Kyoung; Yun, Sung Ji; Kim, Min Sung; Lee, Sun Young; Ha, Jung Min; Kim, Jae Ho; Kim, Chi Dae; Son, Beung Gu; Kang, Jum Soon; Khan, Ikhlas A; Bae, Sun Sik

    2009-06-01

    Lignans are major constituents of plant extracts and have important pharmacological effects on mammalian cells. Here we showed that pinoresinol-4,4'-di-O-beta-D-glucoside (PDG) from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation of mouse embryo fibroblast (MEF) cells with 10 microM PDG resulted in strong stimulation of MEF cell migration and the EC(50) was about 2 microM. Pretreatment with pertussis toxin (PTX), an inhibitor of G(i) protein, completely blocked PDG-induced cell migration demonstrating that PDG evokes MEF cell migration through the activation of the G(i)-coupled receptor. Furthermore, pretreatment of MEF cells with Ki16425 (10 microM), which is a selective antagonist for LPA(1) and LPA(3) receptors, completely blocked PDG-induced cell migration. Likewise, PDG strongly induced calcium mobilization, which was also blocked by Ki16425 in a dose-dependent manner. Prior occupation of the LPA receptor with LPA itself completely blocked PDG-induced calcium mobilization. Finally, PDG-induced MEF cell migration was attenuated by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor such as LY294002. Cells lacking downstream mediator of PI3K such as Akt1 and Akt2 (DKO cells) showed loss of PDG-induced migration. Re-expression of Akt1 (but not Akt2) completely restored PDG-induced DKO cell migration. Given these results, we conclude that PDG is a strong inducer of cell migration. We suggest that the pharmacological action of PDG may occur through the activation of an LPA receptor whereby activation of PI3K/Akt signaling pathway mediates PDG-induced MEF cell migration.

  7. 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.

  8. Changes in mouse liver and chicken embryo yolk sac membrane soluble proteins due to an organophosphorous insecticide (OPI) diazinon linked to several noncholinergic OPI effects in mice and chicken embryos.

    Science.gov (United States)

    Seifert, Josef

    2014-11-01

    The objective of this study was to identify proteins in mouse livers and chicken embryo yolk sac membranes whose quantities were altered by an organophosphorous insecticide (OPI) treatment and which might be linked, based on their functionality, to the recognized noncholinergic effects of OPI. Mice and fertile chicken eggs were treated with an OPI representative diazinon. The quantitative changes in mouse liver and chicken embryo yolk sac membrane soluble proteins caused by diazinon were determined by two-dimensional electrophoresis. Proteins whose quantity was affected by diazinon were identified by the mass spectrometry. In mouse livers, the altered levels of several enzymes of glucose metabolism were considered with regards to amelioration of hyperglycemia due to diazinon; the reduced levels of 3-hydroxyanthranilate 3,4-dioxygenase to the changes in the l-tryptophan to NAD metabolism caused by pyrimidinyl and crotonamide OPI; the reduced levels of catalase, peroxiredoxin and superoxide dismutase to OPI-increased lipid and/or kynurenine oxidation, the latter effect resulting also in increased urinary excretion of xanthurenic and kynurenic acids; and an increase in glutathione S-methyltransferase to OPI detoxification. In chicken embryo yolk sac membranes, the reduced availability of procollagen-proline dioxygenase may be the factor in micromelia caused by OPI in chicken embryos.

  9. In vitro fertilization and embryo culture strongly impact the placental transcriptome in the mouse model.

    Directory of Open Access Journals (Sweden)

    Patricia Fauque

    Full Text Available BACKGROUND: Assisted Reproductive Technologies (ART are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. METHODOLOGY/PRINCIPAL FINDINGS: Blastocysts were transferred either (1 after in vivo fertilization and development (control group or (2 after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. CONCLUSIONS: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.

  10. 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.

  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. Genomic targets of Brachyury (T in differentiating mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Amanda L Evans

    Full Text Available The T-box transcription factor Brachyury (T is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC(n repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species.

  13. 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.

  14. Dynamic imaging and quantitative analysis of cranial neural tube closure in the mouse embryo using optical coherence tomography.

    Science.gov (United States)

    Wang, Shang; Garcia, Monica D; Lopez, Andrew L; Overbeek, Paul A; Larin, Kirill V; Larina, Irina V

    2017-01-01

    Neural tube closure is a critical feature of central nervous system morphogenesis during embryonic development. Failure of this process leads to neural tube defects, one of the most common forms of human congenital defects. Although molecular and genetic studies in model organisms have provided insights into the genes and proteins that are required for normal neural tube development, complications associated with live imaging of neural tube closure in mammals limit efficient morphological analyses. Here, we report the use of optical coherence tomography (OCT) for dynamic imaging and quantitative assessment of cranial neural tube closure in live mouse embryos in culture. Through time-lapse imaging, we captured two neural tube closure mechanisms in different cranial regions, zipper-like closure of the hindbrain region and button-like closure of the midbrain region. We also used OCT imaging for phenotypic characterization of a neural tube defect in a mouse mutant. These results suggest that the described approach is a useful tool for live dynamic analysis of normal neural tube closure and neural tube defects in the mouse model.

  15. Development of neural precursor cells from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    WU Xuan; LI Hai-di; Li Shu-nong; XU Hai-wei; XU Ling

    2001-01-01

    Objective: To explore the serum-free culture conditions for differentiating mouse embryonic stem cells (ES cells)into neural precursor cells (NPC) and compare the effects of human embryonic fibroblasts (HEF) as the feeder layer of ES with that of mouse embryonic fibroblasts (MEF)in vitro. Methods: Mouse ES cells were cultured in or not in feeder layer cells medium containing or not leukemia inhibitory factor to suppress their differentiation. Immunocytochemical method was used to identify NPC by detecting nestin antigen and alkaline phosphatase. Results: The ES cells cultured in HEF were positive to alkaline phosphatase. Serum-free medium allowed the differentiation of ES cells into NPC. Conclusion:HEF could replace MEF and keep the undifferentiated condition of ES cells with more benefits. NPC of high purity could be cultured from ES cells by serum-free culture method.

  16. Memory B cells in mouse models.

    Science.gov (United States)

    Bergmann, B; Grimsholm, O; Thorarinsdottir, K; Ren, W; Jirholt, P; Gjertsson, I; Mårtensson, I-L

    2013-08-01

    One of the principles behind vaccination, as shown by Edward Jenner in 1796, and host protection is immunological memory, and one of the cells central to this is the antigen-experienced memory B cell that responds rapidly upon re-exposure to the initiating antigen. Classically, memory B cells have been defined as progenies of germinal centre (GC) B cells expressing isotype-switched and substantially mutated B cell receptors (BCRs), that is, membrane-bound antibodies. However, it has become apparent over the last decade that this is not the only pathway to B cell memory. Here, we will discuss memory B cells in mice, as defined by (1) cell surface markers; (2) multiple layers; (3) formation in a T cell-dependent and either GC-dependent or GC-independent manner; (4) formation in a T cell-independent fashion. Lastly, we will touch upon memory B cells in; (5) mouse models of autoimmune diseases.

  17. Three-Dimensional High-Frequency Ultrasonography for Early Detection and Characterization of Embryo Implantation Site Development in the Mouse

    Science.gov (United States)

    Peavey, Mary C.; Reynolds, Corey L.; Szwarc, Maria M.; Gibbons, William E.; Valdes, Cecilia T.

    2017-01-01

    Ultrasonography is a powerful tool to non-invasively monitor in real time the development of the human fetus in utero. Although genetically engineered mice have served as valuable in vivo models to study both embryo implantation and pregnancy progression, such studies usually require sacrifice of parous mice for subsequent phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction in silico of high-frequency ultrasound (HFUS) imaging data for early detection and characterization of murine embryo implantation sites and their development in utero. With HFUS imaging followed by 3-D reconstruction, we were able to precisely quantify embryo implantation site number and embryonic developmental progression in pregnant C57BL6J/129S mice from as early as 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. using a VisualSonics Vevo 2100 (MS550S) transducer. In addition to measurements of implantation site number, location, volume and spacing, embryo viability via cardiac activity monitoring was also achieved. A total of 12 dams were imaged with HFUS with approximately 100 embryos examined per embryonic day. For the post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in mesh or solid overlay format enabled visual representation in silico of implantation site location, number, spacing distances, and site volume within each uterine horn. Therefore, this short technical report describes the feasibility of using 3-D HFUS imaging for early detection and analysis of post-implantation events in the pregnant mouse with the ability to longitudinally monitor the development of these early pregnancy events in a non-invasive manner. As genetically engineered mice continue to be used to characterize female reproductive phenotypes, we believe this reliable and non-invasive method to detect, quantify, and characterize early implantation events will prove to be an invaluable investigative tool for the study of female

  18. 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.

  19. Expression of GPR177 (Wntless/Evi/Sprinter), a Highly Conserved Wnt-Transport Protein, in Rat Tissues, Zebrafish Embryos, and Cultured Human Cells

    OpenAIRE

    Jin, Jay; Morse, Megan; Frey, Colleen; Petko, Jessica; Levenson, Robert

    2010-01-01

    GPR177 is an evolutionarily conserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GP...

  20. 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

  1. 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.

  2. 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.

  3. Histological Study on in vitro Co-cultivation of the Myocardium Tissue and Cells with Mouse Embryonic Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    ZHANG Gui-xue; LIU Yan; HU Peng-fei

    2004-01-01

    The histological observation was experimentally conducted on in vitro cultured mouse embryonic myocardium cells and myocardiumoid cell mass. The mouse embryo tissue were cultured and regular pulsatile myocardiumoid tissue could be found. During in vitro culture, the myofilament bundles in the cell were gradually increasing and strongly connectted each other with embryonic age and there were loose muscle fibers initially and intercalated discs were close to each other. The lose myofilament bundles were developed in muscle fibers with age and the distance between intercalated discs was enlarged. There were myofilamentoid structure in inactive cells and filament peripherily.

  4. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  5. "The Role ofL-arginine in Control of Apoptosis in Preimplantation Mouse Embryos Cultured in High Glucose Media "

    Directory of Open Access Journals (Sweden)

    Mohammad Barbarestani

    2004-06-01

    Full Text Available Maternal hyperglycemia causes delay in early stages of embryonic growth and development, higher incidence of congenital malformations and spontaneous miscarriage compared with those of non-diabetic conditions. High glucosis tratogenicity seems to be related to reduction of Nitric Oxide production (NO in hyperglycemic condition. In order to test this hypothesis, 2-cell stage embryos of normal mice were cultured with high concentration of glucose (30mM and different concentrations of L-arginine (5,10,20 mM or L-NAME, an NO syntase (NOS inhibitor. In the end of culture, blastocysts were stained by by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL technique and apoptotic cells were detected by using a Fluorescence microscope. Finally the amount of nitrite in the cultured media was assayed by Griess method. The results indicated that high glucose reduces Nitric Oxide production by preimplantation embryos and increases apoptosis of embryonic cells, but 5-20mM of L-arginine significantly increases Nitric Oxide production and decreases apoptosis. On the contrary L-NAME significantly inhibits the development of pre-implantation embryos. In conclusion, this study indicated that reduced nitric oxide production in high glucosis condition is a main factor for embryonic damage, and supplementation of high glucose media with L-arginine has an important role in prevention of high glucosis embryotoxicity

  6. Isolation and analysis of mouse microglial cells.

    Science.gov (United States)

    Garcia, Jenny A; Cardona, Sandra M; Cardona, Astrid E

    2014-01-01

    Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior, which involves continuous monitoring of neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis as well as inflammatory and immune responses in the brain. This unit describes several microglial cell isolation protocols that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry. Methods for visualizing microglial cells using in situ immunohistochemistry and immunochemistry in free-floating sections are also included.

  7. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    Science.gov (United States)

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  8. Pax4 is not essential for beta-cell differentiation in zebrafish embryos but modulates alpha-cell generation by repressing arx gene expression.

    Science.gov (United States)

    Djiotsa, Joachim; Verbruggen, Vincianne; Giacomotto, Jean; Ishibashi, Minaka; Manning, Elisabeth; Rinkwitz, Silke; Manfroid, Isabelle; Voz, Marianne L; Peers, Bernard

    2012-12-17

    Genetic studies in mouse have demonstrated the crucial function of PAX4 in pancreatic cell differentiation. This transcription factor specifies β- and δ-cell fate at the expense of α-cell identity by repressing Arx gene expression and ectopic expression of PAX4 in α-cells is sufficient to convert them into β-cells. Surprisingly, no Pax4 orthologous gene can be found in chicken and Xenopus tropicalis raising the question of the function of pax4 gene in lower vertebrates such as in fish. In the present study, we have analyzed the expression and the function of the orthologous pax4 gene in zebrafish. pax4 gene is transiently expressed in the pancreas of zebrafish embryos and is mostly restricted to endocrine precursors as well as to some differentiating δ- and ε-cells but was not detected in differentiating β-cells. pax4 knock-down in zebrafish embryos caused a significant increase in α-cells number while having no apparent effect on β- and δ-cell differentiation. This rise of α-cells is due to an up-regulation of the Arx transcription factor. Conversely, knock-down of arx caused to a complete loss of α-cells and a concomitant increase of pax4 expression but had no effect on the number of β- and δ-cells. In addition to the mutual repression between Arx and Pax4, these two transcription factors negatively regulate the transcription of their own gene. Interestingly, disruption of pax4 RNA splicing or of arx RNA splicing by morpholinos targeting exon-intron junction sites caused a blockage of the altered transcripts in cell nuclei allowing an easy characterization of the arx- and pax4-deficient cells. Such analyses demonstrated that arx knock-down in zebrafish does not lead to a switch of cell fate, as reported in mouse, but rather blocks the cells in their differentiation process towards α-cells. In zebrafish, pax4 is not required for the generation of the first β- and δ-cells deriving from the dorsal pancreatic bud, unlike its crucial role in the

  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. Elimination of paternal mitochondria in mouse embryos occurs through autophagic degradation dependent on PARKIN and MUL1

    Science.gov (United States)

    Rojansky, Rebecca; Cha, Moon-Yong; Chan, David C

    2016-01-01

    A defining feature of mitochondria is their maternal mode of inheritance. However, little is understood about the cellular mechanism through which paternal mitochondria, delivered from sperm, are eliminated from early mammalian embryos. Autophagy has been implicated in nematodes, but whether this mechanism is conserved in mammals has been disputed. Here, we show that cultured mouse fibroblasts and pre-implantation embryos use a common pathway for elimination of mitochondria. Both situations utilize mitophagy, in which mitochondria are sequestered by autophagosomes and delivered to lysosomes for degradation. The E3 ubiquitin ligases PARKIN and MUL1 play redundant roles in elimination of paternal mitochondria. The process is associated with depolarization of paternal mitochondria and additionally requires the mitochondrial outer membrane protein FIS1, the autophagy adaptor P62, and PINK1 kinase. Our results indicate that strict maternal transmission of mitochondria relies on mitophagy and uncover a collaboration between MUL1 and PARKIN in this process. DOI: http://dx.doi.org/10.7554/eLife.17896.001 PMID:27852436

  11. Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule

    Science.gov (United States)

    Tesařová, M.; Zikmund, T.; Kaucká, M.; Adameyko, I.; Jaroš, J.; Paloušek, D.; Škaroupka, D.; Kaiser, J.

    2016-03-01

    Imaging of increasingly complex cartilage in vertebrate embryos is one of the key tasks of developmental biology. This is especially important to study shape-organizing processes during initial skeletal formation and growth. Advanced imaging techniques that are reflecting biological needs give a powerful impulse to push the boundaries of biological visualization. Recently, techniques for contrasting tissues and organs have improved considerably, extending traditional 2D imaging approaches to 3D . X-ray micro computed tomography (μCT), which allows 3D imaging of biological objects including their internal structures with a resolution in the micrometer range, in combination with contrasting techniques seems to be the most suitable approach for non-destructive imaging of embryonic developing cartilage. Despite there are many software-based ways for visualization of 3D data sets, having a real solid model of the studied object might give novel opportunities to fully understand the shape-organizing processes in the developing body. In this feasibility study we demonstrated the full procedure of creating a real 3D object of mouse embryo nasal capsule, i.e. the staining, the μCT scanning combined by the advanced data processing and the 3D printing.

  12. 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.

  13. The effects of folic acid on carbon black toxicity in mouse embryo in vivo

    Directory of Open Access Journals (Sweden)

    Roshdy, H.M and Bibars, M.H

    2004-03-01

    Full Text Available The wide commercial use of carbon black oil (CBO to produce asphalt and other commercial product has resulted in numerous environmental problems and harmful effects on human health especially during the pregnancy. This study, examining the effect of maternal low and high dietary folate intake and to protect the pregnant women from the developmental toxicity of CBO. Virgin females CD--1 mice were assigned to diets containing either low 500 or 1300 high (control nmol folic acid/kg for 6 weeks prior to mating and thought out breeding and gestation. From gestation day (GD 6 to 18 females were given by gavage corn oil or CBO at 500 mg/kg body weight, once daily. On CD 18, mice were weight and killed and the liver removed and weighed. Implantation sites, live and dead fetuses, and resorptions were counted, fetuses were weighed individually and examined for external malformations. The low dietary FA treatment alone and with CBO treatment resulted in low maternal liver as well as low fetal liver folate concentrations relative to the high FA dietary groups. Low FA treatment alone resulted in malformed embryos; there were no embryos affected with malformed in the adequate FA-control group. Low folic acid-CBO treatment resulted in a further increase in the malformed embryos. The percent of malformed embryos in high folic acid-CBO treatment was very low compared to the low folic acid-CBO group. The frequency of chromosomal aberrations in maternal and their fetuses was increased significantly in the low folic-CBO group than high folic acid-CBO group. These results show that the low folate dietary diet with the exposure to the high levels of CBO toxic material in pregnant women significantly increases the developmental and mutagenic toxicity in the small fetuses.

  14. Retinoic Acid Controls the Bilateral Symmetry of Somite Formation in the Mouse Embryo

    OpenAIRE

    Vermot, Julien; Llamas, Jabier Gallego; Fraulob, Valérie; Niederreither, Karen; Chambon, Pierre; Dollé, Pascal

    2005-01-01

    A striking characteristic of vertebrate embryos is their bilaterally symmetric body plan, which is particularly obvious at the level of the somites and their derivatives such as the vertebral column. Segmentation of the presomitic mesoderm must therefore be tightly coordinated along the left and right embryonic sides. We show that mutant mice defective for retinoic acid synthesis exhibit delayed somite formation on the right side. Asymmetric somite formation correlates with a left-right desyn...

  15. Germ cell transplantation in infertility mouse

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    This work investigated the spermatogenesis in an infertility BALB/c-nu mouse model by reinfusing germline stem cells into seminiferous tubules.Donor germ cells were isolated from male FVB/NJ-GFP transgenic mice.Seminiferous tubule microiniection was applied to achieve intratubular germ cell transfer.The germ cells were injected into exposed testes of the infertility mice.We used green fluorescence and DNA analysis of donor cells from GFP transgenic mice as genetic marker.The natural mating and Southern blot methods were applied to analyze the effect of sperm cell transplantation and the sperm function after seminiferous tubule microinjecUon.The spermatogenesis was morphologically observed from the seminiferous tubules in 41/60(68.33%)of the injected recipient mice using allogeneic donor cells.In the colonized testes,matured spermatozoa were seen in the lumen of the seminiferous tubules.In this research,BALB/c-nu infertility mouse model,the recipient animal,was used to avoid immunological rejection of donor cells,and germ cell transplantation was applied to overcome infertility caused by busulfan treatment.These results demonstrate that this technique of germ cell transplantation is of great use.Germ cell transplantation could be potentially valuable to oncological patients.

  16. Tbx6 regulates left/right patterning in mouse embryos through effects on nodal cilia and perinodal signaling.

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    Anna-Katerina Hadjantonakis

    Full Text Available BACKGROUND: The determination of left/right body axis during early embryogenesis sets up a developmental cascade that coordinates the development of the viscera and is essential to the correct placement and alignment of organ systems and vasculature. Defective left-right patterning can lead to congenital cardiac malformations, vascular anomalies and other serious health problems. Here we describe a novel role for the T-box transcription factor gene Tbx6 in left/right body axis determination in the mouse. RESULTS: Embryos lacking Tbx6 show randomized embryo turning and heart looping. Our results point to multiple mechanisms for this effect. First, Dll1, a direct target of Tbx6, is down regulated around the node in Tbx6 mutants and there is a subsequent decrease in nodal signaling, which is required for laterality determination. Secondly, in spite of a lack of expression of Tbx6 in the node, we document a profound effect of the Tbx6 mutation on the morphology and motility of nodal cilia. This results in the loss of asymmetric calcium signaling at the periphery of the node, suggesting that unidirectional nodal flow is disrupted. To carry out these studies, we devised a novel method for direct labeling and live imaging cilia in vivo using a genetically-encoded fluorescent protein fusion that labels tubulin, combined with laser point scanning confocal microscopy for direct visualization of cilia movement. CONCLUSIONS: We conclude that the transcription factor gene Tbx6 is essential for correct left/right axis determination in the mouse and acts through effects on notch signaling around the node as well as through an effect on the morphology and motility of the nodal cilia.

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

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    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

  18. 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

  19. A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

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    Graciana Diez-Roux

    Full Text Available Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org, consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

  20. Study on the in vitro Fertilization and Embryo Cryopreservation in Carcinogenesis Transgenic C57-ras Mouse Model%C57-ras癌症转基因小鼠模型胚胎冷冻保种技术研究

    Institute of Scientific and Technical Information of China (English)

    左琴; 刘甦苏; 周舒雅; 刘佐民; 王金恒; 范昌发

    2013-01-01

    目的 建立C57-ras癌症转基因小鼠模型胚胎的冷冻保种技术.方法 通过直接收集体内发育的2-细胞胚或者通过体外受精方法获得2-细胞胚,采用玻璃化冷冻法冷冻、复苏、移植,后代经PCR检测.结果 体内发育至2细胞期胚胎90枚,解冻后的平均复苏率是67.78%,移植产仔率是21.31%,后代的阳性率是23.08%;体外受精的2细胞期胚胎203枚,解冻复苏60枚,平均复苏率是63.33%,移植产仔率是15.78%,后代的阳性率是33.33%.结论 两种胚胎冷冻途径均获得了C57-ras癌症转基因动物模型阳性小鼠,建立了该模型动物的低温保存方法.%Objective Investigate the application of in vitro fertilization (IVF) and embryo cryopreservation in C57-ras transgenic mice.Methods 2-cell embryos were collected in two ways,first of which is by 4-week superovulated C57BL/6J female mice mated with C57-ras transgentic male mice,and the other is by IVF.The 2-cell embryos were cryopreserved after that.Then the cryopreserved 2-cell embryos were thawed and transplanted to caculate the ratio.Genetype of offsprings were analysed by PCR.Results The average recovery rate of cryopreserved normal 2-cell embryo was 67.78% and 21.31% were born after thawed 2-cell embryos transplanted.23.08% of the offsprings were positive of human c-Ha-ras gene; the average recovery rate of cryopreserved IVF 2-cell was 63.33%,and 15.78% were born after transplanted,and 33.33% of the offspring were positive.Conclusion The results suggested that there was no significant difference in cryopreservation of normal and IVF C57-ras 2-cell embryos.The research provided the cryopreservation method for C57-ras transgenic mouse model.

  1. 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.

  2. 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

  3. E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

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    Satoshi Ohtsuka

    Full Text Available Mouse epiblast stem cells (mEpiSCs are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

  4. E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

    Science.gov (United States)

    Ohtsuka, Satoshi; Nishikawa-Torikai, Satomi; Niwa, Hitoshi

    2012-01-01

    Mouse epiblast stem cells (mEpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs) in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

  5. Effect of Different Concentrations of Melatonin on Live Births Resulting from the Transfer of Two-Cell Embryos of NMRI Mice

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    Mahdi Saadati

    2014-12-01

    Full Text Available Background & objectives : Infertility is a global problem affecting millions of men and women in developed and developing countries. In this regard, in-vitro fertilization (IVF plays an important role in improving the quality of life in infertile patients. However, studies have shown that the implantation failure in IVF is the main challenge of this procedure. Melatonin can increase the survival rate of embryos and IVF success rate through eliminating free radicals and removing reactive oxygen species. So, this study is conducted to investigate the effects of different concentrations of melatonin on the rate of newborns of mice following transfer oftwo-cell embryos .   Methods : In this study, female mice with average age of six to eight weeks were superovulated by administering pregnant mares serum gonadotropin (PMSG intraperitoneally (7.5 IU. ip, and followed after 48h by human chorionic gonadotropin (hCG (7.5 IU. ip. Two-cell mouse embryos were obtained from female mice oviduct after 48 h. The embryos transferred bilaterally into pseudopregnant mice of the same strain through surgical procedure and 8-14 embryos were transferred to each tube. The study included 4 treatment groups and one control group (6 mice in each group. The treatment groups were exposed to subcutaneous injection of concentrations of 100 µm , 10 µm , 1 µm and 100 nm of melatonin. After the cesarean on 18th day of pregnancy, the percentage of live births was assessed. The outcomes of the live birth rate were as­sessed using the chi-square test and statistical analyses were carried out using SPSS version 16.0. Percentage of live birth was calculated and compared with the control group.   Results: A total of 701 two-cell mouse embryos were transferred into one control group and four experimental groups. The number and percentage of live births at concentrations of 100 µm and 10 µm of melatonin and the control groups were 21 (15.55%, 13 (9.15% and 9 (6

  6. Effect of potential role of p53 on embryo development arrest induced by H2O2 in mouse.

    Science.gov (United States)

    Hu, De-Bao; Li, Zhong-Shu; Ali, Ihsan; Xu, Li-Jie; Fang, Nan-Zhu

    2017-01-27

    During mammalian embryo development in vitro, mechanism of embryonic development arrest caused by oxidative stress has not been clear so far. The tumor suppressor protein p53 controls cell cycle and programmed cell death by regulating relevant signal pathway. Recent researches revealed that the concentration and distribution of p53 are closely related with reactive oxygen species (ROS). The main objective of this experiment was to explore the role of p53 on embryonic development arrest caused by oxidative stress. Results showed that embryo arrest at two-four-cell stage was significantly increased in the presence of 50 μM H2O2 (39.01 ± 2.74 vs. 77.20 ± 5.34%, p p53 and p53-ser15. Further increased transcription of GADD45a and p21, a downstream of p53, has an especially significant effect on the mRNA expression of GADD45a. However, expressions of cdc2 were reduced by H2O2. In addition, using Pifithrin-α (PFT-α), the suppresser of p53, the result showed that GADD45a and p21 were significantly downregulated, but the cell cycle gene cdc2 was significantly upregulated, while the protein level of p53 and p53-ser15 was significantly decreased. Taken together, these results demonstrate that ROS could activate p53 and regulate p53 target genes to influence early embryo development in in vitro culture.

  7. Mouse cell culture - Methods and protocols

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    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  8. A mouse model for monitoring islet cell genesis and developing therapies for diabetes

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    Yoshinori Shimajiri

    2011-03-01

    Transient expression of the transcription factor neurogenin-3 marks progenitor cells in the pancreas as they differentiate into islet cells. We developed a transgenic mouse line in which the surrogate markers secreted alkaline phosphatase (SeAP and enhanced green florescent protein (EGFP can be used to monitor neurogenin-3 expression, and thus islet cell genesis. In transgenic embryos, cells expressing EGFP lined the pancreatic ducts. SeAP was readily detectable in embryos, in the media of cultured embryonic pancreases and in the serum of adult animals. Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later. By contrast, treatment with growth differentiation factor 11 (GDF11 reduced SeAP secretion rates. In adult mice, partial pancreatectomy decreased, whereas duct ligation increased, circulating SeAP levels. This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

  9. 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

  10. 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.

  11. 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%).

  12. 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...

  13. 冻融后小鼠休眠胚胎超微结构的变化%Ultrastructural observation of dormant mouse embryos cultured in vitro after freezing-thawing

    Institute of Scientific and Technical Information of China (English)

    顾美超; 卢天罡; 刘云海; 倪和民; 张劭俣; 翟椿东; 邢书涵; 郭勇

    2014-01-01

    Objective The aim of this study was to investigate the differences of the cell ultrastucture of normal mouse hatched blastocysts and their dormant ones cultured in vitro after freezing-thawing, and to explore whether the dor-mant embryos have a better anti-freezing shock property than the normal hatched mouse embryos .Methods By transmis-sion electron microscopy , the ultrastructure of these two types of mouse embryos was observed and analyzed .Results By comparative analysis of their ultrastructure , the results showed that the dormant embryos before freezing are being austerity and with lower energy metabolism at a ‘ground state ’ .After freezing-thawing and culture , their cellular structure seemed to be similar to that of the normal embryos cultured in vitro before freezing.However, after freezing-thawing and culture, the number of mitochondria decreased , the nuclei were loose , and their heterochromatin also increased .Conclusions From the ultrastructural observation , compared with the normal mouse hatched embryos , the cellular state of dormant mouse em-bryos after freezing-thawing is more favorable for material storage and energy metabolism , thus, indicating that they have a better anti-freezing property than normal hatched embryos .%目的:从亚细胞超微结构的角度揭示其抗冻能力优于正常孵化胚胎的原因。方法利用透射电子显微镜观察小鼠休眠胚胎与正常孵化期胚胎在细胞连接和各细胞器形态与分布上的差异,以及冻融培养后的变化,并进行相关比较分析。结果通过亚细胞结构对比分析发现:冷冻前小鼠休眠胚胎为紧缩状,处于能量代谢较低的“基态”,通过冻融后培养,细胞器结构恢复与正常孵化胚胎冷冻前相似;而正常孵化胚胎经过冻融后,线粒体数量减少,细胞核松散,异染色质增多。结论小鼠休眠胚胎与正常孵化胚胎冻融后相比,其细胞状态更有利于物质储存及能

  14. A device for the simple and rapid transcervical transfer of mouse embryos eliminates the need for surgery and potential post-operative complications.

    Science.gov (United States)

    Green, Michael; Bass, Shannon; Spear, Brett

    2009-11-01

    We describe a novel device that can be used for the transcervical transfer of embryos into pseudopregnant female mice. This nonsurgical embryo transfer (NSET) device is as efficient as standard surgical embryo transfer in the production of transgenic mice, and can also be used for the transfer of embryonic stem cell-containing chimeric blastocysts and cryopreserved embryos. In addition to the elimination of surgery, recipient females do not have to be anesthetized. The NSET device eliminates a painful surgical procedure as well as potential complications associated with anesthesia/post-operative care, reduces the technical expertise and equipment needed for surgical transfer, and represents substantial cost savings and regulatory reduction. NSET technology provides an easy and rapid alternative to surgical embryo transfer. Address correspondence to Brett Spear, Room 210, Combs Building, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY, 40536-0298, USA. email:

  15. Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver

    Science.gov (United States)

    Orelio, Claudia; Peeters, Marian; Haak, Esther; van der Horn, Karin; Dzierzak, Elaine

    2009-01-01

    Background Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. Design and Methods We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1−/− embryos. Results We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1−/− embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. Conclusions The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells. PMID

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Site-Specific Expression of Gelatinolytic Activity during Morphogenesis of the Secondary Palate in the Mouse Embryo

    Science.gov (United States)

    Gkantidis, Nikolaos; Blumer, Susan; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx. PMID:23091646

  1. Site-specific expression of gelatinolytic activity during morphogenesis of the secondary palate in the mouse embryo.

    Directory of Open Access Journals (Sweden)

    Nikolaos Gkantidis

    Full Text Available Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs. As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5, we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP, but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx.

  2. Cellular and genetic analysis of mouse blastocyst development

    Energy Technology Data Exchange (ETDEWEB)

    Pedersen, R A; Spindle, A I

    1979-01-01

    The development of mouse embryos was studied by both cellular and genetic approaches. In the cellular analysis, determination of cell fate in blastocysts and in cell populations derived from them was studied in an attempt to estimate the time that these cells become committed to their fate. In the genetic analysis, existing mutations that are lethal to mouse embryos were used to discern essential features of early development. In this review, the timing of cell determination in the inner cell mass and the primary ectoderm, and the manifestation of defects in mouse embryos that are homozygous for the A/sup y/ allele of the agouti locus were considered.

  3. The evaluation of quality control on the new human IVF laboratory with mouse embryo testing%小鼠胚胎实验对新建人类体外受精实验室质量控制的评价

    Institute of Scientific and Technical Information of China (English)

    李弘; 袁彩霞; 秦琴; 宋海霞; 曹莹丽; 孙喜娟

    2013-01-01

    Objective To carry out the quality control of new human in vitro fertilization (IVF) laboratory,IVF of mouse were studied.Methods The gametes of mouse were got by surgery,treated by IVF and intracytoplasmic sperm injection(ICSI),the embryos were cultured for five days in vitro,fertility rate,grade 1-2 embryo rate and rate of cleavage to blastulas were investigated.Results 11 cycles of IVF were finished,271 eggs were gained,the fertility rate was 82.2% and grade 1-2 embryo rate was 91.3%,the rate of 2-cell was growing to blastulas was 85.3%.Ten cycle of ICSI were finished,206 oocytes at metaphase Ⅱ (M Ⅱ)were gained,the fertility rate was 84.5% and grade 1-2 embryo rate was 92.9%,the rate of 2-cell was growing to blastulas was 89.9%.Conclusion The quality control of culture condition in human IVF technology is tested by mouse embryo,the data accord with standards.%目的 利用小鼠体外受精技术对我院新建人类体外受精实验室进行质量控制.方法 手术获取小鼠配子,经体外受精或卵胞浆内单精子显微注射后,形成胚胎体外培养5d,观察受精率、优胚率、囊胚率.结果 本研究进行11个常规体外受精周期,取卵371枚,受精率82.2%(305枚),优胚率91.3% (274枚),2-细胞囊胚形成率85.3%(256枚);10个周期卵胞浆内单精子显微注射,取卵子206枚,受精率84.5%(174枚),优胚率92.9%(157枚),2-细胞囊胚形成率89.9%(152枚).结论 通过鼠胚实验对我科新建IVF实验室进行质量控制,结果符合标准.

  4. 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.

  5. Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

    Science.gov (United States)

    Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

    2011-06-01

    The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

  6. 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...

  7. 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.

  8. 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...

  9. Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogencially activated and fertilized embryos.

    Science.gov (United States)

    Martin, Jacinta H; Bromfield, Elizabeth G; Aitken, R John; Lord, Tessa; Nixon, Brett

    2016-09-01

    These data document the vitality of mature mouse oocytes (Metaphase II (MII)) and early stage embryos (zygotes) following exposure to the genotoxic chemotherapeutic agent, etoposide, in combination with PSC833, a selective inhibitor of permeability glycoprotein. They also illustrate the vitality of parthenogencially activated and fertilized embryos following incubation with the calcium chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)), cycloheximide (an antibiotic that is capable of inhibiting protein synthesis), and hydrogen peroxide (a potent reactive oxygen species). Finally, they present evidence that permeability glycoprotein is not represented in the proteome of mouse spermatozoa. Our interpretation and discussion of these data feature in the article "Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes" (Martin et al., in press) [1].

  10. 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.

  11. Effect of PCNA in the mouse uterus during embryo implantation%PCNA在小鼠胚胎着床过程中的作用

    Institute of Scientific and Technical Information of China (English)

    孟君; 魏莎莉; 覃致远; 张谭; 杜芳; 王庆跃

    2009-01-01

    Objective:To investigate the effect of PCNA in the mouse uterus during embryo implantation.Methods:Real-time fluorescence quantitative PCR (FQ -PCR) was used to detect the PCNA mRNA of the mouse of unpregnancy, pregnancy at d2, d3, d4, d5, d6 and d7. PCNA monoclonal antibody was injected in horn of uterus at pregnant d3. Results: The expression of PCNA mRNA showed gradual hoist trend as days passed , and at pregnant d4, d5 it reached the maximum, at pregnant d6,d7 it showed gradual descent trend. The number of blastocysts in horn of uterus which was injected PCNA monoclonal antibody was smaller than in that of injected with Saline. Conclusion: PCNA is persistently expressed in mouse endometrium during the early pregnancy and maybe participates in the regulation process of mouse blastodysts implantation .%目的:探讨增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)在小鼠胚胎着床过程中的作用.方法:实时荧光定量聚合酶链反应(Real-time Fluorescence Quantitative PCR,Real-time FQ-PCR)技术检测未孕及孕2、3、4、5、6、7 d(分别记为d0、d2、d3、d4、d5、d6、d7)小鼠子宫内膜PCNA mRNA的表达;子宫角注射PCNA单克隆抗体观察胚泡着床数.结果:FQ-PCR测得随着小鼠妊娠天数的增加,PCNA mRNA表达量也逐渐增高,在孕d4、d5达到峰值,孕d6、d7逐渐下降.子宫角注射PCNA单克隆抗体后胚泡着床数明显减少.结论:PCNA在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.

  12. Technique of the `in vitro` fertilization and the culture of mouse embryos at preimplantation; Tecnica de fertilizacao `in vitro` e cultura de embrioes de camundongo durante a pre-implantacao

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, Olivia Kimiko [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Yamada, Takeshi [National Inst. of Radiological Sciences, Chiba (Japan)

    1993-03-01

    The mammal embryo is an intensive cellular proliferating system, very radiosensitive and therefore adequate to the study of the biological effects of ionizing radiation. The technique of the in vitro fertilization and the culture of mouse embryos at preimplantation period, modified by Yamada et al (1982) to improve the efficiency of more than 95% of blastocyst formation is described. (author) 2 refs., 7 figs.

  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. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    Science.gov (United States)

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-05-15

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

  15. Distribution of some Glycoconjugates in the Notochord and Developing Gut during Early Morphogenesis in Balb/c Mouse Embryos

    Directory of Open Access Journals (Sweden)

    Mohammad M. Hassanzadeh-Taheri

    2012-03-01

    Full Text Available Background: Embryonic endoderm germinal layer, affected by notochord inductions, forms the primary gut epithelium and parenchyma of its derived organs. This study aims to determine some expressed glycoconjugates and their potential function in notochord and developing gut.Materials and Methods : In this descriptive-analytical study, 9 and 10 embryonic days (ED of Balb/c mouse embryos were fixed in formalin and microscopic sections were prepared from them. These sections were processed for histochemical studies and then they were incubated with 6 different HRP conjugated lectins, including VVA, SBA, and PNA specific to identify terminal sugar (N-acetylgalactosamine (GalNac and lectins of GSA1-B4, LTA and WGA were respectively to identify the terminal sugars of galactose, fructose and sialic acid.Results: The study results showed that the reactions of notochord and developing gut to VVA lectin were moderate on the 9ED and on the 10ED, they showed a significant difference (p < 0.001 from the day before and were severely assessed. Other GalNac specific lectins react severely and almost similarly to notochord and developing gut on the studied days. The other lectins in these two organs did not react similarly.Conclusion: According to the findings of this study, it seems that glycoconjugates with GalNac-terminal sugar probably have played a key role in differentiations of notochord and developing gut and may be involved in the interactions between these two organs.

  16. 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.

  17. 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.

  18. 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.

  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. 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...