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Thyroid hormones alter the transcriptome of in vitro-produced bovine blastocysts
- Fazl A. Ashkar, Tamas Revay, NaYoung Rho, Pavneesh Madan, Isabelle Dufort, Claude Robert, Laura A. Favetta, Chris Schmidt, W. Allan King
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Thyroid hormones (THs) have been shown to improve in vitro embryo production in cattle by increasing blastocyst formation rate, and the average cell number of blastocysts and by significantly decreasing apoptosis rate. To better understand those genetic aspects that may underlie enhanced early embryo development in the presence of THs, we characterized the bovine embryonic transcriptome at the blastocyst stage, and examined differential gene expression profiles using a bovine-specific microarray. We found that 1212 genes were differentially expressed in TH-treated embryos when compared with non-treated controls (>1.5-fold at P < 0.05). In addition 23 and eight genes were expressed uniquely in control and treated embryos, respectively. The expression of genes specifically associated with metabolism, mitochondrial function, cell differentiation and development were elevated. However, TH-related genes, including those encoding TH receptors and deiodinases, were not differentially expressed in treated embryos. Furthermore, the over-expression of 52 X-chromosome linked genes in treated embryos suggested a delay or escape from X-inactivation. This study highlights the significant impact of THs on differential gene expression in the early embryo; the identification of TH-responsive genes provides an insight into those regulatory pathways activated during development.
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- By Mitchell Aboulafia, Frederick Adams, Marilyn McCord Adams, Robert M. Adams, Laird Addis, James W. Allard, David Allison, William P. Alston, Karl Ameriks, C. Anthony Anderson, David Leech Anderson, Lanier Anderson, Roger Ariew, David Armstrong, Denis G. Arnold, E. J. Ashworth, Margaret Atherton, Robin Attfield, Bruce Aune, Edward Wilson Averill, Jody Azzouni, Kent Bach, Andrew Bailey, Lynne Rudder Baker, Thomas R. Baldwin, Jon Barwise, George Bealer, William Bechtel, Lawrence C. Becker, Mark A. Bedau, Ernst Behler, José A. Benardete, Ermanno Bencivenga, Jan Berg, Michael Bergmann, Robert L. Bernasconi, Sven Bernecker, Bernard Berofsky, Rod Bertolet, Charles J. Beyer, Christian Beyer, Joseph Bien, Joseph Bien, Peg Birmingham, Ivan Boh, James Bohman, Daniel Bonevac, Laurence BonJour, William J. Bouwsma, Raymond D. Bradley, Myles Brand, Richard B. Brandt, Michael E. Bratman, Stephen E. Braude, Daniel Breazeale, Angela Breitenbach, Jason Bridges, David O. Brink, Gordon G. Brittan, Justin Broackes, Dan W. Brock, Aaron Bronfman, Jeffrey E. Brower, Bartosz Brozek, Anthony Brueckner, Jeffrey Bub, Lara Buchak, Otavio Bueno, Ann E. Bumpus, Robert W. Burch, John Burgess, Arthur W. Burks, Panayot Butchvarov, Robert E. Butts, Marina Bykova, Patrick Byrne, David Carr, Noël Carroll, Edward S. Casey, Victor Caston, Victor Caston, Albert Casullo, Robert L. Causey, Alan K. L. Chan, Ruth Chang, Deen K. Chatterjee, Andrew Chignell, Roderick M. Chisholm, Kelly J. Clark, E. J. Coffman, Robin Collins, Brian P. Copenhaver, John Corcoran, John Cottingham, Roger Crisp, Frederick J. Crosson, Antonio S. Cua, Phillip D. Cummins, Martin Curd, Adam Cureton, Andrew Cutrofello, Stephen Darwall, Paul Sheldon Davies, Wayne A. Davis, Timothy Joseph Day, Claudio de Almeida, Mario De Caro, Mario De Caro, John Deigh, C. F. Delaney, Daniel C. Dennett, Michael R. DePaul, Michael Detlefsen, Daniel Trent Devereux, Philip E. Devine, John M. Dillon, Martin C. Dillon, Robert DiSalle, Mary Domski, Alan Donagan, Paul Draper, Fred Dretske, Mircea Dumitru, Wilhelm Dupré, Gerald Dworkin, John Earman, Ellery Eells, Catherine Z. Elgin, Berent Enç, Ronald P. Endicott, Edward Erwin, John Etchemendy, C. Stephen Evans, Susan L. Feagin, Solomon Feferman, Richard Feldman, Arthur Fine, Maurice A. Finocchiaro, William FitzPatrick, Richard E. Flathman, Gvozden Flego, Richard Foley, Graeme Forbes, Rainer Forst, Malcolm R. Forster, Daniel Fouke, Patrick Francken, Samuel Freeman, Elizabeth Fricker, Miranda Fricker, Michael Friedman, Michael Fuerstein, Richard A. Fumerton, Alan Gabbey, Pieranna Garavaso, Daniel Garber, Jorge L. A. Garcia, Robert K. Garcia, Don Garrett, Philip Gasper, Gerald Gaus, Berys Gaut, Bernard Gert, Roger F. Gibson, Cody Gilmore, Carl Ginet, Alan H. Goldman, Alvin I. Goldman, Alfonso Gömez-Lobo, Lenn E. Goodman, Robert M. Gordon, Stefan Gosepath, Jorge J. E. Gracia, Daniel W. Graham, George A. Graham, Peter J. Graham, Richard E. Grandy, I. Grattan-Guinness, John Greco, Philip T. Grier, Nicholas Griffin, Nicholas Griffin, David A. Griffiths, Paul J. Griffiths, Stephen R. Grimm, Charles L. Griswold, Charles B. Guignon, Pete A. Y. Gunter, Dimitri Gutas, Gary Gutting, Paul Guyer, Kwame Gyekye, Oscar A. Haac, Raul Hakli, Raul Hakli, Michael Hallett, Edward C. Halper, Jean Hampton, R. James Hankinson, K. R. Hanley, Russell Hardin, Robert M. Harnish, William Harper, David Harrah, Kevin Hart, Ali Hasan, William Hasker, John Haugeland, Roger Hausheer, William Heald, Peter Heath, Richard Heck, John F. Heil, Vincent F. Hendricks, Stephen Hetherington, Francis Heylighen, Kathleen Marie Higgins, Risto Hilpinen, Harold T. Hodes, Joshua Hoffman, Alan Holland, Robert L. Holmes, Richard Holton, Brad W. Hooker, Terence E. Horgan, Tamara Horowitz, Paul Horwich, Vittorio Hösle, Paul Hoβfeld, Daniel Howard-Snyder, Frances Howard-Snyder, Anne Hudson, Deal W. Hudson, Carl A. Huffman, David L. Hull, Patricia Huntington, Thomas Hurka, Paul Hurley, Rosalind Hursthouse, Guillermo Hurtado, Ronald E. Hustwit, Sarah Hutton, Jonathan Jenkins Ichikawa, Harry A. Ide, David Ingram, Philip J. Ivanhoe, Alfred L. Ivry, Frank Jackson, Dale Jacquette, Joseph Jedwab, Richard Jeffrey, David Alan Johnson, Edward Johnson, Mark D. Jordan, Richard Joyce, Hwa Yol Jung, Robert Hillary Kane, Tomis Kapitan, Jacquelyn Ann K. Kegley, James A. Keller, Ralph Kennedy, Sergei Khoruzhii, Jaegwon Kim, Yersu Kim, Nathan L. King, Patricia Kitcher, Peter D. Klein, E. D. Klemke, Virginia Klenk, George L. Kline, Christian Klotz, Simo Knuuttila, Joseph J. Kockelmans, Konstantin Kolenda, Sebastian Tomasz Kołodziejczyk, Isaac Kramnick, Richard Kraut, Fred Kroon, Manfred Kuehn, Steven T. Kuhn, Henry E. Kyburg, John Lachs, Jennifer Lackey, Stephen E. Lahey, Andrea Lavazza, Thomas H. Leahey, Joo Heung Lee, Keith Lehrer, Dorothy Leland, Noah M. Lemos, Ernest LePore, Sarah-Jane Leslie, Isaac Levi, Andrew Levine, Alan E. Lewis, Daniel E. Little, Shu-hsien Liu, Shu-hsien Liu, Alan K. L. Chan, Brian Loar, Lawrence B. Lombard, John Longeway, Dominic McIver Lopes, Michael J. Loux, E. J. Lowe, Steven Luper, Eugene C. Luschei, William G. Lycan, David Lyons, David Macarthur, Danielle Macbeth, Scott MacDonald, Jacob L. Mackey, Louis H. Mackey, Penelope Mackie, Edward H. Madden, Penelope Maddy, G. B. Madison, Bernd Magnus, Pekka Mäkelä, Rudolf A. Makkreel, David Manley, William E. Mann (W.E.M.), Vladimir Marchenkov, Peter Markie, Jean-Pierre Marquis, Ausonio Marras, Mike W. Martin, A. P. Martinich, William L. McBride, David McCabe, Storrs McCall, Hugh J. McCann, Robert N. McCauley, John J. McDermott, Sarah McGrath, Ralph McInerny, Daniel J. McKaughan, Thomas McKay, Michael McKinsey, Brian P. McLaughlin, Ernan McMullin, Anthonie Meijers, Jack W. Meiland, William Jason Melanson, Alfred R. Mele, Joseph R. Mendola, Christopher Menzel, Michael J. Meyer, Christian B. Miller, David W. Miller, Peter Millican, Robert N. Minor, Phillip Mitsis, James A. Montmarquet, Michael S. Moore, Tim Moore, Benjamin Morison, Donald R. Morrison, Stephen J. Morse, Paul K. Moser, Alexander P. D. Mourelatos, Ian Mueller, James Bernard Murphy, Mark C. Murphy, Steven Nadler, Jan Narveson, Alan Nelson, Jerome Neu, Samuel Newlands, Kai Nielsen, Ilkka Niiniluoto, Carlos G. Noreña, Calvin G. Normore, David Fate Norton, Nikolaj Nottelmann, Donald Nute, David S. Oderberg, Steve Odin, Michael O’Rourke, Willard G. Oxtoby, Heinz Paetzold, George S. Pappas, Anthony J. Parel, Lydia Patton, R. P. Peerenboom, Francis Jeffry Pelletier, Adriaan T. Peperzak, Derk Pereboom, Jaroslav Peregrin, Glen Pettigrove, Philip Pettit, Edmund L. Pincoffs, Andrew Pinsent, Robert B. Pippin, Alvin Plantinga, Louis P. Pojman, Richard H. Popkin, John F. Post, Carl J. Posy, William J. Prior, Richard Purtill, Michael Quante, Philip L. Quinn, Philip L. Quinn, Elizabeth S. Radcliffe, Diana Raffman, Gerard Raulet, Stephen L. Read, Andrews Reath, Andrew Reisner, Nicholas Rescher, Henry S. Richardson, Robert C. Richardson, Thomas Ricketts, Wayne D. Riggs, Mark Roberts, Robert C. Roberts, Luke Robinson, Alexander Rosenberg, Gary Rosenkranz, Bernice Glatzer Rosenthal, Adina L. Roskies, William L. Rowe, T. M. Rudavsky, Michael Ruse, Bruce Russell, Lilly-Marlene Russow, Dan Ryder, R. M. Sainsbury, Joseph Salerno, Nathan Salmon, Wesley C. Salmon, Constantine Sandis, David H. Sanford, Marco Santambrogio, David Sapire, Ruth A. Saunders, Geoffrey Sayre-McCord, Charles Sayward, James P. Scanlan, Richard Schacht, Tamar Schapiro, Frederick F. Schmitt, Jerome B. Schneewind, Calvin O. Schrag, Alan D. Schrift, George F. Schumm, Jean-Loup Seban, David N. Sedley, Kenneth Seeskin, Krister Segerberg, Charlene Haddock Seigfried, Dennis M. Senchuk, James F. Sennett, William Lad Sessions, Stewart Shapiro, Tommie Shelby, Donald W. Sherburne, Christopher Shields, Roger A. Shiner, Sydney Shoemaker, Robert K. Shope, Kwong-loi Shun, Wilfried Sieg, A. John Simmons, Robert L. Simon, Marcus G. Singer, Georgette Sinkler, Walter Sinnott-Armstrong, Matti T. Sintonen, Lawrence Sklar, Brian Skyrms, Robert C. Sleigh, Michael Anthony Slote, Hans Sluga, Barry Smith, Michael Smith, Robin Smith, Robert Sokolowski, Robert C. Solomon, Marta Soniewicka, Philip Soper, Ernest Sosa, Nicholas Southwood, Paul Vincent Spade, T. L. S. Sprigge, Eric O. Springsted, George J. Stack, Rebecca Stangl, Jason Stanley, Florian Steinberger, Sören Stenlund, Christopher Stephens, James P. Sterba, Josef Stern, Matthias Steup, M. A. Stewart, Leopold Stubenberg, Edith Dudley Sulla, Frederick Suppe, Jere Paul Surber, David George Sussman, Sigrún Svavarsdóttir, Zeno G. Swijtink, Richard Swinburne, Charles C. Taliaferro, Robert B. Talisse, John Tasioulas, Paul Teller, Larry S. Temkin, Mark Textor, H. S. Thayer, Peter Thielke, Alan Thomas, Amie L. Thomasson, Katherine Thomson-Jones, Joshua C. Thurow, Vzalerie Tiberius, Terrence N. Tice, Paul Tidman, Mark C. Timmons, William Tolhurst, James E. Tomberlin, Rosemarie Tong, Lawrence Torcello, Kelly Trogdon, J. D. Trout, Robert E. Tully, Raimo Tuomela, John Turri, Martin M. Tweedale, Thomas Uebel, Jennifer Uleman, James Van Cleve, Harry van der Linden, Peter van Inwagen, Bryan W. Van Norden, René van Woudenberg, Donald Phillip Verene, Samantha Vice, Thomas Vinci, Donald Wayne Viney, Barbara Von Eckardt, Peter B. M. Vranas, Steven J. Wagner, William J. Wainwright, Paul E. Walker, Robert E. Wall, Craig Walton, Douglas Walton, Eric Watkins, Richard A. Watson, Michael V. Wedin, Rudolph H. Weingartner, Paul Weirich, Paul J. Weithman, Carl Wellman, Howard Wettstein, Samuel C. Wheeler, Stephen A. White, Jennifer Whiting, Edward R. Wierenga, Michael Williams, Fred Wilson, W. Kent Wilson, Kenneth P. Winkler, John F. Wippel, Jan Woleński, Allan B. Wolter, Nicholas P. Wolterstorff, Rega Wood, W. Jay Wood, Paul Woodruff, Alison Wylie, Gideon Yaffe, Takashi Yagisawa, Yutaka Yamamoto, Keith E. Yandell, Xiaomei Yang, Dean Zimmerman, Günter Zoller, Catherine Zuckert, Michael Zuckert, Jack A. Zupko (J.A.Z.)
- Edited by Robert Audi, University of Notre Dame, Indiana
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- The Cambridge Dictionary of Philosophy
- Published online:
- 05 August 2015
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- 27 April 2015, pp ix-xxx
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Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos
- Rabindranath de la Fuente, W. Allan King
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The mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.
Expression and localisation of heat shock protein 70 in cultured bovine oocytes and embryos
- Sheldon J. Kawarsky, W. Allan King
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Effects of elevated in vitro temperature on in vitro produced early bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (hsp70). In vitro matured bovine oocytes, 2-cell and 8-cell embryos, and day 9 hatched blastocysts subjected to control and elevated temperature conditions were analysed by semiquantitative reverse transcription polymerase chain reaction methods for hsp70 mRNA expression. Results revealed the expression of hsp70 mRNA under control conditions and that early embryos can respond to heat stress by transcribing hsp70 mRNA. Confocal laser scanning microscopy used to localise the hsp70 protein in oocytes and embryos revealed that the distribution of hsp70 in the ooplasm of immature and mature oocytes is unaffected by exposure to elevated temperatures and that this protein was closely associated with the meiotic spindle, indicating its possible role in stabilising this structure. In 8-cell embryos derived under control conditions, hsp70 was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed to elevated temperature. In heat-stressed hatched blastocysts, a more even distribution was noted following heat stress relative to corresponding controls, indicating their competence to respond to elevated temperature.
Apoptosis in the early bovine embryo
- Christie Matwee, Dean H. Betts, W. Allan King
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Cell death occurs during early development in vivo and in vitro, although little is known about the mechanism of blastomere death and the relation to embryonic loss. Apoptosis, characterised by chromatin condensation, DNA fragmentation and membrane blebbing, occurs without damage to surrounding cells in contrast to necrosis. Bovine oocytes and in vitro fertilised embryos (total n = 449) were analysed for (1) DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and (2) morphological features of apoptosis. TUNEL labelling was detected in immature and mature oocytes (7%, n = 57 and 23%, n = 60, respectively), and at least one cell of 8- to 16-cell embryos (5%, n = 57), morulae/early blastocysts (79%, n = 39) and expanded/hatched blastocysts (100%, n = 48). In contrast, TUNEL labelling was not detected in zygotes (n = 61), 2-cell embryos (n = 46) or 3- to 7-cell embryos (n = 81). Chromatin condensation, nuclear fragmentation, absence of neighbouring cell destruction and extrusion of cells was frequent among advanced stage embryos. Although not detected during early cleavage under standard conditions, TUNEL labelling indicative of apoptosis was induced by treatment with 10 μM staurosporine for 30 h in 95% of cleavage stage embryos (n = 59). Determination of the expression and localisation of the p53 tumour suppressor gene using reverse transcription polymerase chain reaction and whole-mount immunofluorescence revealed that although p53 transcripts were present throughout early development, nuclear localisation of p53 protein could not be detected in any blastocyst suggesting p53-independent apoptosis. This study has shown that apoptosis is dependent on embryonic developmental stage after standard culture. This suggests that bovine embryos become more capable of accommodating damaged or abnormal cells as development proceeds.
Effects of follicle-stimulating hormone, bovine somototrophin and okadaic acid on cumulus expansion and nuclear maturation of Blue fox (Alopex lagopus) oocytes in vitro
- Vlastimil Sršeň, Jaroslav Kalous, Eva Nagyova, Peter šutovský, W. Allan King, Jan Motlik
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The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte–cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 μm in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 µm in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.