Hostname: page-component-848d4c4894-tn8tq Total loading time: 0 Render date: 2024-06-27T01:11:19.307Z Has data issue: false hasContentIssue false
  • English
  • Français

Ultrastructure of vitrified rabbit transgenic embryos

Published online by Cambridge University Press:  23 October 2013

P. Chrenek ,
A.V. Makarevich ,
M. Popelková ,
J. Schlarmannová ,
S. Toporcerová ,
A. Ostró ,
J. Živčák  and
Zs. Bosze
P. Chrenek*
Affiliation:
Animal Production Research Centre Nitra, 95141 Luzianky Near Nitra, Slovak Republic. Slovak University of Agriculture, Slovak Republic.
A.V. Makarevich
Affiliation:
Animal Production Research Centre Nitra, Slovak Republic.
M. Popelková
Affiliation:
P.J. Šafárik University, Košice, Slovak Republic.
J. Schlarmannová
Affiliation:
University of Constantine Philosophy, Nitra, Slovak Republic.
S. Toporcerová
Affiliation:
P.J. Šafárik University, Košice, Slovak Republic.
A. Ostró
Affiliation:
P.J. Šafárik University, Košice, Slovak Republic.
J. Živčák
Affiliation:
Technical University, Košice, Slovak Republic.
Zs. Bosze
Affiliation:
Agriculture Biotechnological Centre, Godollo, Hungary.
*
All correspondence to: P. Chrenek. Animal Production Research Centre Nitra, 95141 Luzianky Near Nitra, Slovak Republic. Tel: + 421 37 6546 289. Fax: + 421 37 6546 285. e-mail: chrenekp@yahoo.com
Get access Rights & Permissions [Opens in a new window]

Summary

The aim of the study was to determine the viability of rabbit transgenic (enhanced green fluorescent protein (EGFP)-positive) embryos cultured in vitro and compare with gene-microinjected (Mi) non-transgenic (EGFP-negative) embryos following vitrification. Non-microinjected and non-vitrified embryos were used as the control. Morphological signs of injury to embryo organelles were determined at the ultrastructural level using transmission electron microscopy (TEM). Morphometric evaluation was performed on cellular organelles using microphotographs obtained by TEM. Intact and Mi embryos recovered from in vivo fertilized eggs at 19–20 hours post coitum (hpc) were cultured for up to 72 hpc (morula stage), evaluated for the EGFP gene integration and then vitrified in 0.25 ml insemination straws in modified EFS (40% ethylene glycol + 18% Ficoll 70 + 0.3 M sucrose) vitrification solution. After 1–3 days the embryos were devitrified, a representative selection of embryos was analyzed by TEM and the remaining embryos were subjected to additional in vitro culture. Observations by TEM showed that the vitrified/warmed EGFP-positive and EGFP-negative embryos had a slight accumulation of cellular debris and lipid droplets compared with the control intact embryos. More severe changes were detected in the membrane structures of the treated embryos, mostly in the cytoplasmic envelope, trophoblastic microvilli, junctional contacts and mitochondria. We suggest that the higher proportion of deteriorated cell structures and organelles in the treated embryos may be due to the vitrification process rather than to mechanical violation (the gene-microinjection procedure), as a detailed inspection of ultrastructure revealed that most damage occurred in the cell membrane structures.

Keywords

EGFPMorphometryRabbit embryoTransgenesisUltrastructureVitrification
Type
Research Article
Information
Zygote , Volume 22 , Issue 4 , November 2014 , pp. 558 - 564
Copyright
Copyright © Cambridge University Press 2013 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abe, Y., Hara, K., Matsumoto, H., Kobayashi, J., Sasada, H., Ekwall, H., Rodriguez-Martinez, H. & Sato, E. (2005). Feasibility of nylon-mesh holder for vitrification of bovine germinal vesical oocytes in subsequent production of viable blastocysts. Biol. Reprod. 72, 1416–20.Google Scholar
Almasi-Turk, S., Roozbahi, A., Alibadi, E., Haeri, A., Sadeghi, Y. & Hosseni, A. (2009). Developmental consequences of mouse cryotop vitrified oocytes and embryo using low concentrated cryoprotectants. Iranian J. Reprod. Med. 7, 181–88.Google Scholar
Bettencourt, E.M.V., Bettencourt, C.M., Silva, J.N.C.E., Ferreira, P., de Matos, C.P., Oliveira, E., Ramao, R.J., Rocha, A. & Sousa, M. (2009). Ultrastructure characterization of fresh and cryopreserved in vivo produced ovine embryos. Theriogenology 71, 947–58.Google Scholar
Boonkusol, D., Faisaikarm, T., Dinnyes, A. & Kitiyanant, Y. (2007). Effects of vitrification procedures on subsequent development and ultrastructure of in vitro-matured swamp buffalo (Bubalus bubalis) oocytes. Reprod. Fertil. Dev. 19, 383–91.Google Scholar
Cocero, M.J., Díaz de la Espina, S.M. & Aquilar, B. (2002). Ultrastructural characteristics of fresh and frozen-thawed ovine embryos using two cryoprotectants. Biol. Reprod. 66, 1244–58.Google Scholar
Chrenek, P., Bauer, M. & Makarevich, A.V. (2011). Quality of transgenic rabbit embryos with different EGFP gene constructs. Zygote 19, 85–90.Google Scholar
Chrenek, P., Turanová, Z., Slamečka, J. Jr & Makarevich, A. (2013). Quality of rabbit vitrified/thawed transgenic embryos. Zygote 21, 53–8.Google Scholar
Cuello, C., Bethelot, F., Delaleu, B., Venturi, E., Pastor, L.M. & Vazquez, J.M. (2007). The effectiveness of the stereomicroscopic evaluation of embryo quality in vitrified-warmed porcine blastocysts: an ultrastructural and cell death study. Theriogenology 67, 970–82.Google Scholar
Fabian, D., Gjorret, J.O., Berthelot, F., Martinat-Botté, F. & Maddox-Hyttel, P. (2005). Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts. Mol. Reprod. Dev. 70, 155–65.Google Scholar
Fair, T., Lonergan, P., Dinnyes, A., Cottel, D.C., Hyttel, P. & Ward, F.A. (2001). Ultrastructure of bovine blastocysts following cryopreservation: effect of method of blastocyst production. Mol. Reprod. Dev. 58, 186–95.Google Scholar
Kaidi, S., Van Langendonckt, A., Massip, A., Dessy, F. & Donnay, I. (1999). Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos. Theriogenology 52, 515–25.Google Scholar
Karnovsky, M.J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell. Biol. 27, 137A.Google Scholar
Kasai, M., Hamaguchi, Y., Zhu, S.E., Miyake, T., Sakurai, T. & Machida, T. (1992). High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method. Biol. Reprod. 46, 1042–46.Google Scholar
Kuwayama, M, Vajta, G., Ieda, S. & Kato, O. (2005). Vitrification of human embryos using the CryoTip™ method. Reprod. BioMed. Online 11, 608–14.CrossRefGoogle Scholar
Li, R., Lai, L., Wax, D., Hao, Y., Murphy, C.N., Rieke, A. (2006). Cloned transgenic swine via in vitro production and cryopreservation. Biol. Reprod. 75, 226–30.Google Scholar
Makarevich, A.V., Chrenek, P., Olexikova, L., Popelkova, M., Turanova, Z., Ostro, A. & Pivko, J. (2008). Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification. Theriogenology 70, 675–81.Google Scholar
Morato, R., Izquierdo, D., Paramio, M.T. & Mogas, T. (2008). Cryotops versus open-pulled straw (OPS) as carriers for the cryopreservation of bovine oocytes: Effects of spindle and chromosome configuration and embryo development. Cryobiology 57, 137–41.Google Scholar
Orief, Y., Schultze-Mosgau, A., Dafopoulos, K. & Al-Hasani, S. (2005). Vitrification: will it replace the conventional gamete cryopreservation techniques? Middle East Fertility Society Journal 10, 171–84.Google Scholar
Papis, K., Sypecka, J., Korwin-Kossakowski, M., Wenta-Muchalska, E. & Bilska, B. (2005). Banking of embryos of mutated, paralytic tremor rabbit by means of vitrification. Lab. Anim. 39, 284–89.Google Scholar
Pereira, R.M. & Marques, C.C. (2008). Animal oocytes and embryo cryopreservation. Cell Tissue Banking 9, 267–77.Google Scholar
Popelkova, M., Chrenek, P., Pivko, J., Makarevich, A.V., Kubovicova, E. & Kacmarik, J. (2005). Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification. Zygote 13, 283–93.Google Scholar
Rall, W.F., Schmidt, P.M., Lin, X., Brown, S.S., Ward, A.C. & Hansen, C.T. (2000). Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models. ILAR J. 41, 221–27.Google Scholar
Vajta, G. & Kuwayama, M. (2006). Improving cryopreservation systems. Theriogenology 65, 236–44.Google Scholar
Vajta, G., Hyttel, P. & Callesen, H. (1997). Morphological changes of in vitro-produced bovine blastocysts after vitrification, in-straw direct rehydratation, and culture. Mol. Reprod. Dev. 48, 9–17.3.0.CO;2-N>CrossRefGoogle Scholar
Vicente, J.S. & Garcia-Ximenez, F. (1993). Effects of strain and embryo transfer model (embryo from one versus two donor doses/recipient) on results of cryopreservation in rabbit. Reprod. Nutr. Dev. 33, 5–13.Google Scholar
Visintin, J.A., Martins, J.F.P., Bevilacqua, E.M., Mello, M.R.B., Nicácio, A.C. & Assumpção, M.E.O.A. (2002). Cryopreservation of Bos taurus versus Bos indicus embryos: are they really different? Theriogenology 57, 345–59.Google Scholar
Weibel, E.R. & Bolender, R.P. (1973). Stereological techniques for electron microscopic morphometry. In Principles and Techniques for Electron Microscopy, vol. 3 (ed. Hayat, M.A.), p. 237. New York: Van Nostrand Reinhold.Google Scholar