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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
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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 

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