Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification

Makarevich, A. V.; Chrenek, Peter; Olexíková, Lucia; Popelková, M.; Turanová, Z.; Ostró, A; Pivko, J.

doi:10.1016/j.theriogenology.2008.04.043

Cited by 27 publications

(33 citation statements)

References 22 publications

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“…High concentrations of polymers such as Ficoll and dextran appear to be non-toxic to the embryos and can be used to replace an approximately equal amount of EG without changing the solution glass transition temperature (Visintin et al, 2002). Recently, Makarevich et al (2008) documented significant higher survival rate, lower apoptotic index and better actin filament quality for the vitrified rabbit embryos at morula stage when using EFS-based solution in comparison with equi-molar concentrations of EG plus DMSO.…”

Section: Discussionmentioning

confidence: 99%

Ultrastructure of vitrified rabbit transgenic embryos

Chrenek

Makarevich

Popelková

et al. 2013

Zygote

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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.

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Section: Discussionmentioning

confidence: 99%

Ultrastructure of vitrified rabbit transgenic embryos

Chrenek

Makarevich

Popelková

et al. 2013

Zygote

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“…Transgenesis may influence developmental rate of embryos. Makarevich et al (2008) monitored the effect of the vitrification method on the developmental rate of rabbit embryos, using the vitrification medium with ethylene glycol and Ficoll 70. They showed significantly lower ability (p < 0.05) of hatching blastocyst formation when compared with intact (97.00%) or vitrified rabbit embryos (63.00%).…”

Section: Discussionmentioning

confidence: 99%

Quality of rabbit vitrified/thawed transgenic embryos

Chrenek

Turanová

Slamečka

et al. 2011

Zygote

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The aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous-hFVIII or exogenous-enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

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“…It is unknown whether specific traits of ICM and TE could lead to differences in survival to vitrification, although a detrimental effect (such as apoptosis) of vitrification on the numbers of cells on the ICM has been observed [15,30]. The probable reason for reduced embryo viability following freezing and thawing is the disruption of the cytoskeleton, as a result of intracellular ice formation [8,25].…”

Section: Groupsmentioning

confidence: 99%