Publication: Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos
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Issued Date
2006
Resource Type
File Type
application/pdf
ISSN
1040452X
Other identifier(s)
2-s2.0-33646442312
Rights Holder(s)
Scopus
Bibliographic Citation
Molecular Reproduction and Development. Vol 73, No.6 (2006), p.700-708
Suggested Citation
Boonkusol D., Gal A.B., Bodo S., Gorhony B., Kitiyanant Y., Dinnyes A. Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos. Molecular Reproduction and Development. Vol 73, No.6 (2006), p.700-708. doi:10.1002/mrd.20450 Retrieved from: https://hdl.handle.net/20.500.14740/5696
Author(s)
Abstract
The analysis of differences in gene expression, responding to cryopreservation may explain some of the observed differences in further development of the preimplantation stage embryos. The aim of this study was to create a link, for the first time, between morphological/developmental observations and gene activity changes following cryopreservation of embryos. Efficiency of two vitrification methods, solid surface and in-straw vitrifications for pronuclear-stage mouse zygotes and 8-cell stage mouse embryos was compared based on morphological survival, blastocyst formation, and changes in embryonic gene expression. Both stages of embryos were vitrified by SSV using 35% ethylene glycol (EG) for vitrification solution (VS) and in-straw vitrification using 40% EG for VS. No significant differences were found between immediate survival rates of embryos vitrified by SSV and in-straw vitrification in both stages. Blastocyst rates were significantly higher with SSV and not significantly different from that of control. These results showed that SSV was more efficient than in-straw vitrification. Treatment with cytochalasin-b did not improve cryosurvival during SSV. The quantification of selected gene transcripts from single embryo (6 embryos/treatment group) were carried out by quantitative real-time RT-PCR. It was performed by adding 1/8 of each embryo cDNA to the PCR mix containing the specific primers to amplify housekeeping gene (β-actin), heat shock protein gene (Hsp70), genes related to oxidative stress (MnSOD and CuSOD), cold stress (CirpB, Rbm3), and cell-cycle arrest (Trp53). We found upregulation of all six stress-related genes at 3 hr post-warming in pronuclear stage embryos. Expression of these genes showed much higher level (2-33-fold) in in-straw vitrification than in in vitro control embryos. In SSV-treated embryos we could detect only slight changes (0.3-2-fold). At 10 hr post-warming, all genes were downregulated in embryos vitrified by in-straw method. In SSV-treated group expression of Hsp70 showed slight increase and Trp53 showed decrease. In contrast to pronuclear stage, there was no clear pattern of gene expression changes after vitrification in 8-cell stage embryos. Several genes were upregulated both at 3 and 10 hr post-warming. Moreover, we found upregulation of β-actin gene which we expected to use as a reference gene in in-straw treated embryos in both 3 and 10 hr post-warming, while in pronuclear stage embryos and in SSV treatment there was no effect on β-actin expression level. There was no difference in gene expression between blastocysts developed from fresh or vitrified embryos. In conclusion, the real-time RT-PCR method from single embryo opened new opportunities for the understranding of molecular events following cryopreservation. The upregulation of stress-related genes at 3 hr post-warming in pronuclear stage embryos might have been an early indicator of reduced viability following in-straw vitrification in good correlation with the developmental data to blastocyst stage. © 2006 Wiley-Liss, Inc.
Subject(s)
Beta actin
Cell cycle protein
Cold stress protein
Complementary DNA
Copper zinc superoxide dismutase
Cytochalasin B
Ethylene glycol
Heat shock protein 70
Manganese superoxide dismutase
Protein CirpB
Protein Rbm3
Protein Trp53
Unclassified drug
Vitrification solution
Animal cell
Animal experiment
Animal model
Article
Blastocyst
Cell viability
Controlled study
Correlation analysis
Cryopreservation
Embryo
Embryo development
Female
Gene expression
Gene expression regulation
In straw vitrification
In vitro study
Mouse
Nonhuman
Preimplantation embryo
Priority journal
Pronucleus
Protein expression
Real time polymerase chain reaction
Reverse transcription polymerase chain reaction
Solid surface vitrification
Survival rate
Vitrification
Warming
Zygote
Animals
Cryopreservation
Embryo
Embryo, Mammalian
Female
Gene Expression Profiling
Gene Expression Regulation, Developmental
Humans
Mice
Solutions
Animalia
Sugarcane streak virus
Cell cycle protein
Cold stress protein
Complementary DNA
Copper zinc superoxide dismutase
Cytochalasin B
Ethylene glycol
Heat shock protein 70
Manganese superoxide dismutase
Protein CirpB
Protein Rbm3
Protein Trp53
Unclassified drug
Vitrification solution
Animal cell
Animal experiment
Animal model
Article
Blastocyst
Cell viability
Controlled study
Correlation analysis
Cryopreservation
Embryo
Embryo development
Female
Gene expression
Gene expression regulation
In straw vitrification
In vitro study
Mouse
Nonhuman
Preimplantation embryo
Priority journal
Pronucleus
Protein expression
Real time polymerase chain reaction
Reverse transcription polymerase chain reaction
Solid surface vitrification
Survival rate
Vitrification
Warming
Zygote
Animals
Cryopreservation
Embryo
Embryo, Mammalian
Female
Gene Expression Profiling
Gene Expression Regulation, Developmental
Humans
Mice
Solutions
Animalia
Sugarcane streak virus