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Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to examine the transcript variations and identify genes most affected by the treatment. For this, 8-cell-stage embryos were collected from female ICR mice mated with ICR males. The embryos were washed with CZB-HEPES base medium and suspended briefly in equilibrium medium consisting of 4% ethylene glycol (EG) in base medium at room temperature. Following equilibration, the embryos were vitrified in a 35% EG, 0.4 M trehalose, 5% polyvinylpyrrolidone (PVP) solution by means of a solid-surface vitrification (SSV) technique as described earlier (Dinnyes 2000 Biol. Reprod. 63, 513-518). Then 40 embryos each from the control and the vitrified/warmed groups were cultured in CZB medium for 3 h. Total RNAs were extracted from cultured embryos in each group using TRIzol (Invitrogen, Bio-Science, Ltd., Budapest, Hungary), following the manufacturer's instructions. Two rounds of amplification were employed to produce labeled RNA, using low input RNA amplification kit (Agilent Technologies, Kromat, Ltd., Budapest, Hungary) procedures with modifications. Three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22K oligonucleotide slides with subsequent analysis of the results. Moreover, as an independent analysis tool, real time PCR was used with eight designed primers. All of the vitrified embryos were recovered after warming with no morphological signs of cryodamage and used for analysis. The two rounds of amplification yielded 15-16 _g of cRNA. The analysis of repeated hybridizations by Rosetta luminator software (Agilent) showed 20 183 genes and expressed sequence tags (ESTs) that passed the selection criteria and were identified as common signatures in all of the slides. Unsupervised analysis of the gene expression data identified a total of 631 differentially expressed (P < 0.01) genes. However, to support the reliability of the results, only those variations above 1.5 fold differences were considered as significant in the final analysis. Therefore, with this stringent criterion 183 genes were differentially expressed (P < 0.01), of which 109 were up-regulated and the remainder down-regulated. Although genes have multiple and overlapping functions, most of the differentially expressed genes were functionally classified into various physiological categories. These include stress response (8), apoptosis related (6), metabolism (51), temperature response (4), and transcription regulation (15). Moreover, the independent analysis with real time PCR and unamplified samples verified the results of microarray. Thus, based on confirmation of the results by an independent analysis and support by the previous studies for some of the genes, it is possible to conclude that the expression patterns reflect the true biological image of embryos after vitrification, with most effects on stress- and cell metabolism-related genes.This work was supported by EU FP6 (MEXT-CT-2003-59582), Wellcome Trust Foundation (Grant No. 070246), and National Office of Research and Technology (NKTH) (#BIO-00017/2002, #BIO-00086/2002). |
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