[Frontiers in Bioscience E3, 1162-1168, June 1, 2011]
Gene expression profiles of in vivo derived mouse blastocysts after slow-freezing
Li Wang1, Xinyan Zhang2, Chunhua Yu3, Yuanqing Yao2
1
TABLE OF CONTENTS
1. ABSTRACT
The goals of our study were to analyze change of global gene expression profile of mouse blastocysts after slow-freezing and to explore molecular mechanism underlying the decreased pregnancy rate caused by cryopreservation. The results showed that superovulation differentially regulated the expression of 288 genes with at least 2.0-fold change considerations. Among which, 275 genes were down-regulated and the remainder up-regulated in the cryopreserved group. The independent analysis with real-time PCR fully confirmed the results of microarray. These differential genes were classified into eighteen functional groups belonging to biological process, molecular function, and cellular component. Twenty nine genes could be categorized into one or more of KEGG pathways. The pathways of mitogen-activated protein kinase, Wnt and cell cycle were the most predominantly affected. Thus, the expression pattern reflected a broad spectrum of consequences of slow-freezing on the blastocysts, with most effects on stress-related and cell cycle-related genes.
2. INTRODUCTION
The first human embryos were frozen in the late 1970s, with the first pregnancy reported in 1983 in Australia and a birth occurred the following year (1). Slow-cooled freezing methods have been in existence since 1972 and are considered the "gold standard" for cryopreserving embryos by many cryobiologists (2). Although vitrification is now on the rise, most human ART centers routinely use slow-cooling methods for freezing embryos (3).
Clinics trend to choose cryopreservation at the blastocyst stage, although there is debate as to the developmental stage at which human embryos are best cryopreserved (4, 5). Blastocysts are preimplantation embryos that have successfully passed the critical step of genomic activation and so have a high developmental potential (6). Blastocysts also have the advantage of containing numerous small cells. Loss of some cells during freezing and thawing is probably less harmful for the further development of the embryo (7).
Despite a certain success, cryopreservation techniques cause significant morphological, biochemical alterations and lower the pregnancy rate. During the cryopreservation, embryos are subjected to physical and chemical alterations, including polyploid formations (8), the destruction of the cell membrane integrity, ionic disturbances (9), cellular organelle dysfunctions (10, 11) and increased reactive oxygen species (ROS) productions (10). Such detrimental effects may trigger the apoptotic cascade leading to a decrease in implantational and developmental capabilities of embryos (10, 12).
Moreover, in a long-term study including senescence, Dulioust et. al. compared slow-frozen and control mice for several quantitative traits. They found significant differences in morphophysiological and behavioral features, some of them appearing in elderly subjects. Thus, apart from its immediate toxicity, slow-freezing, without being severely detrimental, may have delayed effects (13).
The intricacies of the cold-induced changes in embryos that could affect the intracellular and developmental processes need to be known (14). In this paper, we analyzed change of global gene expression profile of mouse blastocysts after slow-cooled freezing.
3. MATERIALS AND METHODS
3.1. Blastocysts collection
Young ICR female mice and male ICR mice (provided by the Center for Experimental Animals, Peking University Health Science Center) were housed in a specific pathogen free facility with 12h light/ 12hr dark photoperiod, at a temperature of 23 degree and relative humidity of 44%. All experiments were conducted in accordance with the standards for the care and use of animals approved by Peking University Health Science Center Ethical Committee. Mice were sacrificed by cervical dislocation to collect embryos. Female ICR mice were superovulation by standard hormone treatments using an i.p. injection of 5 IU pregnant mare serum (PMSG, Sigma, USA) followed 48 hr later by an i.p. injection of 10 IU human chorionic gonadotrophin (hCG, Sigma, USA). Blastocysts were flushed from the uterine horns 95 hr after the hCG injection.
3.2. Slow freezing and thawing
A slow-rate freezing protocol was employed, as described by Lassalle et al. (1985), using a programmable freezer (Cryomagic, Mirae Biotech, Seoul, Korea). Blastocysts were cryopreserved using 9 % glycerol and 0.2M sucrose (Quinn's Advantage™ Blastocyst Freeze kit; SAGE, Biopharma, Trumbull, CT, USA). The embryos are taken from a starting temperature of 37 degree to -6 degree at 2 degree per minute. They are then seeded manually and held at -6 degree for a total of 10 to 15 minutes before being cooled at about 0.3degree per minute to around -35 degree. They are then transferred to a storage tank of liquid nitrogen.
Eight weeks later, blastocysts were thawed rapidly by removing cryovials from storage, exposure to air for 30 seconds and immersion in a water bath at 30 degree for 45 seconds. Cryoprotectant was removed by exposing the embryos to decreasing concentrations of cryoprotectant (Quinn's Advantage Thaw Kit, SAGE In-Vitro Fertilization, Inc., USA). Post-warming, viable blastocysts re-expand and were allowed up to six hours of incubation to regain their vitality. The expanded blastocysts were either transferred into uteri of psuedopregnant mice or washed in RNAse free water, pooled in 150 ml of TRIzol (Invitrogen, Carlsbad, CA) per group and stored at -80 degree until RNA extraction.
3.3. Embryo Transfer
Female mice were mated with vasectomized males to generate pseudopregnant recipients. The following morning, plug-positive females were selected. Frozen blastocysts were transferred into one uterine horn and control blastocysts transferred into the other one of the same recipient pseudopregnant mouse. On 5th day after transfer, the number of uterine implantation sites was recorded. On 18th day, the number of live fetuses was counted
3.4. RNA extraction and RNA amplification
Two independent biological replicates of fresh and 8-week cryopreserved blastocysts pool were used for microarray experiments. Total RNA was extracted from pools of 150 fresh or cryopreserved blastocysts in either control or superovulation group with use of Trizol reagent (Invitrogen, Life Technologies, USA) according to the manufacturer's instructions. Total RNA yield was 80-110ng per pool. Eighty nanograms of total RNA was used for linear, two-round amplification by in vitro transcription using MessageAmpTM II aRNA Amplification Kit (Ambion, Huntingdon, UK). Final yield of biotinylated cRNA was 80-120 microgram. The quality and purity of cRNA were determined by RNA integrity examined by electrophoresis on 1 % formaldehyde denaturing gel. These samples were used for the subsequent hybridization.
3.5. Hybridization and slide processing
A total of 15μg labeled cRNA was fragmented and hybridized to the Mouse Genome 430 2.0 array (Affymetrix Santa Clara, CA), which contains 45,000 probe sets analyzing the expression level of over 39,000 transcripts and variants from over 34,000 well characterized mouse genes (Two-cycle Target Labeling Assays). The GeneChip arrays were washed and then stained (streptavidin-phycoerythrin) on an Affymetrix FluidicsStation 450 followed by scanning on a GeneChip Scanner 3000. The procedures were repeated for replicate experiment with independent hybridization and processing.
3.6. Scanning and result analyses
The hybridization data were analyzed using GeneChip Operating software (GCOS 1.4). The scanned images were first assessed by visual inspection then analyzed to generate raw data files saved as CEL files using the default setting of GCOS 1.4. A global scaling procedure was performed to normalize the different arrays using dChip software.
In a comparison analysis, we applied a two class unpaired method in the Significant Analysis of Microarray software (SAM) to identify significantly differentially expressed genes between EM and non-EM groups.
3.7. Quantitative real-time RT-RCR：
Gene expression profiles derived from microarray analyses were confirmed quantitatively in 3 pooled samples by RT-PCR analysis. The selected genes for RT-PCR included following 16 genes. Three replicates were used for each real-time PCR reaction. The primers for selected genes are listed in Table 1. The housekeeping gene, beta-actin, was chosen as the internal control for sample normalization.
Two microgram of total amplified RNA from blastocysts was reverse transcribed into cDNA using SuperScript II (Invitrogen, CA, USA). All qPCR assays were performed with QuantiTectTM SYBR Green PCR kit (Qiagen, Hilden, Germany) as indicated. The 2-delta delta CT relative quantification method was performed to analyze the data from the qPCR experiment as previously described (15)
3.8. Statistical analysis
All statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls method. A probability of p<0.05 was used to indicate a significant difference.
4. RESULTS
4.1 Development potential of cryopreserved blastocysts
We analyzed the impact of cryopreservation on the embryo implantation potential in vivo (Table 2). The results showed that on 5.5dpc, the implantation rates (implantation sites/blastocysts transfered) of cryopreserved embryos were significantly lower than the fresh embryos (38.75% vs. 72.50%, p<0.01). In each group, eighty fetuses were allowed to develop to 18.5dpc. there were significantly less viable fetuses developed from cryopreserved blastocysts (20/80 vs. 47/80, p<0.01) (Table 2)
4.2. The differential expression profile of cryopreserved blastocysts detected by microarray
After SAM analysis of microarray data from the 4 subjects consisting of 2 pooled samples of fresh blastocysts and 2 pooled samples of cryopreserved blastocysts, 288 genes were determined to be significantly differentially expressed with a selection threshold of false discovery rate, FDR = 8.06% and fold change >2.0 in the SAM output result (see supplementary data). The SAM result was visualized in Figure 1 using SAM software and visualized with TreeView tools after unsupervised hierarchical clustering. Totally, 217 out of the 288 genes exhibited down-regulation and the remainder were up-regulated in the cryopreserved group. Sixteen differential genes or transcripts were selected to confirm their expression differences in three samples with qRT-PCR. Among them, nine genes were down-regulated and seven genes up-regulated (Figure 2).
4.3. Gene ontologies (GO) classification of differential genes
All 288 differentially expressed genes were analyzed the association with particular function from the Gene Ontology classification using a free web-based Molecular Annotation System 2.0 (MAS 2.0, www.capitalbio.com). The data were categorized based on three independent GO terms with p<0.05: biological process, molecular function, and cellular component. All the differentially expressed gene probes were classified into eighteen functional groups. The first five categories accounted for approximately 41.5% of the total up- and down- regulated genes indentified. Results are summarized in Figure 3.
4.4. Pathway analysis of differential genes
The GO database does not cover every aspect of biology. The differentially expressed genes were also analyzed by MAS, which integrates three different open source pathway resources-KEGG, BioCarta and GenMAPP. The pathways are ranked with statistical significance by calculating their p-values based on hypergeometric distribution (16, 17). Among the 288 genes differentially expressed, 29 of them could be categorized into one or more of KEGG pathways listed in Table 3 (p<0.05). Among these, the pathways of mitogen-activated protein kinase (MAPK), Wnt and cell cycle were the most predominant.
5. DISCUSSION
In this study, we demonstrated for the first time the global transcript variations between cryopreserved blastocysts and the control groups. We showed the alterations of the genes to different important biological process, molecular function, cellular component and signaling pathways, to enable detailed understandings of the effects of slow-freezing on gene functions after warming. Cold stress affects cellular physiological by inhibiting transcription and translation and by increasing the denaturation and misaggregation of proteins (18). Cold stress also slows progression through the cell cycle, with phase G1 typically being the most sensitive (19, 20). Such reactions are evolutionary conserved among species (21) and expected in mammals. Besides the very few molecular level cold response studies available in mammalian cells, this study provided embryonic evidence to support the conservation of cold response and also revealed the difference between reactions of blastocyst stage embryo and mammalian cells to cold response.
Our findings suggested that 3% (9/288) differential genes belong to MAPK signaling pathway, they changed dramatically upon cold stress to blastocysts. MAPK signaling promotes trophectoderm formation from mouse embryos (22). It is also served as a target of anandamide, as well as agonists that stimulate trophoblast differentiation through intracellular calcium signaling (23). Our data are consistent with the previous study which also showed 3% differential genes of MAPK cascades in 8-cell mouse embryos after vitrification (24). Among these differential genes, growth arrest and DNA-damage-inducible 45 beta (Gadd45b) and heat shock protein 1 (Hspb1) were both up-regulated in vitrified 8-cell embryos and slow-frozen blastocysts. These two genes are highly conserved and induced by stresses. They have been implicated in cell cycle arrest (25-27).
Cells cultivated under hypothermic conditions undergo cell cycle arrest, predominantly in G1, but also in G2 ⁄M (28). It has recently been suggested that this is in part due to the expression of the RNA-binding cold shock proteins (Cirp) and RNA binding motif 3 (Rbm3), as their overexpression under normothermic conditions can lead to cell cycle arrest (29). In this study, several cell cycle genes changed, including Rbl1, Cdc14a, Cdk2, Gadd45b, Chek1. However, we did not detect the change of Cirp or Rbm3. Instead, there were 4-fold and 2.4-fold increase of Gadd45b and transducer of ERBB2, 2 (Tob2) respectively. They are genes induced by stress and their overexpression leads to cell cycle arrest (25, 26) (unpublished data).
Among the 288 differentially-expressed genes, we also focused on the genes encoding secreted proteins (known or predicted). Detection of the secreted proteins in culture medium may help develop diagnostic approach to identify the "best" embryo for transfer. Dickkopf-1 (Dkk1), one of these secreted proteins, was 2.5 folds down-regulated by slow-freezing after thawing. Dkk1 is the most well-characterized member of Dkk family, it inhibits the Wnt signaling pathway (30). As the important antagonist of Wnt, Dkk1 plays roles in blastocyst activation and uterine receptivity during the window of implantation (7). Decreased level of Dkk1 may account for the failure of implantation capacity of cryopreserved embryos.
In summary, the expression pattern reflected a broad spectrum of consequences of slow-freezing on the blastocysts, with most effects on stress-related genes and cell cycle-related genes. As mice are model animals for human embryo development studies (31), this study can fill the knowledge gap for the human model. Moreover, this study allowed the identification of new candidate marker genes to optimize cryopreservation protocol and provide candidates for diagnostic purpose.
6. ACKNOWLEDGEMENTS
Li Wang, Xinyan Zhang and Chunhua Yu equally contributed to this article. This work was supported by grant from the Chinese National 973 Projects (2007CB948102) to Yuanqing Yao and grant from the National Science Foundation for Post-doctoral Scientists of China (Grant No. 2008043027). It was also supported by the National Natural Science Fund (Grant No. 30800425)
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Abbreviations: IVF (in vitro fertilization); ROS: reactive oxygen species; GO: Gene ontologies; MAPK: mitogen-activated protein kinase; Gadd45b: DNA-damage-inducible 45 beta; Hspb1: heat shock protein 1; Cirp: cold-inducible RNA-binding protein; Rbm3: RNA binding motif 3; Tob2: transducer of ERBB2, 2; Dkk1: Dickkopf-1
Key Words: Cryopreservation, Microarray, Blastocyst, Gene Expression
Send correspondence to: Yuanqing Yao, 28 Fuxing Rd. Department of Obstetrics and Gynecology, the General Hospital of the People's Liberation Army, Beijing. P. R. China, Tel: 8610-8280-2164, Fax: 8610-8280-2152, E-mail:yqyaolab@gmail.com