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Reproduction (2004) 128 301-311
DOI: 10.1530/rep.1.00297
Copyright © 2004 Society for Reproduction and Fertility
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RESEARCH

Focus on ART

Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos

Paolo Rinaudo1,2 and Richard M Schultz1

1 Department of Biology and 2 Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104, USA

Correspondence should be addressed to R Schultz; Email: rschultz{at}sas.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Culture of preimplantation embryos affects gene expression. The magnitude of the effect on the global pattern of gene expression, however, is not known. We compared global patterns of gene expression in blastocysts cultured from the one-cell stage in either Whitten’s medium or KSOM + amino acids (KSOM/AA) with that of blastocysts that developed in vivo, using the Affymetrix MOE430A chip. The analysis revealed that expression of 114 genes was affected after culture in Whitten’s medium, whereas only 29 genes were mis-expressed after culture in KSOM/AA. Expression Analysis Systematic Explorer was used to identify biological and molecular processes that are perturbed after culture and indicated that genes involved in protein synthesis, cell proliferation and transporter function were down-regulated after culture in Whitten’s medium. A common set of genes involved in transporter function was also down-regulated after culture in KSOM/AA. These results provide insights as to why embryos develop better in KSOM/AA than in Whitten’s medium, and highlight the power of microarray analysis to assess global patterns of gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Preimplantation embryos can develop in media of composition ranging from simple balanced salt solutions and carbohydrates to very complex constituents (e.g. Ham’s F-10) with the further addition of serum or a feeder layer of somatic cells (Lane 2001, Natale et al. 2001, Niemann & Wrenzycki 2000). The blastocysts that develop after culture are known to be developmentally competent because, after embryo transfer, live offspring are born (Ecker et al. 2004). Nevertheless, embryo culture can perturb gene expression (Ho et al. 1995). Of particular note is that expression of imprinted genes appears particularly sensitive to culture conditions. For example, appropriate maternal monoallelic expression of H19 is observed after culture in K modified simplex optimized medium (KSOM) + amino acids (KSOM/AA), whereas biallelic expression is found after culture in Whitten’s medium (WM) (Doherty et al. 2000). Moreover, biallelic expression is accompanied by loss of DNA methylation of cytosine residues on the paternal allele in the differentially methylated domain essential for repression of the paternal allele (Doherty et al. 2000). We elected to assess the effect of embryo culture on global patterns of gene expression in embryos cultured in these two media using oligonucleotide microarrays, because of these aforementioned differences in maintaining or not maintaining appropriate monoallelic maternal H19 expression.

Until recently, quantitative analysis of high-resolution, two-dimensional protein gels (Latham et al. 1991), mRNA differential display (Ma et al. 2001), analysis of expressed sequence tags derived from libraries of various preimplantation stages (Ko et al. 2000, Sharov et al. 2003) and analysis of selected genes (Ho et al. 1995) have been used. Although these approaches have shed some light on the molecular basis underlying preimplantation development, they offer limited insight because only a small number of genes can be readily analyzed. Microarray techniques provide a powerful approach to study patterns of gene expression on a global scale (Hamatani et al. 2004, Wang et al. 2004). The ability to amplify the small amounts of mRNA present in preimplantation mouse embryos, which can only be isolated in limited numbers, makes it feasible to generate enough material for microarray analysis. We used Affymetrix oligonucleotide arrays (MOE430A chip) containing more than 22 000 transcripts and variants, together with a T7-based linear double amplification method, and report here changes in the global patterns of gene expression that occurred during preimplantation development in mouse blastocysts cultured in WM or KSOM/AA from the one-cell stage. We report that, after culture in WM, 114 genes were mis-expressed, whereas only 29 were mis-expressed after culture in KSOM/AA. Of note is that 14 common genes were mis-expressed in either medium and are involved in ion and water transport.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Acknowledgements
 References
 
Collection of preimplantation mouse embryos and RNA extraction
Mouse CF-1 x B6D2F1/J embryos were isolated from superovulated mice as previously described (Zeng & Schultz 2003). Briefly, CF-1 female mice were injected with 5 IU equine chorionic gonadotrophin and 42–46 h later with 5 IU human chorionic gonadotrophin (hCG); the females were then mated to B6D2F1/J males overnight. The next morning, zygotes were obtained from the ampullae and cultured in either WM (Whitten 1971) or KSOM/AA (Ho et al. 1995) to the blastocyst stage in 5% CO2 –5% O2 –90% N2 at 37 °C. Cultures were initiated with more embryos when WM was used, to compensate for the poorer development in WM compared with that in KSOM/AA. After culture in either medium, late-cavitating blastocysts of similar morphology were harvested at 96 h post-hCG. Embryos that developed in vivo were harvested 96 h post-hCG and those that looked morphologically similar to embryos that developed in vitro were collected and used for analysis. Cell numbers were determined by differential labeling of inner cell mass and trophectoderm cells as previously described (Handyside & Hunter 1984). The numbers of total cells and of inner cell mass cells after culture in WM were 70.6 ± 4.3 (mean±S.E.M.) and 16.6 ± 1.2 respectively. Corresponding numbers after culture in KSOM/AA were 81.8 ± 3.8 and 23.9 ± 1.7 respectively. All animal experiments were approved by the Institutional Animal Care and Use Committee and were consistent with NIH guidelines.

Total RNA was extracted from pools of 80 embryos (120 ng total RNA) using Trizol containing 2 µl Pellet Paint (Novagen, Madison, WI, USA) according to the manufacturer’s instructions (Invitrogen). Total RNA was dissolved in 10 µl sterile water and stored at –80 °C. RNA mass and size distribution were determined using the Agilent Bioanalyzer with RNA 6000 Nano LabChips (Palo Alto, CA, USA).

cDNA preparation for microarray analysis
Total RNA samples were submitted to the Penn Micro-array Facility for target preparation and GeneChip hybridization. Total RNA yield was 74–133 ng per replicate pool. This total RNA was used for linear, two-round amplification by in vitro transcription (Affymetrix Small Sample Target Labeling Assay version II, www.affymetrix.com). cRNA yield after the first amplification was 1.5–5 µg, and 0.5 µg of each replicate was used as input template for the second amplification. Final yield of biotinylated cRNA was 74–138 µg, of which 15 µg per replicate was fragmented and hybridized to Affymetrix GeneChips. cRNA samples were hybridized to MOE430A GeneChip, then washed and stained on fluidics stations and scanned at 3 µm resolution according to the manufacturer’s instructions (GeneChip Analysis Technical Manual, www.affymetrix.com).

Analysis of the microarrays
Microarray Analysis Suite 5.0 (MAS; Affymetrix, Santa Clara, CA, USA) was used to quantify microarray signals with default analysis parameters and global scaling to target mean = 150. Quality control parameters for all samples were within the following ranges: scale factor 1–2.7, background 42–88, percent genes detected 37–44% on MOE430A, actin 3'/5' signal ratio 1.5–2.8, and GAPDH 3'/5' signal ratio 2.3–7.5. The MAS metrics output was loaded into GeneSpring v5 (Silicon Genetics, www.silicongenetics.com) with per-chip normalization to the 50th percentile and per-gene normalization to the median. A filtered list was created of all genes detected (MAS ‘P’ call) in at least five of six replicates of the in vivo group.

Independent analyses were applied to identify genes with statistically significant differences in any of the two culture conditions (WM or KSOM/AA). The Gene-Spring pairwise comparison (Welch t-test with ANOVA, P = 0.05, Benjamini and Hochberg multiple testing correction) was conducted between all the possible pair combinations (in vivo, WM, KSOM/AA). The Gene-Spring multi-class analysis was applied to the entire sample set (Welch t-test with Welch ANOVA, P = 0.05, Benjamini and Hochberg multiple testing correction). A one-way ANOVA for microarrays (lgsun.grc.nia.nih. gov/ANOVA/index.html, default parameters) was also conducted. A non-redundant list was compiled containing candidate genes called significantly different in at least one analysis.

Genes exhibiting altered expression after culture were imported to Expression Analysis Systematic Explorer (EASE) to test for over-representation of annotation classes (Hosack et al. 2003). EASE is a program that provides statistical methods (reported as an EASE score) for discovering biological themes within gene lists, using previously published annotation databases. Over-representation does not refer to abundance of gene expression, but rather describes a class of genes that have similar functions – for example, transcription factors – regardless of their expression level, and appear more often in a list of interest than would normally be predicted by their distribution among all genes assayed. The method incorporates jack-knife iterative re-sampling of Fisher exact probabilities, with Bonferroni multiple testing corrections. An EASE score was calculated for likelihood of over-representation in the annotation categories GO Biological Process, GO Cell Component, GO Molecular Function, KEGG Pathway, and SwisProt keyword. GeneSpring ‘genomes’ were built for each of these annotation categories so that EASE scores from all subset lists could be visualized in parallel across all treatment and control groups, and pattern identification methods similar to those applied at the gene level were used to find functional themes in the data set. All reported expression differences from gene-based or annotation-based profiling were re-tested for statistical significance by multi-class ANOVA as appropriate.

Real time RT-PCR analysis
Blastocysts developed in vivo and in vitro (WM and KSOM/AA) were collected as described above and total RNA was isolated. Two embryo equivalents of template RNA were used for each real-time RT-PCR assay according to the manufacturer’s procedure using ABI Prism Sequence Detection System 7000 (Applied Biosystems, Foster City, CA, USA). To confirm the ability of this microarray analysis to resolve the differences in expression level, three genes that showed a statistically significant decrease were selected. The corresponding ABI TaqMan Assay-on-Demand probe/primer sets used were Mm00431846-m1 (Aqp8), Mm00500526-m1 (Slc7a3) and Mm00451610 (Slc15a2). Three replicates were used for each real-time PCR reaction; a minus template served as control. Quantification was normalized to a constant amount of pEGFPN2 RNA spiked into the RNA cocktail before the RNA extraction. Data were analyzed within the log-linear phase of the amplification curve obtained for each probe/ primer using the comparative CT method (ABI PRISM 7700 Sequence Detection System, User Bulletin no. 2).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Acknowledgements
 References
 
Hierarchical cluster analysis
Blastocysts that developed after culture from the one-cell stage in either WM or KSOM/AA (four replicates of each) or blastocysts that developed in vivo (six replicates) were used for expression profiling. We elected to initiate culture at the one-cell stage, rather than the two-cell stage, as in the previous study that assessed the effect of culture on expression of H19 (Doherty et al. 2000), because the experimental design of many studies incorporates culture initiating at the one-cell stage. Moreover, culture initiated at the one-cell stage more closely mimics the experimental procedures performed in human IVF clinics.

An unsupervised hierarchical clustering was performed using genes that were present at least in five of six in vivo control groups. Of the 22 690 genes present on the MOE430A chip, we found that, on average, 39.2% were present in at least five of six in vivo replicates, and three of four WM or KSOM/AA replicates respectively. Although, the clustering dendrogram revealed that the embryos that developed in vivo clustered together, embryos cultured in either WM or KSOM/AA did not (Fig. 1Go). Analysis of the branch tree distances revealed that only minimal differences existed between the WM and KSOM/AA group and overall between the in vivo group and the cultured groups: the minimal branch distance between the in vivo replicates was 0.354 and the final clustering branch was close to one (0.999). Instead, the cultured groups had a minimal branch distance of 0.544 and the more distant replicates branched at a distance of 1.027. This suggests that, although embryo culture leads to perturbations in global patterns of gene expression, the differences between the expression profiles for embryos cultured under the two different conditions are actually quite small.



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  		Figure 1 Hierarchical clustering analysis of all samples. Unsupervised clustering in GeneSpring was used to analyze similarities among replicate samples across all stages tested. Replicate sample numbers are indicated at the bottom of the figure. Colors correspond to relative RNA abundance for the ~9000 genes detected (Affymetrix ‘Present’ call), each of which is represented by one horizontal bar. W, Whitten’s medium; K, KSOM + amino acids.

 
Effect of culture in WM on gene expression
Expression of 114 genes (114/8868 = 1.28%) was statistically different between the two groups and satisfied the post hoc correction (Fig. 2Go) when GeneSpring pairwise comparison (Welch t-test with ANOVA, P = 0.05, Benjamini and Hochberg multiple testing correction) was conducted between the control group and the WM group. It is possible that some of the differences observed after culture in WM (or KSOM/AA) are sex-specific. Of these genes, only six were up-regulated (Table 1Go), whereas 108 were down-regulated (Table 2Go). It is interesting to note that only 2/6 (33%) and 41/108 (38%) were up-regulated or down-regulated more than twofold – that is, many of the changes were small. It is becoming more widely appreciated that modest changes in the expression of a gene can result in profound differences. An example is that changing the expression of the transcription factor OCT4 by 50% can result in changes in cell differentiation (Niwa et al. 2000). Thus small changes in expression of a critical transcription factor or a gene that acts in the early steps of a signal transduction pathway could function as a molecular switch that would lead to widespread and dramatic changes in gene expression.



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  		Figure 2 Differential expression profiles of genes after culture in either Whitten’s medium (WM) (A) or KSOM/AA (KAA) (B).

 

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  		Table 1 Genes up-regulated after culture in Whitten’s medium.
 

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  		Table 2 Genes down-regulated after culture in Whitten’s medium.
 
To confirm that the differences detected from the micro-array analyses were real, three genes that exhibit decreased expression after culture in either WM or KSOM/AA (see below) were selected for real-time PCR analysis. Results of this experiment confirmed that a decrease in expression was observed (Fig. 3Go), strongly suggesting that the changes in gene expression observed from the microarray analyses are valid.



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  		Figure 3 Real-time PCR verification of microarray data. Three genes exhibiting decreased expression after culture in either WM or KSOM/ AA were selected for analysis. The level of expression relative to embryos that developed in vivo by either microarray analysis or real-time PCR was set as 1. IV MA/RT, relative level of expression by embryos that developed in vivo (IV), assessed by either microarray (MA) or real-time (RT) PCR; KSOM/AA MA and KSOM/AA RT, relative levels of expression after culture in KSOM/AA assessed by MA or RT respectively; WM MA and WM RT, relative levels of expression after culture in WM assessed by MA or RT respectively.

 
EASE analysis using GO Biological Process and other annotation categories was implemented to ascertain if any biological theme(s) could be discovered from the list of genes with expression altered by culture in WM. EASE analysis identified five such biological processes: ribosome biogenesis and protein synthesis, cell organization and biogenesis, apoptosis, regulation of cell cycle and proliferation, and selenium binding (Table 3Go). Note that genes can be common to more than one process (e.g. Rp13).


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  		Table 3 EASE analysis of differentially expressed genes after culture in Whitten’s medium.
 
Formation of a functional ribosome requires exquisite coordinate expression of rRNA and ribosomal proteins. Decreased expression of several ribosomal proteins for both the large and small subunits would adversely affect ribosome biogenesis, which would in turn have a negative effect on the rate of protein synthesis and development. For example, cyclin synthesis is required for cell proliferation and decreasing the rate of protein synthesis would inevitably slow down the rate of cell proliferation. In fact, culture in KSOM/AA results in blastocysts that have greater cell numbers when compared with blastocysts that develop in media that do not contain amino acids (Erbach et al. 1994, Ho et al. 1995, Biggers et al. 2000, Summers et al. 2000). The reduced level of expression of cyclin D1, which regulates cell cycle progression (Stacey 2003), and of Ran, which is involved in spindle formation (Kalab et al. 1999) and essential for the nuclear import and export cycle (Quimby & Dasso 2003), could also contribute to retarded development in vitro. In a similar vein, reduced expression of claudin (Table 2Go) and protein kinase C (PKC){zeta}, which is an atypical PKC (Tables 2Go and 3Go), may perturb tight junction assembly and function. Tight junction formation, which initiates after compaction, establishes a permeability seal in the trophectoderm that is essential for vectorial fluid transport and blastocoel formation (Fleming et al. 2001, Watson & Barcroft 2001). Claudin is a component of tight junctions (Tepass 2003) and PKC{zeta}, in association with PAR3 and PAR6, localizes with the tight junction protein, ZO-1 (Suzuki et al. 2001); the role of this complex in tight junction assembly or function is unknown.

Decreased expression of secreted phosphoprotein 1 (osteopontin) (Tables 2Go and 3Go), could, in principle, contribute to reduced implantation. Osteoponin contains a GRGDS amino acid sequence that can mediate adhesion to specific integrins implicated in implantation (Lessey 2002). It is unlikely that modest reductions in expression of these genes results in compromised implantation, because a recent study noted no decrease in the incidence of implantation and development to term after embryo transfer of WM-cultured embryos (Ecker et al. 2004).

Expression of teratocarcinoma-derived growth factor (Tdgf1) (Tables 2Go and 3Go) and Msx1 (Hox7) (Table 2Go) was also reduced after culture in WM. Tdgf1 may be an important regulatory gene in gastrulation and early specification of tissues and organs (Xu et al. 1999). Tdgf1 deficiency leads to abnormal failure of cardiomyocytes to differentiate and embryo lethality. Msx1 is a homeobox gene the expression of which is regulated by BMP signaling and is expressed in several developing organs in vertebrates, including the facial primordia, particularly at the sites where epithelial–mesenchymal interactions occur during organogenesis (Liu et al. 2004). The function of either Tdgf1 or Msx1 in preimplantation is not known.

Effect of culture in KSOM/AA on gene expression
After culture in KSOM/AA, expression of only 29 genes was affected when compared with embryos that developed in vivo (Fig. 2Go). One gene (CD81) was up-regulated, whereas the others were down-regulated (Table 4Go); the significance of CD81 up-regulation, which is involved in signal transduction and cell adhesion in the immune system (Levy et al. 1998) and possibly sperm–egg fusion (Takahashi et al. 2001), is not apparent. The lower number of genes mis-expressed in KSOM/AA than in WM is consistent with KSOM/AA supporting better development in vitro. EASE analysis revealed that Slc7a3, Slc15a2, Aqp8, Slc2a3, Cd81, Arhu and Asns were over-represented (Table 5Go). The common feature of these genes is that they are involved in membrane transport function. Interestingly, expression of Slc2a3 was altered by culture in KSOM/AA but not in WM. Slc2a3 is also known as Glut3, which is involved in facilitating glucose transport.


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  		Table 4 Genes down-regulated after culture in KSOM/AA.
 

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  		Table 5 EASE analysis of genes differentially expressed after culture in KSOM/AA.
 
Inclusion of amino acids in KSOM has several positive effects on preimplantation development in vitro. For example, blastocyst cell number is increased, cavitation occurs earlier, and a greater fraction of blastocysts hatch (Ho et al. 1995, Biggers et al. 2000). Consistent with these beneficial effects is that there are only minor changes in the global pattern of gene expression after culture in KSOM/AA, although it is formally possible that the differences in salt and energy source concentrations may underlie the changes in gene expression. Recent studies have suggested, however, that ammonia generated by hydrolysis of glutamine in the culture medium may promote deleterious effects on embryo development, and experimentally increasing the ammonia concentrations compromises embryo development (Lane & Gardner 2003). Asparagine synthase (Asns), which catalyzes the ATP-dependent formation of asparagine from aspartate and ammonia, is down-regulated and could impair the ability of the embryo to convert ammonia to a non-toxic form. Nevertheless, results presented here suggest that inclusion of amino acids in KSOM is unlikely to compromise developmental competence – that is, the ability of transferred blastocysts to implant and develop to term –because the global pattern of gene expression is very similar to that of embryos that develop in vivo. Current experiments examining the effect on global patterns of gene expression of including amino acids in WM and omitting them from KSOM should provide information about the role of amino acids in governing gene expression in cultured embryos.

Common genes mis-expressed after culture in either WM or KSOM/AA
Analysis of the data in Tables 2Go and 4Go revealed 14 genes that were mis-expressed after culture in either WM or KSOM (Table 6Go). EASE analysis revealed that five of these 14 down-regulated genes (Slc7a3, Slc15a2, 9030418M0Rik, Prdx2, Aqp8) belong to the GO transporter activity family, with the first three belonging to the child ontological carrier activity family (Table 7Go). Solute carrier family 7 is important in the transport of lysine and arginine, whereas solute carrier family 15 is devoted to the transport of oligopeptides. Aquaporin 8 belongs to a class of membrane channel proteins that facilitate bulk water transport (Agre & Kozono 2003). It is present on the basolateral membranes of the trophectoderm and is probably involved in trans-trophectodermal water movement required for cavitation (Barcroft et al. 2003). It will be of interest to determine if over-expressing Aqp8 stimulates development under either of these culture conditions.


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  		Table 6 Genes differentially expressed after culture in either Whitten’s medium or KSOM/AA.
 

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  		Table 7 EASE analysis of genes differentially expressed after culture in either Whitten’s medium or KSOM/AA.
 
Also of note is the reduced expression of the genes for interleukin-6 (Il6) and for peroxiredoxin 2 (Prdx2). Il6 function in vivo may stimulate preimplantation embryo development, because the addition of interleukin-6 to culture media results in an increased cell number in blastocysts, prolonged gestation and increased weight of the generated pups (Cheung et al. 2003). Peroxiredoxin 2 belongs to a newly defined family of antioxidant proteins that have been implicated in cell proliferation and differentiation, intracellular signaling, protecting proteins from oxidative damage, and apoptosis (Fujii & Ikeda 2002). Prdx2 down-regulation could contribute to the increase incidence of apoptosis that is observed in cultured embryos, when compared with those that develop in vivo (Brison & Schultz 1997).

In summary, results presented here highlight the ability of microarray analysis to provide insights into biological processes, in this case preimplantation development. Genes with expression that is affected by culture provide candidates for a standard hypothesis-driven approach to study their function.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
References
 
This research was supported by a grant from the NIH (HD 44575 to R M S) as part of the NICHD Cooperative Program on Female Health and Egg Quality. P R was supported by a training grant from the NIH (T32-HD40135) and a grant from the American Society of Reproductive Medicine. P R thanks John Tobias of the Penn Biomedical Informatics Facility for basic GeneSpring training and helpful discussions regarding statistical analyses of microarray data, Martin Anger for the pEGFPN2 RNA, and Fanyi Zeng for some material used for analysis of gene expression. P R and R M S thank Daniel Dumesic, John Eppig, Tom Fleming, Dick Tasca and Jeremy Thompson for critically reading the manuscript and for their comments.


   Footnotes
 
Received 30 April 2004
First decision 8 June 2004
Accepted 19 June 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 

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