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Call For Papers: 2nd International Symposium on Animal Functional Genomics

Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients

¹ Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Bonn, Germany
² Centre de Recherche en Biologie de la Reproduction, Department of Animal Sciences, Laval University, Pav. Comtois, Sainte-Foy, Quebec, Canada

ABSTRACT

The purpose of this work is to address the relationship between transcriptional profile of embryos and the pregnancy success based on gene expression analysis of blastocyst biopsies taken prior to transfer to recipients. Biopsies (30–40% of the intact embryo) were taken from in vitro-produced day 7 blastocysts (n = 118), and 60–70% were transferred to recipients after reexpansion. Based on the success of pregnancy, biopsies were pooled in three groups (each 10 biopsies) namely: those resulting in no pregnancy (G1), resorbed embryos (G2), and those resulting in calf delivery (G3). Gene expression analysis of these groups was performed using home-made bovine preimplantation-specific cDNA array (219 clones) and BlueChip (with 2,000 clones). Microarray data analysis results revealed a total of 52 and 58 genes were differentially regulated during comparison between G1 vs. G3 and G2 vs. G3. Biopsies resulted in calf delivery were enriched with genes necessary for implantation (COX2 and CDX2), carbohydrate metabolism (ALOX15), growth factor (BMP15), signal transduction (PLAU), and placenta-specific 8 (PLAC8). Biopsies from embryos resulting in resorption are enriched with transcripts involved protein phosphorylation (KRT8), plasma membrane (OCLN), and glucose metabolism (PGK1 and AKR1B1). Biopsies from embryos resulting in no pregnancy are enriched with transcripts involved inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes which may play an important role in determining the fate of the embryo after transfer.

blastocyst; preimplantation; embryo loss; microarray

EARLY EMBRYONIC MORTALITY is a recognized cause of reproductive failure in cattle leading to the loss of a large number of potential calves, retarded genetic progress, and significant loss of money and time in rebreeding cows (38, 49). Mortality results either because of intrinsic defects within the embryo, an inadequate maternal environment, asynchrony between embryo and mother, or failure of the mother to respond appropriately to embryonic signals (32). With the advent of reproductive technologies, this developmental failure becomes more evident. In in vitro-produced (IVP) embryos most of this mortality is sustained within the first 2–3 wk after fertilization (18, 20, 23, 49, 69). The explanation for this high rate of developmental failure with respect to the defect within the embryo (intrinsic errors) remains unclear. However, the extent and regulation of altered gene expression during preimplantation development are likely to be critical for later development of the conceptus (7). In some cases, even a defect in a single but a critical gene is sufficient to cause implantation failure (14). This is in agreement with what shown from in vitro production of bovine embryos correlated with significant up- or downregulation, de novo induction or silencing of genes critical for undisturbed fetal and neonatal development (5, 16, 79). Studies on gene expression during the critical period of embryo development would yield insights into the molecular pathways controlling developmental events that may be compromised in early embryonic mortality (38). Various evidences are already available reporting differences in developmental competence coupled with altered gene expression in embryos derived from various cultured conditions (17, 42, 58, 79). ¹

Various studies in both mice and bovine have shown that the production of embryos under specific culture environments resulted in not only altered gene expression of transcripts related to metabolic and growth but also altered conceptus and fetal development following transfer (37, 40). Despite the fact that data on transcriptional analysis of transferable blastocysts of various origins have been accumulated (17, 58, 79), so far no direct connection of gene expression and developmental competence has been established. For this, what is needed was a well-established biopsy technique to obtain cells from embryos before transfer without any lethal effect on the embryo during further development. Therefore, the main objective of this work is to analyze the gene expression profile of bovine blastocyst biopsies in relation to the outcome of pregnancy after transfer to recipients thereby to identify direct candidate genes related to embryo developmental competence.

MATERIALS AND METHODS

Bovine ovaries were collected from slaughtered cows at a local abattoir and transported to the laboratory within 2–4 h in a thermos flask containing physiological solution (0.9% NaCl supplemented with 50 µl/100 ml Streptocombin; G. Streuli & Co, Aulendorf, Switzerland) maintained at a temperature of 30–35°C. Subsequently, cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm-diameter follicles using a 10-ml syringe loaded with an 18-gauge needle. COCs with multiple cumulus layers and evenly granulated cytoplasm were selected under microscope, washed three times in prewarmed maturation medium [modified Parker medium supplemented with 12% heat-inactivated estrous cow serum and 10 µg/ml FSH], and finally transferred to wells containing 400 µl of maturation medium.

COCs were selected in the above procedures and maintained in groups of 50 in maturation medium at 39°C in a humidified atmosphere containing 5% CO₂ in air. After 22 h of incubation, mature oocytes were transferred to 400 µl of fertilization medium [Fert-TALP (52) supplemented with 6 mg/ml bovine serum albumin, 2.2 mg/ml sodium pyruvate, and 1 mg/ml heparin to which 10 µl of penicillamine, Hypotaurin, and epinephrine was added to stimulate sperm motility]. For in vitro fertilization, spermatozoa were prepared as mentioned earlier (54). In brief, three straws of frozen-thawed semen from a fertile bull were used, and motile spermatozoa were separated by swim-up procedure in a capacitation medium (52). Finally, the spermatozoa were resuspended and added to a fertilization medium containing a group of 50 oocytes. At the end of co-incubation, the presumed zygotes were mechanically denuded by repeated pipetting to remove the attached sperm and cumulus cells and washed three times in the culture medium [CR-1aa (60) supplemented with 10% estrus cow serum, 10 µl/ml of basal medium Eagle essential amino acid solution, and minimum essential medium nonessential amino acid solution]. The cumulus-free zygotes were then transferred to a 400-µl culture medium in a group of 50 covered with mineral oil and incubated at 39°C in a humidified atmosphere with 5% CO₂ in air until day 7 blastocyst stages.

Recovery of embryo biopsies.
Morphologically good-quality day 7 blastocysts were used for biopsies, which was performed with a Beaver microplade (Minitüb, Tiefenbach, Germany) fixed to a micromanipulator under inverse microscope (Leica Camera, Solms, Germany). In this procedure, 30–40% of blastocysts containing both inner cell mass (ICM) and trophectoderm (TE) cells were be taken as biopsy, and the remaining 60–70% was cultured in vitro for 2 h to allow the reexpansion before transfer to recipients.

Embryo transfer procedure.
All experimental animals were handled according to the Animal Protection Law of Germany. Healthy 2-yr-old Simmental heifers of our experimental herd were estrus synchronized by administration of PGF₂ (2 ml Estrumate; Fa. Essex, Germany) followed by a second administration 11 days later. Reexpanded demiblastocysts were transferred by nonsurgical standard procedures into the uterus at day 7 of the estrus cycle, placed to the side of the uterus where the corpus luteum was located. All recipients were monitored for coming back to estrus at day 21. Those cows returning to estrus at day 21 were considered nonpregnant (group 1; G1). Pregnancies were checked at days 28 and 42 by ultrasonography (Pie Medical, 5 MHz) and by rectal palpation at day 56. Positive pregnancies at day 28 that were lost by day 56 were categorized as resorbed (group 2; G2), pregnancies that were lost after day 56 were judged as aborted. Pregnant recipients were allowed to go to term (group 3; G3).

RNA extraction and amplification.
Messenger RNA (mRNA) was isolated from triplicate pools (each with 10 biopsies) from each of the phenotype groups using Dynabead oligo (dT)₂₅ (Dynal Biotech, Oslo, Norway) following the manufacturer's instructions. First- and second-strand cDNA synthesis were carried out as stated before by Brambrink (6). In brief, 1 µl of T7 oligo d(T)21 primer [TCTAGTCGACGGCCAGTGAATTGTAATACGACTCACTATAGG-GCG(T)₂₁] was added to the mRNA and co-incubated for 5 min at 70°C and placed immediately on ice for 3 min. Eight microliters of reverse transcription reaction mix containing 5x first-strand buffer (50 mM Tris·HCl, pH 8.3; 75 mM KCl; 3 mM MgCl₂), 0.3 mM dNTP, 0.1 mM DTT, 10 U RNase inhibitor (Promega, Madison, WI), and 200 U Superscript II RT enzyme (Invitrogen, Karlsruhe, Germany) were added to the primer and mRNA mix. This reaction was carried at 42°C for 90 min followed by 75°C for 15 min to terminate the reaction. Second-strand synthesis and global PCR amplification were carried out using degenerated oligonucleotides primer (DOP) PCR master kit (Roche Diagnosis, Mannheim, Germany). The following were added and properly mixed: 20 µl first-strand cDNA product, 40 µl 2x DOP PCR master mix, and 1.0 µl DOP primer, to a final concentration of 1.0 µl of 10 µM T7 oligo (dT)₂₁ primer and 16.0 µl of water. This PCR reaction was heated at 95°C for 5 min to denature the sample and activate the polymerase, followed by a cycle of denaturation at 95°C for 30 s and annealing at 30°C for 90 s. Unspecific primer annealing is achieved up to this step through application of relatively low annealing temperature. Subsequently the temperature was increased at the rate of 0.2°C/s until it reaches 72°C and incubated for 3 min at this temperature. To this end, second-strand synthesis was completed, and the global PCR amplification continued for the rest of 15 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 3 min, and final extension at 72°C for 7 min. This cDNA was purified and used for in vitro transcription using AmpliScribe T7 transcription kit (Epicentre Technologies, Oldendorf, Germany) according to the manufacturer's instruction. The amplified RNA (aRNA) was purified using RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations. Finally, the aRNA was eluted in 30 µl of RNase-free water from which 8 µl were taken to estimate the yield and purity of aRNA by UV absorbance reading A260/280 using Ultrospec 2100 pro UV/Visible Spectrophotometer (Amersham Bioscience, Freiburg, Germany).

Probe preparation and array fabrication.
In this study, two different cDNA arrays have been used, namely: BlueChip cDNA array (67) and bovine preimplantation-specific custom array (46). The latter was constructed using 219 probes originated from bovine preimplantation embryo cDNA library construction (55), differential display analysis (72), and suppressive subtractive hybridization (SSH) techniques as mentioned earlier (54). In addition to these, some specific genes that are known to be expressed during embryo preimplantation development stage have also been amplified with gene-specific primers. Sequence-specific products were cloned into DH2 Escherichia coli-competent cells for probe preparation as mentioned in detail by Mamo (46). Once product identity is confirmed by sequencing, subsequent amplification of M13 products was performed by standard PCR using amine-modified M13 forward (5'-[AC12]TGTAAAACGACGACGGCCAGT-3') and M13 reverse (5'-[AC12]CAGGAAACAGCTATGACC-3') primers. The PCR products were purified with a PCR purification kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Finally, products were eluted with 30 µl of Millipore water, and the concentration of the DNA samples was measured by taking an optical density reading at A260/280 using Ultrospec 2100 pro UV/Visible Spectrophotometer (Amersham Bioscience), and the products were then stored at –20°C until spotting.

Array design and microarray printing.
Spotting the array and postspotting treatments of the slides were all done at the Resource Centre and Primary Database, Germany. The arrays were prepared by printing purified M13 products containing the target product onto GAPSII aminosilane-coated glass microscope slides (Corning, NY). Owing to the small number of probes and availability of enough space on the slides, all probes and controls were printed in two independent subarrays on one slide.

cDNA target production and labeling.
aRNA from three independent pools was converted into aminoallyl-labeled cDNA using Amersham postlabeling kit (Amersham Bioscience) according to the manufacturer's instruction. In brief, 3 µg of each contrasting aRNA were used for labeling, and cDNA was synthesized with 1.5 µl of random nonamer and 1.5 µl of anchord oligo (dT) primers. The aRNA and primers were co-incubated at 70°C for 5 min followed by 10 min of incubation at room temperature. Then, 10 µl of reaction mix [containing 4 µl of 5x first-strand buffer, 2 µl 0.1 M DTT, 1.5 µl dNTP mix, 1.5 µl aminoallyl dUTP, and 1 µl CyScript reverse transcriptase] was added to the reaction and incubated at 42°C for 90 min. At the end of this reaction, 2 µl of 2.5 M NaOH to hydrolyze the mRNA template were added and incubated at 37°C for 15 min. Finally, products were purified with a CyScribe GFX purification kit (Amersham Biosciences) after the addition of 10 µl of HEPES (2 M). The purified aminoallyl-labeled cDNA was then eluted in 60 µl of 0.1 M sodium bicarbonate.

Dye coupling was performed by co-incubation of the aminoallyl-labeled cDNA with the corresponding dye (Cy3 or Cy5) for 1.5 h at room temperature in the dark. At the end of incubation, nonreacting dyes were quenched by adding 15 µl of 4 M hydroxylamine solution (Sigma) and incubated further for 15 min at room temperature and in darkness. To avoid variation on dye coupling, aRNA samples from the same group were labeled with either Cy3 or Cy5 for dye-swap hybridization. The reaction was then purified with the CyScribe GFX Purification kit (Amersham Biosciences) according to the manufacturer's instruction and eluted in a prewarmed (42°C) formamide-based hybridization buffer (15 µl hybridization buffer, 30 µl 100% formamide, and 15 µl DEPC water). To this 2.5 µl of yeast tRNA 4 mg/ml (Invitrogen) and 2.5 µl of Cot-human DNA 1 mg/ml (Invitrogen) were added to avoid nonspecific hybridization.

Array hybridization.
Prehybridization of the slides was performed by placing the arrayed slides into a Corning GAPS II slide container and incubated in 55°C heated prehybridization buffer containing 5x SSC, 0.1% SDS (Sigma), and 1% bovine serum albumin (BSA; Roche Diagnostic, Basel, Switzerland) for 20 min. Following prehybridization, slides were rinsed briefly in boiling water to denature probes and wash unbound DNA from the slide surfaces, followed by immediate immersion in water at room temperature and then isopropanol. The slides were then dried by centrifugation at 2,000 rpm for 2 min.

Slides covered with glass coverslips (ROTH, Karlsruhe, Germany) were put in the hybridization cassette (TeleChem International) and fixed well. The cassette was then placed in the hybridization chamber (GFL, Dülmen, Germany) prewarmed to 42°C and incubated for 16–20 h. Hybridization and posthybridization washes were carried out according to Hedge et al. (33). Accordingly, slides were washed twice with 2x SSC/0.1% SDS buffer for 5 min at 42°C then once 1x SSC, 0.2x SSC, and 0.1x SSC for 5 min for each at room temperature. Finally these slides were rinsed in double distilled water and isopropanol for 1 min in each. Slides were then shortly centrifuged to dry and scanned immediately.

Image capture and data analysis.
Slides were scanned using a GenePix 4000B scanner (Axon Instruments, Foster City, CA). Features were analyzed with GenePix Pro Version 4.0 software (Axon Instruments). Array data analysis was performed with the significant analysis for microarray (SAM) software program developed at Stanford University (http//www-stst.stanford.edu/tips/SAM). First, a loess normalization of the data was performed with GPROCESSOR freeware (http://bioinformatics.med.yale.edu/group/) to eliminate uninformative data. A mean of log₂ ratio of the biopsies from various groups (no pregnancy/calf delivery and resorbed/calf delivery) using normalized data was then calculated for the replicates to obtain one value per clone. Finally ratios were submitted to SAM analysis. The above experiments were then repeated with reverse-labeled cDNA samples. Heatmaps were generated using HeatMap Builder (available at http://mozart.stanford.edu/heatmap.htm). Heatmap reflects normalized gene expression ratios and is organized with individual hybridization for each experiment. Hierarchical clustering was carried out for up- and downregulated genes using the clustering program (Cluster & TreeView) written by Michael Eisen at the Eisen lab (http://rana.lbl.gov/index.htm). Average linkage clustering algorithm was employed for hierarchical clustering of only differentially expressed genes.

Quantitative real-time PCR.
Real-time PCR reactions were conducted in an ABI Prism 7000 SDS instrument (Applied Biosystems) and SYBR green as a double-strand DNA-specific fluorescent dye. Quantitative analyses of biopsies cDNA were performed compared with the bovine GAPDH gene (endogenous control) and were run in separate wells. Standard curves were generated for both target and internal control genes using serial dilution of plasmid DNA (10¹–10⁸ molecules). PCRs were performed in a 20-µl reaction volume containing 9 µl 2.5x RealMasterMix/20x SYBR Solution, optimal levels of forward and reverse primers, and 2 µl cDNA. Each PCR was run for a particular biopsy group in duplicate to control the reproducibility of quantitative results. A universal thermal cycling parameter (10 s at 50°C, 10 min at 95°C, 45 cycles of 15 s at 95°C, and 60 s at 60°C) was used for the quantification of each gene. After the end of the last cycle, a dissociation curve was generated by starting the fluorescence acquisition at 60°C and taking measurements every 7 s until the temperature reached 95°C. Results were reported as the relative fold change to the calibrator cDNA after normalization of the transcript amount to the endogenous control (Fig. 1).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Quantitative real-time PCR confirmation of selected transcripts between biopsies from blastocysts resulting in calf delivery vs. no pregnancy (A) and calf delivery vs. resorption group (B) and those resulting in calf delivery vs. resorption or no pregnancy (C).

RESULTS

Embryo production, biopsy, and transfer.
In the present experiment, a total of 138 IVP day 7 blastocysts were biopsied; 85% (118/138) survived and were transferred to recipients. During the production of embryos for biopsy and transfer, the IVP procedure had an average efficiency of 80.1 ± 4.8% cleavage rate and 32.4 ± 2.5% blastocyst rate at day 7. Of the total recipients who received reexpanded biopsied blastocysts, 42% (49/118) returned to estrus on day 21 after transfer and were designated as those that were not pregnant at all. Ultrasound-guided pregnancy tests were carried out at days 25, 32, 40, and 50 for those recipients that did not return to estrus at day 21 posttransfer. A total of 30 recipients (25%) were tested to be positive for pregnancy at days 25 or 32 but negative at day 40 or 50, which we determined to be embryo resorption. Only 3% (3/118) of the recipients showed abortion, while 30% (36/118) of the recipients resulted in calf delivery.

Array characterization.
In the present study we used two different cDNA arrays, namely the BlueChip cDNA array, which was kindly provided by the Department of Animal Science, Laval University, Canada, and the bovine preimplantation embryo-specific array, which was prepared in our laboratory. The former array whose potential and limitations are validated by Ref. 67 contained 4,928 spots in two subarrays. Each subarray is composed of 2,304 randomly selected clones obtained from four different SSH: first SSH, GV oocytes subtracted from somatic tissues; second SSH, GV oocytes subtracted from day 8 blastocysts, third SSH, day 8 blastocysts subtracted from GV oocytes; and fourth SSH, day 8 blastocysts subtracted from somatic tissues. All the clones were spotted two times in each array in a total of four replicates. Eleven more samples, namely vide (32 spots), alien1 (8 spots), alien2 (8 spots), GFP (4 spots), GFP1 (4 spots), GFP^1/2 (4 spots), GFP^1/4 (4 spots), GFP1/8 (4 spots), GFP1/16 (4 spots), and H2O/DMSO (50 spots), were spotted to be used as negative control on the array. Housekeeping genes, including tubulin (8 spots), ubiquitin (8 spots), ß-actin (6 spots), and actin (8 spots), were also added as positive control.

The bovine preimplantation-specific custom cDNA array was constructed being enriched with bovine preimplantation stage-specific clones. The majority of the clones were generated from different developmental stages of bovine preimplantation embryos by stage-specific cDNA library construction (55), differential display (72), and SSH techniques (54). In addition to these, some specific genes that are known to be expressed during embryo preimplantation development stage were also amplified with gene-specific primers and included in the array. A total of 219 genes and EST probes were used. These cDNA clones contain on average 215-base pair-long inserts, and all were sequenced from both 3'- and 5'-ends to confirm size and gene identity and to avoid misrepresentation before being purified and spotted on the array. The array has two meta-rows, each with 16 blocks. Each block is 11 x 6, representing 3,112 spots (or features) to avoid possible missing data and confirm reproducibility in which each gene has been represented by three spots in the block. ß-Actin (8 spots), H2AFZ (16 spots), and GAPDH (8 spots) were printed as positive hybridization controls, where 3x SSC (32 spots) and blank spots were used as a negative hybridization control.

Differential gene expression profiles.
During each experiment, a series of six hybridization experiments (three biological replicates with dye swap) were conducted to minimize the false positive expression changes and to identify genes truly differentially expressed (q 0.10) between biopsies derived from blastocysts resulting in no pregnancy, resorption, or calf delivery. All differentially expressed genes were (ontologically) classified on the basis of their functions according to criteria of gene ontology consortium classifications (http://www.geneontology.org). The resulting data were supplemented with additional information from Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), and the ontological classification results are indicated in Figs. 2 and 3. The heatmap (Fig. 4, A and B) represents an overall view of the expression levels of genes on BlueChip and bovine preimplantation-specific custom arrays during comparison between the two experiments (no pregnancy vs. calf delivery and resorption vs. calf delivery).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Ontology classification for differentially expressed transcripts between biopsies derived from blastocysts resulted in no pregnancy and calf delivery. The known genes were classified functionally based on the Gene Ontology Consortium classification (http://www.geneontology.org).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Ontology classification for differentially expressed transcripts between biopsies derived from blastocysts resulted in resorption and calf delivery. The known genes were classified functionally based on the Gene Ontology Consortium classification (http://www.geneontology.org).

View larger version (90K): [in this window] [in a new window]   Fig. 4. The heatmap shows the normalized gene expression ratios and is organized with individual hybridization for the triplicate hybridizations for each experiments using BlueChip cDNA array (A) and duplicate hybridizations using bovine preimplantation-specific custom cDNA array (B) arranged along the x-axis, with normalized expression ratios depicted by color intensity such that highest expression corresponds to the bright red and the lowest expression corresponds to bright green.

View larger version (42K): [in this window] [in a new window]   Fig. 5. Hierarchical clustering for the differentially expressed genes between biopsies derived from blastocysts resulting in no pregnancy (G1) and calf delivery (G3).The columns represent the replicates. The rows represent 52 genes found to be differentially regulated between G1 and G3.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Results of hierarchical clustering for the differentially expressed genes between biopsies derived from blastocysts resulting in resorption (G2) and calf delivery (G3) identified by microarray analysis. The columns represent the replicates. The rows represent 58 genes found to be differentially regulated between the 2 groups of biopsies.

Immunohistological localization of bovine MSX1 protein.
The bovine MSX1 gene, which was found to be differentially regulated during transcriptional analysis of biopsies derived from blastocysts, resulting in no pregnancy and calf delivery. To further characterize the protein expression pattern of this gene, immunohistochemistry was conducted to localize the protein throughout the preimplantation stage embryos. The protein product was seen to be found dispersed in the cytoplasm in immature and matured oocytes and early zygote stages. The fluorescence intensity was clearly reduced at the matured oocyte stage. Starting from late zygote stage until the morula stage, the protein tends to localize around the nucleus. At the blastocyst stage, more intensive staining was detected in the TE cells compared with the inner cell mass cells (Fig. 7).

View larger version (94K): [in this window] [in a new window]   Fig. 7. Subcellular localization of bovine MSX1 protein in bovine oocytes and in preimplantation embryos [immature oocytes (A), matured oocytes (B), zygote (C), 2-cell (D), 4-cell (E), 8-cell (F), 16-cell (G), morula (H), and blastocyst (I) stages]. Control (J) was stained without primary anti-MSX1 antibody. Red arrows indicate concentrated localization of MSX1 protein. The figure is representative of 8–10 oocytes or embryos stained from each developmental stage. Nuclei are stained by propidium iodide (red). Scale bars represent 20 = µm.

The major reproductive wastage in farm animals is early embryo loss, i.e., the anomalous development of embryos and/or an aberration of placentation (15). Precise knowledge of gene expression profile during preimplantation will enable investigators to gain insight into the molecular mechanisms controlling early embryonic survival and contribute to the effort aimed at improve the ever-declining fertility in cattle (22, 40, 42, 79). In the present study we compared the transcriptional profile of blastocyst biopsies related to the fate of the embryo after transfer resulting in no pregnancy, resorption, or calf delivery. To our knowledge this is the first study of this kind in bovine species that directly correlates the embryo gene expression with the outcome of pregnancy. The study was performed using biopsies after blastocyst splitting, which provides a unique possibility to eliminate genetic variability as a factor potentially affecting the results of gene expression analysis (39). The results showed 46 genes were upregulated in blastocysts resulting in no pregnancy, whereas six genes were downregulated when compared with calf delivery (G1 vs. G3). In the second experiment (G2 vs. G3), 38 and 20 genes were up- and downregulated, respectively, in blastocysts resulting in resorption when compared with blastocysts resulting in calf delivery.

Genes highly abundant in blastocysts resulting in no pregnancy (G1).
TNF- (inflammatory cytokine), which is known to be involved in fetal resorption or embryo loss (9, 10, 27, 64, 65), is found to be upregulated in blastocyst biopsy resulting in no pregnancy. The pathophysiology of TNF-mediated fetal loss remains unclear. Elevated TNF restricts ICM proliferation in blastocysts and changes the ratio of mononucleated to multinucleated trophoblast cells (77). Moreover, TNF- is reported to lead to cell apoptosis through cell-autonomous defects, which is one of the intraembryonic causes of death in the preimplantation period in the mouse, which prevents it from carrying out certain specific housekeeping functions (14, 44). These findings suggest a mechanism by which increased expression of TNF during trophoblast differentiation may be detrimental to pregnancy. It is important to recognize that low levels of TNF may be required for normal fetal and placental development, while elevated levels that occur in pathological settings, e.g., tissue damage or infection, may contribute to fetal effects. Further studies are needed to clarify the compartmentalization and role of this cytokine in both normal and abnormal gestation (66). The ability to resist TNF depends on the de novo induction of specific gene products that can be induced by TNF (28). In the present study, some genes that are related to TNF have been also found to be differentially regulated including elongation factor 1 a1 (EEF1A1), which is the first accessory protein involved in the movement of aminoacyl RNA to the ribosome (71). EEF1A1 has been shown to be upregulated in tumor cells (28, 70). Our result may provide an explanation for the selective antiapoptotic advantage of the elevated levels of EEF1A1 observed in tumors (29, 63).

The CD9 gene, which is expressed on blastocysts and endometrium epithelial cells in human and bovine (80), was newly found to play a role in inhibiting embryo implantation (41). This is in agreement with our result that showed the upregulation of CD9 in biopsies derived from blastocysts resulting in no pregnancy. Given the fact that striking similarities between embryogenesis and biology of cancer cells, especially in the process of invasion, CD9 might be involved in embryo invasive behaviors (41).

Bovine MSX1, which was upregulated in embryos involved in the no-pregnancy group by 2.4-fold compared with those leading to calf delivery, was found to be expressed at sites where cellular proliferation and programmed cell death (PCD) occur, suggesting its participation in PCD (47, 74). Overexpression of MSX1 suppressed cell growth and cell cycle progression in human ovarian cancer cell line as found by Park et al. (51). Our results suggest that altered expression of MSX1 maybe correlated with embryo death before implantation in embryos that ended up as no pregnancy. Further investigation will be necessary to identify the regulatory mechanism of this gene in preimplantation embryo. Immunohistological localization of the MSX1 protein indicated the activity of the protein as early as immature oocyte stage. The possible role of this gene as a transcription factor gene in bovine embryogenesis needs further investigation, especially to identify genes suppressed or activated by the activity of MSX1.

Pituitary tumor transforming gene (PTTG1) is a novel oncogene expressed abundantly in most tumors, including those of the pituitary (81), ovary, and testis (56). Overexpression of PTTG1 induces cellular transformation and promotes tumor formation in nude mice and stimulates expression of the Bax gene, which induces apoptosis in a human embryonic kidney cell line (31). In accordance with this, PTTG1 was found to be upregulated in embryos resulted in no pregnancy by nearly fourfold change compared with those resulted in calf delivery.

Genes upregulated in biopsies of blastocysts resulted in no pregnancy (G1) and resorption (G2) vs. calf delivery (G3).
Similarly, in the present study, the polyubiquitin gene, which has been shown to be upregulated in tumor cells (43), was found to be upregulated in biopsies resulting in no pregnancy and resorption compared with those derived from blastocysts resulting in calf delivery. During development, a large number of cells die in a predicted spatial and temporal pattern known as PCD, or apoptosis. This process is crucial for differentiation and involves programmed regulation of gene expression. One of the first genes known to be involved in PCD is the polyubiquitin gene, which is upregulated during the metamorphosis of the hawk moth (34); in the same study it has been shown that the ubiquitin system was implicated in the immune response, development, and PCD. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation.

The aldose reductase gene (AKR1B1), which is known for its 20-hydroxysteriod dehydrogenase activity, was found to be upregulated in biopsies derived from blastocysts resulting in no pregnancy and resorption. The aldose reductase gene was found to be strongly expressed in the endometrium at the time of luteolysis in bovine (45), suggesting its potential involvement in pregnancy failure. The enzyme of this gene is known to have two different activities, namely, metabolizing progesterone, which is found to be important to implantation (35), and synthesizing PGF2 and, subsequently, terminating pregnancy. Aldose reductase is also known as the cause for cell apoptosis in some types of cells, like cardiomyocytes, as it is induced by sorbitol as a response to hyperosmotic pressure (25). On the contrary, high glucose in culture media could lead to upregulation of aldose reductase and subsequent accumulation of sorbitol in cytoplasm and activate apoptotic pathways (78). From these studies and also from ours we can conclude that aldose reductase may determine the fate of the embryo through its involvement in apoptotic pathways.

Phosphoglycerate kinase (PGK1), a key enzyme in glycolysis, is encoded from the X chromosome and is found to be upregulated in G1 and G2 biopsies compared with the G3 group. High-level glucose concentration, which triggers apoptosis during preimplantation murine embryo (57), has led to the overexpression of this gene (48). Interestingly, protease serine 23 gene, which is strongly upregulated in the G1 and G2 groups compared with the G3 group, is known to encode a member of the trypsin family of serine proteases. However, its specific function is unknown.

Genes upregulated in biopsies from blastocysts resulted in calf delivery (G3) compared with resorption (G2).
PGs, which are involved in the process of blastocyst implantation, are known to be produced by both the endometrium and blastocyst, but the former is thought to be the major source of the prostaglandins involved in implantation (68). PGs produced by embryos may be involved in other functions during the preimplantation period such as modulation of the endometrial implantation site (75). This may support our findings in which higher COX2 was detected in biopsies derived from blastocysts resulting in successful pregnancy and calf delivery.

The study by Charpigny et al. (11) showed that COX2 protein was localized in trophoblast cells, not in the ICM, which did not express COX2 in ovine embryo. This may suggest that this gene is necessary for the elongation process that is the result of an intense proliferation of trophoblastic cells and subsequent implantation. Moreover, higher expression of COX2 during the time of the implantation window suggested an important role for the PGs released by the embryo in mediating interactions with the uterus (11, 76).

The homeobox-containing gene family plays a pivotal role in regulating, patterning, and axial morphogenesis in the developing embryo. The caudal-related homeobox protein CDX2 is a transcriptional regulator essential for trophoblast lineage, functioning as early as implantation. The CDX2 gene is the earliest trophoblast-specific transcription factor reported to date (59, 73). An earlier gene targeting approach has demonstrated that CDX2 null embryos fail to implant, suggestive of a major defect in TE development (12, 61). The same authors showed that the implantation failure was due to loss of TE epithelial integrity and/or increased incidence of apoptosis of TE. Therefore, CDX2 is one of the genes crucial for placental development, by which its aberrations in embryo can result in implantation or placental defect as reported by Hall et al. (30).

Ornithine decarboxylase (ODC1) enzyme, which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis (53). In that study it has been shown that embryos that lack ODC1 develop normally to the blastocyst stage and implant but die shortly thereafter, before the onset of gastrulation. Scheduled administration of difluoromethylornithine DFMO (a potent inhibitor of ODC1) during pregnancy in mice induces resorption of embryos when introduced at gestational days 7 and 8 (24). This means that eliminating ODC1 function by gene targeting compromises early mouse embryonic development (53). Similarly in the present study, ODC1 gene product was found to be abundant at lower levels in biopsies from blastocysts from resorption compared with those from calf delivery.

The plasminogen activators (PAs) are serine proteases, which convert the inactive plasminogen to the potent protease plasmin. Plasminogen and its activators and inhibitors participate in implantation process in human and rat (1, 36). The urokinase type of plasminogen activators (PLAU) was found to be upregulated in G3 by more than fourfold compared with G2 biopsies. Similar studies in mouse and rat (1, 62) have shown the involvement and importance in implantation particularly in the invasion process, where the reduction of this gene is associated with implantation failure in mouse (2).

The PLAC8 gene, which is known as an invasion-specific gene, was found to be upregulated in blastocysts resulting in calf delivery by >26-fold compared with those resulting in resorption. Similar studies in bovine have reported that PLAC8 is highly expressed in endometrium of pregnant cow compared to nonpregnant ones (25, 39), suggesting its potential role in placenta development and fetus maternal interface.

Genes upregulated in biopsies derived from blastocysts resulting in calf delivery (G3) compared with no pregnancy (G1) and resorption (G2).
Thioredoxin (TXN) is a ubiquitous protein disulfide reductase and known to be involved in the implantation of mouse embryos (4). Moreover, it was found to be a response to oxidative stress to protect the in vitro embryo development in bovine and mouse (4, 50). While TXN is believed to be early pregnancy factor (EPF) (8), as it is expressed in the preimplantation embryo (13), others suggest that TXN is part of the components that are required for the expression of EPF (19). These facts, together with our findings, enable us to postulate that this gene may play an important role in embryo survival and continuation of pregnancy to term.

A previous study in our laboratory (21) that employed qPCR to profile the HNRPA1 gene in bovine preimplantation embryos showed the highest expression at the two-cell stage and further downregulation until the morula stage with a slight increase at the blastocyst stage.

In conclusion, we have identified genes with recognized and potential roles in pregnancy and genes whose functions still need to be defined in this event. Several of these genes have been implicated in previous reports as being associated with embryo loss or survival in blastocysts during preimplantation period. Factors discussed in this study may be explained individually, but the simultaneous expression and interactions of these molecules may be important for elucidating how embryo death occurred and subsequent embryo loss. The identification of unique genetic markers for the onset of pregnancy signifies that genome-wide analysis, coupled with functional assays, is a promising approach to resolve the molecular pathways required for successful pregnancy. Normalizing the expression patterns of these genes may improve full-term survivability of IVP cattle embryos.

The authors are grateful to Dr. Andreas Waha (Institute of Neuropathology, University of Bonn). The experiment was conducted in the research station of Frankenforst at the University of Bonn.

FOOTNOTES

Address for reprint requests and other correspondence: D. Tesfaye, Inst. of Animal Science, Univ. of Bonn, Endenicher Allee 15, Bonn, Germany (e-mail: tesfaye{at}itz.uni-bonn.de).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

¹ The 2nd International Symposium on Animal Functional Genomics was held May 16–19, 2006 at Michigan State University in East Lansing, MI, and was organized by Jeanne Burton of Michigan State University and Guilherme J. M. Rosa of University of Wisconsin-Madison (see meeting report by Drs. Burton and Rosa, Physiol Genomics 28: 1-4, 2006).

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