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1 Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin
2 Department of Biotechnology, Institute for Animal Science, Neustadt, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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oocytes; microarrays; cross-species hybridization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Bovine oocytes and blastocysts contain 
2.4 ng and 5 ng of total RNA, respectively (9). Therefore, it is necessary to pool large amounts of biological material and/or amplify the RNA without significantly altering the relative levels of individual mRNAs. Protocols for effective and nonbiased amplification of RNA from single oocytes and embryos have been developed (1, 2, 4, 11), and transcript profiling of murine, porcine, and bovine oocytes and embryos has been reported (21, 27, 30, 52, 58, 64, 70). We previously demonstrated the feasibility and high reproducibility of cross-species analysis using a human cDNA microarray. Messenger RNAs derived from human and bovine fetal brains were compared, and the correlation coefficient of cross-hybridization between orthologous genes was 0.94 (3). We now report the first comparative transcriptome profile of bovine oocytes and blastocysts using a human 15,529 gene chip (the ENSEMBL chip) as an initial step to gain insight into gene expression and regulation during preimplantation development. These stages are representative of maternal and embryo-controlled expression, and specifically the blastocyst represents the first major differentiation event in development (1, 2, 4, 11). We have identified numerous genes expressed in the oocyte and blastocyst and have revealed their associated signaling and metabolic pathways. These results provide a basis for functional studies related to the importance of these genes and related pathways in preimplantation development.
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MATERIALS AND METHODS |
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In vitro production of bovine embryos.
Bovine embryos were produced as described recently with minor modifications (74). Briefly, ovaries from a local slaughterhouse were transported to the laboratory in Dulbecco's phosphate-buffered saline (PBS; no. D6650, Sigma, St. Louis, MO) at 25–30°C. Cumulus-oocyte complexes (COC) were isolated via slicing (29). Category I COC (with a homogeneous evenly granulated cytoplasm possessing at least 3 layers of compact cumulus cells) and category II COC (with <3 layers of cumulus cells or partially denuded but also with a homogeneous evenly granulated cytoplasm) were pooled in TCM-air [TCM 199 containing L-glutamine and 25 mM HEPES (Sigma) supplemented with 22 µg/ml pyruvate, 350 µg/ml NaHCO3, 50 µg/ml gentamicin, and 0.1% bovine serum albumin (BSA; fraction V, no. A9647, Sigma)].
For maturation in vitro, TCM 199 containing L-glutamine and 25 mM HEPES served as basic medium. One milliliter was supplemented with 22 µg of pyruvate, 2.2 µg of NaHCO3, and 50 µg of gentamicin. For oocyte maturation, this medium was supplemented with 10 IU of equine chorionic gonadotropin and 5 IU of human chorionic gonadotropin (Suigonan; Intervet, Tönisvorst, Germany) and 10% estrus cow serum (collected at day 1 of standing estrus). COC were divided into groups of 20–25, transferred into 100-µl maturation drops under silicone oil, and cultivated in a humidified atmosphere composed of 5% CO2 in air at 39°C for 24 h. Maturation was determined by extrusion of the first polar body and expansion of the cumulus cells (29).
After in vitro maturation, COC were rinsed in fertilization medium (Fert-TALP supplemented with 6 mg/ml BSA) and fertilized in Fert-TALP containing 10 µM hypotaurine (Sigma), 1 µM epinephrine (Sigma), and 0.1 IU/ml heparin (Serva) and 6 mg/ml BSA. Frozen semen from one bull with proven fertility in in vitro fertilization (IVF) was used. The final sperm concentration added per fertilization drop was 1 x 106 sperm/ml. Fertilization was initiated during a 19-h coincubation under the same temperature and gas conditions as described for maturation.
Presumptive zygotes were cultured in 30 µl of synthetic oviduct fluid medium supplemented with 6% BSA after complete removal of the adhering cumulus cells by repeated pipetting. Embryos were cultured in vitro in a mixture of 7% O2, 88% N2, and 5% CO2 (Air Products, Hattingen, Germany) in Modular incubator chambers (no. 615300, ICN Biomedicals, Aurora, OH). Blastocysts were harvested at day 7 of development (day 0 = IVF). After being washed three times in PBS containing 0.1% polyvinyl alcohol, oocytes and blastocysts were stored in pools of 10 for oocytes or pools of 3 for blastocysts at –80°C in a minimum volume (
5 µl) of medium until RNA extraction.
Sampling strategy and messenger RNA extraction.
Pooled oocyte and blastocyst samples were made up of 40 in vitro-matured oocytes and 12 in vitro-derived blastocysts, respectively. By including biological material from four different in vitro production runs (4 x 10 oocytes, 4 x 3 blastocysts) we could cover the biological variability and concomitantly obtain sufficient amounts of RNA (60 and 90 ng RNA, respectively) from both developmental stages for the amplification. Template mRNA from pooled samples was obtained with a Dynabead oligo(dT) extraction kit (Dynal Biotech). The procedure included the following steps. Twenty microliters of lysis/binding buffer was added, and the samples were processed according to the manufacturer's protocol with the following modification: beads were washed once with washing Buffer A and three times with washing Buffer B before the elution of mRNA was performed at 65°C in 11 µl of RNase-free water.
First-strand synthesis.
One microliter of 20 µM T7 poly(dT)21 primer (5'-TCTAGTCGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTTTT-3') was added to the eluted template mRNA, and the samples were denatured by a 2-min incubation at 70°C. Samples were immediately placed on ice for 1 min, and then 8 µl of master mix was added, consisting of 2 µl of 10x RT buffer (Applied Biosystems, Foster City, CA), 2 µl of 50 mM MgCl2 (Invitrogen), 2 µl of 10 mM dNTP solution (Amersham Biosciences), 1 µl (20 U) of RNasin (Applied Biosystems), and 1 µl (50 U) of Moloney murine leukemia virus (MMLV) reverse transcriptase (Applied Biosystems), followed by incubation at 42°C for 1 h.
Second-strand synthesis.
For second-strand synthesis, 50 µl of 2x DOP-PCR buffer (Roche Diagnostics, Penzberg, Germany), 1 µl of 40 µM degenerate oligonucleotide primer (5'-CCGACTCGAGNNNNNNATGTGG-3'; Roche Diagnostics), and 23.5 µl of water were added to each 20-µl reverse transcriptase sample. Samples were heated to 95°C for 5 min and then incubated at 30°C for 2 min. After the addition of 5 IU Taq DNA polymerase (Invitrogen), the samples were heated at a rate of 0.2°C/s to 72°C and incubated at 72°C for 3 min.
After second-strand synthesis was completed, 2.5 µl each of T7 poly(dT)21 primers and degenerate oligonucleotide primers were added. Twenty-five PCR cycles were performed as follows: cDNA was denatured at 94°C for 30 s, and primer annealing was performed at 60°C for 30 s, followed by an elongation step at 72°C for 4 min.
Purification and concentration.
PCR products were purified with the GFX-PCR DNA purification kit (Amersham Biosciences) according to the manufacturer's protocol and eluted in 75 µl of 0.1x Tris-EDTA (TE) buffer, and volumes were adjusted to 12 µl by vacuum centrifugation.
In vitro transcription.
Two microliters of buffer was added to the solution after centrifugation, followed by 2 µl each of ATP, CTP, GTP, UTP, DTT, and T7 RNA polymerase (Ampliscribe/Biozym). In vitro transcription was carried out at 42°C for 3 h, followed by DNase I digestion at 37°C for 15 min. Amplified RNA was purified with the RNeasy Mini kit (Qiagen, Hilden, Germany).
This procedure provides a means of amplifying minute amounts of RNA from single embryos with minimal bias; the correlation coefficient is 0.97 for autonomous amplifications of RNA from different embryos (11).
Direct labeling of RNA and hybridizations.
MIAME (Minimum Information About Microarray Experiments) guidelines were adhered to in our experimental design.
Four independent labeling (including dye swaps, Cy3 and Cy5) reactions and hybridizations per amplified RNA (aRNA) sample were carried out, always using 3 µg of aRNA for each biological sample. This corresponds to four technical replicates for oocytes and blastocysts, respectively. Full details of labeling and hybridization reactions, slide washing (0.2x SSC-0.1% SDS, 55°C), and scanning are described elsewhere (3, 4).
Global data analysis.
Data was normalized in two steps. The first step accounted for the dye effects caused by the difference in red/green fluorescence labeling for each single experiment. Here we used the LOWESS method (18). The parameters d, W, t, and f are needed to adjust the procedure. The order of the polynomial d and the number of iterations t were set to 1. The weighting function W was set as recommended; the fitting parameter f was set to 0.3. In the second step of the normalization each single-array experiment was normalized to a common median value, taking into account additional multiplicative noise. The procedure has been described previously (4).
The validity of gene expression of each individual signal was judged by comparison to a negative control sample. To verify whether a given gene was significantly expressed, we compared its signal to a signal distribution derived from negative controls. In our array design, we distributed 3,362 empty spot positions on the array. After quantification, a small, nonzero intensity was assigned to each empty spot reflecting the amount of background signal on the array. Since these positions were spread uniformly over the array, the distribution of signals reflects a global background distribution for the experiment and indicates whether cDNA signals were at or above the background level of expression. For each cDNA, we counted the relative proportion of empty positions on the array that were smaller than the observed intensity [background (BG) tag]. BG tags from replicated experiments for the same cDNA were averaged. Thus high values (close to 1) indicated that the cDNA was expressed in the tissue tested, whereas low values reflected noise. cDNAs were considered "expressed" when their average BG tag was above 0.9, a threshold consistent with the limit of visual detection of the spots.
For each cDNA, we performed statistical tests based on the replicate signals in each oocyte and blastocyst hybridized. Three standard tests were used in parallel: Student's t-test, the Welch test, and Wilcoxon's rank-sum test (37). For each stage, i.e., oocyte vs. blastocyst, we performed at least four independent replicate hybridizations.
Pathway analysis.
Array data were used to test whether entire groups of genes associated with specific pathways showed differential expression. Pathways were taken from the KEGG database (version KEGG2, 01/06/2005). The procedure has been described previously (4).
Independent verification of array data.
A description of the primer sequences, amplicon length, and annealing temperatures is given in Supplemental Table 1.1
End-point RT-PCR.
Conventional end-point PCR was performed with 25 ng of the cDNA for each gene or 50 fg of cDNA of the globin control. The 50-µl reaction mixture consisted of 5 µl of 10x PCR buffer (Invitrogen), 1.5 mM MgCl2, each dNTP at 200 µM, and each primer at 0.5 µM. Amplification was performed in an MJ Research PTC-200. Hot start was accomplished by adding 1 U of Taq DNA polymerase (Invitrogen) while the reaction mixture was maintained at 72°C. The PCR program consisted of denaturization at 97°C for 2 min; 2 min at 72°C, at which point Taq polymerase was added; and then cycles consisting of 95°C for denaturization, annealing at a temperature specific to each primer pair for 15 s, and elongation at 72°C for 15 s. The number of cycles performed was determined for each gene in preliminary experiments to give measurements that came within the linear phase of the amplification curve. PCR products were separated on a 2% agarose gel in Tris-borate-EDTA (TBE) buffer containing 0.2 µg/ml ethidium bromide.
Real-time RT-PCR.
Four biological replicates each consisting of pools of two oocytes or blastocysts were thawed in 40 µl of lysis buffer. Two picograms of rabbit globin mRNA (BRL, Gaithersburg, MD) was added to the solution to serve as an internal standard. Poly(A)+ RNA was isolated with a Dynabeads mRNA Direct Kit (Dynal, Oslo, Norway) and was eluted with 22 µl of distilled (d)H2O. Eleven microliters (equivalent to a single oocyte or blastocyst) was used as input for the reverse transcription reaction, and the other 11 µl was used as input for the negative control (to monitor genomic DNA contamination, without reverse transcriptase).
Reverse transcription was performed in a 20-µl volume consisting of 2 µl of 10x RT buffer (Invitrogen), 2 µl of 50 mM MgCl2 (Invitrogen), 2 µl of 10 mM dNTP solution (Amersham Biosciences), 1 µl (20 U) of RNasin (Applied Biosystems), 1 µl (50 U) of MMLV reverse transcriptase (Applied Biosystems), and 1 µl of hexamers (50 µM) (Applied Biosystems). The samples were incubated at 25°C for 10 min for primer annealing and then incubated at 42°C for 1 h. Finally, the samples were heated to 95°C for 5 min. Two-microliter aliquots of the RT reaction were used as template for real-time PCR, which was performed in 96-well optical reaction plates (Applied Biosystems). The PCR mix in each well included 10 µl of 2x Power SYBR Green PCR Master Mix (Applied Biosystems), 6.4 µl of dH2O, 0.8 µl each of the forward and reverse primers (5 µM), and 2 µl (0.1 oocyte/blastocyst equivalent) of cDNA in a final reaction volume of 20 µl. Rabbit globin (100 fg) was amplified along with the target genes for normalization. Each gene was analyzed four times.
The reaction was carried out in an ABI 7500 Fast Real-Time System (Applied Biosystems) using the following program: denaturation and activation of the Taq polymerase for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, and finally heating with a ramp rate of 2% at 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s to display a dissociation curve of the product. Data generated by Sequence Detection Software 1.3.1 were transferred to Microsoft Excel for analysis. Differential mRNA expression of each gene was calculated by the comparative threshold cycle (Ct) method recommended by the manufacturer.
Functional annotations.
For functional annotations, genes specifically expressed in bovine oocytes and blastocysts were identified by subtracting the overlapping subset from the individual lists of significantly expressed genes. The corresponding lists of Entrez gene IDs were then employed as input for the DAVID gene annotation tool (25). Several GO categories specifying the biological processes (GO: 0008150), molecular functions (GO: 0003674), and cellular component (GO: 0005575) were selected, and relative abundances of genes within these categories were computed to compare the oocyte- and blastocyst-specific gene expression profiles.
Online database.
To enable a global overview of gene expression in oocytes and blastocysts, which can be interrogated, we have presented the expression data as a database for searching for expression levels of specific genes and their related Gene Ontologies (http://goblet.molgen.mpg.de/cgi-bin/stemcell/preimplantation-development.cgi). We used GO terminology taken from the Gene Ontology website (http://www.geneontology.org). This was imported into the sqlite database (http://www.sqlite.org). Data analysis was carried out with R-Statistics software (http://www.r-project.org).
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RESULTS |
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6,000 genes expressed in the human blastocyst, whereas only 1,324 were detected as expressed in the bovine blastocyst.
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Distinct and overlapping gene expression in oocytes and blastocysts.
Despite the fundamental importance of the transition from maternal to embryo-coded gene transcription, progress in understanding how human and bovine preimplantation embryonic cells establish regulation of transcription and their own pattern of gene expression from a completely inactive genome has been hampered by the paucity of molecules known to regulate these processes. To gain further insights into the molecular mechanisms underpinning this process, we analyzed our data set for putative genes essential for oogenesis (maternal transcripts), the transition from maternal to embryo control of gene expression (MET), and further growth of the embryo to the blastocyst stage (embryonic transcripts). To enable identification of these transcripts, three distinct tests (Student's t-test, Welch test, Wilcoxon's rank-sum test) were used to help overcome individual bias (37). This analysis revealed a subset of 1,220 putative marker genes that were differentially expressed at the 0.05 level of significance. A full list of oocyte and blastocyst markers and genes common to both cell types is presented in Supplemental Tables 2, 3, and 4, respectively. The expression patterns of a selection of these oocyte and blastocyst marker genes were verified independently on replicate (4x) unamplified mRNA samples of oocyte and blastocyst origin. Of these genes, only COPS4 showed discordant expression patterns between the array-derived data and real time. The results shown in Fig. 3 confirmed that indeed DAZL and UPF1 were enriched in oocytes, whereas VDAC2 and EI24 were enriched in blastocysts. A description of the primer sequences and amplicon lengths are given in Supplemental Table 1.
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Mitochondria are the most abundant organelles in the mammalian oocyte and early embryo and play a key role in a number of physiological events during development, including the very first stages following fertilization (65). We detected expression of several transcripts encoding mitochondrial ribosomal protein subunits (MRPL2, 17, 18, 27, 28, 39, 48 and MRPS17). This would suggest active translation of mitochondrial proteins. However, the significance of the overrepresentation of the L subunit is unknown. We also found markers for regulators of intracellular pH and maintenance of NAD+/NADH balance and an enrichment of the NADH dehydrogenases (NDUFAB1; NDUFB3, 8, 9, 11; NDUFS2, 4, 8).
Several members of cell cycle-related genes (CCNAR, CCND, CCNE1, CCNF, CCNG, CCNJ, CCNK, CDC16, CDC37, CDK9, CHEK1, CHES1, CLK2, CLK4, GSPT1) were overexpressed in the blastocyst. In mammalian cells, cyclin E (CCNE1)-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to play an essential role in promoting S-phase entry (51, 66). In addition, GSPT1, a GTP-binding protein essential for the G1-to-S phase transition of the cell cycle, is also upregulated (38). Eighteen genes that encode proteins that function as solute carriers were enriched in the blastocyst. This large family of solute carriers facilitates transport of glucose (SLC2A14), nucleotides (SLC25A4 and SLC25A5), thiamine (SLC19A2), cations (SLC7A7), zinc (SLC39A7), and glutamate (SLC1A3). Finally, the trophectoderm markers PSG2 and KRT18 identified in our previous study (4) were 5- and 50-fold enriched, respectively.
Genes common to both oocyte and blastocyst.
Of the 419 commonly expressed genes, 11 (2.6%) are uncharacterized and therefore can be designated as novel. A list of the 20 most abundantly expressed transcripts common to both the blastocyst and the oocyte is presented in Table 3. During mammalian preimplantation development, the gametic epigenetic information is reprogrammed in a process that is essential for the establishment of nuclear totipotency in oocytes and two-, four-, and eight-cell stage embryos, for directing appropriate expression of imprinted genes, and also for establishing differentiation to two distinct cell layers in the blastocyst, the inner cell mass and the trophoblast (4). We looked specifically for the expression of genes implicated in this process (Fig. 5). The gene SMARCA2/BRM, which is a member of the SWI/SNF family highly similar to the brahma protein of Drosophila, was 89-fold enriched in the oocyte. The encoded protein is part of the large ATP-dependent chromatin remodeling complex SNF/SWI, which regulates both transcriptional activation and repression (7). Other epigenetic regulators such as the methyltransferases SET7 and DNMT1 (oocyte form DNMT1o) were enriched in the blastocyst and the oocyte, respectively. Several core histones (HIST1H2BD), histone acetylases (MYST4), and deacetylases (HDAC7A) were differentially expressed in the oocytes and blastocysts, respectively (Fig. 5A). In keeping with epigenetic modifications and the effect on gene transcription, we also screened the data set for differential expression of putatively imprinted genes (Fig. 5B). The data suggest significant overexpression of MEST in oocytes, while GNAS, UBE3A, SNRPN, and PON2 were enriched in the blastocyst. These patterns of expression have also been observed in both human and bovine preimplantation embryos (5, 55, 57).
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Oocyte enriched genes conserved in human, bovine, and mouse.
To identify oocyte-conserved genes we made use of available data sets that compared mouse oocyte transcripts to those of humans (44) and compared these to our oocyte-expressed genes (Supplemental Tables 2 and 4). This resulted in the identification of 31 genes; among these was BUB3, which has been shown in Xenopus egg extracts to be essential for the mitotic spindle checkpoint pathway (14). Other common transcripts were the germ cell-specific transcripts DAZL and NASP and a panel of genes implicated in cell proliferation such as FER, KIT, and MAPRE1. The full list and description of these genes are given in Supplemental Table 5.
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DISCUSSION |
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The ultimate approach for identifying genes solely transcribed by the newly formed embryonic genome and not the female gamete is to block protein synthesis by culturing one- to two-cell embryos in 
-amanitin. This approach has revealed embryonic transcription of Hsp70.1 (16), U2afbp-rs (46), eIF-4C (22), Xist (77), and Tead2 (42) and from the transcription-requiring complex (TRC) (20) in the mouse. While these earlier studies were informative, the advent of new analytical methods has enabled the identification of an increasing number of genes as being characteristic of either maternal or embryonic expression. Among the methods used to study human, bovine, and porcine preimplantation development are subtractive hybridization, expressed sequence tag (EST) libraries, differential display, serial analysis of gene expression (SAGE), in situ data mining, and microarrays (1).
Although each technique has its own unique merits, DNA microarrays have gained precedence because they enable comparative whole genome transcriptome analysis, albeit at a lower sensitivity of detection. A major drawback with the use of microarrays compared with the other technologies for the sort of analysis we describe here is that low-abundance oocyte- and preimplantation embryo-specific transcripts may not be represented as probes on most microarray platforms. Additionally, deadenylated mRNAs, which are known to be stored as inactive transcripts in oocytes, would never be isolated or transcribed with oligo(dT)-mediated mRNA labeling protocols, and therefore these genes within the oocyte pool will never be presented as labeled targets for possible detection on the arrays. This drawback is clearly manifested in our analysis, where we detected only 583 genes expressed in the oocyte compared with 1,743 in the blastocyst (Fig. 2B). Nonetheless, we identified conservation of 31 of these genes in human, bovine, and mouse oocytes. This unexpectedly low number can be attributed to the fact that the mouse and human oocyte data sets were derived from the Affymetrix platform and were not based on a cross-species approach.
The cDNA array used in this study has an advantage for cross-species comparison in that the length of each cDNA probe is sufficiently long that a small degree of mismatch is tolerated. In the present study, high stringency was used, which implies that transcripts with low homology between the bovine and human sequences are not detected as expressed. On the other hand, a strong signal in either the oocyte or the blastocyst hybridization demonstrates high nucleotide sequence homology, which in turn indicates conservation of an important gene component in an essential signaling or metabolic pathway. Another major advantage of our cross-species hybridization approach is that developmentally conserved novel and annotated marker genes could be identified for human and bovine oocytes and blastocysts. In addition, we have assigned each gene to its corresponding GO molecular function, biological process, and cellular component (http://goblet.molgen.mpg.de/cgi-bin/stemcell/preimplantation-development.cgi). The final functional classifications of the observed gene profiles according to biological process will give significant insight into the molecular changes involved in compaction and cell adhesion occurring during the transition from maternal to embryonic control of transcription.
In this study, we have demonstrated the identification of genes, related Gene Ontologies, and pathways relevant in the oocyte and the blastocyst. Gene expression patterns in the oocyte, with the overexpression of ELAVL4, TACC3, and MSY2, confirmed the importance of transcriptional and translation regulation (30, 76). Interestingly, the expression of ELAV4 and TACC3 mirrors observations in mouse oocytes (30, 35). Furthermore, TACC3 and TACC2 are functional homologs of MASKIN and, like the ELAVL gene family, are involved in translational control in oocytes and preimplantation embryos. Because translational control plays a central role during oocyte maturation and early embryogenesis, it is not surprising that these genes show evolutionary conservation.
High-level expression of genes previously identified as germ cell-specific transcripts (DAZL, TTTY12, TEX9, YBX2/MSY2, PHTF1) in oocytes suggests that a developmentally conserved process is operative in both male and female germ cell development (56, 63). Although testes and ovary are functionally nonequivalent, they share a common meiosis machinery, and these transcripts probably play similar roles in male and female germ cells. Lack of DAZL expression leads to embryonic arrest of germ cell development in mice (48), and DAZL is expressed in human oocytes (44, 56). Sequence variants of DAZL and single nucleotide polymorphisms are associated with premature ovarian failure and menopause (63). The gene encoding MSY2 is a member of a multifunctional Y-box protein family implicated in regulating the stability and translation of maternal mRNAs during mouse oogenesis. Mouse Msy2 transcript and protein are expressed in growing oocytes and one-cell embryos, but subsequently are degraded by the late two-cell stage, with no detectable expression in the blastocysts. Moreover, results from RNA interference-mediated suppression of Msy2 function in mouse oocytes support its role in stabilizing maternal mRNAs in growing oocytes, a process essential for generating meiotically and developmentally competent oocytes (76).
Genes indicative of transcriptional, translational control, and posttranslational modifications were found to be active in the blastocyst. This is manifested by the 33-fold enrichment of transcripts for EIF2B2 and the 2.5-fold enrichment of transcripts for HTEAD3. This implies conservation of the function of these gene families in mammalian preimplantation development (40, 41). Also enriched in the blastocysts is XPO1/EXPORTIN, which is involved in signal-mediated transport of proteins from the nucleus and has been shown to be enriched in mouse and swine embryos at the two-cell and four-cell stages, respectively (32). Furthermore, it has been speculated that XPO1 may regulate MET, possibly by controlling the pool of transcription factors present in the nucleus (13). High levels of expression of numerous components of the proteasome, specifically the PSMA, PSMB, and PSMC class of proteins, lends further support to the notion that RNA and protein degradation are an integral part of the regulatory machinery essential for MET, as already shown in the mouse (30). Together, these expression patterns underscore the importance of pre-mRNA processing, stability, mRNA trafficking, and translation seen in other mammalian cell types (39).
Epigenetic regulation and chromatin remodeling as an underlying mechanism of transcriptional control necessary for executing MET (59) have been aptly illustrated by the differential expression of key genes known to be involved in this mode of transcriptional regulation (Fig. 5), for example, the overexpression in the oocyte of SMARCA2/BRM and the oocyte variant of DNMT1, together with overexpression of the methyltransferase (SET7), histone acetylases (MYST4), and deacetylases (HDAC7A). Furthermore, this would indicate that the establishment and maintenance of MET involves a certain degree of methylation of CpG islands, histone methylation, acetylation, and deacetylation resulting in transcriptional programs essential for executing MET and subsequent development to the blastocyst stage. These epigenetic modifications would presumably also account for the observed differential expression of a subset of putatively imprinted genes (Fig. 5B). It is also probable that the proposed epigenetic changes are acting in trans to alter the expression of these putatively imprinted genes. In conclusion, the observed expression patterns reflect the essential roles played by these genes in the regulation of X inactivation, imprinting, maintenance of pluripotency, and establishment of the trophectodermal lineages in preimplantation embryos (4, 31).
The identified signaling and metabolic pathways operative in bovine oocytes and blastocysts mirror observations in human blastocysts (Table 4; Ref. 4). For example, the observed increase in glucose metabolism in the blastocyst of both species is supportive of existing data showing that during the early stages of mammalian preimplantation development embryos are characterized by low levels of respiration and glucose metabolism (8, 26) and glucose may even compromise development of early human embryos (19). After compaction, preimplantation embryos are capable of metabolizing glucose, and this switch seems to be associated with developmental regulation of the expression of the glucose transporters and their isoforms during preimplantation development (6, 15), which is in line with the observed increased expression of genes encoding glucose transporters such as SLC2A14 in the blastocyst. Furthermore, numerous studies in bovine, human, and mouse suggest that phosphatidylinositol 3-kinase signaling regulates glucose utilization in blastocysts and therefore is essential for their survival (4, 6, 54). In contrast, the carrier SLC25A12, a Ca2+-binding mitochondrial aspartate-glutamate carrier, is enriched in the oocyte. Although the significance of this is presently unknown, it could be an indication of the requirement of intracellular Ca2+ as a universal signal to trigger metabolic activity in oocytes (28). Since the function of most of the novel family members is unknown, future research must concentrate on defining their function, e.g., substrate specificity, regulation of subcellular localization, and mechanisms of cellular trafficking during preimplantation development.
Bovine preimplantation development has recently gained significant attention because of its similarity with human development. Bovine embryos are now easily produced in large numbers from slaughterhouse material and serve as a valuable model in assisted reproductive technologies (ARTs) including in vitro embryo production and somatic cloning, which are in an advanced stage in this species (45). Numerous studies have shown that the use of ARTs may be associated with a variety of pathological symptoms that are summarized under the term "large offspring syndrome" (47, 71). Epidemiologic studies in humans have revealed that the use of some reproductive technologies is associated with an increased frequency of imprinting defects, twins, and neurological disorders (24, 53, 60, 61). The causative mechanism is thought to be deviant epigenetic control of mRNA expression patterns during preimplantation development that persist throughout fetal development (50). The present study was performed with pooled in vitro-matured oocytes and in vitro-cultured blastocysts derived from IVF of in vitro-matured oocytes. This unavoidable and commonly used strategy of pooling oocytes and preimplantation-stage embryos for microarray-based gene expression studies does not reveal transcriptional heterogeneity between these samples as a result of culturing in vitro. Furthermore, some of the mRNA expression profiles shown here will need verification with in vivo-matured oocytes and in vivo-produced blastocysts. While such studies cannot be performed with human embryos, the bovine model is a practical alternative. We have initiated such studies by hybridizing RNA from all relevant preimplantation-stage embryos derived in vivo to the bovine Affymetrix array.
In conclusion, our cross-species approach has demonstrated that the transition from maternal to embryonic control of transcription in mammals (bovine and human) is accomplished by the regulated expression of developmentally conserved genes and related Gene Ontologies. This process is influenced and regulated by changes in the oocyte and blastocyst epigenomes, leading to altered chromatin architecture and ultimately induced or repressed gene transcription and associated signaling and metabolic pathways necessary for preimplantation development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
1 The online version of this article contains supplemental material. 
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