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a Department of Physiological Sciences,
b College of Veterinary Medicine, Departments of Statistics
c Animal Science, Division of Agricultural Science and Natural Resources, Oklahoma State University, Stillwater, Oklahoma 74078-2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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400 ng of total RNA), a reverse transcription-polymerase chain reaction (PCR) procedure was used to amplify the mRNA and generate sufficient cDNA to conduct suppression-subtractive hybridization. The subtracted cDNA products were cloned, and 126 cDNAs representing expressed mRNAs were isolated, sized, single-pass sequenced, and compared to known sequences in GenBank. Ninety-two clones provided sequence information for further analysis. Among these, 31 exhibited high homology to known genes. Three, 26S proteasomal ATPase (PSMC3), casein kinase 2 
subunit (CK2), and phosphoglycerate kinase (PGK) were selected and further characterized using real-time quantitative PCR to assess their differential expression in hatched blastocysts. Overall, a 1.3-, 1.6-, and 1.5-fold increase in expression level was observed in hatched blastocysts compared with intact blastocyst for PSMC3, CK2, and PGK, respectively. These results show that construction of subtracted cDNA libraries from small numbers of embryos is feasible and can provide information on gene expression patterns during preattachment embryogenesis.
early development, embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies reveal that most embryo losses in heifers occur before Day 14 after insemination [2]. Contemporary developments in the in vitro production of bovine embryos have fostered basic studies aimed toward understanding the intricate pathways controlling early embryo development. Despite these advances, success rates in terms of blastocysts yields range between 30% and 40% following in vitro culture with 50% being able to initiate a successful pregnancy following transfer [3]. Furthermore, in vitro-produced embryos continue to exhibit conspicuous morphological, biochemical, and metabolic differences compared with their in vivo counterparts [4]. Other detrimental effects of in vitro embryo culture include fetal oversize and significant fetal loss after Day 35 due to failure of normal allantoic development within the conceptus [5]. These negative consequences have substantially hampered the field application of in vitro embryo production in the bovine.
It is presumed that successful preimplantation and early fetal development is reliant on the timely expression of approximately 10 000 genes [4]. Unfortunately, sequence information for only a few of these genes is currently known, meaning that our basic understanding of gene expression patterns driving blastocyst development is very restricted. In many cases, our present knowledge of genes expressed during early embryogenesis in the bovine has been gained from studies using data extrapolated from the mouse as the starting point. Thus, the identification of novel genes and analysis of their function during preimplantation embryogenesis in the bovine is necessary. Several modifications of the reverse transcriptase-polymerase chain reaction (RT-PCR) have been used to quantify the relative abundance of individual gene transcripts [4]. Differential display RT-PCR developed a decade ago [6] was recently applied to compare patterns of RNA expression from preattachment bovine embryos [7]. Large and representative subtractive cDNA libraries have been successfully constructed from preattachment murine embryos to identify novel genes critical for development [8].
The present communication describes the use of a modification of suppression-subtractive hybridization (SSH), originally developed in 1996 [9], to study differential gene expression in the bovine preattachment embryo. Use of SSH is advantageous because it enriches low abundance transcripts that are differentially expressed in the tester population. For a discussion of the basis of SSH and how it leads to the enrichment and isolation of differentially expressed transcripts, see [9]. In the present study, we attempted to identify changes in gene expression between in vitro-produced, intact Day 7–8 blastocysts (driver) and Day 9–10 hatched blastocysts (tester). A better knowledge of gene expression patterns during early preattachment development would yield insights into the molecular pathways controlling early development and as a preamble to understanding events that may be compromised in early embryonic mortality.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were collected from cows at a local abattoir and transported to the laboratory in 0.9% normal saline supplemented with penicillin G (100 IU/ml) and streptomycin sulfate (0.2 µg/ml) at 26–30°C within 5 h. Oocytes were aspirated from follicles ranging in diameter from 2 to 8 mm using an 18-gauge needle and transferred into a modified-PBS solution containing 0.3% BSA. In vitro maturation, fertilization, and culture were performed according to protocols described in Mohan et al. [10].
RNA Extraction
In vitro-produced intact blastocysts (Days 7–8) and hatched blastocysts (Days 9–10) (n = 30) were frozen in 250 µl of denaturing solution (4 M guanidium isothiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarcosyl, and 0.1 M 2-ß mercaptoethanol). Total RNA was extracted according to the method described in Mohan et al. [10] from a pool of 30 embryos at both stages.
Driver and Tester cDNA Synthesis
Due to the small amounts of mRNA extractable from bovine embryos, mRNA was reverse transcribed and the cDNA was subjected to PCR using the SMART cDNA synthesis kit (Clontech Laboratories Inc., Palo Alto, CA). Protocols were followed according to the manufacturer's instructions. Briefly, about 400 ng of total RNA from both intact blastocysts (driver) and hatched blastocysts (tester) was denatured at 70°C for 2 min, then reverse transcribed in the presence of 1 µM of cDNA synthesis primer (CDS), 1 µM SMART II oligonucleotide, 1 mM of 50;ts dNTPs, and 200 units of reverse transcriptase (Superscript, 200 U per reaction; Invitrogen, Carlsbad, CA) at 42° C for 1 h. The reaction was primed using an anchored oligo-dT primer called the CDS primer, carrying an internal PCR primer sequence at the 5' end. The SMART II oligonucleotide anneals with the CCC tailing left by the reverse transcriptase on the newly formed cDNA and contains the same internal primer as the CDS primer. The samples were diluted with 40 µl of Tris-EDTA (TE) buffer and the reaction was stopped by heating to 70°C for 7 min. Approximately 4 µl of diluted driver and tester cDNA were denatured for 1 min at 95°C and subjected to 34 PCR cycles in the presence of 0.2 mM of 50x dNTP; 0.2 mM of PCR primer, which anneals on both the SMART II oligonucleotide; and the CDS primer along with 2 µl of 50;ts Advantage 2 polymerase mix (Clontech). The cycling conditions were as follows: denaturation at 95°C for 5 sec, annealing at 65°C for 5 sec, and extension at 68°C for 6 min. Aliquots (15 µl) were analyzed on a 2% agarose gel. PCR products were extracted once with 150 µl of phenol:chloroform:isoamyl alcohol (25:24:1). Approximately 120 µl of the aqueous phase was removed and concentrated to 40–70 µl using 700 µl of n-butanol. The cDNA was then purified using CHROMA SPIN-350 gel exclusion columns (Clontech) to remove unincorporated dNTPs, primers, and DNA fragments less than 300 base pairs (bp) in length. Both driver and tester cDNAs were then digested with 15 units of RsaI in a 500-µl reaction mixture at 37°C for 3 h, and the reaction was stopped by adding 8 µl of EDTA. RsaI digested driver and tester cDNA were extracted once with phenol:chloroform:isoamyl alcohol. The aqueous phase was removed, precipitated with ethanol, and the pellet was redissolved in 7 µl of TNE buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, and 0.1 mM EDTA). The final concentration of both driver and tester was 300 ng/µl. One microliter of RsaI digested tester cDNA was diluted in 5 µl of sterile water, and 2 µl of the diluted tester was then ligated with 2 µl of adapter 1 and adapter 2R (2 µM) according to the guidelines provided in the PCR-Select cDNA subtraction kit (Clontech) in separate ligation reactions in a total volume of 10 µl at 16°C overnight using 400 units of T4 DNA ligase in 2 µl of 5;ts ligation buffer. The ligation was stopped by adding EDTA/glycogen and heated at 72°C for 5 min to inactivate the ligase. Samples were stored at -20°C. A PCR-based ligation efficiency analysis to verify that at least 25% of the cDNAs had adaptors on both ends was performed according to the instructions detailed in the Clontech PCR-Select cDNA subtraction kit user manual.
Suppression Subtractive Hybridization
SSH [9] was performed using the Clontech PCR-Select cDNA subtraction kit. Briefly, 1.5 µl of driver cDNA (450 ng) was added to each of two tubes containing 1.5 µl of adapter 1 and adapter 2R-ligated tester cDNA (20 ng) in 1 µl of 4;ts hybridization buffer. The samples were denatured at 98°C for 1.5 min, and then allowed to anneal at 68°C for 8 h. Following first hybridization, the two samples were combined in the presence of fresh excess denatured driver cDNA (300 ng) in 1 µl of 4;ts hybridization buffer. The samples were allowed to hybridize overnight at 68°C. The hybridized samples were diluted in 200 µl of dilution buffer (20 mM Hepes pH 8.3, 50 mM NaCl, and 0.2 mM EDTA), heated at 68°C for 7 min, and stored at -20°C.
PCR Amplification of Subtracted Products
Two PCR amplifications of the subtracted cDNAs were performed. The primary PCR contained 1 µl of diluted, subtracted cDNA, and 24 µl of the PCR master mixture prepared using the reagents provided in the kit. PCR was performed at 75°C for 5 min to extend the adaptors; 94°C for 25 sec; and 27 cycles at 94°C for 10 sec, 66°C for 30 sec, and 72°C for 1.5 min. The amplified products were diluted 10-fold in sterile deionized water. The diluted primary PCR product was used as template in a secondary nested PCR for 10 cycles at 94°C for 10 sec, 68°C for 30 sec, and 72°C for 1.5 min using two nested primers, 1 and 2R, provided in the kit. Primary and secondary PCR products were analyzed on a 2% agarose gel. A second PCR-based analysis was performed according to the instructions detailed in the Clontech PCR-Select cDNA subtraction kit user manual to test for the efficiency of subtraction.
Cloning and Analysis of Subtracted cDNA
Following PCR subtraction, the amplified products were cloned into the pCR II vector of the TA cloning kit (Invitrogen) and used to transform competent DH5 Escherichia coli cells. Colonies were grown for 16–18 h at 37°C on Luria broth (LB) agar plates containing ampicillin, X-gal (5-bromo 4-chloro 3-indoyl-ß-D-galactopyranoside) and isopropyl-ß-D-thiogalactopyranoside for blue/white colony selection. Plasmids were extracted and the inserts were subjected to dideoxy chain termination sequencing (Applied Biosystems, Model 373A Automated Sequencer, Oklahoma State University Recombinant DNA/Protein Resource Facility) and the identity of each product was confirmed in a sequence homology analysis using the Basic Local Alignment Search Tool [11].
Quantitative 1-Step RT-PCR
Expression of the 3 clones of interest; namely, 26S proteasomal ATPase (PSMC3), casein kinase 2 subunit (CK2), and phosphoglycerate kinase (PGK) was evaluated by real-time quantitative RT-PCR using a fluorescent reporter and 5' exonuclease assay system [12]. This technique, from our previous experience, is capable of efficiently amplifying and detecting a product from as few as 10 copies of the target. Approximately 20 pooled embryos per group were grouped as early blastocysts, expanded blastocysts, early hatched blastocysts, and late hatched blastocysts (in culture until Day 10). One sample from each of the 4 stages was assayed in triplicate wells. All 4 samples representing each of the 4 stages came from different embryo batches (i.e., embryos from 4 independent runs of the in vitro fertilization protocol were present in each pool). Reverse transcription of total RNA and PCR amplification was performed using the Taqman One-Step RT-PCR Master Mix Reagents Kit, Taqman fluorescent probe, and sequence detection primers (PE Applied Biosystems, Foster City, CA). Taqman probe specific for target was designed to contain a fluorescent 5' reporter dye (FAM) and 3' quencher dye (TAMRA). Each RT-PCR reaction (25 µl) contained the following: 2;ts Master Mix without uracil-N-glycosylase (12.5 µl), 40;ts Multiscribe and RNase Inhibitor Mix (0.63 µl), target forward primer (200 nM), target reverse primer (200 nM), fluorescent-labeled target probe (200 nM) designed from the mRNA sequence isolated from hatched blastocysts using SSH, and total RNA (40 ng) quantified spectrophotometrically based on A260:A280 ratios. Forward and reverse primer and probe sequence for all 3 targets are shown in Table 1. The PCR amplification was carried out in the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). Thermal cycling conditions were 48°C for 30 min and 95°C for 10 min, followed by 40 repetitive cycles at 95°C for 15 sec and 60°C for 1 min. As a normalization control for RNA loading, parallel reactions in the same multiwell plate were performed using 18S ribosomal RNA as target (18S Ribosomal Control Kit, PE Applied Biosystems).
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Quantification of gene amplification was made following RT-PCR by determining the threshold cycle (CT) number for FAM fluorescence within the geometric region of the semilog plot generated during PCR. Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. The relative quantification of target gene expression across treatments was evaluated using the comparative CT method [13]. The 
CT value was determined by subtracting the ribosomal CT value for each sample from the target CT value of that sample. Calculation of CT involved using the highest sample CT value (i.e., the sample with the lowest target expression) as an arbitrary constant to subtract from all other CT sample values. Fold changes in the relative gene expression of target was determined by evaluating the expression, 2- CT.
Statistical Analysis
Quantitative RT-PCR CT values were analyzed using SAS Proc Mixed as a completely randomized design with 4 treatments [14]. Blastocysts grouped as early and late (expanded) and hatched blastocysts grouped as early and late (in culture until Day 10) were considered as 4 independent treatments. A probability value of P < 0.05 was considered significant. Results are presented as arithmetic means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After 34 PCR cycles using the SMART cDNA synthesis kit, agarose gel electrophoresis revealed cDNA bands ranging in size from 250 bp to 1.5 kilobases (kb) for both blastocyst and hatched blastocyst (data not shown). Following size exclusion chromatography and RsaI digestion, approximate yields of cDNA for the tester and driver ranged around 5–8 µg. Two different adaptors were ligated to the tester cDNA, and ligation efficiency was confirmed using a PCR-based assay employing 2 different sets of primers in 2 independent PCR reactions. The first set of primers included a glyceraldehyde-3-phosphate dehydrogenase-specific (G3PDH 3' primer) primer and PCR primer 1, which bound specifically to the adaptor sequence (product size 1.2 kb). The second set of primers bound internally to the G3PDH gene (G3PDH 3' and 5' primer) (product size 500 bp). The PCR product using both sets of primers resulted in bands of the expected size (data not shown). Further, the PCR products amplified using both primer sets were of the same intensity, indicating that the adaptor ligations worked successfully. Finally, a PCR-based subtraction efficiency analysis was performed using specific primers provided in the kit by comparing the abundance of G3PDH before and after subtraction. For the unsubtracted sample, a G3PDH PCR product (500 bp) was observed after 18 cycles (data not shown). However, the same product in the subtracted sample appeared after 23 cycles (data not shown), indicating that G3PDH levels were reduced several fold in the subtracted product.
A total of 126 clones were selected after SSH, and we obtained partial sequence information on 92 clones following dideoxy chain termination sequencing. After restriction digestion with EcoRI and agarose gel electrophoresis it was found that approximately 18% of the clones were in the 100–300 bp size range, 40% in the 301–500 bp size range, 27% in the 501–800 bp size range, and 15% ranged in size from 801–1500 bp. The partial sequences obtained from all 92 clones were compared with known sequences in the GenBank (National Center for Biotechnology Information, Bethesda, MD) database. Their putative identity, nucleotide homologies with other known sequences, and insert size are shown in Table 2. Sequence data were submitted to the dbEST database (National Center for Biotechnology Information).
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RT-PCR Quantitation Using Taqman PCR
The mRNA expression of PSMC3, CK2, and PGK was quantified using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The relative abundance of mRNAs encoding PSMC3, CK2, and PGK were calculated using the comparative CT method (Table 3).
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Ribosomal 18S RNA was used to normalize each sample for variation in RNA loading. As shown in Table 3, 18S rRNA was variable across developmental stages as a result of the dynamic nature of the RNA populations in the developing embryonic cells. We are not aware of any product suitable as a stable normalizer for this type of analysis in the preattachment embryo. Results with G3PDH (not shown) reveal that it is also dynamic in these embryos, as are structural elements such as actin.
Based on normalization with 18S rRNA levels, expression of PSMC3 mRNA in early intact blastocysts was significantly different from late hatched blastocysts (P < 0.05). Similarly, expression of CK2 mRNA in late intact blastocysts was significantly different from late-hatched blastocysts (P < 0.05). However, for PGK, early hatched blastocysts exhibited significant differences compared with late hatched blastocysts (P < 0.05). Overall, hatched blastocysts had higher expression levels than intact blastocysts for all three genes examined as well as for 18S rRNA. Differences in expression for all three genes based solely on RNA loading in the PCR reactions were several fold greater between intact and hatched blastocysts (4- to 8-fold; Table 3) than when analysis was based on normalization. This can be evaluated based simply on the differences in average CT values. We report the more conservative approach above, and recognize the technical limitations of using a normalization control.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we report the construction of a subtracted cDNA library from bovine hatched blastocysts. Approximately 92 from a total of 126 clones isolated provided sequence information. Homology searches revealed the identities of 31 of these expressed sequences (expressed sequence tag; EST) with known genes (Table 2), among which three, PSMC3, CK2, and PGK with predicted roles during early embryogenesis were further characterized using real-time quantitative PCR. Overall, the largest group of identified ESTs, roughly 40%, was homologous to coding regions for products involved in protein synthesis, and included several ribosomal proteins. The remaining identified ESTs were related to transcription (about 20%), metabolism (20%), and signaling (20%).
Protein synthesis in the early preattachment embryo has been subjected to a great deal of scrutiny in the recent past [20–22]. The protein content of in vivo-derived preattachment cattle embryos from the 2-cell through to the elongated blastocyst at Day 16 has been described [20]. The protein content increased 2-fold from the morula to the expanded blastocyst stage followed by a 160-fold increase to the hatched blastocyst stage on Day 13. Although protein synthesis is critical, proteolysis, on the other hand, is equally vital to the upkeep of appropriate levels of short-lived and regulatory proteins that are mostly involved in basic cellular processes such as regulation of cell cycle and cell division, cell differentiation, metabolism, stress response, modulation of the immune and inflammatory responses, modulation of cell-surface receptors and ion channels, transcription, and signaling factors [23, 24].
In eukaryotic cells, degradation of intracellular proteins is mediated by the 26S proteasome, a nonlysosomal ATP-dependant protease complex. This complex present in both the nucleus and cytoplasm consists of a proteolytic cylinder-shaped particle (20S proteasome) and an ATPase-containing complex (19S cap complex). The ubiquitin-conjugated proteins are unfolded by the 19S regulatory subunit, thereby facilitating their entry into the 20S proteasome cylinder particle [25, 26]. Several ATPases with a highly conserved ATPase domain [27] such as PSMC1 (S4), PSMC2 (MSS1), PSMC3 (TBP1), PSMC4 (TBP7), and PSMC5 (TRIP1) comprise the 19S complex. One of the subtracted products isolated and further characterized in the present study revealed 90% similarity to the conserved ATPase domain of PSMC3 as well as TAT binding protein 1 (TBP1). PSMC3, which is synonymous with TBP1, was earlier identified as a component of human immunodeficiency virus TAT-binding protein and negatively regulated TAT-mediated transcriptional activity. In Table 3, the CT values for PSMC3 decreased in the hatched blastocysts, indicating that the target copy numbers were higher, which agrees with the fact that SSH enables isolation of differentially expressed/up-regulated sequences. After normalization with the internal control 18S rRNA, we observed that expression increased by approximately 1.3-fold in the hatched blastocysts compared with late blastocysts (Table 3). The differential expression of this ATPase may be indicative of an energy-dependant active protein degradative process in the hatched blastocyst to eliminate abnormal proteins along with various cell cycle regulatory proteins so that development can continue uninterrupted. From recent studies in mice it appears that PSMC3 may be indispensable for early preattachment development, because PSMC3 knockout mice fail to implant, owing to defective blastocyst development [28]. E3.5 PSMC3-/- embryos when cultured in vitro for 5 days exhibited shrinking of embryonic cells and failed to differentiate into trophectoderm and inner cell mass cells. Taken together, these findings lead to the suggestion of a specific role for PSMC3 in blastocyst formation, hatching, and during posthatching preattachment development in the bovine.
Casein kinase 2, a pleiotropic serine-threonine-specific growth-related protein kinase is known to regulate a myriad of intracellular processes fundamental to maintaining cell viability, cell proliferation and differentiation, signal transduction, transcriptional control, apoptosis, cell cycle, etc. [29]. Not only it is ubiquitously expressed in every eukaryotic tissue, but also in every cellular compartment [30]. Described to exist as a spontaneous heterotetramer (130 kDa), it is composed of two catalytic subunits; namely, (42–44 kDa), ' (38 kDa), or both; and two noncatalytic ß subunits whose molecular size in animals is approximately 26 kDa. In contrast to other protein kinases, CK2 is unique in its ability to utilize GTP in addition to ATP as a phosphate donor. Approximately 160 proteins are known to be phosphorylated by CK2 and include several proteins involved in cell cycle control, and transcriptional and translational processes [29].
Following quantification, an approximately 1.6-fold increase in CK2 mRNA levels was detected in hatched blastocysts compared with intact blastocysts (Table 3). A possible "proliferation marker," the high expression pattern of CK2 in hatched blastocyst is consistent with this stage during embryonic development being one in which high proliferation rates prevail. An increase in CK2 has been reported during late embryogenesis in mice [31] and early embryogenesis in nematodes [32] and insects [33]. In sea urchins, almost all of the increase in phosphorylation during early development has been attributed to CK2-like activity [34]. Embryonic stage-specific changes in protein phosphorylation have been described for mice [35] and more recently in elongating blastocysts in cattle [36]. In mice, protein phosphoryation is associated in critical events such as zygotic genome activation [37] and blastocyst expansion [38], and is required for preimplantation embryo development [39]. Several growth factors have also been shown to satisfy this essential role at least in part by regulating the expression and activity of CK2 [40].
Blastocyst formation involves the establishment of one or more transtrophectoderm ion gradients, contributed equally by Na/K-ATPase, which pumps water through water channels called aquaporins [41]. Aquaporins belong to a critical group of genes whose members are integral membrane proteins and function to channel the movement of water through the cell membrane [42]. A role for CK2 in blastocyst formation is further strengthened by the presence of supposed CK2 phosphorylation sites in the primary amino acid sequence of aquaporins [42]. Recently, it was reported that CK2 may be involved in the phosphorylation of the proteasome and is critical for its association with the 19S regulatory complex and activity [43]. In sea urchins, inhibition of CK2 activity delayed hatching of the blastula from the fertilization envelope and the transition from blastula to gastrula [34]. It is an interesting question to ask whether a similar cause-and-effect relationship would be applicable to the bovine blastocyst. Taken together, the hitherto known functions of CK2 fit well with the events leading to the formation and possible hatching of the blastocyst, implying that CK2 activity has important physiological roles during early embryogenesis in the bovine.
Two metabolic pathways, the pentose phosphate pathway and the Embden-Meyerhof pathway, play essential roles during embryo development [44]. Cattle embryos use very little glucose until the 16-cell stage [3, 44, 45]. However, utilization of glucose significantly increases at the morula stage [44] through to blastocyst expansion [45]. PGK catalyzes the first ATP-generating reaction in glycolysis by transferring a phosphoryl group from the acyl phosphate of 1,3-bisphosphoglycerate to ADP, forming 3-phosphoglycerate and ATP. A third subtracted cDNA clone representing PGK was isolated and selected for further characterization using real-time PCR. Similar to the previous two products, the expression of PGK was 1.5-fold higher in hatched blastocysts compared with intact blastocysts (Table 3). The expression of PGK would suggest active use of glycolytic substrates such as glucose to meet the energy requirements of the early embryo. An increase in the utilization of glucose has been reported as a means to meet the growing energy demands of Na+-K+ ATPase required for the formation and maintenance of the blastocele [45] and one other report suggested it to be essential for hatching of the bovine blastocyst [46]. Higher glucose utilization has also been directly correlated with greater blastocyst viability [47] and better developmental potential in vitro in Day 10 cattle embryos [46]. The trend toward higher expression of PGK as embryo development advances seem to agree with the aforementioned reports on glucose utilization/metabolism by bovine embryos in vitro.
In summary, we have isolated, sequenced, and identified several differentially expressed mRNAs from in vitro-produced bovine hatched blastocysts. Expression levels of three of these mRNAs—PSMC3, CK2, and PGK—known to be associated with early embryogenesis, were shown to increase in the hatched blastocyst using the Taqman real-time quantitative PCR assay. Quantitative comparisons using normalization to 18S rRNA were made difficult, however, by the apparent fluctuation in amounts of the 18S rRNA itself. It is unclear whether any transcript might be a stable reference even over short time periods. The comparative CT method used allows for determination of relative differences in transcript concentration using small amounts of starting material. One disadvantage is the dependence on a normalizer, which may itself be in a dynamic state. Similarly, attempts at absolute measurement using a standard curve would be difficult to interpret given the dynamic nature of the developing embryo.
The characterizations in the present study pertain to in vitro-derived embryos and need to be extended to their in vivo counterparts. The subtracted cDNAs analyzed in the present study are not solely specific to the hatched blastocyst. Quantitative differences cannot be inferred without validation by methods such as quantitative PCR. A different approach to follow in future studies would be to perform SSH in both the forward and reverse direction, and to identify the true positive clones (i.e., transcripts unique to one or the other stage) using a differential hybridization screening protocol.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the Oklahoma Agricultural Experiment Station. We thank the Lew Wentz Foundation, Wentz Project Scholarship program for support for S.R. 
2 Correspondence: J.R. Malayer, Department of Physiological Sciences, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078-2006. FAX: 405 744 7110; malayer{at}okstate.edu
Accepted: March 1, 2002.
Received: November 21, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amanitin sensitivity during bovine preattachment development. Mol Reprod Dev 2000; 55:152-163[CrossRef][Medline]This article has been cited by other articles:
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  A. El-Sayed, M. Hoelker, F. Rings, D. Salilew, D. Jennen, E. Tholen, M.-A. Sirard, K. Schellander, and D. Tesfaye Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients Physiol Genomics, December 13, 2006; 28(1): 84 - 96. [Abstract] [Full Text] [PDF]
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 A.G.B. Hurst, D.W. Goad, M. Mohan, and J.R. Malayer Independent Downstream Gene Expression Profiles in the Presence of Estrogen Receptor {alpha} or {beta} Biol Reprod, October 1, 2004; 71(4): 1252 - 1261. [Abstract] [Full Text] [PDF]
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J. Yao, X. Ren, J. J. Ireland, P. M. Coussens, T. P. L. Smith, and G. W. Smith Generation of a bovine oocyte cDNA library and microarray: resources for identification of genes important for follicular development and early embryogenesis Physiol Genomics, September 16, 2004; 19(1): 84 - 92. [Abstract] [Full Text] [PDF]
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