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BOR - Papers in Press, published online ahead of print January 8, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.012104
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BIOLOGY OF REPRODUCTION 68, 2073–2080 (2003)
DOI: 10.1095/biolreprod.102.012104
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

Timing of Blastocyst Expansion Affects Spatial Messenger RNA Expression Patterns of Genes in Bovine Blastocysts Produced In Vitro1

C. Wrenzycki, D. Herrmann, and H. Niemann2

Department of Biotechnology, Institute for Animal Science (FAL), Mariensee, 31535 Neustadt, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blastocyst formation and expansion are dependent on the differentiation and function of a proper transport of nutrients through the trophectoderm (TE) enclosing the inner cell mass (ICM). Coincident with compaction and cavitation, glucose becomes the preferred energy substrate of the early embryo. These hallmarks in early development require well-orchestrated gene expression patterns specifically with regard to timing and localization. The present study investigated the relative abundance (RA) of gene transcripts in the two lineages of in vitro-produced expanded bovine blastocysts in relation to timing of development, i.e., blastocyst expansion and localization of specific mRNAs. Expanded blastocysts from either Day 7 or Day 8 or isolated ICMs derived thereof were analyzed with the aid of a semiquantitative reverse transcriptase-polymerase chain reaction assay for gene transcripts, which are thought to play a pivotal role in blastocyst expansion, i.e., Na/K-ATPase {alpha}1 subunit (Na/K), E-cadherin (E-cad), zonula occludens protein-1 (ZO-1), desmocollin II (Dc II), plakophilin (Plako), trophoblastic function (interferon {tau} [IF{tau}]), and glucose transport (glucose transporter-1, -3, -4 [Glut-1, -3, -4]). Total cell number, ICM cell number, or ICM/total cells ratio were similar in Day 7 and Day 8 expanded blastocysts. Significant differences were determined in the RA for Na/K, E-cad, Dc II, Plako, and ZO-1 transcripts between TE cells of expanded blastocysts derived from either Day 7 or Day 8. The RA of Dc II, Glut-1, and Glut-4 was significantly decreased in the ICM compared with the TE at Day 7. Similarly, the RA of Na/K, Dc II, Glut-1, and Glut-4 at Day 8 of development was significantly decreased in the ICM compared with the TE. Interestingly, no differences were observed when comparing ICMs originating from blastocysts expanded at either Day 7 or Day 8. Plako and IF{tau} transcripts were not detected in isolated ICMs, indicating that expression of these mRNAs is restricted to the TE. In contrast, similar expression patterns within the ICM and TE were determined for Na/K, E-cad, ZO-1, and Glut-3 mRNA. Dc II, Glut-1, and Glut-4 were more abundant in the TE than in ICM. Results show that expression of developmentally important genes is related to the two cell lineages in the early embryo and emphasize the critical role of a well controlled spatial gene expression pattern for regular preimplantation development.

early development, embryo, gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Timing of development has been proposed as an important criterion for evaluating the quality of in vitro-produced embryos [1, 2]. Faster developing embryos are considered to be developmentally more competent and more viable than their slower developing counterparts, although the slower developing embryos may eventually reach the blastocyst stage as well. Transfer of embryos with an early appearance of the blastocoelic cavity has a higher probability to result in live offspring [3, 4]. In addition, in vitro culture systems have been shown to affect the sex ratio of the developing embryo, with male embryos developing faster and a higher percentage reaching the blastocyst stage [5–8]. Furthermore, transfer of frozen/thawed in vivo or in vitro Day 7-expanded blastocysts resulted in higher pregnancy rates than that of Day 8-expanded blastocysts [9, 10].

Blastocyst formation initiated at compaction and represented by cell polarization and the onset of cellular differentiation [11] is mediated by fluid transfer across the intercellular connections between the outer blastomeres. During subsequent cavitation, the blastomeres differentiate into trophectoderm (TE) and inner cell mass (ICM) cells. The ICM cells contribute to all embryonic tissues and are part of the extraembryonic membranes, whereas the TE cells mainly form the outer layer of the placenta [12]. A proper ratio of both cell lineages is crucial for undisturbed embryonic and fetal development. In the bovine embryo, compaction occurs around the 16- to 32-cell stage [13] and is dependent on the well-orchestrated expression of genes mediating cell adhesion and TE differentiation [14]. Perturbations in the expression of genes involved in compaction and cavitation have been observed in in vitro-produced bovine morulae and blastocysts compared with their in vivo counterparts [15, 16].

Coincident with compaction and cavitation and the accompanying first differentiation, the embryo undergoes marked changes in energy substrate utilization as glucose replaces pyruvate and lactate as the preferred energy substrates [17]. With the formation of the blastocyst, glucose consumption increases and glucose transport is upregulated by the early embryo. The expression pattern of the facilitative glucose transporters has recently been studied in in vitro-produced bovine embryos [18], and alterations in their expression pattern attributed to different culture systems have been determined [19]. Expression patterns of genes critically involved in the formation and expansion of a viable blastocyst and its metabolism have not been investigated with regard to developmental timing and localization of mRNAs.

Here we report for the first time the spatial distribution of mRNAs of genes in bovine blastocysts related to developmental timing, i.e., blastocyst expansion. The relative abundance (RA) of gene transcripts thought to play important roles in blastocyst formation and expansion, i.e., Na/K-ATPase {alpha}1 subunit (Na/K), E-cadherin (E-cad), zonula occludens protein-1 (ZO-1), desmocollin II (Dc II), plakophilin (Plako); trophoblastic function, i.e., interferon {tau} (IF{tau}); and glucose transport, i.e., glucose transporter-1, -3, -4 (Glut-1, -3, -4) were analyzed in intact in vitro-produced blastocysts from Days 7 and 8 and isolated ICM derived thereof.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Production of Bovine Embryos

Bovine embryos were produced as described recently [16]. Briefly, ovaries from a local slaughterhouse were transported to the laboratory in Dulbecco PBS (D6650, Sigma Chemical Co., St. Louis, MO) at 25°C–30°C. Cumulus-oocyte complexes (COCs) were isolated via slicing [20]. Category I COC (with a homogenous evenly granulated cytoplasm possessing at least three layers of compact cumulus cells) and category II COC (with less than three layers of cumulus cells or partially denuded but also with a homogenous evenly granulated cytoplasm; [21]) 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, A9647, Sigma]).

For maturation in vitro, TCM 199 containing L-glutamine and 25 mM Hepes served as the basic medium. One milliliter was supplemented with 22 µg pyruvate, 2.2 µg NaHCO3, and 50 µg gentamicin. For oocyte maturation, this medium was supplemented with 10 IU eCG and 5 IU hCG (Suigonan; Intervet, Tönisvorst, Germany) and 10% oestrus cow serum (collected at Day 1 of standing oestrus). COC were divided in 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.

Following 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), 0.1 IU/ml heparin (Serva, Heidelberg, Germany), and 6 mg/ml BSA. Frozen semen from one bull with proven fertility in in vitro fertilization (IVF) was used. For IVF, semen was prepared by the modified "swim-up" procedure [22, 23]. Briefly, semen was thawed in a waterbath at 37°C for 1 min. After swim-up separation in Sperm-TALP containing 6 mg/ml BSA for 1 h, the semen was washed twice by centrifugation at 350 g and 36°C for 10 min before being resuspended in Fert-TALP supplemented with heparin and BSA. 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 (SOF) supplemented with 10% estrous cow serum 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 (615300, ICN Biomedicals, Inc., Aurora, OH). Expanded blastocysts were harvested at either Day 7 or Day 8 of development (day 0 = IVF) as intact embryos or for isolating the ICM. Only blastocysts of morphological grade I [24] were included in this study. After washing three times in PBS containing 0.1% polyvinyl alcohol (PVA), all intact embryos or isolated ICM were stored individually at -80°C in a minimum volume (5 µl or less) of medium until RNA extraction.

Differential Staining of ICM and TE Cells and ICM Isolation

For determination of total cell numbers and the ratio of ICM and TE cells in in vitro-produced bovine embryos, the differential staining procedure was performed with a representative sample of Day 7 and Day 8 blastocysts according to the protocol described by Eckert and Niemann [25] with minor modifications. Briefly, after removal of the zona pellucida with 1.0% pronase (Type XXV, Sigma, P6911) in PBS (w/v), the embryos were incubated in trinitrobenzene sulfonic acid (Sigma, P883) for 10 min on ice, then washed in TCM air and incubated in antidinitrophenyl BSA (anti-DNP BSA; 61-006, ICN Biochemicals, Eschwege, Germany) diluted 30:70 in PBS at 37°C for 30 min. After washing in PBS plus PVA, complement lysis was induced by incubating the embryos in guinea pig complement (Sigma, S1639) diluted 1:4 in PBS plus PVA supplemented with propidium iodide (Sigma, P4170, 10 µg/ml) at 37°C for 20 min, followed by brief washing and fixation in ice-cold ethanol. The inner nuclei were stained with Hoechst 33342 (Bisbenzimid, Sigma, B2261, 10 µg/ml in ethanol) for 10 min. Stained embryos were mounted into 100% glycerol. Under a fluorescence microscope (excitation filter at 420 nm, barrier filter at 365 nm), the outer TE cells were identified by the pink fluorescence of propidium iodide, whereas the ICM cells were recognized by their blue fluorescence of the bisbenzimide. Embryos in which clear evaluation failed or that were disrupted during the staining procedure were discarded.

ICMs were isolated employing the same protocol as described above. After removal of the zona pellucida, incubation in trinitrobenzene sulfonic acid and anti-DNP BSA, lysis of the outer cells was induced via guinea pig complement diluted 1:4, but without propidium iodide. To ensure complete removal of adhering TE cells from the ICMs, only embryos in which all TE cells were evenly lysed were processed. The ICMs were liberated from lysed TE cells by gentle passage through a fine hand-drawn pipette. Finally, the isolated ICMs were washed three times in PBS supplemented with 0.1% PVA before being frozen at -80°C. Intact blastocysts from the same IVP run were sham-treated in parallel by removing the zona pellucida.

Determination of the Relative Abundance of Developmentally Important Gene Transcripts in Bovine Embryos

Poly(A)+RNA was isolated from single blastocysts or single isolated ICMs as described recently [26] and was used immediately for reverse transcription (RT), which was carried out in a total volume of 20 µl using 2.5 µM random hexamers (Perkin-Elmer, Vaterstetten, Germany). Prior to RNA isolation, 1 pg of rabbit globin RNA (BRL, Gaithersburg, MD) was added as an internal standard and was analyzed simultaneously. The rabbit globin mRNA represents a mixture of the {alpha}- and ß-chains derived from polyribosomes of reticulocytes and was purified via oligo-dT cellulose chromatography. No products were obtained when the transcripts for the gene of choice were analyzed. Globin with and without preparation (globin ± prep) served as recovery control. Previously, the validity of this approach had been proven and revealed efficient amplification of the globin standard and the RNA of choice [27]. The reaction mixture consisted of 1x RT buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, Perkin-Elmer); 5 mM MgCl2; 1 mM of each dNTP (Amersham, Brunswick, Germany); 20 IU RNase inhibitor (Perkin-Elmer); and 50 IU MuLV reverse transcriptase (RT) (Perkin-Elmer). The mixture was overlaid with mineral oil to prevent evaporation. The RT reaction was carried out at 25°C for 10 min, 42°C for 1 h, followed by a denaturation step at 99°C for 5 min and flash cooling on ice. Polymerase chain reaction (PCR) was performed with embryo equivalents (percentage of the volume from the RT reaction employing one embryo in a defined volume) as described in Table 1 from different embryos or ICMs generated in different IVP runs in a final volume of 50 µl of 1x PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl, Gibco BRL, Eggenstein, Germany); 1.5 mM MgCl2; 200 µM of each dNTP; and 1 µM of each sequence-specific primer (globin, 0.5 µM) using a PTC-200 thermocycler (MJ Research, Watertown, MA). To ensure specific amplification, a "hot start" PCR was employed by adding 1 IU Taq DNA polymerase (Gibco) at 72°C. PCR primers were designed from the coding regions of each gene sequence using the oligo program. The sequences of the primers used, the annealing temperatures, the fragment sizes, and the sequence references have been summarized in Table 1.


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  		TABLE 1. Primers used for PCR

The PCR program employed an initial step of 97°C for 2 min and 72°C for 2 min (hot start) followed by different cycle numbers (see Table 1) of 15 sec each at 95°C for DNA denaturation, 15 sec at different temperatures for annealing of primers, and 15 sec at 72°C for primer extension. Due to the very low amount of starting material (~5 ng RNA), 35–39 PCR cycles are required for an efficient amplification of embryonic transcripts [27]. The last cycle was followed by a 5 min extension at 72°C and cooling to 4°C. As negative controls, tubes were prepared in which RNA or reverse transcriptase was omitted during the RT reaction (data not shown).

The RT-PCR products were subjected to electrophoresis on a 2% agarose gel in 1x TBE buffer (90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8.3) containing 0.2 µg/ml EtBr. Further EtBr in the same concentration was added to the running buffer. The image of each gel was recorded using a CCD camera (Quantix, Photometrics, München, Germany) and the IP Lab Spectrum program (Signal Analytics Corporation, Vienna, VA). The intensity of each band was assessed by densitometry using an image analysis program (IP Lab Gel; Scananalytics, Fairfax, VA). The relative amount of the mRNA of interest was calculated by dividing the intensity of the band for each developmental stage by the intensity of the globin band for the corresponding stage. Experiments were repeated with at least 10 embryos or ICMs for each mRNA. The RA in the ICM isolated from blastocysts from that of contemporary intact blastocysts (RA TE = RA intact blastocyst RA ICM) in the same replicate IVP run. RA was calculated on a per cell basis for intact embryos or isolated ICMs, respectively, taking into account the average cell number found for blastocysts from Days 7 and 8. Blastocysts from the same IVP run were used either as intact embryos or after ICM isolation. The calculated repeatability (0.90) and the average accuracy (0.70) of the assay allowed calculation of statistically significant differences between treatment groups for each transcript from a minimum of eight replicates [28].

For each pair of gene-specific primers, semilog plots of the fragment intensity as a function of cycle number were used to determine the range of cycle number over which linear amplification occurred, and the number of PCR cycles was kept within this range [26]. Since the total efficiency of amplification for each set of primers during each cycle is not known, such assays can only be used to compare RAs of one mRNA among different samples [29]. This semiquantitative RT-PCR assay yields sensitive and highly reproducible results from pooled and single embryos and compares favorably with real-time PCR [30].

Statistical Analyses

Cell numbers and RAs were analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA) software package. After testing for normality (Kolmogorov-Smirnov test with Lilliefor correction) and testing for equal variance (Levene median test), an analysis of variance (ANOVA) followed by multiple pairwise comparisons using the Tukey test was employed. Differences of P <= 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Allocation of ICM Versus TE Cells in Expanded IVP Blastocysts at Day 7 or Day 8 of Development

A total of 30 Day 7 and 19 Day 8 expanded IVP blastocysts were differentially stained and possessed an average of 147 ± 6 and 146 ± 8 cells, of which 58 ± 4 and 64 ± 5 were allocated to the ICM, corresponding to an ICM/total cells ratio of 39 ± 2% and 43 ± 2%, respectively. No significant difference was detected between the two different groups. These average cell numbers were used when calculating the RA for intact embryos and ICMs on a per cell basis.

Spatial Expression of Transcripts

Representative gel photographs of a semiquantitative RT-PCR assay of the analyzed gene transcripts in single expanded bovine blastocysts from Day 7 or Day 8 of development and ICMs isolated thereof, as well as the corresponding globin bands, are shown in Figure 1.



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  		FIG. 1. Representative gel photographs of a semiquantitative RT-PCR analysis of various developmentally important gene transcripts in intact in vitro-produced bovine blastocysts ([A1], [A3], [B1], [C1]) or isolated ICMs ([A2], [A4], [B2], [C2]). Blastocyst expansion occurred in these embryos at either Day 7 ([A1], [A2], [B1], [B2]) or Day 8 ([A3], [A4], [C1], [C2]). Globin ± prep (globin + prep = globin present during RNA extraction procedure; globin - prep = globin RT-PCR without RNA extraction procedure) indicates the control of RNA recovery

As shown in Figure 2, the examined transcripts showed different patterns of distribution within the two cell lineages. Plako and IF{tau} transcripts were not detected in isolated ICMs, indicating that these mRNAs are restricted to the TE. Similar expression patterns within the ICM and TE were detected for Na/K, E-cad, ZO-1, and Glut-3 mRNA, whereas Dc II, Glut-1, and Glut-4 mRNA showed a higher (P <= 0.05) level of expression within the TE than in the ICM.



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  		FIG. 2. Distribution of the various transcripts within the two cell lineages (TE = dark gray, ICM = light gray) of Day 7 or Day 8 blastocysts

Effect of the Age of the Blastocyst During Expansion on the Relative Abundance of Developmentally Important Gene Transcripts

Alterations in mRNA abundances of the specific gene transcripts in relation to the occurrence of blastocyst expansion, either at Day 7 or Day 8, are depicted in Figure 3. Significant differences between TE cells from blastocysts that showed expansion at either Day 7 or Day 8 were determined for the RA of Na/K, E-cad, Dc II, Plako, and ZO-1 transcripts. The RA of Dc II, Glut-1, and Glut-4 were significantly decreased in the ICM compared with the TE at Day 7. Similarly, the RA of Na/K, Dc II, Glut-1, and Glut-4 at Day 8 of development was significantly decreased in the ICM versus TE. Interestingly, no differences were observed in ICMs originating from blastocysts expanded at either Day 7 or Day 8.



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  		FIG. 3. Effects of timing of blastocyst expansion on relative mRNA abundances (means ± SEM) in intact expanded bovine blastocysts from Day 7 (open bars) or Day 8 (black bars) as well as in ICMs isolated from Day 7 (light gray bars) or Day 8 (dark gray bars) blastocysts. The relative abundance of each mRNA in the TE of Day 7 (horizontal stripe) or Day 8 (vertical stripe) blastocysts was calculated by subtracting the amount of the ICM from that of the intact embryo. Bars with different superscripts within each gene transcript differ significantly ([A]:[B] ICM vs. TE in Day 7 blastocysts; [a]:[b] ICM vs. TE in Day 8 blastocysts; P <= 0.05). Significant differences (P <= 0.05) in the TE of blastocysts expanding at either Day 7 or Day 8 are indicated by an asterisk (*). The relative abundances have been calculated on a per cell basis for intact embryos and isolated ICMs as well


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study is the first to investigate the effects of timing of blastocyst expansion on the spatial expression pattern of genes specifically involved in compaction/cavitation, trophoblastic function, and glucose transport. No differences were observed in the cell number of blastocysts with an expanded blastocoele either at Day 7 or Day 8, indicating differences in the speed of developmental progress. Previously, Day 7 blastocysts had a significantly higher cell number than those appearing at Day 8 [2, 31, 32]. We have shown that bovine IVP-expanded blastocysts from Day 7 are superior in their in vivo survival after freezing and thawing than their Day 8 counterparts [10]. The cell numbers found in the present study for IVP embryos compared favorably with those determined for in vivo-derived expanded blastocysts [33], indicating that expanded blastocysts of excellent quality had been analyzed in the present study and no major differences between Day 7 and Day 8 embryos could be found. However, sex-related differences cannot be ruled out.

Accumulation and composition of fluid in the blastocoele during early murine embryonic development is critically regulated by Na/K-ATPase activity and is essential for differentiation of ICM and TE cell types [34]. In bovine embryos produced in vitro, transcripts encoding multiple isoforms of Na/K-ATPase are expressed throughout early development [35]. In the present study, Na/K transcripts were detected in the ICM and TE from expanded blastocysts, which is consistent with the finding of {alpha}1-subunit polypeptide in TE and ICM cells of bovine blastocysts [36].

E-cad-mediated cell to cell adhesion is associated with compaction [11, 37]. E-cad transcripts and protein were found in the ICM and TE of expanded bovine blastocysts [38, 39]. In the present study, mRNA for ZO-1 was found in both the TE and ICM cells of the bovine blastocyst. ZO-1 protein was detected as a continuous ring at the apical points of TE cell contact in bovine and murine embryos [39, 40]. ZO-1 mRNA within ICM cells may be inherited from the parent cell undergoing differentiation into TE or stem from de novo transcription.

In contrast to ZO-1, in the present study desmocollin II (Dc II) was derived predominantly from the TE cells of the bovine blastocyst. A similar expression pattern has been detected in murine embryos [41]. Desmocollin II is involved in the formation of desmosomal junctions that play a critical role in stabilizing the TE during blastocyst expansion [41, 42]. Plako 1 is a major component of the desmosomal plaque from stratified and epithelia [43] and is involved in desmosome organization and stability as well as in the regulation of desmosome assembly [44]. In expanded bovine blastocysts, Plako mRNA is restricted to the TE. Protein data are not yet available.

Bovine embryos begin to express IF{tau} as soon as the blastocyst forms [45]. IF{tau} production is primarily dependent on the presence of a functional TE [46]. These findings are consistent with the present results as no differences were detected in the RA of IF{tau} mRNA in expanded blastocysts produced either at Day 7 or Day 8. However, a negative relationship between early IF{tau} production and developmental competence has been reported [47]. Comparing in vivo- and in vitro-derived blastocysts, it was suggested that an early and high amount of IF{tau} mRNA indicates poor quality of the bovine embryo [16, 48].

Starting with the blastocyst stage, bovine embryos become dependent on aerobic metabolism and acquire the capacity to utilize glucose [17]. These changes in the role of glucose during preimplantation development indicate a close relationship between glucose metabolism and the expression of glucose transporters [48]. In the present study, we have shown that bovine Glut-1 and Glut-4 transcripts are expressed at higher levels in TE cells compared with ICM cells, whereas a similar expression pattern was found for Glut-3 transcripts in the two lineages. Glut-3 and Glut-4 were selected for the present study because recent data indicated that both transcripts are susceptible to environmental changes, suggesting a critical role in the formation and maintenance of the bovine blastocyst [19]. In the mouse, the well-orchestrated spatial expression pattern of glucose transporter mRNAs plays a critical role throughout preimplantation development [49]. Glut-1 protein was detected in the ICM and TE of murine blastocysts, whereas Glut-3 protein was exclusively found in TE cells, and Glut-4 was never determined in early murine and human embryo development [50–53]. Glut-8 expression and translocation are correlated with blastocyst survival in the mouse [54]. These data indicate that spatial glucose transporter expression is regulated in a species-specific manner. Murine and bovine embryos have a different temporal expression pattern with regard to glucose transporters [18, 52]. But it has to be taken into account that protein data are not yet available for preimplantation bovine embryos. Recently, we have shown that glucose transporter mRNA expression is affected by culture conditions with a decrease of Glut-1 and an increase in Glut-3 compared with in vivo embryos [16, 19, 28]. A persistent decrease in glucose transport may result in enhanced apoptosis that is potentially associated with fetal malformation or miscarriage [55, 56]. Experiments blocking Glut-3 expression via antisense oligodeoxynucleotides have shown that Glut-3 expression might have physiological functions additional to its activity in glucose transport in murine blastocyst formation [51].

The genes involved in compaction and cavitation analyzed in the present study show a significantly higher transcription in TE cells isolated from Day 8-expanded blastocysts compared with TE cells from Day 7 embryos, indicating that expression of these genes is correlated with the timing of blastocyst expansion. Increased transcription is indicative for stress response and poor quality of the early embryo [26]. The present results support this observation. Previously, it was shown that in vitro-produced expanded blastocysts appearing at Day 7 have a better cryosurvival than their in vivo- and in vitro-derived Day 8 counterparts [4, 10], which is likely attributed to a favorable ultrastructure of the TE [57–59]. These findings suggest a different inherent quality of expanded blastocysts from Day 7 versus those from Day 8.

A well-controlled expression of genes is essential for the regular formation of the two lineages of the blastocyst. Alterations in the expression pattern of leukemia inhibitory factor (LIF) and its receptor subunits in in vitro-produced bovine blastocysts have been detected and were associated with a perturbated status of early differentiation [25]. During blastocoele formation the LIF-LIF receptor system is downregulated probably to slow down proliferation and to allow enough time for the organization of cell differentiation [2, 32]. Perturbations in cell allocation in the blastocyst may lead to either early embryonic death or the formation of fetal anomalies associated with early abortions [60]. In cloned bovine blastocysts an aberrant allocation of ICM and TE cells has been determined [61]. A higher incidence of placental anomalies is observed in both early and late gestation and in pregnancies derived from cloned or in vitro-produced embryos [62], which may have originated from early perturbations in blastocyst formation. Similarly, expression of genes that were exclusively found in the TE was affected by in vitro culture and nuclear transfer [16, 28]. Recently, an unequal methylation pattern was found between ICM and TE regions, suggesting a widespread gene dysregulation in extra embryonic regions, thereby resulting in placental dysfunction [63]. Collectively, the above findings indicate that deviations from the normal spatial expression pattern early in development may have profound effects for the outcome of pregnancy.

In conclusion, the present study has identified genes that are exclusively expressed in the ICM or TE. Their expression was affected by timing of blastocyst expansion. Future studies employing in vivo IVP embryos from different culture systems or nuclear transfer-derived embryos will aid in elucidating whether in vitro culture or cloning alter the spatial expression pattern of genes in both cell lineages necessary for normal development. The need for further studies also arises from the numerous unsuccessful attempts to generate and cultivate true embryonic stem cells from the ICMs of preimplantation bovine embryos [64, 65]. The findings of the present study emphasize the critical role of an adequate spatial gene expression in preimplantation bovine embryo development.


   ACKNOWLEDGMENTS
 
The authors are grateful to K. Korsawe and S. Wilkening for their skilled technical assistance.


   FOOTNOTES
 
1 Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/12-2). Back

2 Correspondence: Heiner Niemann, Dept. of Biotechnology, Institute for Animal Science (FAL), Mariensee, 31535 Neustadt, Germany. FAX: 49 5034 871 101; Niemann{at}tzv.fal.de Back

Received: 9 October 2002.

First decision: 23 October 2002.

Accepted: 7 January 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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