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BOR - Papers in Press, published online ahead of print May 26, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028639
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BIOLOGY OF REPRODUCTION 71, 1108–1119 (2004)
DOI: 10.1095/biolreprod.104.028639
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Oxygen-Regulated Gene Expression in Bovine Blastocysts1

A.J. Harvey2,3, K.L. Kind3, M. Pantaleon4, D.T. Armstrong3, and J.G. Thompson3

Research Centre for Reproductive Health,3 Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia Department of Physiology and Pharmacology,4 University of Queensland, St. Lucia, Queensland 4072, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIF{alpha} protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1{alpha}, HIF2{alpha}) was unaltered by oxygen concentration. HIF2{alpha} protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1{alpha} protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2{alpha}.

developmental biology, early development, embryo, environment, gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The level of oxygen (O2) utilization by the preimplantation-stage embryo is stage dependent, whereby increased consumption coincides with the initiation of compaction and with blastocyst formation, as these processes are energy demanding [1]. This increase is associated with an increased dependence on glycolysis, through increased glucose uptake and lactate production [1]. Alterations in the requirement for O2 by the preimplantation embryo coincide with theoretical reductions in the availability of O2, with the transition from the oviduct to the uterus. This agrees with the only adequate study of O2 concentration in the reproductive tract that demonstrated a concomitant reduction in O2 tension, with passage from the oviduct to the uterus in the hamster, rabbit, and rhesus monkey [2].

Oxygen concentration has a significant effect during embryo development in vitro. Several studies report that lowered (5–7%) concentrations enable greater numbers of embryos to develop to the blastocyst stage in vitro in several species [3–9]. Recently, it was shown that this is particularly evident postcompaction, as bovine embryo development and quality were further enhanced when O2 levels were reduced from 7% to 2% [10]. Nevertheless, other studies have failed to show any effect of O2 concentration during development [11, 12].

Hypoxia-inducible factors (HIFs) are heterodimeric DNA-binding complexes that modulate the expression of several genes and regulate adaptive responses to alterations in O2 [13]. The main complex is composed of two basic helix-loop-helix protein subunits, HIF1ß (also known as aryl hydrocarbon nuclear translocator or ARNT), which is constitutively expressed, and an alpha subunit that is stable only in cells cultured under low O2 conditions [14, 15]. Additional alpha subunits have been identified (HIF2{alpha}, HIF3{alpha}), which show varying degrees of homology with HIF1{alpha} [16, 17], although their exact functions and target genes, especially under reduced O2 conditions, remain elusive. HIF{alpha} proteins are continuously synthesized but are rapidly degraded by the ubiquitin-proteasome system under normoxic conditions [18, 19]. Ubiquitination is dependent on Fe2+ ions and can be inhibited with iron chelators [19, 20]. A variety of genes possess hypoxic response element sites, facilitating regulation by low O2 conditions, including glucose transporters (e.g., GLUT1, GLUT3), glycolytic enzymes [21], vascular endothelial growth factor (VEGF), insulin-like growth factor-II (IGF-II), and nitric oxide synthases (NOS) [reviewed in 22, 23]. The expression of several of these genes is known to be altered in in vitro-produced embryos [24–27]. Of particular importance to embryo development is the regulation of genes involved in glycolytic metabolism, pathways that have increasing importance during postcompaction stages [28, 29]. Furthermore, studies of HIF1a knockout mouse models have demonstrated embryonic lethality at Day 10.5 involving poor fetal and yolk sac vascularization [30, 31], while HIF2a knockouts die between embryonic Days 9.5 and 12.5, developing severe vascular defects in both the yolk sac and embryo proper [32], suggesting that HIFs are regulators of critical developmental processes associated with normal postimplantation embryo development. Additionally, expression of HIFs within placental tissues has been demonstrated to be involved in differentiation, particularly during the first trimester [33]. HIFs may therefore function as an important mechanism prior to, and following, implantation, for the induction of the correct cascade of genes required to support further development, which has particular relevance to the atmospheric conditions used during culture.

The aim of this study was to examine the expression of HIF subunits HIF1{alpha} and HIF2{alpha} and the oxygen responsive genes GLUT1 and VEGF by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and to examine protein localization of HIF1{alpha} and HIF2{alpha} by immunofluorescence in blastocysts cultured under low O2 conditions postcompaction. Additionally, analysis of developmental capacity and cell numbers within the trophectoderm and inner cell mass of blastocysts following the various O2 culture treatments were undertaken to determine the effect of O2 on development and cell allocation in our culture system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Oocyte Maturation, Fertilization, and Embryo Culture

Cumulus-oocyte-complexes were aspirated from antral follicles (2- to 8-mm diameter) from abattoir-derived bovine ovaries in Hepes-buffered Tissue Culture Medium-199 (TCM-199) (ICN Biomedicals, Costa Mesa, CA) supplemented with 50 µg/ml kanamycin (Sigma-Aldrich, St. Louis, MO), 50 µg/ml heparin (Sigma-Aldrich) and 4 mg/ml fatty acid-free bovine serum albumin (FAF-BSA) (ICN Biomedicals) and subsequently washed twice in Hepes-TCM199 supplemented with 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA) and 4 mg/ml FAF-BSA. Oocytes with an intact cumulus investment and homogenous cytoplasm were selected and matured for 24 h in bicarbonate-buffered TCM199 supplemented with 0.1 IU/ml hCG (Serono, Australia), 1 IU/ml recombinant human FSH (Organon, The Netherlands), 10% FCS, and 4 mg/ml FAF-BSA (ICP Bio, New Zealand) at 38.5°C under a 6% CO2 in air atmosphere. Fertilization was then performed in 500 µl of fertilization media (Cook Bovine Fert medium; Cook Australia, Australia), supplemented with 0.2 mM penicillamine (Sigma-Aldrich), 0.1 mM hypotaurine (Sigma-Aldrich), and 2 mg/ ml heparin (Sigma-Aldrich) [10]. Cumulus cells were removed by gentle pipetting 23–24 h postinsemination and presumptive zygotes were transferred to 20-µl drops of Cook Bovine Cleave medium (modified SOFaa, Cook Australia) and cultured under mineral oil at 38.5°C in 7% O2, 6% CO2, balance N2. On Day 5, compact morulae were randomly allocated to treatments of 2%, 7%, or 20% O2 (6% CO2, balance N2), in groups of 5–6 in 20-µl drops of modified SOFaa medium (Cook Bovine Blast medium; Cook Australia) at 38.5°C. All Cook Bovine (Wash, Fert, Cleave, and Blast) media was supplemented with 4 mg/ml FAF-BSA (ICP Bio). Resulting blastocysts were collected at Day 7 and treated for PCR analysis, immunofluorescence, or cell-count determination. Embryos were also assessed for quality at Day 7 according to the definitions presented in the Manual of the International Embryo Transfer Society.

Collection of In Vivo-Derived Bovine Embryos

In vivo-derived blastocyst-stage embryos were removed from mature Fresian and Limousin cows following superovulation using 400 mg of NIH-FSH-P1 (Folltropin-V, Bioniche Animal Health A/Asia Pty. Ltd., Australia) and artificial insemination using the same source of semen as for in vitro-produced embryos. Prior to FSH treatment, the estrous cycle of all cows was synchronized using a CIDR-B device (Inter-Ag, New Zealand). Embryos were removed by nonsurgical flushing procedures in EmCare medium (ICP Bio) on Day 7 following onset of estrus. A total of 69 in vivo blastocyst-stage embryos were recovered from 23 donor cows. Embryos were divided into three groups of 23 and placed in 500 µl of TriReagent (Sigma-Aldrich) and quick frozen for PCR analysis. Embryos were collected in compliance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and under the guidelines approved by The University of Adelaide Animal Ethics Committee.

RNA Extraction

RNA was extracted from pools of 23–33 blastocysts placed in 500 µl of TriReagent (Sigma-Aldrich). Additionally, RNA was extracted from bovine liver for standard curve generation. Chloroform separation was performed at 14 000 rpm for 10 min at 4°C following 15 min on ice. The aqueous phase was transferred to fresh tubes and mixed with 25 µl isopropanol (Sigma-Aldrich) to which 1 µg of GlycoBlue (Ambion, Austin, TX) was added and centrifuged for 10 min at 4°C at 14 000 rpm. The supernatant was then transferred to a fresh tube containing 225 µl isopropanol and stored at –80°C overnight to precipitate.

Isolated RNA was Dnase treated to eliminate contaminating DNA according to the manufacturer's specifications (Promega Corporation, Australia). RNA was then re-extracted using phenol and chloroform:isoamyl alcohol and precipitated using 2.5x the volume of the aqueous phase with 100% ethanol and 0.1x the volume of the aqueous phase with 3 M sodium acetate, pH 5.2.

Reverse Transcription

RNA from embryos was reverse transcribed into cDNA following pelleting, washing of RNA with 75% ethanol, drying of the pellet, and resuspension in 11 µl PCR-grade water. Liver RNA was analyzed by spectrophotometry to determine the concentration of RNA at 260 nm. RNA was incubated at 70°C with random (hexamer) primers (Roche Applied Science), then quick chilled on ice, followed by addition of a mixture containing 1x reaction buffer, 10 mM dithiothreitol (Invitrogen), and 0.5 mM dNTPs (Applied Biosystems, Foster City, CA) and incubated at 25°C for 10 min and then 42°C for 2 min. For a final volume of 20 µl, 200 U of Superscript (Invitrogen) was added to samples and incubated at 42°C for 50 min, followed by 15 min at 70°C to inactivate the enzyme. The cDNA samples were then stored at –20°C until required.

RT-PCR and Confirmation of PCR Products

Aliquots of the reverse transcription reaction were amplified with 1U Taq polymerase (Qiagen, Australia) in a final volume of 50 µl containing 1x buffer, 1.5 mM MgCl2, 10 pmol of each sequence-specific primer (refer to Table 1) and 10 mM of each dNTP. The mixture was overlaid with mineral oil and then amplified for 40 cycles in a PTC-100 thermal cycler (MJ Research Inc., Waltham, MA), where each cycle included denaturation at 94°C for 1 min, reannealing primers to target sequences for 1 min at 60°C, and primer extension at 72°C for 1 min followed by a final extension at 72°C for 7 min after the 40th cycle. PCR products were analyzed by electrophoresis through 2% agarose gels containing 0.5 µg/ ml ethidium bromide and were photographed using Kodak Digital Science software (Eastman Kodak Co., Rochester, NY). To confirm the identity of the PCR products, the DNA bands were excised and purified using a QIAquick gel purification kit (Qiagen) according to the manufacturer's instructions. DNA was then sequenced using an Automated Sequencer ABI 373 XL, with DYEnamic ET Terminator chemistry (Applied Biosystems).


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  		TABLE 1. Primer sequences designed using Primer Express (Applied Biosystems), expected product length, GenBank sequence details, and cycle threshold (Ct) ranges for each gene

PCR Primers

Primers were designed using Primer Express (Applied Biosystems) according to specific requirements for real-time analysis. Primers were obtained from Geneworks (Adelaide, SA, Australia). The sequences of the primers and the sizes of the expected PCR amplicon are shown in Table 1.

Real-Time PCR

Samples were analyzed by real-time RT-PCR using an ABI-PRISM 5700 Sequence Detection System (Applied Biosystems). Standard curves were generated using serial dilutions of 100 ng/µl liver cDNA. Reactions were undertaken using SYBR green Master Mix (Applied Biosystems), as a double-stranded DNA-specific fluorescent dye, with the appropriate primer set. PCR was initiated with 2 min at 50°C, then 10 min at 95°C. The program continued with 40 cycles of 15 sec at 95°C and 60 sec at 60°C. Each assay, for each respective gene of interest, included duplicates of each cDNA sample (single embryo equivalents) or liver standard, a no-template control, and a negative RT sample. The parameter Ct (cycle threshold) is defined as the cycle number at which fluorescence intensity exceeds a fixed threshold. Relative mRNA expression for the genes of interest were calculated using the standard curves produced from the serial liver dilutions. The expression of 18S rRNA was used to normalize samples for the amount of cDNA used per reaction. ß-Actin, previously reported to respond to oxygen in some cell types and not others [34], was also analyzed. Four to six pools of embryos recovered from independent in vitro-embryo production (IVP) experiments were analyzed. Resulting real-time RT-PCR products were analyzed by gel electrophoresis. Dissociation curve analysis was also performed to confirm the amplification of a single product.

Immunofluorescence

Blastocysts, cultured under varying O2 concentrations (2%, 7%, or 20%), were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature and washed three times with PBS before placing on Cell-Tak (Beckton Dickinson Biosciences, Franklin Lakes, NJ)-coated coverslips. Embryos were then permeabilized in a 0.25% Triton-X in PBS solution, then washed briefly in PBS. To minimize nonspecific binding, embryos were incubated for 1 h at 25°C in blocking solution (1% Tween 20 [ICN Biomedicals], 0.5% BSA, in PBS containing 5% serum, according to secondary antibody host). Following application of the primary antibody (HIF1{alpha} polyclonal, 1:50 dilution; Novus Biologicals 100-134, Littleton, CO; HIF2{alpha} monoclonal, 1:200 dilution; Novus Biologicals 100-132) overnight at 4°C in humidified chambers, embryos were washed in PBS and exposed for 1 h at 25°C to fluorescein isothiocyanate (FITC)-conjugated sheep-anti-rabbit IgG for HIF1{alpha} (AMRAD Biotech, Boronia, Australia); or goat-anti-mouse IgG for HIF2{alpha} (EMD Biosciences-Calbiochem, San Diego, CA) diluted 1:400 with blocking solution. Embryos were counterstained with propidium iodide (10 µg/ml, for 5 min). Coverslips were mounted on cavity slides in glycerol, following brief exposure to 20%, 50%, and 80% (v/v) glycerol in PBS, and examined using a Bio-Rad MRC-1000 confocal laser scanning microscope mounted on a Nikon Diaphot 300 inverted microscope with a 40x water immersion objective. Controls were performed by 1) omission of the primary antibody, 2) omission of the secondary antibody, and 3) omission of both the primary and secondary antibodies. A minimum of 10 blastocysts were stained per treatment from each of six independent IVP replicates.

Differential Staining

Differential cell counts were performed using a modified technique of that reported by Sjöblom et al. [35]. Briefly, expanded/hatched blastocysts were incubated in acid tyrodes (either briefly for hatched blastocysts or for an extended period of time to remove zona of expanded blastocysts), followed by a brief wash in 4 mg/ml poly-vinyl alcohol (PVA) in PBS (PBS/PVA) (Sigma-Aldrich). Zona-free embryos were then incubated in 10 mM trinitro-benzene-sulfonic acid (Sigma-Aldrich) in PBS/PVA on ice for 10 min. Embryos were subsequently incubated with 0.1 mg/ml anti-dinitrophenol-BSA antibody (Molecular Probes, Eugene, OR) in SOF at 37°C for 10 min. Following complement-mediated lysis using guinea pig complement (Sigma-Aldrich) in KSOM, supplemented with 10 µg/ml propidium iodide (Sigma-Aldrich) for 20 min at 37°C (to stain the trophectoderm [TE]), embryos were placed in absolute ethanol containing 4 µg/ ml bisbenzimide (Hoechst 33342, Sigma-Aldrich) at 4°C overnight (stains both the inner cell mass [ICM] and trophectoderm). Embryos were then whole mounted in a drop of 80% glycerol in PBS on microscope slides, coverslipped, and sealed with nail polish. Embryos were examined under a fluorescence microscope (Olympus, Japan) equipped with an ultraviolet filter with a digital camera attached to determine total and compartment cell counts.

Statistical Analysis

Real-time PCR data and cell counts were analyzed by ANOVA using SigmaStat software (SPSS Inc., Chicago, IL), and significant differences between means were determined using the Tukey-Kramer post-hoc test for comparison of multiple means. All developmental and cell proportional data were arcsine transformed prior to analysis. Differences were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preliminary PCR Analysis of Bovine Blastocysts

RT-PCR analysis of bovine IVP embryos using HIF1{alpha}, HIF2{alpha}, and HIF1ß primers revealed an amplified cDNA fragment of appropriate size (Fig. 1, a, b, and c, respectively), consistent with observations in bovine arterial endothelial cells ([36], Fig. 1). Bovine liver, ovary, and follicular cells also displayed the amplification product. As a result, to ensure that blastocyst samples were not contaminated with cumulus cells, all samples were assayed for the amplification of CYP19 (aromatase; data not shown). The presence of transcripts specific for all other genes listed in Table 1 was also verified in bovine embryos (Fig. 1d). Purification and sequencing of all PCR products confirmed the identity of the expected cDNA fragments. Controls performed by using non-reverse-transcribed RNA as a template did not reveal any amplification products.



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  		FIG. 1. Gel electrophoresis illustrating RT-PCR products of HIF1{alpha} (a), HIF2{alpha} (b), and HIF1ß (c) for bovine arterial endothelial cells (AEC), liver (LIV), ovary (OV), granulosa (G), cumulus (CC) cells, and blastocysts (BL). d) ß-actin (A, B), GLUT1 (C, D), CYP19 (E, F), VEGF (G, H), and 18S rRNA (I, J) for bovine blastocysts (A, C, E, G, I) and liver (B, D, F, H, J). For fragment sizes, refer to Table 1

Oxygen-Responsive mRNA Expression in the Preimplantation Bovine Blastocyst

The relative abundance of mRNA encoding HIF1{alpha}, HIF2{alpha}, GLUT1, VEGF, and 18S rRNA was measured by real time RT-PCR using the standard curve method. All data are presented as mean ± SEM arbitrary units, normalized to 18S rRNA. The expression of GLUT1 was increased in bovine blastocysts following 2% O2 culture, when compared with 7% and 20% O2 (P < 0.01, Fig. 2a). VEGF expression followed a similar trend in vitro, where relative mRNA abundance tended to be increased following culture under 2% O2, compared with 7% and 20% O2, although not significantly (P = 0.081, Fig. 2b). Comparisons between groups indicated that VEGF expression was higher in embryos cultured under 2% O2, compared with those cultured under 20% O2 (P < 0.01). Furthermore, regressional analysis determined a significant association between VEGF expression and O2 concentration (P = 0.05). HIF1{alpha} and HIF2{alpha} mRNA expression was unaltered by O2 concentration (Fig. 2, c and d, respectively). ß-Actin mRNA expression was unaltered by O2, and was not significantly different from the level observed within in vivo-derived blastocysts (Fig. 2e). Expression of these genes by in vivo-derived embryos was highly variable. Expression of VEGF, HIF1{alpha}, and HIF2{alpha} by in vitro-produced embryos did not differ from that observed in in vivo-derived embryos. However, GLUT1 expression was significantly lower following in vitro culture under 7% and 20% O2, when compared with both in vivo-derived embryos and embryos cultured under 2% O2 (Fig. 2a).



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  		FIG. 2. Real-time PCR analysis of GLUT-1 (A), VEGF (B), HIF1{alpha} (C), HIF2{alpha} (D), and ß-actin (E) mRNA following in vitro culture (n = 4–6 pools of embryos; white bars) under 2%, 7%, or 20% oxygen or derived in vivo (n = 3 pools of embryos; grey bars). a and b, Different letters above bars indicate significant differences (P < 0.05)

HIF{alpha} Protein Localization Following In Vitro Culture

The presence of HIF protein was determined by immunolocalization using specific antibodies raised against the HIF1{alpha} and HIF2{alpha} subunits. All epifluorescence data were detected using the same microscope settings, and visual assessment made on the intensity observed in merged images (degree of yellow coloration). HIF1{alpha} (Fig. 3) was undetectable in in vitro-produced bovine blastocysts, regardless of the O2 concentration used postcompaction (2%, Fig. 3, a–c; 7%, Fig. 3, d–f; 20%, Fig. 3, g–i). HIF2{alpha} protein was detected in in vitro-produced bovine blastocysts cultured under all O2 conditions, predominantly localized to the nucleus (Fig. 4). Increased nuclear HIF2{alpha} protein localization was evident following postcompaction culture under 2% O2, particularly within the inner cell mass cells (Fig. 4, a– c), in comparison with control (7%) cultured blastocysts (Fig. 4, d–f). In contrast, blastocysts cultured under 20% O2 postcompaction displayed increased cytoplasmic localization in addition to reduced staining within inner cell mass cells (Fig. 4, g–i) compared with blastocysts cultured under either 2% or 7% O2 postcompaction. All controls performed were negative (Fig. 5). Mature bovine and mouse spermatozoa were also stained as a further positive control and clear staining was evident in the midpiece, as previously described ([37], data not shown).



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  		FIG. 3. Absence of HIF1{alpha} protein (undectable FITC staining; b, e, h) in bovine blastocysts cultured under a 2% (a–c), 7% (d–f), or 20% (g–i) oxygen atmosphere postcompaction, with propidium iodide staining of nuclei for each blastocyst (a, d, g), and merged images (c, f, i) presented. Original magnification x600



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  		FIG. 4. Localization of HIF2{alpha} protein in bovine blastocysts cultured under a 2%, 7%, or 20% oxygen atmosphere postcompaction (b, e, h, respectively), with propidium iodide staining of nuclei for each blastocyst (a, d, g), FITC-labeled HIF2{alpha} protein (b, e, h), and merged images (c, f, i) presented. Original magnification x600



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  		FIG. 5. Negative control staining, by omission of the primary antibody (a–c) or omission of the secondary antibody substituted with nonimmune serum (d–f), of bovine blastocysts (b, e), with propidium iodide staining of nuclei for each blastocyst (a, d) and merged images (c, f) presented. Original magnification x600

Following observations that HIF1{alpha} protein could not be detected in bovine blastocysts after exposure to 2% O2, bovine compacting morulae were cultured in the presence or absence of 1 µM desferrioxamine (DFO) under a 7% O2 atmosphere. DFO is an iron chelator [38] that has been extensively used in HIF protein studies, as it interferes with hydroxylation of HIF protein normally required for ubiquitination targeting under normoxic conditions, thereby stabilizing HIF{alpha} subunits [20, 39, 40]. HIF1{alpha} protein remained undetectable in bovine embryos cultured under a 7% O2 atmosphere (Fig. 6, a–c), and in the presence of DFO (Fig. 6, d–f). HIF2{alpha} protein was detectable following culture in the presence of 1 µM DFO (Fig. 7, d–f), with nuclear localization of protein in both the inner cell mass and trophectoderm, contrasting with the nuclear localization predominantly in the trophectoderm of blastocysts cultured under 7% O2 (Fig. 7, a–c).



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  		FIG. 6. Absence of HIF1{alpha} protein (b, e) in bovine blastocysts cultured in the presence (d–f) or absence (a–c) of DFO postcompaction, with propidium iodide staining of nuclei for each blastocyst (a, d) and merged images (c, f) presented. Original magnification x600



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  		FIG. 7. Localization of HIF2{alpha} protein (b, e) in bovine blastocysts cultured in the presence (d–f) or absence (a–c) of DFO postcompaction, with propidium iodide staining of nuclei for each blastocyst (a, d), FITC-labeled HIF2{alpha} protein (b, e), and merged images (c, f) presented. Original magnification x600

To establish a positive control for immunofluorescent detection of HIF{alpha} proteins, bovine cumulus-oocyte complexes were a) matured as described for 24 h (control), b) matured for 20 h under normal conditions (20% O2), followed by 6 h under anoxic atmosphere conditions, or c) followed by 6 h in the presence of 1 µm DFO. Immunolocalization of HIF1{alpha} and HIF2{alpha} protein was then undertaken.

HIF1{alpha} was readily detected in cumulus cells of control-matured oocytes, localized to the cytoplasm (Fig. 8, a–c). However, nuclear localization of HIF1{alpha} was evident in cumulus cells following acute anoxic culture (Fig. 8, d–f) and in the presence of DFO (Fig. 8, g–i). Likewise, HIF2{alpha} protein was detected in the cytoplasm of cumulus cells of control-matured oocytes (Fig. 9, a–c); however, nuclear localization was evident following acute anoxic culture (Fig. 9, d–f) and in the presence of DFO (Fig. 9, g–i). HIF{alpha} proteins remained undetectable in the oocyte, regardless of treatment.



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  		FIG. 8. Localization of HIF1{alpha} protein in bovine cumulus-oocyte complexes matured under normal (20% O2) conditions (a–c), matured for 20 h under normal conditions followed by a 6-h exposure to anoxia (d–f) or a 6-h exposure to DFO (g–i), presented as propidium iodide staining of nuclei (a, d, g), FITC-labeled HIF1{alpha} protein (b, e, h), and a merged image of both channels (c, f, i). Original magnification x600



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  		FIG. 9. Localization of HIF2{alpha} protein in bovine cumulus-oocyte complexes matured under normal (20% O2) conditions (a–c), matured for 20 h under normal conditions followed by a 6-h exposure to anoxia (d–f) or a 6-h exposure to DFO (g–i), presented as propidium iodide staining of nuclei (a, d, g), FITC-labeled HIF2{alpha} protein (b, e, h), and a merged image of both channels (c, f, i). Original magnification x600

Effect of Oxygen Atmosphere on Development and Cell Numbers

The development of embryos to the blastocyst stage, from cleaved, was not affected by O2 concentration (Table 2). Blastocysts cultured under 20% O2 tended to have higher total cell numbers; however, no significant difference was observed between the three O2 concentrations (Table 3) with an average of 177.8 ± 6.7 cells per blastocyst. This total cell number is comparable with that reported by Van Soom et al. [41] for in vivo-derived expanded and hatched blastocysts. In contrast, allocation to the trophectoderm and inner cell mass compartments of the blastocyst was significantly altered. Blastocysts produced following 2% O2 culture had proportionately more ICM cells than those produced following 7% and 20% O2 culture (P < 0.05, Table 3). Conversely, allocation to the trophectoderm was significantly increased after culture under 7% and 20% O2 (P < 0.05). Although the increase in ICM proportion following 2% culture tended to reflect a decrease in mean trophectoderm cell number, there was no statistically significant difference in mean ICM (P > 0.05) and TE (P > 0.05) number between treatments.


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  		TABLE 2. Development of in vitro-produced embryos following culture under different oxygen treatments from 23 independent IVP replicates (one-way ANOVA).*


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  		TABLE 3. Number of total, ICM, and TE cells (mean ± SEM) of Day 7 expanded and hatched blastocysts following culture under different oxygen concentrations from four independent IVP replicates


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Reduced developmental capacity of mammalian embryos following culture under atmospheric conditions has often been attributed to an increase in reactive oxygen species, leading to detrimental effects on development through increased oxidative stress and DNA damage [42, 43]. However, to date, no studies have addressed the molecular mechanisms whereby effects of O2 concentration on embryo development are mediated. Results of this study indicate that the bovine blastocyst has the ability to detect and respond to low O2 conditions by altering expression levels of GLUT1. Parallel alterations in HIF2{alpha} protein localization within the inner cell mass suggest that alterations in gene expression and embryo quality may be a HIF2{alpha}-mediated effect. Additionally, blastocyst quality is significantly altered by O2 concentration. Therefore, O2 is a mediator of gene expression in the bovine preimplantation embryo and a contributor to embryo quality, in terms of cell differentiation, following in vitro culture.

In the present study, analysis of mRNA expression in the bovine blastocyst revealed that relative GLUT1 mRNA abundance significantly increased following 2% O2 culture. In other studies, GLUT1 mRNA levels have been found to differ between in vivo- and in vitro-derived blastocysts, with bovine in vivo-derived embryos expressing significantly higher levels of GLUT1 mRNA than those produced in vitro under 7% O2 conditions [25], although Lazzari et al. [44] observed comparable GLUT1 mRNA expression regardless of origin. Moreover, further studies in our laboratory have shown that the addition of DFO, used to stabilize HIF{alpha} protein, postcompaction significantly increased GLUT1 mRNA levels in the bovine blastocyst [45]. Glucose becomes the predominant energy substrate postcompaction, where uptake is regulated through facilitative glucose transporters, including GLUT1, GLUT2, and GLUT3. Pantaleon et al. [46] and Moley [47] have demonstrated the importance of glucose transporters as regulatory molecules critical for early embryo development, where downregulation results in decreased cell numbers and increased apoptosis.

VEGF mRNA expression has not previously been reported in bovine blastocysts, and its role during early development has not yet been elucidated. Previous studies have reported expression of VEGF in the mouse [48], both in vivo and in vitro, and the human [49, 50] blastocyst. The specific role of VEGF expressed by the blastocyst is unknown, but it has been suggested that it may be important for induction of angiogenesis during early implantation in the human embryo [49, 50]. However, the involvement of VEGF in fetal development is particularly evident from the resulting embryonic lethality in mouse knockout models [51].

In the present study, the level of VEGF mRNA tended to be higher following 2% O2 culture in vitro; however, it was not significantly different from that observed following culture in other O2 concentrations. Direct comparison between 2% and 20% O2 cultured blastocysts indicated that expression of VEGF was increased by culture under 2% O2. The level of VEGF mRNA in vivo was considerably lower than may have been expected and not significantly different from VEGF levels observed in in vitro-produced embryos. This may be due to the high instability (rapid degradation) of VEGF mRNA under atmospheric O2 levels, which is well established [52]. The mRNA levels of VEGF within in vivo-derived embryos, as reported here, may not accurately reflect absolute levels, as it is likely that mRNA degradation occurred with exposure to air-equilibrated flushing and handling solutions for up to several hours during in vivo embryo recovery. The current study suggests that there is a minimal degree of O2 regulation of VEGF mRNA in bovine blastocysts. However, it is important to note that the primers designed to amplify VEGF were constructed to identify regions common to all isoforms, therefore masking any individual effects on specific isoforms. A focus on the isoforms VEGF121, VEGF165, and VEGF189, which are known to be highest in abundance in reproductive tissues [53–55] and human blastocysts [50], may be required. Similarly, investigation of the VEGF receptors flt-1, known to be regulated by O2 [56, 57], and flk-1, recently shown to be activated by HIF2{alpha} [58], would help elucidate specific roles of VEGF in bovine blastocysts.

Consistent with previous reports in somatic cells [59, 60], no difference in the expression of HIF1{alpha} or HIF2{alpha} mRNA was observed following exposure to different O2 concentrations. This is likely due to the regulation of HIFs by O2 primarily at the posttranscriptional level. DNA binding and protein levels of the HIF1{alpha} subunit increase exponentially as cells are exposed to decreasing O2 [61]. Although maximal expression of HIF1{alpha} protein occurs under conditions of 0.5% O2 [61], protein stabilization has been previously reported at 2% O2 in a number of cell lines [61]. In the present study, HIF1{alpha} protein was undetected by immunofluorescence staining in bovine blastocysts, regardless of the O2 concentration used. Moreover, supplementation of culture media with DFO, a compound that normally stabilizes the HIF{alpha} protein, failed to elicit HIF1{alpha} protein stabilization in the bovine blastocyst.

The results described here are in marked contrast with observations in mouse blastocysts, where HIF1{alpha} protein is detectable in in vivo-derived blastocysts (unpublished results), with HIF2{alpha} protein expression yet to be examined in vivo, while both HIF1{alpha} and HIF2{alpha} protein are detectable following culture under 2%, 7%, and 20% O2 (unpublished results). These results suggest a species-specific difference in the mechanisms of protein stabilization. It remains to be determined if HIF1{alpha} stabilization in the bovine blastocyst is regulated by significantly reduced (lower than 2%) O2 concentrations or alternative mechanisms. Conversely, in the mouse, pronounced responses to O2, with 3- to 4-fold increased expression of GLUT-1 and VEGF in blastocysts, following postcompaction culture under 2% O2, compared with embryos cultured under 7% or 20% O2 or developed in vivo, have been observed [62]. Such responses in the mouse blastocyst are significantly larger than observed here for bovine embryos.

In contrast, nuclear localization of HIF2{alpha} protein was readily detected at the blastocyst stage in the bovine embryo, with increasing intensity following postcompaction culture under 2% O2. The presence of DFO similarly increased HIF2{alpha} nuclear protein localization within the inner cell mass of blastocysts (Fig. 7). More apparent was a reduction in HIF2{alpha} protein within the inner cell mass with increasing O2 concentration (Fig. 4). Furthermore, DFO treatment and anoxic culture for the final 6 h of oocyte maturation resulted in both HIF1{alpha} and HIF2{alpha} protein nuclear localization within cumulus cells, compared with normal matured (20% O2) oocytes, consistent with increased nuclear accumulation under these conditions. Results of the present study are consistent with previous observations where detection of HIF2{alpha} protein, in the absence of HIF1{alpha} protein, has been observed in distinct populations of cells within a variety of tissues, in response to reduced oxygen [63, 64].

It remains unclear if the absence of detectable HIF1{alpha} protein, despite detectable levels of HIF1{alpha} mRNA, is an artifact of culture or a real phenomenon. A variety of stimuli can induce the expression of HIF1{alpha} protein, including a range of cytokines and growth factors, as well as redox molecules [reviewed in 22]. In vitro culture systems, including that used in the present study, commonly lack growth factors and other mitogenic stimuli known to be important components of the reproductive-tract environment. Therefore, the lack of HIF1{alpha} protein expression in the present study may reflect a deficiency of the culture environment, where a more dynamic milieu is required by the embryo to stimulate HIF1{alpha} synthesis and stabilization. Conversely, whether HIF2{alpha} expression by the bovine embryo is influenced by the presence or absence of growth factors and other stimuli in the in vitro environment remains to be determined. The collection and analysis of in vivo-derived embryos is required to examine this further. However, collection of in vivo-derived embryos under reduced O2 conditions remains a considerable technical and financial challenge.

An alternative explanation is that bovine embryos have mechanisms to either prevent HIF1{alpha} protein translation, or a nonhypoxic mechanism for HIF1{alpha} protein degradation. This raises two immediate questions: 1) Why would bovine (and not mouse embryos) require a mechanism to prevent HIF1{alpha} stabilization? 2) When does HIF1{alpha} stabilization occur during bovine embryo development? One possible answer to the first question is that, unlike mouse embryos, bovine (indeed, all ruminant) embryos following hatching remain unattached to the endometrium and undergo significant development without the assistance of a developing functional endometrial exchange interface for nutrient transfer such as the hemochorial placental unit. The answer to the second question requires further examination, but it is tempting to speculate that HIF1{alpha} is likely to play a role in angiogenesis associated with yolk sac and/or allantoic development. With this in mind, it is of interest that recent studies demonstrate severe developmental abnormalities in placental development, including vasculature, associated with in vitro culture of bovine embryos [65]. Further investigation characterizing the timing of HIF1{alpha} protein stabilization in posthatching development in utero of embryos derived from both in vivo and in vitro systems may prove fruitful in further unraveling the nature of in vitro culture-induced abnormalities.

In the current study, development of grade 1 and 2 blastocysts of cleaved embryos was not significantly altered by the postcompaction O2 concentration used, which contrasts with the results of Thompson et al. [10], although development rates in the present study, particularly that of control (7% O2) cultured embryos, were higher than those reported by Thompson et al. [10]. Total cell number was not affected by any postcompaction O2 treatments and numbers were comparable with those reported by Van Soom et al. [41] for in vivo-derived expanded and hatched blastocysts. Significantly, the proportion of total cells in the inner cell mass or trophectoderm was altered by O2 concentration. The proportion of cells within the inner cell mass were significantly higher following 2% O2 culture, compared with culture under 7% or 20% O2. Increased allocation to the inner cell mass is thought to be associated with improved posttransfer outcomes [66]. These results suggest that O2-regulated gene expression has an effect on inner cell mass development, where changes in the localization of HIF2{alpha} protein were most apparent. That is, results suggest that the inner cell mass cells, rather than the trophectoderm, are most affected by the change in O2 postcompaction. Findings of differential localization of HIF2{alpha} between the inner cell mass and trophectoderm under differing O2 levels are also consistent with the proposal that large mammalian embryos would develop an O2 gradient from the outside to the center of the blastocoel cavity, whereby the inner cell mass is exposed to a more reduced O2 concentration [67], particularly with lower external O2 concentrations [68].

Results of the present study suggest that environmental changes in O2 concentration regulate the gene expression pattern of the O2-sensitive gene GLUT1, with physiological consequences also evident in cell allocation. Results support the hypothesis that a reduced O2 concentration postcompaction is beneficial for bovine in vitro-produced embryos. Genetic responses may be mediated by HIF2. The expression of other genes, in addition to GLUT1, with important roles in the regulation of embryonic development and metabolism, may also be activated following culture under low O2 conditions. However, as HIF2{alpha} target genes are largely unknown, this is difficult to assess. Further studies are required to determine the range of genes regulated by O2 in the preimplantation bovine embryo and to compare these results with a greater sample of in vivo-derived embryos. Although HIF1ß mRNA expression was investigated in the current study, further investigations are also required to demonstrate the presence of the HIF1ß protein subunit in the bovine blastocyst. Additionally, protein localization of HIF proteins in in vivo-derived embryos requires further study. However, the necessity to collect embryos under low O2 conditions to maintain HIF protein localization is a major hurdle.

Hypoxia-inducible factor 2 is suggested to act as a possible mediator of responses of the preimplantation embryo to environmental changes in oxygen concentration through alterations in gene expression, with physiological consequences also evident in cell allocation. Thus, oxygen-regulated gene expression is a potential mechanism through which the oxygen concentration of the preimplantation environment can influence embryo quality. Importantly, oxygen is a physiological stimulus for the embryo. Results of this study suggest that HIF2 may facilitate oxygen-dependent gene expression during bovine preimplantation embryo development, over the levels of oxygen to which embryos were exposed in this study. Furthermore, the bovine embryo presents a model of oxygen-mediated gene expression through HIF2, while HIF1{alpha} was undetected.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Shane Ashworth (Total Livestock Genetics, Camperdown, Victoria) for assistance with the collection of fresh in vivo-derived bovine embryos. The authors would also like to thank Dr. Daniel Peet and Dr. Murray Whitelaw (School of Molecular and Biomedical Science, University of Adelaide) for helpful discussion.


   FOOTNOTES
 
1 Supported by a National Health and Medical Research Council Project Grant (157941) and by a TQEH Research Foundation Postgraduate Research Scholarship to A.J.H. Back

2 Correspondence. FAX: 61 8 82227521; alexandra.harvey{at}adelaide.edu.au Back

Received: 19 February 2004.

First decision: 11 March 2004.

Accepted: 25 May 2004.


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 MATERIALS AND METHODS
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 DISCUSSION
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