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Research Centre for Reproductive Health,4 Discipline of Obstetrics and Gynecology, Medical School, The University of Adelaide, Adelaide, South Australia 5005, Australia
Discipline of Agriculture and Animal Science,3 The University of Adelaide, Adelaide, South Australia 5005, Australia
ABSTRACT
Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction—with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.
developmental biology, early development, embryo, environment, gene regulation
The level of oxygen (O2) utilization by the preimplantation embryo is stage-dependent. An increased consumption coincides with the initiation of compaction and blastocyst formation, as these processes are energy demanding [1] and dependent on oxidative phosphorylation [2]. This increase in energy demand 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. Fischer and Bavister [3] reported oxygen concentrations significantly lower than atmospheric (20%) in the reproductive tract, with a concomitant reduction in O2 tension with passage from the oviduct to the uterus in the hamster, rabbit, and rhesus monkey. A similarly low O2 concentration is found in the human reproductive tract [4].
Oxygen concentration has a significant effect during embryo development in vitro. While there are studies that show no effect of oxygen during development [5, 6], the majority of reports reveal that lowered (5%–7%) concentrations enable greater numbers of embryos to develop to the blastocyst stage in vitro in several species [7–13]. Our laboratory has also provided evidence that further, albeit small, improvements in bovine embryo development and quality can be observed when O2 levels were reduced from 7% to 2% during postcompaction development [2, 14]. A small, yet significant, increase in the expression of glucose transporter 1 (SLC2A1, previously known as GLUT1) in bovine blastocysts cultured under 2% oxygen, relative to culture under 7% or 20% oxygen has been observed, which (at 2% oxygen) is not significantly different from SLC2A1 expression in in vivo-derived embryos [14].
Oxygen-regulated gene expression in somatic cells is largely mediated by the hypoxia-inducible factor (HIF) family of transcription factors [15], first identified in Hep3B cells under reduced oxygen conditions [16]. The DNA-binding complex is composed of two basic helix-loop-helix protein subunits, the constitutively expressed HIF1ß (also known as aryl hydrocarbon nuclear translocator [ARNT]), and the oxygen-controlled 
subunit, stable only in cells cultured under low-O2 conditions [17, 18]. Many genes respond to low-O2 conditions, including glucose transporters (e.g., SLC2A1, SLC2A3), glycolytic enzymes [19], insulin-like growth factors (IGF2), and nitric oxide synthases [20, 21]. Previous studies have examined the localization of HIF proteins to determine whether the effects of low oxygen during blastocyst development were under HIF-mediated regulation. While HIF1A and HIF2A mRNA were detected in bovine blastocysts, only HIF2 protein was detectable under low oxygen [14]. This suggested that regulation of gene expression by low oxygen in the bovine blastocyst is most likely mediated by HIF2 transcription factor activity, independent of the major O2-regulator HIF1. Moreover, differential display PCR comparing bovine embryos incubated in either 2% or 7% O2 during postcompaction development failed to reveal regulation of well-known HIF1-regulated genes but did establish anaphase-promoting complex (ANAPC1) and myotrophin (MTPN) as genes regulated by oxygen in bovine blastocysts [22].
Regulation of HIF alpha subunits involves prolyl and asparaginyl hydroxylation under normoxic conditions, which results in degradation of the protein. These reactions are dependent on the presence of 2-oxoglutarate, ascorbate, and iron [23]. Under reduced oxygen conditions, the residues remain unaltered, enabling stabilization of HIF1 and HIF2 alpha subunits, and regulation of oxygen-responsive genes. In somatic cells, an environment favorable to stabilization of the alpha proteins can be mimicked by the addition of factors, such as certain iron chelators that sequester iron and prevent the enzymatic modification that targets the protein for degradation. Stabilized HIF proteins are then able to bind to DNA and induce gene activation. The iron chelator desferrioxamine (DFO) has been used extensively in the determination of the regulatory pathways involved in oxygen signaling, and the responses of HIF-regulated genes [24–27]. Therefore, the addition of DFO to embryo culture may result in altered gene expression as observed in somatic cells.
Additional known HIF regulated-genes in the bovine blastocyst remain to be characterized. While in somatic cells most genes examined to date are proposed to be largely regulated by HIF1, few genes have been identified as specifically being regulated by HIF2. Mouse knockout studies, while variable depending on genetic background, suggest that antioxidant gene expression is perturbed in HIF2 null offspring [28]. Furthermore, the in vitro embryo culture environment has previously been associated with alterations in the gene expression of antioxidant enzymes [29].
Therefore, the purpose of this study was to determine the expression of key genes previously identified as regulated by HIF1 (such as lactate dehydrogenase, glucose transporter 1, inducible nitric oxide synthase, cyclooxygenase 2, and vascular endothelial growth factor), and potential HIF2 candidate genes, such as antioxidant enzymes, in blastocysts cultured under various oxygen atmospheres postcompaction. In addition, the expression of these genes, and others previously reported as regulated by oxygen in bovine blastocysts [14, 22], was examined in the presence or absence of the iron chelator DFO.
Unless otherwise stated, chemicals were obtained from Sigma-Aldrich (St Louis, MO).
In Vitro Oocyte Maturation, Fertilization, and Embryo Culture
Cumulus-oocyte-complexes (COCs) were recovered from antral follicles measuring 2–8 mm in diameter from abattoir-derived bovine ovaries. COCs were matured and fertilized as previously described [14]. Cumulus cells were removed by gentle pipetting 22 h postinsemination, and presumptive zygotes were transferred to 20-µl drops of Cook Bovine Cleave medium (modified SOFaa, Cook Australia, Eight Mile Plains, Queensland) and cultured under mineral oil at 38.5°C in 7% O2, 6% CO2, balance N2. All Cook Bovine (Wash, Fert, Cleave, and Blast) media are supplemented with BSA.
Experiment 1: Effect of Oxygen Concentration on HIF-Regulated Gene Expression
Compact morulae were randomly allocated on Day 5 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. Resulting blastocysts were collected on Day 7.
Experiment 2: Effect of Desferrioxamine on Gene Expression by Bovine Blastocysts
Day 5 compact morulae were randomly allocated to culture in the presence of 0 µM, 100 nM, or 1 µM desferrioxamine (DFO) under a 7% O2, 6% CO2, balance N2 atmosphere at 38.5°C until Day 7. Resulting blastocysts were collected on Day 7. Initial experiments utilized higher (1, 10, and 100 µM) concentrations.
RNA was extracted from pools of 35–40 bovine blastocysts (n = 6 pools per treatment) placed in 500 µl of TriReagent as previously described [14]. RNA was also extracted from bovine liver for standard curve generation. Liver RNA was analyzed by spectrophotometry to determine the concentration of RNA, using absorbance at 260 nm.
RNA from embryos was reverse transcribed into cDNA as previously described [14] following DNase-treatment (Ambion, Austin, TX). RNA suspended in 15 µl of PCR-grade water was incubated at 70°C with random hexamer primers (Boehringer Ingelheim, Germany), then quick chilled on ice, followed by addition of a mixture containing 1x reaction buffer, 10 mM DTT (Invitrogen), and 0.5 mM dNTPs (Applied Biosystems, CA), and incubated at 25°C for 10 min, then at 42°C for 2 min. For a final volume of 25 µl, 200 U of Superscript (Invitrogen) was added, and samples were incubated at 42°C for 50 min, followed by 15 min at 70°C to inactivate the enzyme. Resulting cDNA was stored at –20°C until required for real-time PCR analysis.
Primers for all genes were designed using Primer Express software (Applied Biosystems) according to specific requirements for real-time analysis. Primers were synthesized by Geneworks (Adelaide, South Australia). The sequences of the primers and the sizes of the expected PCR amplicon are shown in Table 1. Other primers not listed were as previously described [14, 22].
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Samples were analyzed by real-time 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. Each PCR was initiated with 2 min at 50°C, then 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 60 sec at 60°C. Each assay included duplicates of each sample or liver standard for each respective gene of interest, a no-template control, and a negative RT sample. Relative mRNA expression for the genes of interest was 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. Six pools of embryos recovered from independent IVP experiments were analyzed for each experiment. Dissociation curve analysis was performed to confirm the amplification of a single product.
Immunofluorescence of blastocysts, cultured in the presence or absence of DFO, was carried out as previously described [14]. Primary antibodies were obtained from Novus Biologicals, CO, (HIF1A polyclonal: 100–134, 1:50 dilution; HIF2A monoclonal: 100–132, 1:200 dilution) incubated with FITC-conjugated sheep-anti-rabbit IgG for HIF1A (AMRAD Biotech, Boronia, Australia); or goat anti-mouse IgG for HIF2A (EMD Biosciences, Calbiochem, CA). Embryos were counterstained with propidium iodide (10 µg/ml, for 5 min). Mounted embryos were 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.
Differential cell counts were performed as previously described [14, 30]. Briefly, expanded and hatched blastocysts were incubated in acid tyrodes followed by a brief wash in 4 mg/ml polyvinyl alcohol (PVA) in PBS (PBS/PVA). Zona-free embryos were then incubated in 10 mM trinitro-benzene-sulfonic acid in PBS/PVA and subsequently incubated with 0.1 mg/ml antidinitrophenol-BSA antibody (Molecular Probes, Eugene, OR) in SOF. Following complement-mediated lysis using guinea pig complement in KSOM, supplemented with 10 mg/ml propidium iodide (to stain the trophectoderm [TE]), embryos were placed in absolute ethanol containing 4 mg/ml bisbenzimide (Hoechst 33342; stains both the inner cell mass [ICM] and TE). Embryos were then whole-mounted in glycerol 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.
Real-time PCR data 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. Cell count data were analyzed by t-test. Differences were considered statistically significant at P < 0.05.
Expression of HIF1-Regulated Genes in the Preimplantation Bovine Blastocyst
Examination of known HIF1-regulated and potential HIF2-regulated genes in bovine blastocysts by real-time PCR was undertaken. The expression of lactate dehydrogenase A (LDHA) displayed a 4-fold increase under 2% oxygen, compared with 7% and 20% oxygen (P < 0.001; Fig. 1A). Assessment of expression of other genes known to be regulated by HIFs revealed no oxygen-mediated alteration in expression of aldose reductase (Fig. 1B), cyclooxygenase 2 (PTGS2; Fig. 1F), or inducible nitric oxide synthase (NOS2; Fig. 1G). Similarly, expression of the antioxidant enzymes CuZn- (Fig. 1C), Mn- (Fig. 1D) superoxide dismutase (SOD1 and SOD2, respectively) and glutathione peroxidase (GPX1; Fig. 1E) was not influenced by oxygen concentration postcompaction. Messenger RNA for the vascular endothelial growth factor receptor flt-1 was virtually undetectable in the bovine blastocyst regardless of the oxygen concentration used (data not shown).
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Desferrioxamine Treatment and Embryo Development
Development to the blastocyst stage in the presence of DFO concentrations above 1 µM significantly reduced the percentage of cleaved embryos developing to grade 1 and 2 blastocysts (P = 0.007; Fig. 2A). Development at lower concentrations was unaltered, although a small trend towards improved development was observed in the presence of 100 nM DFO (Fig. 2B). Total cell number was not significantly altered by the addition of 1 µM DFO postcompaction, compared with blastocysts cultured in the absence of DFO (Table 2). However, the number of cells in the TE was significantly reduced with the addition of DFO (P = 0.022; Table 2), while inner cell mass cell number was unaltered by the addition of DFO, leading to a significant increase in inner cell mass proportion (P = 0.001; Table 2).
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Regulation of Gene Expression Through the Addition of Desferrioxamine
The expression of the metabolic genes SLC2A1 and LDHA were significantly increased in the presence of 1 µM DFO (P = 0.046 and 0.012, respectively; Fig. 3, A and B, respectively), with 100 nM DFO-treated embryos exhibiting an intermediate, although not significantly different, level of expression of both genes. DFO treatment also increased the expression of MTPN and NOS2 mRNA, whereby expression was significantly increased in the presence of 1 µM DFO (P = 0.003 and 0.035, respectively; Fig. 3, E and F, respectively). Expression of ANAPC1, PTGS2, VEGF, and ß-actin were not significantly different in the presence or absence of DFO (Fig. 3, C, D, G, and H).
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HIF1A and HIF2A mRNA expression was compared between blastocysts cultured in the presence or absence of 1 µM DFO. HIF1A mRNA was not altered by the addition of 1 µM DFO. In contrast, mRNA expression of HIF2A was significantly reduced by the addition of 1 µM DFO to culture postcompaction (data not shown, P = 0.021).
Desferrioxamine and HIF Protein Localization
HIF1A protein was not detected in the bovine blastocyst in the presence or absence of 1 µM DFO (data not shown). HIF2 protein localized to the nuclei of blastomeres within both the ICM and trophectoderm in embryos cultured under 7% oxygen with or without DFO addition (Fig. 4). A tendency towards increased protein intensity was observed in the ICM of DFO treated blastocysts.
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The detrimental effect of atmospheric (20%) oxygen culture continues to be largely attributed to increased reactive oxygen species production. However, insufficient (or absent) induction of oxygen-sensitive transcription factors that mediate gene expression changes in response to alterations in external oxygen concentrations may be a determinant of reduced embryonic development and quality following culture at nonphysiological oxygen levels. Oxygen concentrations have a significant influence on gene expression patterns in both somatic cells [31], mouse embryos [32], and bovine embryos [14, 22]. Oxygen regulated gene expression in somatic cells is largely mediated through hypoxia-inducible transcription factor activity. However, while HIF2 protein is readily observable in bovine blastocysts, HIF1 protein, identified in somatic cells as the predominant responsive subunit to alterations in oxygen, has not been detected in bovine oocytes, early embryos, or blastocysts [14]. Moreover, specific target genes for HIF2 regulation have not been thoroughly investigated. Here, we have examined the degree to which exposure of bovine embryos to low oxygen, or an iron chelator, alters the expression of known HIF1 target genes, as well as genes proposed to be regulated by HIF2, to further investigate the molecular mechanisms involved in regulating development of preimplantation embryos under reduced oxygen conditions.
In the presence of oxygen and iron, proline residues in two degradation domains are modified by HIF prolyl hydroxylases, resulting in ubiquitination and degradation of HIF subunits [33]. Since both molecular oxygen and iron are elements required for this hydroxylation process, the actions of the prolyl hydroxylase enzymes can be inhibited by iron chelation, hence preventing the HIF proteins from being targeted for degradation. The iron chelator desferrioxamine (DFO) is commonly used in somatic cell studies of HIF regulation. In the present study, protein for HIF1A remained undetectable in the presence or absence of DFO (data not shown). Immunofluorescence confirmed HIF2A protein localization, with expression in DFO treated embryos being similar to that observed in embryos cultured in the absence of DFO (i.e., 7% oxygen only). A tendency towards increased HIF2A staining in the inner cell mass of DFO treated embryos was observed; however, further analysis is required to determine quantitatively whether levels of HIF2 protein are different. Studies have demonstrated that DFO can activate HIF-mediated transcriptional activity, even in the absence of increased protein stabilization [34], eliciting changes in gene expression through increased DNA binding [16, 34].
While iron chelators, including DFO, have been used extensively to assess the mechanism of regulation of HIFA proteins and their target genes in a number of cell types, recent studies suggest that the degree of inhibition of the prolyl hydroxylases may vary between iron chelators [35]. The activation of HIFs in response to both oxygen [36] and "mimic" substrates [37] is also likely to be cell-type specific. Additionally, limited overlap in the gene expression response to different iron chelators and hypoxia has been observed in recent studies of Hep3B cells [37] and mouse embryonic fibroblasts [38]. This data is supported by our observations that NOS2 mRNA expression is altered by DFO treatment, but unaffected by reduced oxygen conditions, and the reverse was observed for ANAPC expression in bovine embryos. It is also important to consider that many studies investigate the effects of exposing cells to short-term stimuli (18–24 hours or less), whereas embryos in the present study are exposed to oxygen or DFO for a period of 72 h. It is likely that the addition of DFO to culture results in conditions that increase HIF- DNA-binding activity, mimicking a more reduced (0%–1%) oxygen environment, which under conditions of 2% oxygen would not be as active. Indeed, increases in bovine blastocyst gene expression of SLC2A1, LDHA, and MTPN observed following DFO supplementation exceed that observed following culture under 2% oxygen conditions, supporting this proposal. Therefore, the specific mechanisms through which DFO influences HIF-mediated gene expression, in the current study, may be more complex than specific inhibition of the activity of the PHDs, and may involve changes in redox state components.
The absence of oxygen-mediated regulation of genes such as PTGS2 and NOS2, previously identified as HIF1-regulated genes in a range of somatic cells [39, 40], further support our earlier observations that bovine embryos lack active HIF1 machinery, where only moderate alterations in the expression of specific genes are detected. Identification of further HIF2 target genes has been difficult, particularly as few cells express only HIF2A, in the absence of the primary oxygen-regulatory factor HIF1. Efforts have largely focused on exploring functional redundancy with the production of HIF2A knockout mice, although the resulting phenotypes are highly dependent on the genetic background. Scortegagna et al. [28] suggested that antioxidant enzymes were regulated by HIF2A, as expression of antioxidant enzymes in HIF2A–/– offspring was impaired. In the present study, the expression of the superoxide dismutases (SOD1 and SOD2), as well as glutathione peroxidase, were unaltered by oxygen concentration, suggesting that at least in the bovine embryo, these genes are not specific targets of HIF2. This may again reflect species-specific, or mechanistic, differences in HIF regulation in the preimplantation embryo. Microarray analysis of bovine embryos may be a useful tool in further determining potential HIF2 specific targets. Relatively few HIF2A candidate genes were recently identified by microarray analysis of MCF7 cells that had been exposed to HIF2A siRNA [41]. A similar approach may be useful to determine the effects of elimination of HIF2A on bovine embryo development.
Increased expression of LDHA, SLC2A1, and MTPN in bovine blastocysts cultured under 2% oxygen further supports a role for oxygen-mediated gene expression as a mechanism through which oxygen influences embryo development. In the present study, lactate dehydrogenase A, which catalyses the interconversion of pyruvate and lactate with nicotinamide adenine dinucleotide (NADH) as a coenzyme, displayed a 2- to 4-fold increase in bovine blastocysts following 2% oxygen culture, relative to 7% and 20% oxygen culture. In anaerobic cells conversion of pyruvate to lactate regenerates NAD+ and is essential for continued glycolytic flux. Lactate is also essential for mitochondrial respiration [42]. Previous studies have established that expression of both LDHA mRNA [43] and protein are inducible by reduced oxygen in somatic cells [44, 45]. All five isoforms of lactate dehydrogenase have been detected in mouse embryos [46], suggesting that lactate dehydrogenases may have important roles during early embryo development. Recently, expression of LDHA-C mRNA has also been reported in bovine oocytes and embryos [47]. Furthermore, the addition of 1 µM DFO to culture also resulted in a significant increase in LDHA mRNA expression, consistent with observations where increased LDHA expression was observed in the presence of DFO in somatic cells [48]. Increased LDHA activity may be associated with an improved redox state, through the catalysis of NADH to NAD+ [49], and may therefore be an indicator of embryo quality. Oxygen regulation of SLC2A1, both in the present and previous study [14], and regulation by DFO in the present study further suggest that glycolytic metabolism of bovine embryos under reduced oxygen conditions, or with the addition of specific iron chelators, may be more active than in blastocysts cultured under higher oxygen concentrations or in the absence of DFO. Increased expression of SLC2A1 in the presence of DFO is also consistent with observations in somatic cells [50]. An increasing preference for glycolytic metabolism as development progresses is thought to occur [51], and our observations here support this. However, examination of energy metabolism of blastocysts would more accurately determine the influence of oxygen on glycolytic activity.
Myotrophin is a cytosolic protein that stimulates protein synthesis and myocardial cell growth associated with increased levels of hypertrophy marker genes [52], and also functions in regulating c-myc, c-fos, and c-jun mRNA levels, as well as gap-junction expression [53]. MTPN was identified as an oxygen-regulated gene following differential display PCR of bovine blastocysts [22]. Results of the present study further support an oxygen/HIF responsive activation of MTPN, whereby addition of DFO significantly increases MTPN mRNA expression in bovine blastocysts. Expression of MTPN has been observed in ovarian follicular and corpus luteum development in rats [54]. Yamakuni et al. [55] have also reported a role for MTPN in regulating Ca2+ oscillations. Further investigation of MTPNs role in bovine embryo development is required, particularly with respect to its involvement in gap junctional expression and calcium signaling roles.
The presence of 100 nM and 1 µM concentrations of DFO did not significantly affect development to the blastocyst stage, although there was an approximately 10% increase in the percentage of grade 1 and 2 blastocysts developing in the presence of 100 nM DFO. This is consistent with our observations, following culture under various oxygen concentrations, that development to the blastocyst stage is unaffected by alterations to our system [14] and is likely a result of the heterogeneity of oocyte populations, as well as the formulation of commercial culture media, which supports high development rates under control conditions (7% O2). Concentrations of greater than 1 µM DFO, however, significantly reduced development of grade 1 and 2 blastocysts, suggesting a potentially toxic effect at these concentrations. Iron chelators act as reducing agents, which form a key component of the cellular redox state. Oversupply of reducing substrates may result in reduced cellular function and changes in gene expression, resulting in reduced blastocyst development.
While development rates were not significantly altered by the addition of 100 nM or 1 µM DFO to culture, an increased percentage of ICM was observed. This is consistent with an increased percentage of ICM following low (2%) oxygen culture [14], potentially a predictor of improved posttransfer outcomes [56]. However, this is in contrast to observations by Fischer-Brown et al. [57], where reduced (5%) oxygen conditions led to a suggested beneficial increase in TE cell numbers and therefore a reduced inner cell mass proportion. However, Fischer-Brown et al. [57] assessed Day 9-hatched blastocyst cell numbers, at which point it is likely these embryos are initiating elongation, as compared with late Day 7 expanded and hatched blastocysts in our studies. Significantly, the proportions reported in their study for IVP-generated embryos far exceed that observed in our culture system, which are more consistent with that reported for in vivo derived embryos (34% ICM, [58]), particularly under 2% oxygen conditions. A more recent study by the same group demonstrates altered fetal and placental growth in response to oxygen culture [59], in the absence of obvious differences in development to the blastocyst stage [57], supporting a beneficial role posttransfer for reduced oxygen conditions.
In conclusion, the current study suggests that typical HIF-regulated genes are not influenced by alterations in the external oxygen environment in the bovine embryo, complementing previous observations that HIF1A protein is not detectable in blastocyst stage bovine embryos. It remains to be determined whether this is a functional absence, necessary for prolonged exposure to an in utero environment during elongation, or a culture-induced deficiency in the absence of other factors, such as growth factor support. Indeed, HIFs are responsive to nonoxygen stimuli [60]. Our data provide several lines of evidence that HIF2 is a regulator of the expression of MTPN, LDHA, and SLC2A1 in the bovine blastocyst, in response to both oxygen and the iron chelator DFO. Our data further support the hypothesis that reduced oxygen concentrations or activation of HIF through the addition of DFO postcompaction provides measurable changes in gene expression of some, but not all, oxygen-sensitive genes. Furthermore, small shifts in gene expression following low oxygen exposure, or more significant changes with the addition of 1 µM DFO, appear to have (if anything) more beneficial rather than detrimental effects on bovine embryos. Recently, Covello et al. [61] reported that Oct4 expression was regulated by HIF2A (but not HIF1A) in mouse embryos, through a HIF1A knockout/HIF2A knock-in strategy. This suggests a role for oxygen concentration in determining early differentiation events and supports our data that HIF2A activity may be linked to increases in the ICM cell population. Differences in the magnitude of gene expression changes between oxygen and DFO administration may reflect different underlying mechanisms within the HIF pathway that are yet to be elucidated. Redox regulation of HIFs has previously been proposed [49, 62]. Whether more significant differences in gene expression would be observed following even more prolonged culture (from the zygote stage) may further elucidate oxygen-mediated mechanisms of cellular control. Further work is underway to investigate other signaling mechanisms that may interact with the HIF regulatory pathway.
FOOTNOTES
1Supported by National Health and Medical Research Council Project Grant 157941. A.J.H. supported by TQEH Research Foundation Postgraduate Research Scholarship. 
Correspondence: 2Correspondence and current address: Department of Biological Sciences, University of New Orleans, 2045 Lakeshore Drive, New Orleans, LA 70122; and Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808. FAX: 225 763 3030; e-mail: ajharvey{at}uno.edu
Received: 9 November 2006.
First decision: 27 December 2006.
Accepted: 27 February 2007.
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