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Symposium-Related Papers |
1 Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia, 5011 Australia
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(Received 14 December 2005;
accepted after revision 14 February 2006;
first published online 16 February 2006)
Corresponding author K. Kind: Department of Obstetrics and Gynaecology, University of Adelaide, Level 4, Maternity Building, The Queen Elizabeth Hospital, Woodville Road, Woodville, South Australia, 5011 Australia. Email: karen.kind{at}adelaide.edu.au
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Introduction |
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In addition, embryos appear to encounter a decreasing oxygen gradient as they travel from the oviduct to the uterus (Fischer & Bavister, 1993), with post-compaction blastocyst development occurring in the lower oxygen environment of the uterus. Exposure to a lower oxygen environment in vitro may be most beneficial during the post-compaction period. Decreasing the oxygen concentration, from 7 to 2%, during the compaction to blastulation stages of bovine embryo in vitro production, increases the development rate of grade 1 and 2 blastocysts (Thompson et al. 2000). Furthermore, this suggests that, in the cow, a 2% oxygen environment during the post-compaction period is beneficial for in vitro development. However, our studies suggest that the response of the embryo to oxygen environments less than 5% varies between species (Harvey et al. 2004; Kind et al. 2005). Culture of mouse embryos under 2% oxygen, during the post-compaction period, does not alter blastocyst development rates, when compared with 7 or 20% oxygen (Kind et al. 2005). However, mouse embryos cultured post-compaction under 2% oxygen have 3- to 4-fold higher levels of expression of the oxygen-regulated genes Slc2a1 (formerly known as Glut-1), Slc2a3 (formerly known as Glut-3) and Vegf, when compared with embryos cultured under 7 or 20% oxygen, or embryos developed in vivo (Kind et al. 2005). This contrasts with the bovine embryo, where Slc2a1 expression is increased by 30–50% in bovine embryos cultured at 2% oxygen post-compaction, when compared with embryos cultured under 7 or 20% oxygen, but is not different from in vivo developed embryos (Harvey et al. 2004). Thus, 2% oxygen represents a hypoxic environment for the mouse blastocyst, and is associated with perturbations in gene expression.
Perturbed gene expression in the mouse embryo in response to a low oxygen environment raises the question of whether this may be associated with longer-term consequences for subsequent development. Altered embryonic environments, including suboptimal culture conditions, have been implicated in adverse fetal and placental developmental outcomes (Fleming et al. 2004; Sjöblom et al. 2005). Therefore, the aim of this study was to determine the effects of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development. In addition, placental morphometry and gene expression were examined to further investigate placental development.
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Methods |
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For embryo culture, potassium simplex optimised media (KSOM) (Erbach et al. 1994) containing 0.2 mM glucose and modified to contain 0.05 mM phosphate (Biggers & McGinnis, 2001) and 100 µM EDTA was used (Kind et al. 2005). Fatty acid-free BSA was purchased from ICPbio, Auckland, New Zealand. L-Alanyl-L-glutamine (1.0 mM) (GlutaMax, Invitrogen, Carlsbad, CA, USA) was substituted for glutamine. All other reagents were from Sigma Chemical Company.
Embryo collection and culture
Female 3- to 4-week-old CBAB6F1 mice were injected subcutaneously at 16.00 h with 5 IU of equine chorionic gonadotropin (Folligon, Intervet, Boxmeer, Holland), followed 48 h later by 5 IU of human chorionic gonadotropin (hCG) (Pregnyl, Organon, Sydney, Australia). Following hCG injection, females were caged individually, overnight with a male of the same strain. Twenty-two hours post hCG females were humanely killed by cervical dislocation, and 1-cell embryos were collected by flushing the oviducts into Hepes-buffered handling media, containing 50 U ml–1 hyaluronidase. Cumulus cells were removed and embryos were washed twice in handling media and once in KSOM media. All procedures were approved by the Animal Ethics Committee, University of Adelaide.
Embryos were cultured in Petri dishes in 20 µl drops of modified KSOM medium under mineral oil in 37°C humidified atmospheres at 7% O2, 6% CO2, 87% N2 in modular incubator chambers (Billups-Rothenburg, Del Mar, CA, USA). Fifteen embryos were included per drop and embryos were randomly assigned to one of three separate Petri dishes in each experiment. After 48 h culture (
72 h post hCG), when embryos had reached the morula stage, dishes were transferred to 2, 20 or 7% O2, containing 6% CO2 and the balance N2, in modular incubators. Embryos were cultured for a further 40 h (112 h post hCG), to the blastocyst stage. In vivo developed blastocysts were collected from superovulated mice at 88 h post hCG injection.
Embryo transfers
Naturally ovulating female Swiss Albino mice (10–12 weeks of age), mated with vasectomised CBAB6F1 males, were used as recipients for embryo transfers. Mating was confirmed by the presence of a vaginal plug the following morning (day 1 of pseudopregnancy). Blastocyst transfers were performed on day 4 of pseudopregnancy. Recipients were anaesthetized by intraperitoneal injection of 2% Avertin (2,2,2-Tribromoethanol in 2-methyl-2-butanol, diluted to 2% solution in H2O; 0.015 ml (g body weight)–1, 0.3 mg (g body weight)–1), and level of anaesthesia was assessed by suppression of the withdrawal reflex and monitoring respiration rate. Blastocysts from one of the three oxygen treatment groups, or in vivo derived blastocysts, were randomly allocated to individual uterine horns of the female recipient, with six blastocysts transferred to each horn.
Fetal and placental analysis at day 18 of pregnancy
Embryo transfer recipients were humanely killed on day 18 of pregnancy by cervical dislocation. Number of viable or resorbing implantation sites was recorded. Fetuses and placentas were weighed and fetal crown–rump length was measured. Placentas were either immediately snap-frozen for later gene expression analysis, or fixed in 4% paraformaldehyde–2.5% polyvinyl pyrrolidone in 70 mM sodium phosphate buffer (pH 7.4) overnight at 4°C. The following day placentas were bisected sagittally, washed in four changes of PBS over 2 days, and processed and embedded in paraffin wax with cut surfaces down.
Thirty serial mid-sagittal 7 µm sections were cut and placed on 10 3-aminopropyltriethoxysilane-coated slides and the first slide with a full placental face was stained with Masson's trichrome, as previously described (Roberts et al. 2001, 2003; Sjoblom et al. 2005). Sections were examined on an Olympus BH2 microscope equipped with a Video Image Analysis system using Video Pro software (Leading Edge, Adelaide, Australia). The total cross-sectional areas of junctional and labyrinthine zones were measured, and expressed as a percentage of the total midsaggital area. Placentas from in vivo (n= 9), 20% (n= 11), 7% (n= 13) and 2% (n= 13) groups from 5 to 9 dams were analysed.
Placental labyrinthine morphometry
Placental sections were double immunolabelled, as previously described (Roberts et al. 2001, 2003; Sjoblom et al. 2005), with rat anti-mouse monoclonal antibodies MTS12 (kind gift of Professor Richard Boyd, Department of Pathology and Immunology, Monash University, Melbourne, Australia), to label murine fetal endothelial cells, and rabbit anti-human pan-cytokeratin (Zymed, San Francisco, CA, USA) to label trophoblast. A triple layer technique was used as previously described (Roberts et al. 2003; Sjoblom et al. 2005). Sections were deparaffinized and brought to water. Sections were incubated at 37°C for 15 min in 0.03% protease (Sigma) for antigen retrieval. Endogenous peroxidase activity was quenched by incubating with 6% hydrogen peroxide for 40 min, then sections were washed in three changes of PBS for 5 min each. Non-specific binding was blocked by incubation with phosphate-buffered saline containing 10% swine serum and 1% BSA (Sigma) for 10 min without washing.
Sections were then incubated overnight in a humidified chamber at room temperature with MTS-12 supernatant with 10% swine serum and 1% BSA. Sections were washed as before and incubated for 60 min in biotinylated rabbit anti-rat secondary antibody (DakoCytomation, Denmark; 1: 200 in PBS containing 10% swine serum and 1% BSA). Sections were washed again and streptavidin–horseradish peroxidase (Zymed) was added for 60 min. Following washing, sections were incubated with diaminobenzidine containing 2% ammonium nickel (II) sulphate (Sigma), to form a black precipitate.
Sections were washed as before and then incubated overnight as above with rabbit anti-human pan-cytokeratin diluted 1: 100 in PBS containing 10% swine serum and 1% BSA. The entire process was repeated as before, except that a biotinylated goat anti-rabbit antibody (1: 200, in PBS containing 10% swine serum and 1% BSA) was used, and nickel was omitted from the chromogen, leaving a brown precipitate. Negative controls, omitting the primary antibody, were included in all analyses.
The sections were examined on an Olympus Vanox AHBT3 microscope with a 2.5 x ocular lens and 20 x objective lens, equipped with a Video Image Analysis System with IP Laboratory Image analysis program. Volume densities of trophoblast, fetal capillaries and maternal blood space in the labyrinth were quantified by point counting with an isotropic L-36 Merz transparent grid placed on the monitor screen (Weibel, 1979). Ten fields (360 points) were counted in randomly selected sections for each placenta. The first field was chosen randomly and adjacent sections were systematically selected 0.5 mm apart using a stage micrometer. The volume density of each labyrinthine component was calculated as volume density, Vd=Pa/PT, where Pa is the total number of points falling on that component and PT is the total number of points counted (Weibel, 1979; Roberts et al. 2001, 2003). The surface density (surface area per gram of placenta) of trophoblast was measured by line intercept counting on the same grid and the same fields, and calculated taking into account the total magnification on the monitor using the formula: surface density, Sv= 2 xIa/LT, where Ia is the number of intercepts with the line, and LT is the total length of the lines applied (Weibel, 1979). The arithmetic mean barrier to diffusion was calculated as BT (barrier thickness) =Vd/SV, where Vd is the volume density of trophoblast, and Sv is the surface density of trophoblast. Mass of labyrinthine tissue (ML) was calculated as percentage labyrinth x placental weight (g). Surface area of trophoblast was then calculated according to the formula SA =SVxML cm2. Placentas from in vivo (n= 9), 20% (n= 10), 7% (n= 13) and 2% (n= 11) from 5 to 9 dams per group were analysed.
Placental gene expression
Frozen placentas were bisected and total RNA was extracted from half of each placenta using TriReagent (Sigma), according to the manufacturer's instructions. Final RNA pellets were redissolved in 50 µl sterile water, and samples were DNAse treated (DNA-free, Ambion, Austin, TX, USA) according to the manufacturer's instructions. RNA concentration was calculated from the absorbance at A260.
Total RNA (2 µg) from each placenta was reverse transcribed to cDNA using Superscript III reverse transcriptase (Invitrogen). RNA was combined with 250 ng random primers and 500 µM of each dNTP (Invitrogen) and incubated at 65°C for 5 min, followed by incubation on ice for 1 min. Reverse transcription was then performed in a final volume of 20 µl, containing First-Strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 5 mM DTT, and 200 U Superscript III. Reactions were incubated at 25°C for 10 min, 50°C for 60 min and 70°C for 15 min. cDNAs were stored at –20°C until use. RNA from an in vivo developed day 18 placenta was used to generate a pooled standard cDNA. Ten 2 µg RNA aliquots were reverse transcribed, pooled, then diluted to the equivalent of cDNA generated from 25 ng starting RNA per microlitre and stored as aliquots at –20°C.
Real-time PCR was performed in an Applied Biosystems 5700 GeneAmp Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Oligonucleotide primers were designed using Primer Express (Applied Biosystems) and were synthesized by GeneWorks, Adelaide, Australia. Primer sequence details are described in Table 1. PCR was performed in 20 µl volumes containing 1 x SYBR Green Master Mix (Applied Biosystems) and 500 nM forward and reverse primers. cDNAs were diluted in water to a concentration per microlitre equivalent to cDNA generated from 0.5 ng starting RNA. For Igf2, 2 µl diluted cDNA was analysed (1 ng equivalent); for Igf2r, H19, Slc2a1 and Slc2a3, 4 µl diluted cDNA was analysed (2 ng equivalent) and for Rn18S, 1 µl diluted cDNA was analysed (0.5 ng). The thermal cycling program consisted of 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. A cycle threshold (Ct) was calculated for each sample using the GeneAmp 5700 software. Dissociation curves, to detect non-specific amplification, were generated for all reactions, and the presence of a single amplified product was also confirmed by gel electrophoresis. No template samples containing water substituted in place of cDNA were included in all assays to confirm the absence of non-specific amplification products. Placentas from in vivo, 2% and 20% (all n= 10) and 7% (n= 11) groups from 6 to 8 mothers per group were analysed.
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All data are presented as mean ±S.E.M. Implantation and fetal development data were analysed by GLIM statistical package (version 4.0, National Algorithm Group, Oxford, UK) by general linear modelling using the log likelihood statistic. All other statistical analyses were carried out using SPSS for Windows (SPSS, Chicago). All comparisons between treatment groups were performed using one-way ANOVA. Litter size was included as a covariate in analysis of fetal and placental outcome measures as indicated in the text. Bonferroni post hoc tests were used for all comparisons between groups. Significance was accepted at P < 0.05.
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Results |
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Embryo development rates were assessed following culture for 48 h (development to morula) and 88 h (development to blastocyst) (Table 2). There were no differences between groups in overall development to the morula or blastocyst stages. Exposure to differing oxygen concentration during the post-compaction period did not alter development rates from morula to blastocyst (Table 2).
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Embryos were transferred to a total of 39 recipients. Twenty-seven of the recipients were pregnant in at least one horn at day 18 of pregnancy. Non-pregnant animals contained embryos from all treatment groups (2%: n= 7 horns; 7%: n= 7 horns; 20%: n= 8 horns; in vivo: n= 2 horns). These animals were not included in further analysis. In addition, two uterine horns that exhibited uterine adhesions were excluded from analysis. Therefore, a total of 52 uterine horns were studied (2%: n= 15; 7%: n= 14; 20%: n= 15; in vivo: n= 8). No implantation sites were detected in seven horns (2%: n= 1; 7%: n= 3; 20%: n= 1; in vivo: n= 2). Implantation rates were not different between culture groups or when compared with in vivo derived blastocysts (Table 3). However, development of a viable fetus following implantation (viable fetuses/blastocyst implanted) was reduced when embryos were cultured under 2% oxygen, compared with 7% oxygen (Table 3).
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Effect of oxygen concentration on fetal and placental weights
Fetal weight was reduced following embryo culture at 2% oxygen post-compaction, compared with culture at 7 or 20% oxygen (Table 3). Culture under 2% oxygen also reduced fetal weight when compared with embryos developed in vivo (Table 3). Culture under 7 or 20% oxygen did not alter fetal weight when compared with in vivo development (Table 3). Fetal crown–rump length was also reduced following embryo culture under 2% oxygen, when compared with 20% oxygen, or in vivo development (Table 3). Differences in fetal size remained significant when corrected for number of fetuses within the treatment horn, or total litter size for the mother (combined number of fetuses for both horns).
There was no difference in placental weights across all groups (Table 3). Fetal: placental weight ratios in the 2% oxygen treatment group were reduced when compared with 7 or 20% cultured embryos, but were not different from in vivo developed embryos (Table 3).
Effect of oxygen on placental structure
Oxygen concentration used during embryo culture did not significantly alter the mid-sagittal cross sectional total area or the proportion of labyrinthine or junctional zone of the placenta (Table 4).
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No differences were observed in expression of Slc2a1, Slc2a3, Igf2, Igf2r or H19, in day 18 placentas developed from embryos cultured under 2, 7 or 20% oxygen, or developed in vivo and transferred into pseudopregnant recipients (Fig. 1).
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Discussion |
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Post-implantation mortality in embryos cultured at 2% oxygen post-compaction was increased and the weight of viable fetuses was reduced following embryo culture at 2% oxygen. Thus, although similar numbers of embryos cultured under different oxygen conditions developed to blastocyst, perturbations associated with exposure to low oxygen impact on post-implantation viability. Reduced fetal weights have been observed previously in the mouse following embryo culture (Harlow & Quinn, 1979; Khosla et al. 2001a; Sjoblom et al. 2005). However, fetal weight was not reduced in embryos cultured at 7 or 20% oxygen in the current study, when compared with in vivo developed embryos. Others have reported a significant decrease in fetal development per blastocyst transferred and fetal development per implantation site when embryos were cultured at 20%, compared with 5% oxygen (Karagenc et al. 2004). However, unlike the current study, the exposure to different oxygen conditions commenced at the 1-cell stage in that study (Karagenc et al. 2004). Previous studies have demonstrated reduced blastocyst development rates when embryos are cultured under 20%, compared with 5 or 7% oxygen from the zygote stage (Pabon et al. 1989; Gardner & Lane, 1996; Orsi & Leese, 2001). Similarly, culture under 20% oxygen reduces blastocyst total cell number when commenced at the zygote or 2-cell stage, but not the 8-cell stage (Karagenc et al. 2004). Inner cell mass number was reduced by culture at 20%, compared with 5% oxygen, regardless of when the culture commenced, although these effects were most significant with treatment from the 1-cell stage (Karagenc et al. 2004). Nevertheless, no difference in fetal weights at day 15 gestation were observed following embryo culture for 95 h under 20%, compared with 5% oxygen (Karagenc et al. 2004). Similarly, culture of embryos from the 2-cell stage to blastocyst under 20% or 5% oxygen was not associated with differences in fetal or placental weight at day 18 of pregnancy (Harlow & Quinn, 1979).
Successful implantation and development of mouse embryos following exposure to oxygen concentrations ranging from 2 to 20%in vitro demonstrates that the pre-implantation embryo is able to adapt to significant variation in environmental conditions. Indeed, no difference in fetal weight was observed when embryos were cultured at 7 or 20% oxygen, or developed in vivo and then transferred. Variations in oxygen concentration were imposed during the post-compaction period in the current study, and the ability of the embryo to adapt to environmental perturbations, including oxygen, may be greater in the post- compared with precompaction embryo (Karagenc et al. 2004; Lane & Gardner, 2005; Zander et al. 2006). Increased expression of key genes in the blastocyst under low oxygen (Kind et al. 2005) demonstrates that the embryo can detect and respond to environmental change, and may be one mechanism through which the embryo acts to maintain metabolic and cellular homeostasis when exposed to perturbed environments (Lane & Gardner, 2005). Nevertheless, post-implantation viability was decreased by 2% oxygen culture, and while remaining embryos implanted and successfully underwent fetal and placental development, fetal, although not placental, weight was decreased. Whether these perturbations of fetal growth and development are associated with longer-term consequences for adult health (Barker, 1998; Gluckman & Hanson, 2004; McMillen & Robinson, 2005) remains to be determined.
Perturbations in embryonic metabolism may be one mechanism through which low oxygen influences embryonic viability and subsequent development. Alterations in gene expression in blastocysts cultured under 2% oxygen include a 3- to 4-fold increase in the expression of Slc2a1 (Glut-1) and Slc2a3 (Glut-3) (Kind et al. 2005) and glucose uptake is increased in embryos cultured under 2% oxygen, when compared with culture at 7% oxygen (Kind et al. 2005). Low oxygen also stimulates the expression of glycolytic enzymes (Semenza, 1999), although this has not been examined in the blastocyst. It has been suggested that up-regulation of embryonic metabolism is associated with a reduction in viability of the embryo (Leese, 2002). Mouse blastocysts with elevated glycolytic activity have a reduced ability to develop into a viable fetus (Lane & Gardner, 1996). However, higher rates of glucose uptake by the blastocyst are also associated with an increase in successful fetal development (Lane & Gardner, 1996; Gardner et al. 2001). Metabolic changes associated with culture under low oxygen, in the current study, may influence subsequent development; however, more complete analysis of metabolism in embryos cultured under 2% oxygen is required to assess this further. Blastocyst cell number is also associated with subsequent fetal viability (Lane & Gardner, 1997). Embryos cultured under 2% oxygen post-compaction have a reduction in total cell number in the blastocyst (Kind et al. 2005), which may also contribute to deficits in fetal development.
No significant difference in placental weights was observed after culture at different oxygen concentrations. However, fetal: placental weight ratio was reduced by post-compaction embryo culture at low oxygen. Previous studies have reported a decrease in the relative proportion of the placental labyrinth, the region responsible for exchange of substrates in the mouse placenta, and an increase in the relative proportion of junctional zones in placentas developed from cultured embryos (Sjoblom et al. 2005). No difference in the proportion of labyrinthine and junctional zones in the placenta following embryo culture was observed in the current study. Surface density of the trophoblast apical membrane available for exchange between maternal and fetal blood in the labyrinth was lowest in placentas from 2% cultured embryos, although this was only significantly different from placentas developed following embryo culture under 7% oxygen, and not when compared with in vivo developed embryos. Similarly, arithmetic mean barrier thickness of the trophoblast tended to be highest in placentas from 2% oxygen-cultured embryos but this difference was not significant. In contrast, others have reported reduced thickness and increased surface density of the trophoblast layer following in vitro embryo culture, when compared with in vivo developed embryos (Sjoblom et al. 2005). However, these results were obtained in a culture system utilizing 20% oxygen (Sjoblom et al. 2005). No differences were observed in placentas from embryos cultured at 20% oxygen, when compared with those developed in vivo, in the current study; however, other components of the embryo culture system may also influence placental development (Sjoblom et al. 2005). Thus, the current study suggests that alterations in placental structure may be induced by culture under different oxygen conditions; however, further analysis is required to assess this. In addition, the contribution of these changes to the decrease in fetal weight, observed following culture under low oxygen conditions, requires further investigation.
Expression of a selection of key placental genes was also examined in the current study. Placental expression levels of Igf2, Igf2r, H19 and the glucose transporter genes, Slc2a1 and Slc2a3 were not altered at day 18 following embryo culture under different oxygen conditions. A role for SLC2A1 (GLUT-1) in taking up glucose for use as a placental fuel, and for SLC2A3 (GLUT-3) in glucose transfer to the fetus has been suggested in the rodent placenta (Zhou & Bondy, 1993). IGF2 has been identified as an important regulator of placental growth (DeChiara et al. 1990; Baker et al. 1993; Constancia et al. 2002). Deletion of the Igf2 gene (De Chiara et al. 1990; Baker et al. 1993), or a placenta-specific transcript of Igf2, expressed predominantly in labryrinthine trophoblast (Constancia et al. 2002), is associated with retarded placental growth, and altered placental transfer function in mice (Sibley et al. 2004). In the current study, total Igf2 expression was measured, and analysis of expression from different promoters may be of further interest. In addition, Igf2, Igf2r and H19 are imprinted genes. Disruption of H19 imprinting has been reported in mouse blastocysts (Doherty et al. 2000; Mann et al. 2004), fetuses (Khosla et al. 2001a) and in the placenta (Mann et al. 2004) following culture of embryos in Whitten's medium (Doherty et al. 2000; Mann et al. 2004) or in the presence of serum (Khosla et al. 2001a). Similarly, disruption of imprinting of the Igf2, Igf2r and H19 gene, and altered expression of the P0 specific Igf2 transcript have been reported in association with placental overgrowth in placentae from cloned mice (Ogawa et al. 2003). In the current study no differences in expression levels of these genes were observed. However, specific methylation analysis is required to confirm the absence of imprinting defects. Nevertheless, it is possible that insults imposed on the embryo during the post-compaction period only, such as the low oxygen exposure in the current study, exert their effects on subsequent development through mechanisms such as altered gene expression, rather than perturbations in imprinting or epigenetic mechanisms.
Therefore, the current study has demonstrated that reducing the oxygen concentration used during the post-compaction period of embryo culture, to a level that represents a hypoxic stimulus for the mouse embryo, is associated with increased post-implantation mortality and reduced fetal weight. Although the mouse embryo was able to adapt to significant variation in oxygen concentration with no effect on placental weight or gene expression, fetal weight was decreased and some measures of placental structure were altered following embryo culture under 2% oxygen. Altered fetal development has been associated with adverse adult health outcomes, including increased risk of cardiovascular and metabolic diseases (Barker, 1998; Gluckman & Hanson, 2004; McMillen & Robinson, 2005). This study further supports the suggestion that perturbation of the embryonic environment can influence subsequent fetal, and possibly placental development, and thus may also have longer-term consequences for adult health (Khosla et al. 2001b; Ecker et al. 2004; Fleming et al. 2004; Sjoblom et al. 2005). The current results indicate that oxygen is an important component of the embryonic environment, and demonstrates that reductions in the oxygen level experienced by the embryo during the post-compaction period can alter both embryonic gene expression (Kind et al. 2005) and subsequent fetal development.
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