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research-article |
Human Reproduction Unit, Disciplines of Physiology3 and Medicine,4 University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
ABSTRACT
The expression of TRP53 in blastocysts that had been cultured from the zygote stage in vitro for 90 h was compared with that in blastocysts collected from the uterus in C57BL6 (B6) and in F1 hybrid (B6CBF1) strain mice. In both strains, there was little TRP53 detected in blastocysts collected from the uterus. There was some increased expression in cultured embryos from B6CBF1 mice and marked increased expression in cultured B6 blastocysts. In cultured B6 embryos, there was obvious accumulation of TRP53 within the nuclear region of embryonic cells. Cultured B6 zygotes had significantly poorer rates of blastocyst formation and of capacity to undergo implantation or form viable fetuses than cultured zygotes from B6CBF1 mice or B6 blastocysts collected from the uterus. Trp53–/– zygotes (B6 background) were significantly more likely to form blastocysts than sibling wild-type embryos, with Trp53+/– embryos having an intermediate level of viability (P < 0.01). On transfer of blastocysts to recipient females, Trp53–/– blastocysts were more likely to form viable fetuses than wild-type or heterozygous sibling blastocysts when the embryos resulted from culture of zygotes (P < 0.001). This shift in viability did not occur when embryos were only subjected to 24 h of culture from the compacted embryo stage. Culture in vitro in the B6 strain caused a marked increase in the expression and nuclear accumulation of TRP53. This expression was a significant cause of the loss of viability that occurs on culture of zygotes from this strain in vitro.
assisted reproductive technology, cell survival, early development, embryo, implantation, in vitro fertilization, Mdm2, TRP53, zygote
The use of animal models, particularly the mouse, has contributed much to our knowledge of the processes of preimplantation embryo development. These models have been particularly useful for development of embryo culture techniques. In the mouse, it is well known that embryo manipulation and culture result in a characteristic phenotype of slow embryo development in vitro [1]. After several days in culture, embryos typically have fewer cells and more cells within each embryo undergoing death than corresponding-stage embryos collected from the reproductive tract [2–4]. On transfer of cultured mouse embryos to the reproductive tract, there is commonly a characteristic reduction in the rate of formation of normal fetuses [5]. This phenotype is particularly severe within some inbred strains of mice [6].
The underlying cause of this phenotype remains to be defined. A number of cellular stressors induced by the culture environment have been identified and include growth and survival factor deprivation [4, 7], metabolic and substrate imbalances [8, 9], oxidative stress [10], and genotoxic damage [11]. In somatic cells, all such stresses are capable of activating the transformation-related protein 53 (TRP53) stress response pathway. TRP53 is a transcription factor that has many functions [12], including reducing the rate of cell cycle progression (e.g., by the induction of cyclin-dependent kinase inhibitors such as Cdkn1a) and inducing cell death (by the synthesis of prodeath mediators such as Bax). Trp53 mRNA is expressed in mouse [13] and human [14] preimplantation embryos. In human embryos produced by in vitro fertilization, its expression was higher in embryos with poor morphology following culture, as assessed by the degree of cytoplasmic fragmentation [15]. There is much anecdotal evidence that transgenic overexpression of Trp53 is incompatible with early mouse embryo development, such that no ubiquitously expressing TRP53 transgenic lines have been reported, to our knowledge. The loss of TRP53 latency (due to the deletion of Mdm2 [16–18]) also results in early mouse embryonic lethality. This can be overcome by simultaneous deletion of Trp53 [16–18]. Furthermore, diabetes-induced early embryopathy was partially ameliorated by Trp53 deletion in a mouse model [19]. These lines of evidence suggest that levels of Trp53 expression may affect embryo viability. However, it is generally recognized that expression of TRP53 is largely regulated after transcription [20]. Therefore, assessment of any potential effect of TRP53 expression on embryo viability should be performed at the level of protein expression.
This study examines the effects of zygote culture on the expression of TRP53 in embryos that are highly susceptible to culture stresses (C57BL6 [B6]) compared with those that are resistant (F1 hybrid [B6CBF1]). Culture in vitro induced the expression of TRP53 in both strains but to a much higher degree in B6. The inhibition of TRP53 by its genetic deletion (B6 background) reduced the loss of embryo viability following culture. In embryos that are susceptible to some loss of viability following embryo culture, this study shows that an effector of this reduced viability is the expression of TRP53 in the embryo.
The use of animals was in accord with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and was approved by the institutional animal care and ethics committee. The strains of mice used in experiments were the following: B6, B6CBF1 (B6 x CBA/He and CBA/He x B6), Trp53–/–, Trp53+/–, Trp53+/+ (B6.129S2-Trp53tm1Tyj strain extensively backcrossed with B6), and Quackenbush outbred strain. The genotype of all Trp53 knockout mice was confirmed by PCR before use in experiments. All animals were housed and bred in the Gore Hill Research Laboratories, St. Leonards, NSW, Australia, under a 12L:12D cycle and had access to food and water ad libitum. Four to eight-wk-old females were superovulated by intraperitoneal injection of 10 IU of eCG (Folligon; Intervet International, Boxmeer, the Netherlands) followed 48 h later by 10 IU of hCG (Chorulon; Intervet International). Females were paired with males of proven fertility. Pregnancy was confirmed by the presence of a copulation plug the following morning (Day 0.5).
Mouse Embryo Collection and Culture
Embryos were flushed from the reproductive tract with Hepes-buffered modified human tubal fluid medium (Hepes-HTF) and were cultured in modified HTF medium (mod-HTF) [7]. All components of the media were tissue culture grade (Sigma Chemical Company, St. Louis, MO) and contained 3 mg/ml of BSA unless otherwise stated (CSL Ltd., Melbourne, Victoria, Australia). Zygotes were collected 20–21 h after hCG injection and were freed from their cumulus cells by brief exposure to 300 IU of hyaluronidase (Sigma Chemical Company) in Hepes-HTF. Embryos were recovered in minimal volume and were assigned to various treatments as required in mod-HTF. Embryos were cultured in 10-µl volumes in 60-well human leukocyte antigen plates (LUX 5260; Nunc Inc., Naperville, IL) overlaid by approximately 2 mm of heavy paraffin oil (Sigma Chemical Company). Embryos were cultured in groups of 10. Culture was at 37°C in 5% CO2 for the periods indicated in individual experiments.
The developmental stage and morphology of embryos were assessed by visualizing the embryos using an inverted phase-contrasted microscope (Diaphot; Nikon, Kanagawa, Japan) at 24-h intervals after zygote collection. After culture, cell counts and integrity of nuclei were assessed by visualization of cell nuclei following staining with 4 µg/ml of Hoechst dye 33342 (Sigma Chemical Company). Embryos were left in this solution for 40 min and were then prepared as wet mounts on a glass microscope slide under a coverslip. Nuclei were visualized using mercury lamp ultraviolet illumination and epifluorescence on a Nikon Optiphot microscope with an Olympus DPlanApo 40 ultraviolet objective. Individual nuclear morphology was categorized as normal (round, ovoid, or mitotic) or as abnormal (punctuate, pyknotic, or fragmented).
Morphologically normal, zona pellucida-intact blastocysts were used in embryo transfer experiments. Groups of 10 blastocysts were surgically transferred to each uterine horn on Day 2.5 in pseudopregnant Quackenbush outbred recipient female mice, as previously described [21]. Nine days after embryo transfer, recipients were killed, and the numbers of implantation sites and viable fetuses were identified. The fetus, yolk sac, and placenta were isolated.
Whole preimplantation-stage embryos, yolk sacs of viable fetuses (it was impossible to genotype aborting fetuses because of the extensive infiltration of maternal leucocytes), or 5-mm tails from 3–6-wk-old mice were used for genotyping. Preimplantation embryos were thoroughly washed 3 times; the zona pellucida was removed by incubation using 0.5% (w/v) pronase (Sigma Chemical Company) in PBS and was then washed a further 3 times in sterile 1x PBS solution. Embryos were subjected to several rounds of freeze thawing in liquid nitrogen. Yolk sac and tail tissue had DNA extracted using the BuccalAmp DNA Extraction Kit (Epicentre, Madison, WI). DNA was then subjected to PCR genotyping. Three primers were used (SigmaGenosys Australia Pty. Ltd., Castle Hill, NSW, Australia), including two sense-oriented primers from exon 6 of Trp53 (5' AT GAG CCA CCC GAG GTT) and the neomycin gene (5' TCC TCG TGC TTT ACG GTA TC) and an antisense primer from exon 7 of Trp53 (5' TAT ACT CAG AGC CGG CCT).
Western blot was performed as previously described [22]. Embryos were collected and washed 3 times in cold PBS and transferred in a maximum volume of 1.5 µl of PBS into 1.5 µl of 2x extraction buffer supplemented with protease and phosphatase inhibitors (2x PBS, 2% [v/v] Triton X-100, 24 mM deoxycholic acid, 0.2% [w/v] SDS, 20 mM NaF, 20 mM Na4P2O7, 2 mM PMSF, 3.08 µM aprotinin, 42 µM leupeptin, and 2.91 µM pepstatin A, all from Sigma Chemical Company). The embryos were lysed by three cycles of freezing in liquid nitrogen and thawing (with vortexing). Protein samples were diluted with 1 µl of 5x Laemmli buffer (50 mM Tris-HCl, 5 mM EDTA [pH 8.0], 12.5% [w/v] sodium dodecyl sulfate, 0.05% [w/v] bromophenol blue, and 25% beta-mercaptoethanol), incubated 10 min at 60°C and size separated using 20% homogenous SDS PAGE (Pharmacia, Uppsala, Sweden) on a PhastSystem separation and control unit apparatus (Pharmacia) or using minigels (Bio-Rad Laboratories, Hercules, CA). Proteins were blotted into polyvinylidene fluoride membranes (Hybond-P; Pharmacia) in a semidry blotting apparatus overnight using transfer buffer (12 mM Tris [pH 7.0], 96 mM glycine, and 20% [v/v] methanol). Nonspecific binding was blocked by 5% [w/v] skim milk in PBS supplemented with 0.05% [v/v] Tween-20 (PBST) at room temperature for 1 h. Membranes were probed overnight at 4°C in 5% skim milk in PBST with primary antibody. A second horseradish peroxidase-conjugated antibody was applied for 1 h at room temperature. Membranes were developed using Femto Maximum chemiluminescent substrates (Pierce, Rockford, IL) for 5 min at room temperature. To reduce the background while maintaining maximum signal intensity, the Femto Maximum chemiluminescent substrate was diluted 1:2 using PBST. The membranes were dried briefly on 3-mm paper and were exposed under transparency to an x-ray film (CL-XPosure Film; Pierce) for various times.
The primary antibody was 1:200 anti-TRP53 (Ab-7) polyclonal antibody (PC35; Oncogene Research Products). Analysis was performed on groups of 30 embryos.
Embryos were washed 3 times in PBS using 0.1% (w/v) BSA, 0.1% (v/v) Tween-20, and 0.2% (w/v) sodium azide (washing buffer) and were then fixed using freshly prepared 2% (v/v) paraformaldehyde (Sigma Chemical Company) in PBS (pH 7.4) for 30 min and permeabilized with 2% paraformaldehyde using 0.3 % Tween-20 (Sigma Chemical Company) at room temperature for 30 min. Embryos were washed 3 times in washing solution and were then were blocked in PBS containing 2% BSA and 30% serum for 3 h. They were stained overnight at 4°C using primary antibodies (1:300 Ab-7 polyclonal antibody [PC35; Oncogene Research Products]) or an equivalent concentration of isotype control immunoglobulin (negative control). Primary antibody was detected by incubation of embryos using secondary antibodies coupled to fluorescein isothiocyanate in PBS with 2% BSA for 1 h at room temperature. Optical sectioning was performed using a Bio-Rad Radiance confocal microscope with a Nikon PlanApo 60x/1.4 oil emersion objective, as previously described [23]. Images were captured using Lasersharp 2000 version 4.0 (BioRad Laboratories). Microscope and laser settings were adjusted such that no fluorescence was observed using nonimmune controls. All test specimens were observed using these same settings.
Statistical analyses were performed using SPSS statistical package version 11.5 (SPSS Inc., Chicago, IL). The proportion of embryos developing to a given developmental landmark following culture in vitro was assessed using binary logistic regression analysis, treating the proportion developing to a given developmental landmark as the dichotomous dependent variable and the treatment and experimental replicates as covariates in the model. The treatment effects on the numbers of cells in the resulting blastocysts were assessed using univariate regression analysis within the general linear model. Full factorial analysis was performed using significant factor contrasts performed to assess differences between individual treatments. Using chi-square test, the genetic distribution was assessed compared with expected mendelian segregation and the effects of treatment, strain, or genotype on the success of implantation and fetal development after embryo transfer.
Embryos were collected at the zygote stage (Day 0.5) and were cultured in vitro for 90 h (cultured) or were collected directly from the uterus at the blastocyst stage (Day 3.5). Cultured B6 zygotes were less likely to develop to the blastocyst stage than B6 embryos collected fresh from the reproductive tract or cultured B6CBF2 zygotes (collected from B6CBF1 x B6CBF1 mating) (P < 0.001) (Fig. 1A). A subset of blastocysts from these treatments was assessed for cell number. Blastocysts derived from cultured B6 zygotes had fewer cells per blastocyst than B6 blastocysts collected fresh from the reproductive tract (P < 0.001) (Fig. 1B) or cultured hybrid zygotes (P < 0.001).
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On transfer of the blastocysts resulting from these treatments to the uterus of recipient females, there was a profound strain-dependent effect of embryo culture on the implantation rate and the capacity to form fetuses (Fig. 1C). Culture of B6 zygotes for 90 h caused an approximate 50% reduction in the capacity of the resulting blastocysts to implant into the uterus following embryo transfer compared with cultured B6CBF2 blastocysts (P < 0.01) or B6 blastocysts collected fresh from the reproductive tract (P < 0.01). In the case of fresh blastocysts and cultured B6CBF2 zygotes, more than half of the implanted blastocysts formed viable fetuses, while only approximately 1 in 10 of the cultured B6 blastocysts that successfully implanted formed viable fetuses (P < 0.01). Therefore, small decrements in the rate of preimplantation embryo viability corresponded to profound reductions in the long-term developmental capacity. The high viability of cultured B6CBF2 hybrid zygotes under identical culture conditions shows that the low viability was a strain effect of culture.
Western blot showed that an anti-TRP53 antibody detected a single band of 53 kDa in B6 blastocysts cultured for 90 h from the zygote stage (Fig. 2A). By contrast, there was much less expression in embryos cultured for 48 h (8-cell) or for 72 h (morulae stage). There was no expression in Trp53–/– blastocysts (cultured from the zygote stage), confirming the specificity of the antibody and the Western blot. Immunolocalization (Fig. 2B) showed that cultured B6 blastocysts had high levels of TRP53 staining compared with B6 blastocysts collected fresh from the reproductive tract. Much of the increased staining was localized to the nuclear region of the cells. By contrast, the expression of TRP53 in cultured embryos from B6CBF1 parents was less prominent than that in B6 embryos (Fig. 2B). There was no obvious accumulation of staining within the nuclear region of the cells. Staining in cultured B6CBF2 blastocysts seemed to be greater than that observed in blastocysts collected from the reproductive tract, but they did not show the same degree of up-regulation as that observed in B6 embryos. The results are representative of three replicate experiments with at least eight embryos per treatment in each experiment. We found little variability of TRP53 expression in fresh blastocysts in either strain, with consistently low levels of expression and no apparent accumulation in the nuclei. Cultured B6CBF2 embryos were also consistent in their low level of expression. There was a small proportion of blastocysts (<10%) in which higher levels of expression were observed, and in these a proportion of the nuclei also showed staining. However, this was not representative of the pattern of TRP53 expression in B6CBF2 blastocysts in the culture model used. All cultured B6 embryos examined had higher levels of TRP53 expression than the levels observed in B6 blastocysts collected from the uterus. In some cultured B6 embryos (~20%), there were variable levels of localization of TRP53 in the nuclear region, such that some cells had high levels of staining in the nuclear region and other cells had lower levels of nuclear staining.
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The effect of Trp53 dosage on embryo development was assessed by mating Trp53+/– x Trp53+/– parents (Fig. 3A). Embryos collected from the reproductive tract on Day 3.5 were at the blastocyst stage and showed no difference from expected mendelian segregation (P > 0.05). Embryos collected on Day 0.5 and cultured from the zygote stage for 90 h demonstrated a small excess of Trp53+/– embryos compared with the expected mendelian ratio (P < 0.05). Fifty-four percent of these zygotes formed blastocysts or hatching blastocysts after 90 h of culture. When the developmental fate of embryos was assessed against their genotype, it was observed following culture for 90 h from the zygote stage that Trp53–/– embryos were more likely to develop to the blastocyst stage (hatching plus intact zona pellucida) and were less likely to have degenerate or retarded morphology (P < 0.001). By contrast, Trp53+/+ embryos were more likely to be in the retarded or degenerate classification (P < 0.001). Trp53+/– embryos showed an intermediate level of resistance to culture stress. There was a further clear effect of Trp53 gene dosage on the likelihood of embryos beginning the hatching process. Therefore, under conditions that caused a marked increase in the expression of TRP53 (culture of B6 background zygotes), the genetic inhibition of TRP53 improved the development of embryos in vitro.
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To determine if an interaction existed between the duration of culture, the Trp53 expression, and the competence of the embryo to form a viable fetus, embryos from Trp53+/– x Trp53+/– matings were collected from the reproductive tract as zygotes on Day 0.5 and cultured for 90 h or as compacted embryos on Day 2.5 of pregnancy and cultured for 24 h. The culture of compacted embryos for 24 h resulted in 80% of the resulting embryos being at the zona pellucida-intact blastocyst stage. By contrast, the culture of zygotes resulted in a greater diversity of embryo development stage (Fig. 3B). To compare the viability of embryos from these two groups, embryos of equivalent development stages were chosen, namely, zona pellucida-encased blastocysts. Morphologically normal, zona pellucida-intact blastocysts were transferred to the uterus of pseudopregnant recipient females. The culture duration had no effect on the implantation rate of embryos (P > 0.05) but adversely affected the proportion of implanting embryos that were capable of forming viable fetuses following implantation (P < 0.001) (Fig. 4A). Following 24 h of culture, fetuses were found in expected mendelian proportions; however, there was a significant shift in favor of Trp53–/– fetuses following culture of zygotes for 90 h (P < 0.001) (Fig. 4B). This result indicates a preferential survival of embryos of the null genotype following culture from the zygote stage but not following culture from the morulae stage.
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The results of this study show that zygotes that developed poorly in vitro (B6 strain) had marked up-regulation and nuclear accumulation of TRP53 in the resulting blastocysts. This up-regulation did not occur during development in vivo. The greater embryo viability of Trp53–/– blastocysts after extended culture of zygotes demonstrated that the increased TRP53 expression was responsible for a significant amount of the lost developmental potential of B6 embryos subjected to culture in vitro in this model. Embryos from B6CBF2 mice were resistant to the effects of culture as assessed by their growth rate in vitro and by their viability on embryo transfer. The amount of TRP53 expressed in such blastocysts was modest. This generation of differential expression of TRP53 by embryo culture provides the first described mechanistic basis for the well-known strain-dependent differences in the susceptibility of embryos to culture stress, to our knowledge.
Our results also demonstrate that in this model the culture of zygotes in vitro resulted in the preferential survival of Trp53-deficient embryos. This selection occurred at two levels as follows: 1) The fastest growing preimplantation-stage embryos of best morphology were more likely to be Trp53–/– or Trp53+/– (Fig. 3) (in clinical practice, embryos with these growth properties are the most likely to be transferred [24]). 2) The transferred Trp53–/– blastocysts were more likely to develop to viable fetuses following culture in vitro compared with their wild-type or heterozygous siblings (Fig. 4). The absence of Trp53 in some genetic backgrounds in mice (e.g., 129/sv) results in developmental defects in a subset (~16%) of fetuses [25]. This is most commonly due to the development of exencephaly, and this occurs predominantly in female fetuses. In such affected strains, the excess of Trp53–/– fetuses that occurs in response to culture may be mitigated to some degree by this increase in late-onset developmental defects and lethality.
The comparison of the long-term viability of embryos subjected to culture for a prolonged period with that of embryos that have not presents a challenge for experimental design. In susceptible species and strains, zygotes subjected to culture have retarded development compared with controls. They also show greater variability in their development rates. Therefore, comparison of embryos of the same developmental stage requires that zygotes subjected to culture be older (from the time of fertilization) than controls. Blastocysts produced by culture of zygotes are approximately 24 h older than their equivalent-stage uncultured (or 24-h cultured) controls. To produce cohorts of developmentally equivalent blastocysts from cultured zygotes and embryos cultured for a shorter duration (or not cultured at all) requires the selection of appropriate embryos by the investigator. In our experiments that assessed the viability of embryos after embryo transfer, we chose to compare the viability of zona pellucida-intact blastocysts. This entailed the selection of the most developmentally advanced embryos in the case of the controls (collected from the uterus in the experiment comparing the viability of B6 and hybrid embryos or from the 24-h culture group in the Trp53 gene dosage experiment) and the exclusion of embryos that had commenced hatching from the zona pellucida (and embryos that had not achieved the blastocyst stage) in the 90-h culture group. In all groups, selection was from among all zona pellucida-intact blastocysts available. In the Trp53 gene dosage experiment, the genotypic distribution of the transferred 90-h cultured blastocysts was probably similar to that shown in Figure 3B. However, it was impossible to genotype individual embryos before transfer. Therefore, the rate at which transferred embryos of a given genotype formed viable fetuses could not be directly measured. As a consequence, we cannot exclude the possibility of some confounding effects of this design.
The reduced viability of embryos following culture in vitro is commonly considered a consequence of their response to a range of cellular stresses imposed on them by the culture environment. These stresses include growth and survival factor deprivation [4, 7], metabolic and substrate imbalances [8, 9], and oxidative stress [10] and may involve gross or minor chromosome aberrations [11]. In somatic cells, this range of stressors is capable of activating the TRP53 pathway [12, 26]. This study did not directly assess which of the potential stressors caused by culture induced the observed TRP53 response. The culture conditions and media used are similar to those widely utilized throughout the history of human assisted reproductive technologies. In recent years, there has been some evolution of media formulations, and it will be of interest to determine how various culture conditions and procedures (e.g., cryopreservation and micromanipulation) affect TRP53 expression. Indeed, monitoring TRP53 expression in embryos may be a tool for assessing the effects of competing culture techniques on embryo viability.
This study shows that increased TRP53 accounts for a significant amount of the loss of viability of B6 zygotes following culture. Canonically, the expression of TRP53 is regulated by the E3 ubiquitin protein ligase MDM2. MDM2 leads to ubiquitination and rapid degradation by the 26S proteasome of its targets, including TRP53 [27, 28]. This has the net effect of maintaining low levels of TRP53 in cells at most times. During periods of stress, this rapid turnover is suppressed, leading to a rapid increase in TRP53 levels within the cell. The preimplantation embryo lethality of Mdm2–/– embryos is reversed by the double-knockout Mdm2–/– Trp53–/– [16, 17], suggesting that the loss of TRP53 latency via reduced MDM2 is a plausible cause of the increased TRP53 expression of embryos following culture. The MAPK8 (previously known as Jnk) stress pathway is also activated by embryo culture [29], and this pathway can also act to breach the latency of TRP53 expression [30].
Our study also demonstrates three defined phases of embryo development during which the adverse effects of culture are manifested in cultured B6 embryos based on defects associated with the following rates: 1) the rate of development to morphologically normal blastocysts, 2) the rate of successful embryo implantation, and 3) the rate at which implanted embryos form viable fetuses. The deletion of TRP53 in B6 background mice resulted in partial amelioration of the first and third of these defects. TRP53 exerts its effects via many downstream effectors [12], but our study did not inform us of the likely targets, nor did it identify whether the TRP53 effectors are the same during the preimplantation phase of growth and during the formation of the fetus. It has been shown using a nontransformed mouse trophoblast cell line that giant cell transformation requires the down-regulation of TRP53 [31], and this requirement may account for some of the effects of the Trp53–/– genotype.
The use of inbred strains such as B6 in embryological studies has the advantage that many genetically modified lines are produced in this background. The notoriously poor long-term viability of embryos from inbred lines following culture and manipulation in vitro has limited their usefulness for some forms of embryological research, including the formation of embryonic stem cell lineages. It remains to be investigated whether this observation of enhanced TRP53 expression in B6 embryos is universal for all susceptible strains and species. It has been reported that TRP53 is not associated with early developmental arrest in the bovine embryo during culture in vitro [32]; however, the bovine embryo seems to be resistant to culture stress, and its response may more closely resemble that of embryos from B6CBF2 animals. Furthermore, there are considerable differences between bovine culture systems and those used herein.
This study provides a new model for the investigation of the effects of culture on the cell biology of the preimplantation embryo. It offers an accessible tool for investigating how to ameliorate the stresses of culture environments and demonstrates a model for studying the long-term developmental consequences of such treatments.
ACKNOWLEDGMENTS
We thank G. Lozano, M. D. Anderson Cancer Center, for assistance with this study and for comments on the manuscript; Dr. R. Thomas for maintaining animal pedigrees; and the staff of the Gore Hill Research Laboratories for the breeding and care of animals.
FOOTNOTES
1Supported by grants from the Australian Health and Medical Research Council. 
Correspondence: 2FAX: 61 2 9926 6343; e-mail: chriso{at}med.usyd.edu.au
Received: 29 August 2006.
First decision: 11 September 2006.
Accepted: 26 October 2006.
REFERENCES
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