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Research Article |
Research Centre for Reproductive Health,3 Department of Obstetrics and Gynaecology, University of Adelaide, Woodville, South Australia 5011, Australia
Repromed,4 Dulwich, South Australia 5065, Australia
ABSTRACT
The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry; however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 µM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage; however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.
assisted reproductive technology, early development, embryo, gene regulation, in vitro fertilization
INTRODUCTION
Embryo culture systems are a vital component of assisted reproductive technology (ART) for the production of animal and human embryos. Traditionally, the in vitro development of the mammalian preimplantation embryo is associated with decreased embryo viability and development, along with severe alterations in metabolism and gene expression [1, 2]. However, more significantly, it has been established that the conditions to which the preimplantation-stage embryo is exposed can also affect both the ability to establish a pregnancy and affect fetal growth and also adult health in animal models [3, 4]. As a result of such studies, human IVF procedures have involved growing embryos for a short term before replacement, at the four- to eight-cell stage, into the reproductive tract. This is due to the suggestion that extended culture of embryos, to the blastocyst stage, may increase abnormal outcomes after transfer by escalating levels of cellular perturbations [3, 5, 6]. However, there is little direct evidence to support these statements and it is currently unknown at what stage the embryo is most vulnerable to stress that can cause downstream effects on the embryo and the resultant fetus.
The pre- and postcompaction-stage embryos differ greatly in many aspects, including preferred metabolic substrate [7–9], genome control (maternal or embryonic) [10], mitochondrial structure [11], and level of metabolic activity [12]. The precompaction stage embryo also lacks efficient regulatory mechanisms for pH [13–16] and reactive oxygen species (ROS) [17]. In contrast, following the formation of a transporting epithelium at compaction, there is an increase in the ability of the embryo to regulate homeostatic processes such as intracellular pH [18]. Also the mitochondria of the early embryo are ovoid in shape and have low numbers of transverse cristae and reduced surface area [11] and are less able to regulate metabolic activity in vitro [19] compared with the later stage embryo. This suggests that the precompaction-stage embryo may be vulnerable to perturbations in the surrounding environment. However, whether this results in differences in the response of the embryo to in vitro stress is currently unknown.
One in vitro stress that has been demonstrated to influence the ability of the embryo to develop is ammonium [20]. Ammonium levels increase significantly during the culture period due to the deamination and spontaneous breakdown of amino acids, especially glutamine, in the medium [2, 21]. Ammonium has been shown to decrease embryo cleavage and blastocyst development, decrease blastocyst cell number, alter metabolism, and increase apoptosis [2, 21]. Additionally, exposure to ammonium during the entire preimplantation stage, from the zygote to the blastocyst, also decreases implantation rates and fetal development as well as increasing the incidence of fetal abnormalities [20–22]. Despite the obvious effect of ammonium on the mammalian preimplantation embryo, it is currently unknown at what stage the embryo is most vulnerable to ammonium and how this stress induces its effect on the embryo and subsequent fetus. Therefore, the aim of this study was to investigate the effect of exposure to ammonium at varying stages of embryo development and discern at what stage the embryo is most susceptible to this stress.
MATERIALS AND METHODS
Culture Media
The media used for embryo culture were G1.2 and G2.2, and MOPS-G1 was used as a collection and handling medium [23]. G1.2 and G2.2 is a sequential culture system and was used, as it results in high rates of embryo development and fetal outcomes according to the literature [24]. This sequential media also contains a dipeptide of glutamine (the most volatile amino acid), which prevents its spontaneous breakdown, which results in a negligible ammonium buildup (<9 µM) [21]. All media components were purchased from Sigma Chemical Co. (St. Louis, MO) and prescreened for ability to support embryo development with a one-cell mouse bioassay [23]. Ammonium chloride was added to medium at a concentration of 300 µM where indicated.
Embryo Collection and Culture
Embryos were collected from prepubescent F1 female mice (C57Bl6 x CBA) following superovulation with 5 IU of eCG (Folligon; Intervet, Bendigo, Victoria, Australia) and 5 IU hCG (Pregnyl; Organon, Oss, The Netherlands) 48 h later. Immediately following the second injection, females were placed with males from a Swiss outbred strain. Mating was determined the following morning by the presence of a vaginal plug. At 23 h post-hCG, zygotes were collected into MOPS-G1 medium and were denuded by incubation with 1 mg/ml hyaluronidase (Sigma Chemical Co.). Zygotes were then washed twice in MOPS-G1 and once in G1.2 before being placed into culture. Embryos were cultured in groups of 10 in 20-µl drops of medium under mineral oil (Sigma Chemical Co.). All embryos were cultured for 43 h in G1.2 at 37°C in 6%CO2:5%O2:89%N2 and then washed in G2.2 and cultured in G2.2 for a further 48 h to the blastocyst stage. All embryo culture dishes were prepared 4 h before embryo culture to allow gas and temperature equilibration. All procedures were conducted in accordance with the National Institutes of Health guide for the care and use of laboratory animals [25].
In Vivo Blastocyst Collection
At 87 h post-hCG, uterine horns were isolated and blastocysts were flushed from the tract with MOPS-G1 medium. Blastocysts were washed twice in MOPS-G1 medium and placed into culture drops.
Assessment of Morphology
Embryo morphology was assessed at 19, 43, 74, and 91 h of culture using phase-contrast microscopy (100x). Embryos were classified as follows: two- to eight-cell, morula (fully compacted embryo), and blastocyst or hatching blastocyst (clear herniation of the zona pellucida by the trophectoderm).
Differential Staining Protocol
Allocation of cells in the blastocyst to the inner cell mass (ICM) or trophectoderm (TE) was assessed using a differential staining protocol described by Gardner et al. [26]. Briefly, all blastocysts were placed into 0.5% pronase (Sigma Chemical Co.) to dissolve the zona, and then were incubated in 10 mM TNBS (2,4,6-trinitrobenzenesulfonic acid; Sigma Chemical Co.) at 4°C for 10 min. Following this, blastocysts were transferred to 0.1 mg/ml 
-DNP BSA (anti-dinitrophenyl-BSA; Sigma Chemical Co.) for 10 min at 37°C and placed in guinea pig serum with propidium iodide (Sigma Chemical Co.) for 5 min at 37°C. Blastocysts were then stained with bisbenzimide in ethanol overnight. The following day, the embryos were washed in 100% alcohol, then mounted in a glycerol drop on a siliconized slide. Blastocysts were then viewed on a fluorescent microscope at 400x under a UV filter where the ICM nuclei appeared blue and TE nuclei stained pink.
Levels of Apoptosis in Blastocysts
The number of apoptotic cells in each blastocyst was determined using a TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) technique using an In Situ Cell Death Kit (Roche Molecular Biochemicals, Indianapolis, IN) as described by Kamjoo et al. [27]. Apoptotic cell nuclei are labeled with FITC using this procedure and all cell nuclei were counterstained with propidium iodide. The number of apoptotic cells was expressed as a percentage of the total cell number of each blastocyst to give the apoptotic cell index.
Real-Time RT-PCR
The relative levels of expression of the genes Slc2a1, Slc2a3, and 18S were determined by real-time RT-PCR (ReTi RT-PCR). Thirty blastocysts per replicate were snap frozen in 500 µl Tri reagent (Sigma Chemical Co). RNA was extracted and reverse transcription performed following the protocol outlined by Sigma Chemical Co. as modified by Kind et al. [28].
ReTi RT-PCR was performed following the protocol also described by Kind et al. [28] using SYBR Green Mix (Applied Biosystems, Foster City, CA) in the master mix. The oligonucleotide primers used were designed using Primer Express (Applied Biosystems) and synthesized by Geneworks (Adelaide, Australia) and have been previously described by Kind et al. [28]. The cDNA was diluted to 0.6 embryo equivalent per 1 µl using nuclease-free water. Real-time PCR was carried out on a GeneAmp 5700 Sequence Detection System with a thermal cycling program of 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. A cycle threshold (Ct) was calculated for each sample using GeneAmp 5700 software and the levels of gene expression were compared with 18S and normalized for the control embryos grown without ammonium [29].
Glucose Uptake by Blastocysts
Cultured blastocysts were incubated in 50-nl drops of G2.2 media where the glucose concentration was reduced to 0.8 mM (0.5 mM glucose and 0.3 mM of 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG) (Molecular Probes, Eugene, OR). Blastocysts were incubated for 1.5 h at 37°C in 6%CO2:5%O2:89%N2. At the end of the incubation, glucose uptake was assessed by determining the levels of fluorescence in the blastocysts from the 6-NBDG using a photomultiplier with photometer attachment. The level of fluorescence with this dye was determined to reflect glucose uptake as measured by quantitative microfluorescence (data not shown). The distribution of glucose within the blastocysts was assessed using confocal microscopy.
Embryo Transfers
Female Swiss mice aged between 8 and 12 wk were mated to vasectomized males and embryos transferred on Day 4 of pseudopregnancy. Females were anaesthetized with 2% Avertin (0.015 ml/g body weight) and six blastocysts were transferred to each uterine horn, with each treatment being allocated to a different horn. A total of 10 transfers were performed for each treatment group. Treatment groups were assigned to each uterine horn using random numbers. On Day 15, the number of implantations and fetuses were recorded as well as fetal and placental weight and crown-rump length. The maturity or growth rates of fetuses were assessed using the technique of Wahlsten [30]. Development of four features (limbs, ears, eyes, and skin) was assessed and compared with development in fetuses derived from naturally mated females. Using this procedure, fetal growth can be estimated to one quarter of a day.
Statistical Analysis
For binomial data, each replicate was expressed as a proportion for analysis. Embryo development and cell numbers were analyzed using a univariate general linear model (SPSS 11.5; SPSS Inc., Chicago, IL). Development for each replicate experiment was expressed as a proportion, and day of replicate was treated as a covariate. Differences between treatments were assessed using the least significant difference method (LSD). Levels of apoptosis in blastocysts were not normally distributed and were analyzed using a nonparametric Mann-Whitney U-test. Fetal data were analyzed using GLIM statistical package (version 4.0; National Algorithm Group, Oxford, UK) by general linear modeling, where a binomial distribution was assumed and differences between treatments assessed using the log-likelihood statistic. Differences in gene expression were assessed using a method described by Livak and Schmittgen [29], which calculates the fold increase (+) or decrease (–) in gene expression compared with the control. Differences in expression were assessed on normalized data using a univariate general linear model and LSD.
Experimental Design
Embryos were randomly allocated to each different treatment group and exposed to 300 µM ammonium during a certain stage of development. Treatment 1 was the control group and was cultured in sequential culture media with no ammonium exposure (total of 91 h of culture). Treatment 2 was exposed to 300 µM ammonium from the zygote to the two-cell stage (from 0 to 19 h of culture). Treatment 3 was exposed from the two-cell to the eight-cell stage (19–43 h of culture). Treatment 4 was exposed to ammonium from the eight-cell to blastocyst stage (43–91 h of culture). Treatment 5 was exposed to 300 µM ammonium for the entire culture period (total of 91 h of culture).
RESULTS
Effect of Ammonium on Embryo Development
Zygotes were exposed to either control media G1.2 (up to eight-cell stage) and G2.2 (eight-cell to blastocyst stage) or media containing 300 µM ammonium chloride. Embryos were randomly allocated to each treatment group and exposed to 300 µM ammonium during a certain stage of development (see Experimental Design). Exposure of embryos to ammonium did not affect development to the two-cell stage after 24 h of culture or the percentage of embryos that reached the eight-cell stage after 48 h of culture (data not shown). Ammonium exposure also did not affect development to the morula and blastocyst stage after 74 h (data not shown) or blastocyst stage after 91 h of culture (Table 1). The morphology of the blastocysts from all groups cultured with ammonium was similar to that of control blastocysts.
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Effect of Ammonium on Allocation of Cells to the ICM and TE
There was a significant reduction in total blastocyst cell number and ICM number in embryos exposed to ammonium from the zygote to the two-cell stage (P < 0.05) or from the two-cell and eight-cell stages (P < 0.05), as well as those cultured with ammonium for the entire preimplantation period from the zygote to the blastocyst stage (P < 0.05; Table 2). There was no difference in total blastocyst cell number and ICM cell number between the group incubated with ammonium from the eight-cell to the blastocyst stage and the control embryos cultured in the absence of ammonium, and ammonium exposure at any stage of development did not affect TE cell number. Analysis of the percentage ICM cells of total cell number in the blastocysts determined that ammonium exposure from the zygote to the two-cell stage or for the entire period from the zygote to the blastocyst stage significantly reduced the percentage of ICM cells in the blastocysts compared with the control blastocysts (P < 0.05; Table 2).
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Effect of Ammonium on Levels of Apoptosis in Cultured Blastocysts
There was a significant increase in the levels of apoptosis in embryos exposed to ammonium from the zygote to the two-cell stage (P < 0.05) and those incubated with ammonium continually throughout development (P < 0.05) compared with control embryos (Fig. 1). There was no significant difference in apoptotic cell index in treatments where ammonium exposure was from the two-cell to the eight-cell stage (P = 0.07) or from the eight-cell to the blastocyst stage compared with control blastocysts.
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Effect of Ammonium on Gene Expression
The level of Slc2a1 mRNA expression in blastocysts was not altered by exposure to ammonium at any stage of development. In contrast, culture with ammonium at any stage of development resulted in a significant decrease in Slc2a3 expression when compared with control blastocysts (P < 0.01, Table 3).
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Effect of Ammonium on Glucose Uptakes by Blastocysts
Culture of embryos with ammonium at any stage of development significantly reduced the levels of glucose uptake at the blastocyst stage compared with blastocysts cultured in the absence of ammonium (P < 0.001; Figs. 2 and 3). Analysis of the distribution of glucose within the blastocysts determined that glucose levels were significantly lower within the blastocoel and the inner cell mass in embryos cultured with ammonium (Fig. 3).
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Effect of Ammonium on Blastocyst Viability
Culture with ammonium at any stage of development did not affect implantation rates (Table 4). However, exposure to ammonium throughout the entire preimplantation period resulted in a significant decrease in the percentage of fetuses that developed per embryo transferred as well as inducing a significant reduction in the percentage of fetuses that developed per implantation (Table 4; P < 0.05). Similar negative effects of ammonium on fetal development were observed when the exposure period occurred between the zygote and two-cell or between the two-cell to the eight-cell stage. There was no significant effect of ammonium exposure postcompaction on fetal development, although rates were lower than that observed for the control blastocysts. All cultured blastocysts resulted in lower fetal developmental rates compared with in vivo-developed blastocysts (Table 4).
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Culture with ammonium from the zygote to the blastocyst resulted in a significant reduction in fetal weights and crown-rump length compared with blastocysts cultured without ammonium (Table 5). In contrast, continual exposure to ammonium did not affect crown-rump length or fetal weight (Table 5; P < 0.05). Incubation with ammonium at the postcompaction stage did not affect fetal weight or crown-rump length (P < 0.05). Fetal maturity was reduced in all groups that were cultured with ammonium (P < 0.05). Pregnancy rates for all cultured treatments were significantly reduced compared with in vivo-developed embryos (Table 4).
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DISCUSSION
The data presented in this study support the hypothesis that the cleavage stage embryo is more sensitive to stress than the postcompaction stage embryo. Additionally, this study highlighted that, although there were no morphological differences between any of the blastocysts exposed to ammonium, ammonium had a significant effect at the cellular level. The observed negative effects of culture with ammonium from the zygote to the blastocyst stage in this study on blastocyst cell number, ICM cell number, and apoptosis is in agreement with previous studies [1, 21, 22]. It has been shown that culture with ammonium at levels of 18.8–300 µM throughout development decreases blastocyst cell number, selectively inhibits ICM development, and increases cellular apoptosis in blastocysts [1, 21, 22]. Additionally, culture with 300 µM ammonium from the zygote to the blastocyst stage has been shown to decrease the ability of blastocysts to develop into a viable fetus as well as increasing the incidence in fetal abnormalities [1, 21, 22]. Similarly, in this study, culture with 300 µM ammonium throughout development decreased fetal development as well as inhibiting fetal growth. However, what we observed in this study was that the same detrimental effects on the development of the blastocyst and pregnancy rates and fetal growth were observed when the culture period was for only 19 h at the precompaction stage. In contrast, when the exposure to ammonium occurred postcompaction, most parameters were unaffected. These data therefore imply that there may be an increasing ability of the embryo to adapt or become tolerant to ammonium as development progresses beyond compaction. There are significant changes in physiology that occur in the embryo around the stage of compaction. Prior to compaction, the early embryo has a reduced capacity to regulate metabolism and cellular homeostasis compared with the postcompaction stage embryo. Edwards et al. showed that the ability of embryos to regulate a pH stress increases significantly with compaction [31]. Embryos exposed to a weak acid at the precompaction stage show a decrease in intracellular pH, while those exposed postcompaction were able to maintain pH at the physiological level [31]. Similarly, the ability of the embryo to maintain normal development when exposed to other stresses, such as osmotic stress in the medium, are significantly increased following compaction [32]. Together, these data support that there is an increased ability of the embryo to cope with an in vitro-induced stress following compaction.
As well as inducing physiological changes to the embryo, ammonium has previously been shown to induce alterations in gene expression in the blastocyst. Exposure to ammonium throughout development causes general suppression in gene expression, as well as perturbing H19 gene expression [3, 21]. In this study, ammonium was shown to significantly decrease the expression of the glucose transporter Slc2a3, although Slc2a1 levels were not altered. Slc2a3 is responsible for the uptake of glucose by the blastocyst from the maternal environment. In contrast, Slc2a1 is located on the basolateral membrane and is responsible for the transport of glucose from the trophectoderm into the blastocoel cavity [33, 34]. This decrease in Slc2a3 expression would be expected to result in a decrease in glucose uptake by the blastocyst. Indeed, levels of glucose uptake were reduced in the blastocysts cultured with ammonium [21]. A reduction in the levels of glucose uptake and metabolism would reduce the capacity for ATP production and subsequently decrease viability. In this study, blastocysts from culture treatments that reduced Slc2a3 expression and glucose uptake also reduced fetal development rates after transfer. Interestingly, the expression of Slc2a3 is not activated until compaction [34], indicating that exposure to ammonium precompaction can affect levels of genes that are not expressed until later in development. This therefore implies that ammonium is causing a disruption in cellular function that can alter gene expression at later developmental stages. The differential effect on the expression levels of the glucose transporters by ammonium implies that there may be different pathways and sensitivities involved in the regulation of these transporters in the blastocyst. In support of these, there is evidence in other cell types that express more than one facilitated glucose transporters that the isoforms are differentially regulated [35, 36]. The mechanisms for this longer term disruption are currently unknown and require further investigation.
Incubation with ammonium at the precompaction stage or throughout development resulted in a significant decrease in the percentage of viable fetuses that were formed as well as causing a significant delay in fetal maturity and decreased crown-rump length. The detrimental effect of ammonium in the culture medium during the entire preimplantation period has previously been reported [1, 37]. The data presented in this study have further demonstrated that an exposure of embryos to ammonium for only a short time period, especially during the precompaction stage, can have a significant effect on fetal health by not only reducing pregnancy rates but by also affecting the actual growth rate of the fetus. This observation provides further evidence that ammonium is inducing an effect on the precompaction-stage embryo that may not be evident until later in development. Exposure at the postcompaction stage also resulted in decreased fetal maturity, indicating that the postcompaction-stage embryo is also vulnerable to the in vitro-induced stress, but to a lesser extent. Therefore, both gene expression and postimplantation outcomes are affected by ammonium exposure at all stages of development; however, it is clear that aberrant outcomes are increased when embryos were exposed to ammonium precompaction. This is likely due to the reduced ability of the precompaction-stage embryo to regulate metabolic and cellular homeostasis.
The mechanism for the negative effect of ammonium has not yet been explained. One hypothesis is that effects may be due to changes in intracellular pH. Indeed, exposure of two-cell embryos with 300 µM ammonium significantly decreases intracellular pH [21]. Studies in the sea urchin embryo have indicated that pH alteration can profoundly affect cellular events governing early development [38]. Similarly, in mammalian embryos, alterations in intracellular pH disrupt mitochondrial distribution, where mitochondria were seen to disperse away from the nucleus and into the intermediate cytoplasm. It has been hypothesized that this dispersion disrupts efficient delivery of ATP to the nucleus, which leads to a loss of energy needed to support transcriptional activity and subsequently delayed development [39–41]. Whether the abnormal affects of ammonium on development and metabolism are mediated by changes in pH regulation are yet to be established. Alternatively, ammonium may affect embryo development by having a direct effect on the ability of the embryo to produce ATP. In other cell types, ammonium has been shown to inhibit mitochondrial shuttle activity [42, 43], as well as altering the activity of glycolytic enzymes [44, 45]. Whether ammonium has these same effects on metabolic homeostasis in the embryo is as yet unknown.
In conclusion, the results from this study clearly indicate that the precompaction stage, in particular between the zygote to two-cell, is extremely sensitive to ammonium, which implies that this is a crucial stage of development for environmental stress. It has also been determined that even a short-term exposure of the precompaction-stage embryo to ammonium can result in significant disruptions in later gene expression and development. This indicates that ammonium may cause an irreversible change in the embryo. This has significant implications for the in vitro production of embryos and highlights the need for care to be taken in conditions for the growth of embryos, especially for those at the precompaction stage.
ACKNOWLEDGMENTS
The authors would like to gratefully acknowledge the expertise and assistance of Dr. Karen Kind in the gene expression studies, David Froiland for assistance with the imaging, and Dr. Hamish Hamilton for his comments on the manuscript.
FOOTNOTES
1 Supported by the Australian NHMRC project grant 299037. D.L.Z. is a recipient of a TQEH postgraduate scholarship, J.G.T. a NHMRC Senior Research Fellowship, and M.L. is a recipient of a R.D. Wright Fellowship from the NHMRC. 
2 Correspondence: Michelle Lane, Research Centre for Reproductive Health, University of Adelaide, Level 4, Maternity Building, The Queen Elizabeth Hospital, Woodville Road, Woodville, South Australia 5011, Australia. FAX: 61 8 8222 7521; michelle.lane{at}adelaide.edu.au
Received: 1 August 2005.
First decision: 17 August 2005.
Accepted: 11 October 2005.
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20 blastocysts examined per treatment. Data represents mean ± SEM. * Significantly different from control (P < 0.05)
