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BOR - Papers in Press, published online ahead of print May 28, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.018093
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BIOLOGY OF REPRODUCTION 69, 1109–1117 (2003)
DOI: 10.1095/biolreprod.103.018093
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

Ammonium Induces Aberrant Blastocyst Differentiation, Metabolism, pH Regulation, Gene Expression and Subsequently Alters Fetal Development in the Mouse

Michelle Lane1, and David K. Gardner

Research Department, Colorado Center for Reproductive Medicine, Englewood, Colorado 80110


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of ammonium in the culture medium has significant detrimental effects on the regulation of embryo physiology and genetics. Ammonium levels build up linearly over time in the culture medium when media containing amino acids are incubated at 37°C. Ammonium in the culture media significantly reduces blastocyst cell number, decreases inner cell mass development, increases apoptosis, perturbs metabolism, impairs the ability of embryos to regulate intracellular pH, and alters the expression of the imprinted gene H19. In contrast, the rate of blastocyst development and blastocyst morphology appear to be normal. The transfer of blastocysts exposed to ammonium results in a significant reduction in the ability to establish a pregnancy. Furthermore, of those embryos that manage to implant, fetal growth is significantly impaired. Embryos exposed to 300 µM ammonium are retarded by 1.5 days developmentally at Day 15 of pregnancy. It is therefore essential that culture conditions for mammalian embryos are designed to minimize the buildup of ammonium to prevent abnormalities in embryo physiology, genetic regulation, pregnancy, and fetal development.

assisted reproductive technology, early development, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Formulations for embryo culture media have traditionally consisted of a balanced salt solution, such as Kreb Ringer Salts, supplemented with carbohydrates and a protein source, such as serum albumin [1–3]. However, development and viability of embryos cultured in such media lagged significantly behind in vivo embryo development. During the last several years, significant research has been conducted regarding the physiology and culture of mammalian embryos. Such studies have demonstrated that optimal embryo development requires gradients of carbohydrates, amino acids, and vitamins [1–3]. Clearly, amino acids are an essential component of embryo culture media and are regulators of embryo physiology [4–18]. Amino acids stimulate embryo development by acting as intracellular pH regulators [19], osmolytes [20–23], energy substrates [24], regulators of metabolism [25, 26], and chelators [27]. Development of the cleavage-stage embryo is stimulated by the presence of the amino acids alanine, asparagine, aspartate, glutamate, glycine, proline, and serine [10, 11, 14, 15]. However, after the embryo has compacted, forms the blastocoel, and differentiates into the inner cell mass (ICM) and trophectoderm (TE), development is stimulated by a more complex array of amino acids that have specific effects on the two cell types [14, 15, 28–32]. Perhaps of greatest significance is that the inclusion of specific amino acids increases implantation rates of blastocysts similar to those of in vivo-developed embryos [15].

Amino acids have been shown to be key regulators of embryo development and viability, but they spontaneously break down in culture to produce ammonium [10, 12, 16]. Also, the embryo metabolizes amino acids, resulting in the additional production of ammonium in the medium [10]. Ammonium in the culture medium has been shown to be detrimental to blastocyst development. However, most significantly, when ammonium is present in the medium for embryos from an F1 hybrid mouse, significant fetal retardation and an induction of the birth defect exencephaly occur following embryo transfer [12]. The incidence of this birth defect increases linearly with the ammonium concentration. The mechanism(s) by which ammonium affects embryo development and viability is currently unknown. The aim of the present study was to determine the effects of ammonium in the culture medium on embryo differentiation, apoptosis, physiology, metabolism, and gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media Composition

The medium for embryo collection was a 4-morpholinepropanesulfonic acid (MOPS)-buffered modification of medium G1.2 (MOPS-G1) (Table 1) with a pH of 7.35. Media for embryo culture were G1.2 and G2.2 supplemented with 5 mg/ml of human serum albumin (HSA) (Table 1) [33, 34]. All salts were Analar grade and purchased from BDH (Dorset, U.K.). Pyruvate, lactate, taurine, alanine, asparagine, aspartate, glycine, glutamate, proline, serine, alanyl-glutamine, and MOPS were purchased from Sigma Chemical Co. (St. Louis, MO). Amino acid and vitamin solutions were obtained from ICN (Aurora, OH). Both EDTA and HSA were obtained from Vitrolife AB (Gothenburg, Sweden). All media, media components, and disposables used for embryo culture were screened for ability to support embryo development with a 1-cell mouse embryo bioassay (zygote development to the blastocyst stage in a protein-free medium of >80% and blastocyst cell numbers of >65 on Day 5) before use [2].


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  		TABLE 1. Composition of culture media (mM).a

Embryo Collection and Culture

Embryos were collected from CF1 female mice following superovulation with 5 IU of eCG (Sigma) and 5 IU of hCG 48 h later (Pregnyl; Organon, Inc., West Orange, NJ). Immediately following the second injection, females were placed with males of the same strain to generate CF1 x CF1 embryos. Mating was indicated by the presence of a vaginal plug the following morning. All experimental protocols were approved by the Institutional Animal Care and Use Committee.

Zygotes were collected at 21 h post-hCG in MOPS-G1 and denuded by incubation with hyaluronidase (0.5 mg/ml; bovine testes, type IV; Sig-ma) for less than 1 min. Zygotes were washed twice in MOPS-G1 and once in medium G1.2 before placement in culture. Embryos were cultured in groups of 10 in 20-µl drops of medium under paraffin oil (BDH) [35]. All embryos were cultured in medium G1.2 at 37°C in 6% CO2:5% O2:89% N2. After 48 h of culture in medium G1.2, all embryos were washed twice in G2.2 and then cultured in G2.2 for a further 48 h to the blastocyst stage.

Allocation of Cells to the ICM and TE

Allocation of cells in blastocysts to the ICM and TE was assessed using a modification of the technique reported by Hardy et al. [36], as described by Gardner et al. [37].

Levels of Apoptosis in Blastocysts

The number of apoptotic cells in blastocysts was determined by the TUNEL method using an In Situ Cell Death Detection Kit (Roche Molecular Biochemicals, Indianapolis, IN) as previously described [38]. The number of cells that demonstrated TUNEL labeling was expressed as a percentage of the total cell number of each blastocyst to give the apoptotic cell index.

Analysis of Pyruvate Oxidation and Glycolysis

Pyruvate oxidation and glycolytic activity were assessed by incubation with radiolabeled substrates in a microcentrifuge tube [39]. Pyruvate oxidation and glycolytic activity by single embryos were determined by incubation in medium G1.2 for zygotes and 2-cell embryos or G2.2 for blastocysts with [2-14C]pyruvate (0.32 mM, 0.085 mCi/ml) and [5-3H]glucose (0.25 mCi/ml) as previously described [24, 39]. Oxidation of labeled pyruvate was determined from the recovery of CO2, and glycolytic activity was similarly determined using the recovery of tritiated water [24, 39]. Metabolism was expressed in terms of picomoles per embryo per hour (pmol embryo-1 h-1).

Measurement of Intracellular pH

Intracellular pH levels were determined by ratiometric fluorescence analysis using the fluorochrome SNARF 1-acetoxymethyl ester (SNARF-1-AM; Molecular Probes, Eugene, OR). Two-cell embryos were incubated with 5 µM of SNARF-1-AM for 30 min at 37°C in either medium G1.2 or G1.2 supplemented with NH4+ at 6% CO2:5% O2:89% N2. Embryos were washed twice in the same medium without the fluorochrome. The excitation wavelength was set to 535 nm, and the ratio of fluorescence intensities of images obtained at emission wavelengths of 640 nm (pH sensitive) and 600 nm (pH insensitive) was obtained for each embryo. Intracellular pH levels were calibrated using an in situ, four-point standard with the ionophores nigericin (10 µg/ml) and valinomycin (5 µg/ml) run on each day of experiment [40–42].

Assessment of Blastocyst Viability

Blastocysts derived from CF1 x CF1 matings were cultured for 96 h and then transferred to Day 4 pseudopregnant F1 (C57BL/6xCBA) female mice (-1 day asynchronous). Embryos from each treatment were allocated to each uterine horn using random numbers. Six blastocysts were transferred to each uterine horn. On Day 15 of pregnancy, females were killed, and implantation sites and numbers of fetuses were determined. Weights of resultant fetuses were also determined, and growth rates were assessed (see below).

Determination of Fetal Growth

The growth rates of fetuses were assessed using the technique of Wahlsten and Wainwright [43], as described by Lane and Gardner [12]. Development of four features (limbs, ears, eyes, and skin) was assessed and compared to development in fetuses derived from naturally mated females. Using this procedure, fetal growth can be determined to one-quarter of a day.

Analysis of Ammonium Levels in Media

Ammonium levels in the media were assessed using an ultramicrofluorometric technique [10, 44], based on the following equation:Glutamate dehydrogenase

The reaction reagents had the following composition: 0.24 mM NADH, 0.75 mM NaHCO3, 0.63 mM ADP, 14.15 mM {alpha}-ketoglutarate, and 3 U/ml of glutamate dehydrogenase (EC 1.4.1.3) in 157 mM triethanolamine buffer, pH 8.0. A calibration curve of ammonium chloride concentrations between 0 and 0.5 mM was run with each series of assays. Media were incubated at 37°C in either 20-µl drops under oil or in 500 µl in a well without oil, and 5-nl samples of media taken at 24-h intervals.

Uridine Incorporation

Levels of uridine incorporation into RNA were determined by incubating blastocysts in medium G2.2 with the appropriate level of NH4+ containing 10 µM [3,4,5-3H]uridine for 2 h. The amount of labeled uridine that was incorporated was determined using the procedure of Piko and Clegg [45], and levels were determined by scintillation counting and expressed as levels of dpm.

Assessment of Levels of Gene Expression

The relative levels of expression of the genes Na+/K+ ATPase, Igf2, NAT-2, and H19 were determined by reverse transcription-polymerase chain reaction (RT-PCR). The levels of expression were expressed as a percentage of ß-actin. Primers and PCR conditions for all genes were as previously described [46].

Statistical Analysis

Blastocyst development, implantation, and fetal development were assessed by general linear modeling using the log likelihood statistic. Day of experiment was fitted as a cofactor. Cell numbers, levels of apoptosis, intracellular pH levels, metabolism, fetal growth, uridine incorporation, ammonium levels, and gene expression levels were initially assessed for normality using the Kolmogorov and Smirnov test. For data sets that were found to be normally distributed, between-treatment differences were determined by analysis of variance followed by the Bonferroni procedure for multiple comparisons. For nonparametric data sets, between-treatment differences were assessed using a Kruskall-Wallis test followed by the Dunn test [47].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of Ammonium in Embryo Culture Media

Twenty-microliter drops of the following media, KSOMAA [17], G1.2/G2.2 (Table 1), P1/Blastocyst (Irvine Scientific, Irvine, CA), and Quinn Advantage Cleavage/Blastocyst (Sage Biopharma, Sacramento, CA), were set up under oil. For the sequential media systems, the first medium was measured for the first 48 h, and the second medium was measured for the remainder of the culture period. In addition, to examine the effects of volume or oil overlay, KSOMAA was also incubated as 500-µl drops of media in a four-well plate without an oil overlay. All media were supplemented with 5 mg/ml of HSA. All dishes were placed in the incubator at 37°C in 6% CO2:5% O2:89% N2. Dishes were placed in the incubator at 1600 h to mimic the procedure of placing the embryos in the culture media at 1000 h the following day. The following day at 1000 h, 5-nl samples of media were removed and analyzed for ammonium levels. This sampling was repeated each morning to simulate 0 (time that embryos are placed in the media, 18 h of incubation at 37°C), 24, 48, 72, 96, and 120 h of culture. For the sequential media series, the second medium of the sequential system was set up on the afternoon of Day 2 to simulate a changeover on the morning of Day 3, as per the manufacturer's instructions.

The levels of ammonium produced in the media are shown in Figure 1. For KSOMAA, the levels of ammonium produced by incubation at 37°C were significantly higher than the levels for all other media tested from the time of culture (24 h, 178.1 ± 15.2 µM, P < 0.05) through 120 h of culture (545.2 ± 24.7 µM, P < 0.001). The ammonium levels produced in KSOMAA increased linearly throughout the incubation period (r = 0.983, P < 0.05), and this phenomena was independent of incubation volume and oil overlay. Levels of ammonium in Quinn Advantage medium and G1.2/G2.2 medium were significantly lower than produced in P1 and Blastocyst medium (P < 0.01). No significant difference was observed in the levels of ammonium between Quinn Advantage and G1.2/G2.2 medium (P > 0.05). In both cases, the highest levels of ammonium that were measured were 20.2 ± 2.1 and 10.5 ± 2.3 µM, respectively. The substitution of glutamine for alanyl-glutamine in G1.2/G2.2 and Quinn Advantage media resulted in significantly lower ammonium levels.



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  		FIG. 1. Effect of incubation of culture media at 37°C on the levels of ammonium buildup. For all media, 20-µl culture drops were set up under oil. For KSOMAA, the medium was also prepared in 500-µl drops of media in a four-well plate without oil. All dishes were incubated at 37°C at 6% CO2:5% O2:89% N2. Culture drops were prepared to simulate a culture and, therefore, were set up at 1600 h on the evening before a culture that was to be performed the following morning. Media was sampled at the time equivalent of 24, 48, 72, 96, and 120 h of culture. For each media system at each time point, a minimum of 10 samples were analyzed (two replicates). No difference was observed in the data collected from the two replicates. Open triangles represent the medium G1.2/G2.2. Open diamonds represent the medium Quinn Advantage. Open circles represent the medium P1/Blastocyst. Crosses represent the medium KSOMAA in 20-µl drops under oil, and closed triangles represent this medium in a volume of 500 µl in wells without oil. At all time points after 0 h, culture medium KSOMAA in drops and wells and the medium P1/Blastocyst had significantly higher levels of ammonium than did the medium G1.2/G2.2 (P < 0.05). The levels of ammonium in KSOMAA were significantly higher than in all other media at all time points (P < 0.05). No difference was observed in the levels of ammonium produced in KSOMAA between drop incubation and well incubation

Effect of Ammonium on Embryo Development and ICM Formation

Zygotes were cultured in medium G1.2/G2.2 supplemented with ammonium chloride at either 0, 18.8, 37.5, 75, 150, or 300 µM. Embryos were cultured to the blastocyst stage, and total cell numbers as well as ICM and TE development of the blastocysts were assessed.

Blastocyst development was not affected by the presence of ammonium up to 300 µM in the medium (Table 2). Similarly, the morphology of the resultant blastocysts was not different when embryos were cultured with ammonium. In contrast, a significant decrease was observed in total cell numbers of blastocysts when the ammonium concentration was 37.5 µM. Increasing the concentration of ammonium in the culture media up to 150 µm did not further decrease the cell number of the resultant blastocysts (Table 2). Total cell numbers, number of ICM cells (Table 2), and percentage of ICM cells (Fig. 2) within the blastocysts were significantly reduced by the presence of ammonium in the media.


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  		TABLE 2. Effect of ammonium on CF1 x CF1 mouse embryo development in culture.a



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  		FIG. 2. Development of the ICM of blastocysts cultured in the presence of ammonium. At least 220 embryos were cultured per treatment. **Significantly different from blastocysts cultured in the absence of ammonium (P < 0.01)

Effect of Ammonium Levels on the Levels of Apoptosis in Blastocysts

Zygotes were cultured in either medium G1.2/G2.2 or in G1.2/G2.2 media supplemented with 18.8, 75, or 300 µM ammonium and cultured to the blastocyst stage. At the blastocyst stage, the level of apoptosis was assessed. Blastocysts that were cultured in media G1.2/G2.2 had low levels of apoptosis, with 2.9 ± 0.4 apoptotic cells in each blastocyst (Table 3). Culture with 18.8 µM ammonium resulted in a significant increase in the number of apoptotic cells in the blastocysts (Table 3). Increasing the ammonium concentration to 150 and 300 µM further increased the apoptotic cell index of the blastocysts such that 15.5% ± 2.7% of all cells in blastocysts cultured in 300 µM ammonium were apoptotic (Table 3).


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  		TABLE 3. Effect of ammonium on the number of apoptotic cells in blastocysts.a

Effect of Ammonium on Embryo Metabolism

Incubation of 2-cell embryos with 18.8 µM ammonium resulted in a significant decrease in the levels of pyruvate oxidation (Fig. 3A). Increasing the ammonium concentration to 75 and 300 µM further decreased the levels of pyruvate oxidation. Ammonium in the incubation medium at a concentration of 18.8 µM did not affect glycolytic metabolism; however, 2-cell embryos incubated in the presence of 75 or 300 µM ammonium had significantly increased levels of glycolysis compared to control embryos (P < 0.01) (Fig. 3A).



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  		FIG. 3. Effect of ammonium concentration on embryo metabolism. A) Two-cell embryos. B) Eight-cell embryos. C) Blastocysts. Twenty embryos were examined per treatment. Solid bars represent glycolytic activity. Open bars represent pyruvate oxidation. *Significantly different from embryos incubated in the absence of ammonium (P < 0.05). **Significantly different from embryos incubated in the absence of ammonium (P < 0.01). Values are mean ± SEM

Culture of 8-cell embryos in the presence of 75 or 300 µM resulted in a significant reduction in the levels of pyruvate oxidation (Fig. 3B). All concentrations of ammonium significantly increased the levels of glycolytic activity compared to embryos cultured in the absence of ammonium (Fig. 3B).

Blastocysts incubated in the presence of all concentrations of ammonium significantly reduced the levels of pyruvate oxidation compared to blastocysts incubated in medium G1.2/G2.2 without ammonium (Fig. 3C). Glycolytic activity of blastocysts was reduced in the presence of 300 µM ammonium (Fig. 3C).

Effect of Ammonium on the Regulation of Intracellular pH

Two-cell embryos collected from the oviduct and incubated in medium G1.2 had an intracellular pH of 7.28 ± 0.02. Embryos that were cultured with 18.8 µM ammonium had a significantly decreased level of intracellular pH compared with control embryos (Fig. 4). Increasing the levels of ammonium to 75 or 300 µM resulted in a further significant decrease in intracellular pH levels (Fig. 4).



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  		FIG. 4. Effect of incubation with ammonium on intracellular pH of 2-cell embryos. Twenty embryos were examined per treatment. *Significantly different from embryos incubated in the absence of ammonium (P < 0.05). **Significantly different from embryos incubated in the absence of ammonium (P < 0.01). Values are mean ± SEM

Effect of Ammonium on Fetal Development and Fetal Growth Rates

Culture of zygotes to the blastocyst stage in the presence of ammonium concentrations up to 150 µM did not affect subsequent implantation rates (Table 4). However, culture in the presence of 300 µM ammonium significantly decreased implantation rates following transfer (P < 0.05) (Table 4). Fetal development rates were significantly reduced when blastocysts were cultured in an ammonium concentration of 75 µM or higher. Increasing the ammonium concentration in the media to 300 µM further decreased fetal development rates. An ammonium concentration of 75 µM or higher resulted in a significant decrease in the percentage of implantations that resulted in fetuses.


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  		TABLE 4. Effect of ammonium on blastocyst viability and fetal outcome.a

When ammonium was present at 75 µM or greater, a percentage of all fetuses were morphologically abnormal (Table 4). Abnormalities observed included significant underdevelopment, abnormal cranial development, and stunted limb development. Fetal weight was only affected when blastocysts were cultured in the presence of 300 µM ammonium. However, it was not possible to obtain weights on some of the grossly abnormal fetuses, and this may have affected the overall mean of the groups cultured with ammonium at 75 µM or greater.

Interestingly, culture with 18.8 µM resulted in fetuses with longer crown-rump lengths that those obtained after culture in medium G1.2/G2.2 (Table 4). Increasing the concentration of ammonium to 75 or 300 µM significantly reduced crown-rump length of the fetuses (Table 4).

Using a morphological scoring system to stage the fetuses, those resulting from blastocysts cultured in media G1.2/G2.2 exhibited normal fetal development of 15.0 ± 0.2 days (Fig. 5). Fetal growth was significantly retarded at all ammonium concentrations (Fig. 5), and at a concentration of 300 µM, fetal development was retarded by 1.5 days.



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  		FIG. 5. Effect of culture of embryos with ammonium to the blastocyst stage on subsequent fetal growth rates in utero. Forty-eight blastocysts were transferred per treatment. *Significantly different from blastocysts cultured in the absence of ammonium (P < 0.05). **Significantly different from blastocysts cultured in the absence of ammonium (P < 0.01). Values are mean ± SEM

Effect of Ammonium on Gene Expression of Blastocysts

Zygotes were cultured in the presence of ammonium to the blastocyst stage, and the levels of uridine incorporation were assessed. Culturing embryos with ammonium concentrations of 18.8 or 75 µM did not affect the levels of uridine incorporation in blastocysts (1.32 x 105 and 1.39 x 105, respectively) compared to those cultured in medium G1.2/G2.2 (1.49 x 105). However, blastocysts cultured with 300 µM had significantly reduced levels of uridine incorporation (0.89 x 105, P < 0.01). This decrease in the levels of uridine incorporation when blastocysts were cultured with 300 µM was still evident when the data were corrected for average cell number.

No difference was observed in the levels of gene expression evaluated using semiquantitative RT-PCR of Na+/K+-ATPase, Igf2, neural transferase NAT-2, or ß-actin when embryos were cultured with 300 µM ammonium. In contrast, blastocysts cultured with 300 µM ammonium had significantly increased expression levels of H19 compared to the control blastocysts cultured in media G1.2/G2.2 (Fig. 6).



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  		FIG. 6. Effect of culture with 300 µM ammonium on levels of gene expression in blastocysts. Six replicate experiments were conducted. *Significantly different from blastocysts cultured in the absence of ammonium (P < 0.05). Values are mean ± SEM. Solid bars represent no ammonium. Open bars represent 300 µM ammonium


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data in the present study demonstrated that ammonium in the culture media resulted in rates of blastocyst development and blastocyst morphology equivalent to those observed when embryos were cultured in media without added ammonium. However, the presence of ammonium at very low concentrations, as low as 18.8 µM, during development from the zygote to the blastocyst stage has significant detrimental effects on embryo physiology. Transfer of these blastocysts to pseudopregnant recipients resulted in a significant reduction in the ability to establish a pregnancy and to produce fetuses of normal size after transfer. Embryos cultured with ammonium had significantly delayed development in utero. Exposure of embryos to 300 µM ammonium resulted in perturbed expression of the imprinted gene H19. Transfer of these embryos produced fetuses that were delayed in development by 1.5 days at Day 15 of pregnancy. The significance of the effects of ammonium on embryo development are highlighted by the levels of ammonium that appear in some culture media from the spontaneous breakdown of amino acids when incubated at 37°C. Culture media such as KSOMAA have levels of ammonium produced in the medium after only 24 h of culture that can affect all aspects of embryo physiology and also gene expression. These data indicate that ammonium is highly toxic to the murine embryo. Therefore, in studies assessing embryo normalcy after in vitro culture, it is essential that the conditions used minimize the levels of ammonium that build up in the culture media.

Blastocyst development of embryos from CF1 mice was not affected by the presence of ammonium in the culture media up to 300 µM. Neither was any difference observed in the morphology of blastocysts that were produced in the presence or the absence of ammonium. However, similar to previous studies [10, 48], blastocysts in the present study that were cultured with ammonium had significantly lower blastocyst cell numbers. Interestingly, the inhibitory effect of ammonium was greatest on the development of the ICM of the blastocysts, because a significant linear decrease was observed in the percentage development of the ICM in blastocysts cultured with ammonium. The significance of this observation is that development of the ICM of the blastocyst is proportional to the ability to establish a pregnancy and develop into a normal fetus [15]. Therefore, this decrease in the development of the ICM suggests that these blastocysts would have a reduced capability to produce fetuses of normal size after transfer. This hypothesis was supported as increasing the concentration of ammonium in the medium resulted in a decrease in the rates of fetal development.

In addition to a reduction in blastocyst cell numbers, embryos that were cultured with ammonium had significantly higher rates of apoptosis compared to blastocysts cultured in media G1.2/G2.2. It has been previously demonstrated that suboptimal culture conditions increase the levels of apoptotic cells in blastocysts [38] and that culturing embryos in KSOM without glutamine reduces the level of apoptosis in blastocysts [49]. Because glutamine breaks down in culture to produce significant levels of ammonium [10], this decrease in the apoptotic cell index of embryos cultured in the absence of glutamine would seem to be the direct result of a decrease in the levels of ammonium produced in the media [49].

Ammonium in the medium also perturbed the metabolic activity of embryos. At the 2- and 8-cell stages, incubation with ammonium increased the levels of glycolysis and decreased pyruvate oxidation. Early cleavage-stage embryos use very low levels of glucose. The oxidation of carboxylic and amino acids are the major energy-generating pathways [32, 50]. Glucose does not become the preferred carbohydrate until the blastocyst stage [51–54]. Premature elevation of glycolytic activity coupled with a decrease in oxidative capacity, as observed when ammonium was present in the medium, is associated with decreased developmental competence of cleavage-stage embryos, presumably caused by a crabtree-like effect, in which elevated glycolytic activity inhibits respiratory activity and oxidative metabolism [25, 55]. At the blastocyst stage, ammonium at all concentrations caused a similar decrease in oxidative capacity of the blastocysts. However, glycolysis was only affected by 300 µM ammonium. Ammonium has been shown to reduce the oxidative capacity of tissues by interfering with the transport of reducing equivalents between the cytoplasm and the mitochondria by disrupting the malate-aspartate shuttle activity [56]. Disruption of this shuttle results in an inability of cells to maintain the balance of NAD+/NADH within the cytoplasm and mitochondria and results in a decrease in tricarboxylic acid-cycle activity [57, 58]. Ammonium also causes changes in the mitochondrial NAD+:NADH ratios [59]. It is possible that ammonium may inhibit oxidative metabolism of the embryo by interfering with the activity of the malate-aspartate shuttle, which regulates carbohydrate utilization in embryos [60]. Additionally, because glucose metabolism is also regulated by the activity of the malate-aspartate shuttle, the reduction in glucose metabolism at the blastocyst stage likely results from an inability to maintain cytoplasmic NAD+ pools and, therefore, utilization of glucose by the Embden-Meyerhof pathway (metabolism of glucose to pyruvate).

The ability to tightly regulate intracellular pH is essential for normal cellular function. Ammonium, which is a weak acid, significantly inhibited the ability of embryos to regulate intracellular pH. Therefore, the sodium hydrogen antiporter, which regulates embryo intracellular pH in the acid range, was not able to overcome the effects of ammonium in the culture medium. Because intracellular pH is a universal regulator of cell function, an inability to maintain normal levels of intracellular pH in embryos results in a reduction in developmental competence [61, 62].

In addition to the vast perturbations in embryo physiology, culturing embryos in the presence of ammonium, not surprisingly, resulted in a general suppression in the levels of gene expression. This is evidenced by blastocysts cultured with 300 µM ammonium having significantly reduced levels of uridine incorporation. However, most significantly, the expression of the imprinted gene H19 was perturbed by incubation with ammonium. It has previously been demonstrated that H19 expression in blastocysts is sensitive to the culture conditions employed [63]. Collection of embryos in suboptimal conditions, using a tissue culture medium in the absence of bicarbonate, followed by culture in either a simple medium (Whitten) or KSOMAA, has been shown to affect the parental-specific expression of the H19 gene [63]. Because KSOMAA generates levels of ammonium in excess of the 300 µM ammonium tested here, the observed perturbed (leaky) imprinting of H19 in blastocysts cultured in KSOMAA probably resulted from the excessive buildup of ammonium in the culture conditions used [63]. This is currently under investigation in our laboratory. Previously, it has been reported that although high rates of blastocyst development can be obtained in the medium KSOMAA, the rate of embryo development in KSOMAA slows as the incubation period increases, presumably because of the ammonium buildup [46].

The transfer of blastocysts cultured in the presence of ammonium to pseudopregnant recipients revealed reduced fetal development rates, but the most significant observation was that the growth rates of the fetuses in utero were delayed compared to those of the controls. Therefore, in agreement with previous studies, the presence of ammonium in the culture medium during the preimplantation period had significant effects on the postimplantation development of the embryo [12]. This observation has significant implications for the in vitro production of embryos, because it clearly demonstrates that the environment to which the preimplantation embryo is exposed can affect subsequent postimplantation development, not only by reducing pregnancy rates but also by actually affecting the growth rate of any resulting fetus.

The significance of the observed effects of ammonium on embryo physiology and gene expression is that it is possible to generate morphologically normal-appearing blastocysts that are severely compromised. Therefore, it is essential that culture conditions and protocols with low levels of ammonium are used. The data from the initial experiment in the present study demonstrated that the media systems used in human-assisted conception contain very low levels of ammonium, because they routinely supplement glutamine with the more stable form of alanyl-glutamine, thereby markedly reducing ammonium buildup. However, in contrast, media that are routinely used for animal embryo production, such as KSOMAA or synthetic oviduct fluid medium with amino acids (SOFaa), contain amino acids, such as glutamine, that result in the production of high levels of ammonium. This production of ammonium is independent of the volume of medium used and of whether the medium is covered in oil (present study and unpublished observations), and it happens spontaneously when the medium is incubated at high temperatures of approximately 37–39°C. In fact, after less than 24 h of incubation, a medium such as KSOMAA resulted in the production of ammonium at detrimental levels. Therefore, when this medium is employed, it is quite plausible that any detrimental effect demonstrated on the physiology or genetics of the resulting blastocysts is a direct result of the very high levels of ammonium produced.

In conclusion, amino acids are essential regulators of embryo physiology and function, but they break down in culture media to produce ammonium. Ammonium has detrimental effects on all aspects of embryo physiology that were examined in the present study. Therefore, it is essential that in culture of the mammalian embryo, conditions are used that result in only minimal production of ammonium in the medium, thereby maintaining normal embryo function. This can be achieved by using media containing more stable forms of amino acids, by changing media drops every 24–48 h, and by only incubating media for limited times at 37°C. When embryos are cultured in media with minimal ammonium produced, in vivo rates of development and ICM formation can be obtained [64].


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the technical contribution of Jeff Maybach and Elizabeth Hewitt. We also thank Dr. John Gibbons and Elizabeth Hewitt for their comments on the manuscript, Organon, Inc., for the gift of Pregnyl, and Vitrolife AB for an unrestricted Research Grant.


   FOOTNOTES
 
1 Correspondence: Michelle Lane, michelle.lane{at}adelaide.edu.au Back

Received: 10 April 2003.

First decision: 2 May 2003.

Accepted: 21 May 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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