Disruption of Mitochondrial Malate-Aspartate Shuttle Activity in Mouse Blastocysts Impairs Viability and Fetal Growth

Abstract

The nutrient requirements and metabolic pathways used by the developing embryo transition from predominantly pyruvate during early cleavage stages to glucose at the blastocyst; however, the complexities involved in the regulation of metabolism at different developmental stages are not clear. The aims of this study were to examine the role of the malate-aspartate shuttle (MAS) in nutrient metabolism pathways in the developing mouse blastocyst and the consequences of impaired metabolism on embryo viability and fetal and placental growth. Eight-cell-stage mouse embryos were cultured in the presence of the MAS inhibitor amino-oxyacetate, with or without pyruvate as an energy substrate in the media. When the MAS was inhibited, the rate of glycolysis and lactate production was significantly elevated and glucose uptake reduced, relative to control cultured embryos in the presence of pyruvate. Despite these changes in embryo metabolism, this did not influence development to the blastocyst stage, but it did reduce the number of inner cell mass and trophectoderm cells. When these embryos were transferred to psuedopregnant females, inhibition of the MAS significantly reduced the proportion of embryos that implanted and developed into fetuses on Day 18 of pregnancy. Finally, fetal growth was reduced while placental weight was maintained, leading to a decreased fetal:placental weight ratio relative to control embryos. These results suggest that impaired metabolism of glucose in the blastocyst via the MAS alters the ability of the embryos to implant and form a pregnancy and leads to reduced fetal weight, likely via altered placental development and function.

Disruption of the malate-aspartate shuttle activity in the mouse blastocyst impairs glucose metabolism and embryo development and viability, and reduces fetal development, likely via altered placental function.

INTRODUCTION

There is emerging evidence that there are significant developmental consequences of perturbing the environment surrounding the developing oocyte and/or early embryo. Several studies have determined that deliberate perturbation to the conditions to which the mammalian preimplantation embryo is exposed alters both survival to the blastocyst stage and ability to establish a pregnancy. However, more significantly, there is evidence that a stress imposed on the early embryo either in vivo or in vitro can also alter the normal developmental program for fetal growth and health. Examples of this include fetal overgrowth in domestic animal embryos after culture in the presence of serum from the zygote to the blastocyst stage [1, 2], as well as slowed fetal growth in rodent species [3–6] and impaired cognitive behavior of the offspring [7] following culture in the presence of extreme in vitro stresses, such as hypoxia and elevated ammonium. Despite these striking developmental observations as a consequence of such perturbations, crucial information regarding the mechanisms that lead to these outcomes is lacking.

In other cellular models it has been determined that mitochondrial dysfunction caused by changes in mitochondrial bioenergetics alters the metabolic state of the cell, modifying the ATP:ADP ratio and the redox state (NAD⁺:NADH ratio), resulting in permanent developmental consequences to cellular health (for review, see Knudsen and Green [8]). We therefore propose that mitochondrial dysfunction in preimplantation embryos may be similarly involved in the permanent developmental consequences observed after exposure to an environmental stress.

Mitochondrial reducing equivalent shuttles play an important role in most cells in regulating a balance in NAD⁺:NADH levels between the cytoplasm and the mitochondria. These shuttles result in the net transfer of NADH across the inner mitochondrial membrane for ATP production while regenerating NAD⁺ within the cytoplasm to enable further metabolism of glucose and lactate. One of these shuttles, the malate-aspartate shuttle (MAS) is found in most cells [9, 10]. This shuttle transfers NADH from the cytoplasm into the mitochondria via a series of reactions catalyzed in the cytoplasm by malate dehydrogenase 1 (MDH1) and glutamate oxaloacetate transaminase 1 (GOT1), and in the mitochondria by mitochondrial malate dehydrogenase 2 (MDH2) and glutamate oxaloacetate transaminase 2 (GOT2). The activity of mitochondria shuttles is essential for cells to metabolize glucose and lactate as energy sources.

It has been established that the MAS is present in two-cell mouse embryos and is responsible for the ability of embryos at this stage of development to use lactate as an energy source [11]. Interestingly, mouse zygotes have been shown to have an absolute requirement for pyruvate as an energy source in culture [12]. The inability to use lactate as an energy source was shown to be the result of a lack of mitochondrial shuttle activity, which could be restored in vitro if the conditions were modified. Therefore, the activity of this mitochondrial shuttle affected the ability of the embryos to effectively use different carbohydrates for ATP production and development [11].

Mouse blastocysts preferentially use glucose as an energy substrate, with glucose metabolized by both oxidation in the mitochondria and also by aerobic glycolysis [13, 14]. The balance between mitochondrial and cytoplasmic utilization of glucose is altered by suboptimal incubation conditions, such as culturing embryos in a medium lacking amino acids, and results in the embryo converting the majority of the glucose taken up by the embryo to lactate [14–16]. This is a relatively energetically inefficient route for glucose metabolism and results in a significant reduction in the net ATP production by the blastocysts, which may effect essential cellular signaling and function and contribute to the observed reduction in viability [15]. Although the role of the MAS has been examined for earlier-stage embryos, a function for the MAS in blastocyst development has not been previously reported. Therefore, the aim of this study was to determine whether disruption of the MAS activity in mouse blastocysts affects the ability to balance glucose metabolism and to determine the consequences of MAS activity inhibition on pregnancy outcome and fetal development in the mouse.

MATERIALS AND METHODS

Handling and Embryo Culture Media

The composition of all media used for these experiments is outlined in Table 1 [17]. G-MOPS handling medium was used for zygote collection and manipulation, and cleavage-stage embryos were cultured in modified G1.2 until the eight-cell stage. From the eight-cell stage, embryos were cultured in modified G2.2: the control medium contained 3.15 mM glucose, 5.87 mM lactate, and 0.1 mM pyruvate as carbohydrate energy sources, and a no-pyruvate (-P) medium contained 3.15 mM glucose and 5.87 mM lactate but no pyruvate (-P). A third treatment group used the modified medium with no pyruvate but with the addition 0.5 mM of a MAS inhibitor, amino-oxyacetate (-P + AOA; Sigma). All media were prepared in house and were tested in a proven mouse embryo assay prior to use [18]. Human serum albumin (HSA; 5 mg/ml; Vitrolife) and Phenol red (0.005 g/l; Sigma Chemical Co., St. Louis, MO) were added to all culture media.

TABLE 1.

The composition of embryo handling and culture media used (mM).

Embryo Collection and Culture

Mouse embryos were obtained from F1 hybrid (C57BL6 × CBA) females, in compliance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and under the guidelines approved by The University of Adelaide Animal Ethics Committee. Females at 3–4 wk of age were superovulated with 5 IU eCG (Folligon; Intervet Australia Pty Ltd., Bendigo, Australia), and 48 h later with 5 IU hCG (Pregnyl; Organon, Oss, The Netherlands). Females were placed overnight with males of the same strain. Zygotes were collected at 24 h after hCG, were denuded from the surrounding cumulus by incubation for 1 min with 0.5 mg/ml hyaluronidase, and were washed twice in handling medium. All embryos were cultured in groups of 10–15 in 20-μl drops of medium at 37°C in 6% CO₂:5% O₂:89% N₂. Zygotes were cultured in G1.2 for 44 h and then in either G2.2 (control) or G2.2 with modified carbohydrates (-P and -P + AOA treatments) for a further 48 h to the blastocyst stage. Embryo cleavage was assessed after 20 h of culture, and following 92–93 h of culture embryos were classified as two-eight cells, morula (compact structure), blastocyst (blastocoel cavity comprised less than three quarters of the embryo), expanded blastocyst, and hatching blastocyst (clear hemiation of the zona pellucida). In vivo-developed blastocyst-stage embryos were collected at 88 h after hCG, then incubated with or without AOA for 3 h prior to measurement of glucose and lactate uptake and oxygen consumption.

Assessment of Glucose Uptake and Lactate Production

Individual blastocysts were placed into 40-nl drops of metabolism medium (G2.2 modified to contain 0.5 mM glucose as the sole energy substrate and lacking amino acids and vitamins) at 37°C, and serial 1-nl samples of media were taken over a 1.5-h period. The 1-nl samples of media were assessed for levels of glucose and lactate using microfluriometry using methods described previously [19]. Linear rates of glucose uptakes (pmol/embryo/h) and lactate production (pmol/embryo/h) were assessed, and the ratio of lactate production to glucose uptake was determined using the assumption that 2 mol lactate is produced per mole of glucose taken up by the embryo.

Assessment of Oxygen Consumption

Rates of oxygen consumption by embryos were assessed by a noninvasive pyrene method [20]. Blastocyst-stage embryos were incubated at 37°C in 2 μl G-MOPS handling medium with or without AOA in a 5-μl PCR micropipette loaded with 1 μl pyrene (dissolved in paraffin oil; Sigma Chemical Co.) that had been pre-gassed overnight in a gas phase of 20% oxygen. After loading, each end of the tube was sealed with wax, and oxygen consumption of embryos was determined by measuring the changes in fluorescence of the pyrene over a 5-h period using a fluorescence microscope with photometer attachments (Leica, Wetzlar, Germany). For each experiment a control of 1 mg/ml yeast that had been incubated overnight in 60 mM glucose for zero oxygen and a 20% oxygen control where the PCR tube was loaded with medium and no embryos were included. The levels of oxygen consumption were then calculated using a program that relates the linear change in pyrene fluorescence to changes in oxygen levels and models the diffusion of oxygen along the length of medium and pyrene [21]. Oxygen consumption was expressed as picomoles of oxygen consumed per embryo per hour.

Differential Staining of Blastocyst-Stage Embryos

The number of inner cell mass (ICM) and trophectoderm (TE) cells in the blastocysts was determined using a previously described staining procedure [22]. Briefly, the zona pellucida was removed by incubating blastocysts in 0.5% pronase followed by incubation in 10 mM picrylsulfonic acid at 4°C for 10 min. Embryos were transferred to 0.1 mg/ml anti-dinitrophenyl-BSA (anti-DNP-BSA) for 10 min at 37°C and then placed into 10 μg/ml propidium iodide in guinea pig serum for 5 min at 37°C. Blastocysts were then stained in 6 μg/ml bisbenzimide in ethanol overnight and washed in 100% ethanol prior to mounting in glycerol on a microscope slide the following day. Stained blastocysts were visualized using a fluorescent microscope under an ultraviolet filter, allowing TE (appearing pink) and ICM (appearing blue) cell nuclei to be counted independently.

Embryo Transfer and Pregnancy Outcome

To determine the ability of the embryos to establish a pregnancy, blastocysts were transferred to pseudopregnant female mice. Swiss female mice between 8 and 12 wk of age were placed with vasectomized males to establish pseudopregnancy, and blastocysts were transferred surgically to the uterus on the morning of Day 4 of pseudopregnancy. To each uterine horn six blastocysts were transferred, and a different treatment was transferred to each uterine horn as determined by random numbers. A total of 10–13 transfers were performed for each treatment group. On Day 18 of pregnancy, the number of fetuses and resorption sites were recorded, and fetal and placental measurements were taken.

Statistical Analysis

All data were analyzed using a univariate generalized linear model, and differences between treatments were determined using LSD (SPSS 15.0; SPSS Inc., Chicago, IL). For binomial data each replicate was expressed as a proportion for analysis. For embryo assessments the day of replicate was treated as a covariate, and for fetal development, litter size and recipient were additionally fitted as covariates.

RESULTS

Effect of Inhibiting MAS Activity on Metabolic Activity of In Vivo Blastocysts

In vivo-developed blastocysts were collected and incubated in either control medium G2.2 or medium containing the MAS inhibitor AOA for 3 h prior to assessment of metabolism. The ratio of the rate of lactate produced per amount of glucose taken up was determined and is indicative of glycolytic activity. The ratio was 0.5 for in vivo-developed mouse blastocysts incubated in medium G2.2, whereas inhibition of MAS with AOA significantly increased this such that all of the glucose taken up by the blastocyst could be accounted for by lactate production (P < 0.05; Table 2). This change in the metabolic fate of glucose was also coupled with a significant decrease in overall glucose uptake by blastocysts cultured in the presence of AOA relative to controls (P < 0.05).

TABLE 2.

The effect of short-term inhibition of MAS activity on the glycolytic activity and oxygen consumption of in vivo developed blastocysts.*

To further determine the role of MAS on the maintenance of oxidative capacity, the levels of oxygen consumption were determined in blastocysts. In vivo-developed blastocysts incubated with AOA had significantly decreased levels of oxygen consumption compared with those incubated in medium G2.2 without AOA (Table 2).

Effect of Inhibition of MAS Activity on Blastocyst Development

The role of MAS activity on development to the blastocyst stage was determined by culturing eight-cell embryos in either control medium or in G2.2 medium lacking pyruvate with or without AOA (-P + AOA and -P, respectively). Eight-cell control embryos reached the blastocyst stage at high rates, and restricting the carbohydrates in the medium by removing pyruvate in either the presence or absence of AOA did not affect development to the blastocyst stage (Table 3).

TABLE 3.

Effect of MAS activity on in vitro blastocyst development and cell differentiation.*

Assessment of the quality of the blastocysts by examining cell number and differentiation into the ICM and TE revealed that removal of pyruvate (-P) from the culture medium reduced the total cell number of the blastocysts, with a significant reduction in both ICM and TE cell numbers; however, the proportion of ICM:TE cells in the blastocyst was not affected. This was exacerbated with the addition of AOA (-P + AOA), with a further reduction in both the total cell numbers and those of the ICM and TE, whereas the proportion of ICM:TE cells did not differ in either treatment (Table 3).

Effect of In Vitro Culture on Blastocyst MAS Activity

The ratio of the rate of lactate produced per amount of glucose taken up was determined. The ratio in control cultured blastocysts was 0.40, and a similar conversion was measured in blastocysts when pyruvate was removed from the medium (0.48). The addition of AOA to this medium significantly increased this ratio to 1.07, indicating that all of the glucose taken up was converted to lactate (Table 4). There was a significant increase in glucose uptake relative to control embryos when pyruvate was removed from the medium (6.9 vs. 5.3 pmol/embryo/h), but the addition of AOA to this modified medium conversely resulted in a significant decrease in glucose uptake (3.7 pmol/embryo/h; Table 4).

TABLE 4.

Effect of MAS activity on in vitro blastocyst metabolism.*

Lactate production by the blastocyst was increased when pyruvate was removed from the medium, with or without the presence of AOA, when compared to blastocysts cultured in the control medium (Table 4).

Effect of MAS Inhibition on Blastocyst Viability and Postimplantation Development

Blastocyst viability and development after implantation were assessed following embryo culture and transfer into pseudopregnant females. Blastocysts cultured in control medium or in medium containing no pyruvate (-P) implanted at similar rates (Table 5). Addition of AOA to the medium from the eight-cell to the blastocyst stage (-P + AOA) significantly reduced both implantation rates and the proportion of fetuses that developed (P < 0.05; Table 5). There was also a significant reduction in the percentage of implantations that developed into fetuses for this treatment relative to control and -P treatments (P < 0.05; Table 5).

TABLE 5.

The effect of MAS activity on in vitro blastocyst viability following transfer to pseudopregnant females.

Examination of the resultant fetuses revealed that culture of embryos in the absence of pyruvate, with or without the MAS inhibitor AOA, significantly reduced fetal weight when compared to embryos cultured in control media (Fig. 1A). However, there was no significant difference in the average weight of placentas recovered from the same females between the different embryo culture treatments (Fig. 1B). The ratio of fetal:placental weight was significantly lower when embryos were cultured in the absence of pyruvate with the addition of AOA prior to transfer compared with control embryos (P < 0.05), and this trend approached significance when comparing the ratios between control and -P culture embryos (P = 0.07; Fig. 1C).

FIG. 1.

Effect of inhibition of MAS activity in the in vitro blastocyst on placental and fetal characteristics following embryo transfer. A) Fetal weight. B) Placental weight. C) Fetal:placental weight. Data represent n = 23, 33, and 7 paired fetuses and placentas for the conrol, -P, and -P + AOA treatments, respectively. Data are expressed as mean ± SEM. Different lowercase letters represent significant differences between treatments, P < 0.05. In C, there was no significant difference between treatments.

DISCUSSION

It is possible for many early embryos to develop to the blastocyst stage under various culture conditions, but a high proportion of these blastocysts are unable to implant and develop into viable fetuses when transferred to the uterus. Although our understanding of the cellular mechanisms that regulate the viability of these blastocysts is limited, it was hypothesized that perturbed metabolism may contribute to the reduced viability. The data presented in this paper demonstrate for the first time that the balance of mitochondrial metabolism with cytoplasmic metabolism, which is maintained by MAS, is important for the development of the mouse blastocyst and the formation of a viable pregnancy. There was reduced fetal size when the function of the MAS was impaired, but there also was preservation of placental size across all treatments; these suggest some form of intrauterine growth restriction as a consequence of impaired metabolism of the blastocyst. These observations indicate that there is a permanent change in the physiology of the TE cells that is maintained in the cells of the placenta and provides a mechanism whereby fetal development may be programmed.

The mouse embryo transitions from using pyruvate as the predominant energy source during early cleavage stages to glucose at the blastocyst stage [13]. It has been demonstrated that the MAS is important for controlling carbohydrate utilization in the early embryo, enabling metabolism of lactate as an energy source to support development [11]. However, the requirement for pyruvate to support the first cleavage division in the mouse zygote results from inadequate activity of the MAS, which can be overcome by the addition of aspartate, thus permitting the utilization of lactate [11]. At the two-cell and eight-cell stages there is increased activity of the MAS in vivo and in vitro, which enables the transport of reducing equivalents across the mitochondrial membrane, thereby maintaining the redox balance between the cytoplasm and mitochondria and enabling the use of lactate and glucose as energy substrates [11]. Although the role of the MAS has been established for the earlier-stage embryo, a function for the MAS in blastocyst development has not been reported previously.

In the absence of functional MAS activity, NAD⁺ is used rapidly in the cytoplasm without being regenerated, causing a redox imbalance between the cytoplasm and the mitochondria. Because cells require a balance between these two compartments, in cell types that either lack or have low MAS activity, a metabolic adaptation allows the continued use of glucose as an energy substrate; that is, they instead convert the glucose taken up to lactate, which results in the replenishment of NAD⁺ within the cytoplasm by the conversion of NADH to NAD⁺ by lactate dehydrogenase. Although this maintains the NAD⁺ levels within the cytoplasm, it is at a significant energy cost because there is a reduction in the levels of ATP generated. This conversion of glucose to lactate occurs in the presence of sufficient oxygen for oxidation to occur, as is termed aerobic glycolysis. The removal of pyruvate from the medium would result in the embryo becoming reliant on the MAS to transport NADH across the mitochondrial membrane, although the further addition of AOA to the medium would further limit mitochondrial transport of NADH, essentially ensuring all energy would be derived from the energetically inefficient pathway of glucose to lactate.

The observation that reduced MAS activity in many cell types results in a significant portion of glucose being converted to lactate rather than entering the mitochondria for oxidation is interesting, given reports that suboptimal embryo culture conditions also perturb the levels of lactate produced when glucose is used as the preferred energy substrate [14]. This led to the hypothesis that adaptation of metabolism in the mammalian blastocyst may result from a reduction in the functional capacity of the MAS. The studies presented here exposed mouse embryos to the MAS inhibitor AOA to elucidate the metabolic and developmental consequences of the activity of this shuttle in the eight-cell to blastocyst stage embryo. Initial experiments exposed in vivo-derived blastocysts to the inhibitor for a short period of time (3 h) and confirmed that inhibition of MAS activity resulted in a similar change in glucose metabolism, such that all of the glucose taken up by the blastocysts was converted to lactate via aerobic glycolysis (lactate:glucose ratio) and reduced oxygen consumption.

Embryo culture with this inhibitor from the eight-cell to the blastocyst stage with only glucose and lactate in the media for metabolism also effected glucose uptake and utilization in this way. These effects are similar to what was observed when blastocysts were cultured in medium without physiological regulators, such as amino acids and vitamins [16]. Interestingly, blastocysts cultured without pyruvate had levels of lactate production and glycolysis similar to those in the control blastocysts, but had significantly higher levels of glucose uptake. This indicates that there was some compensation for the loss of pyruvate uptake by increasing the levels of glucose utilization, as has been reported for the sheep blastocyst [23]. The reduction in oxygen consumption and glucose uptake in the presence of AOA could be viewed as a quietening of metabolism, as postulated by Leese in 2002 [24]. However, the impaired fetal viability of the embryos that ensues following transfer suggests that metabolism of these embryos is below that of the hypothesized quiet level and that which promotes viability. The significance of a relationship between respiration rate and embryo viability remains unclear and inconclusive [25], particularly in light of the variability between species in metabolism, for the later-stage preimplantation embryo.

Despite these metabolic changes when the MAS was inhibited in later-stage mouse embryos with AOA, the ability to develop to a blastocyst itself was not affected. Eight-cell embryos developed to the blastocyst stage at high rates even in the presence of AOA, when all of the glucose was converted to lactate. However, the numbers of cells of the blastocysts were significantly reduced in these conditions, as was also seen when pyruvate was absent in the media, although to a lesser extent. Similarly, studies that have grown embryos in conditions lacking amino acids where there appears to be a reduction in the activity of MAS, also demonstrated a reduction in blastocyst cell number [26, 27].

The ICM cells of the blastocyst have been shown previously to rely entirely on aerobic glycolysis for their energy requirements, converting 100% of the glucose taken up to lactate [28]. Our own studies (data not shown) have confirmed this finding. Therefore, ICM cells would appear to have limited MAS activity, and therefore the addition of the inhibitor to the medium would be expected to cause minimal disruption to the ICM's capacity to generate energy. In contrast, the TE cells generate a significant proportion of their energy from oxidation, approximately 55% [28]. Therefore, it is likely that the addition of AOA to inhibit MAS activity would significantly reduce glucose oxidation and the metabolic capacity of the cells forming the trophectoderm, and therefore may mediate the majority of its effects on viability via these cell types.

The reduced metabolic capacity of the TE cells of the blastocysts cultured with AOA and the increased dependence of glucose conversion to lactate for energy generation also were associated with a significant reduction in the viability of the blastocyst. It has been determined previously that in both the mouse and human that blastocysts that maintained a capacity to oxidize glucose, thereby reflecting an operational MAS, had significantly greater ability to develop into a fetus compared with those that relied entirely on glucose conversion to lactate [15, 29, 30]. Similarly, the data in this study demonstrated that inhibition of the MAS, and therefore glucose oxidation, resulted in a significant impairment to implantation, with significantly fewer blastocysts being able to implant and, once implantation was initiated, significantly more resorptions. Therefore, it would appear that blastocysts that had low or no activity of the MAS were less able to form a viable implantation than those that had some MAS activity. However, interestingly, the reduced blastocyst cell number when glucose and lactate were the only substrates (-P) indicates that MAS activity on its own does not have sufficient capacity to maintain oxidation; rather, exogenous pyruvate is also required for optimal metabolism and development. There are several reports that indicate that there is a synergistic interaction between pyruvate and glucose for the development of the embryo to the blastocyst stage [31, 32], and the data from our study confirm these earlier studies.

An interesting observation from this study is that embryos grown in conditions lacking pyruvate (-P) were able to implant and produce a fetus at the same rate as the controls, yet those fetuses that did form were significantly compromised—that is, they were smaller. In contrast, embryos that were also exposed to AOA with no pyruvate implanted and developed at significantly lower rates, but also had significantly compromised fetal size. Of note at this point is that the embryos that were transferred (on Day 4.5) differed significantly in cell number as a result of treatment relative to controls, and all embryos were transferred into recipients that were synchronized at Day 3.5 at the time of transfer. Therefore, the differences in fetal weight may simply reflect an immaturity, relative to the controls, of the blastocysts derived from the -P + AOA treatment and the -P treatment. The fact that there was no effect of treatment on the ability of an embryo to develop to the blastocyst or on placental weight makes this an interesting model to study the time of implantation relative to blastocyst development.

For both the -P and -P + AOA treatments, the corresponding placentas did not differ in weight relative to the controls, suggesting a functional or structural change in the placenta. During normal placentation, the trophoblast cells adhere to and invade the uterine stroma, and the cells differentiate into villous or extravillous components. Placental deficiency, leading to intrauterine growth restriction, is a phenomenon observed across species, and the underlying causes are numerous [33]. It is conceivable that the metabolic insufficiency of embryos as a consequence of MAS inhibition and culture media lacking pyruvate impaired the ability of trophoblast cells to differentiate and invade the uterine stroma. Such an inability could have all manner of consequences—these include reduced functional capacity of the placenta for exchange of gas and important nutrients, such as amino acids and glucose; malformation of placental vasculature, required to facilitate increased blood flow from the mother through to the placenta; and, finally, impaired development of the syncitiotrophoblast cell layer, required for the production of hormones to support pregnancy, such as placental lactogen [34]. Thus, the observations reported in this study are indicative that there may be some permanent change in the physiology of the TE cells that is maintained in the cells of the placenta. This is an intriguing observation, and work is currently underway in our laboratory to examine placental transport of glucose and amino acids.

In conclusion, this study has demonstrated an important role for the MAS in regulating nutrient metabolism in the developing mouse blastocyst. Furthermore, by perturbing the normal operation of this shuttle in the embryo, the growth trajectory of the developing fetus can be programmed, likely via alterations in the function of the placenta. Exploration of the consequences of dysfunctional metabolism in the embryo on the normal developmental pathways in placental and fetal tissues will provide further mechanistic insight into the phenomena of developmental programming of adult health.