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a Institute of Reproduction and Development, Monash University, Monash Medical Centre, Clayton, Victoria 3168, Australia
b Colorado Center for Reproductive Medicine, Englewood, Colorado 80110
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The enzyme lactate dehydrogenase (LDH), a reversible near-to-equilibrium enzyme, converts lactate to pyruvate to enable its entry into the tricarboxylic acid cycle for subsequent oxidation. The activity of LDH in mouse embryos has been assessed in several studies. LDH activity was shown to be highest at the zygote stage, decreasing at the blastocyst stage [9]. While studies have compared the activity of LDH in extracts from embryos [9–11], there is no information regarding the kinetics of LDH in the preimplantation embryo. Moreover, the effect of lactate concentration on the regulation of LDH activity and therefore pyruvate and lactate utilization has not been determined.
The aim of this study was to assess the metabolism of lactate and its role in the regulation of pyruvate and glucose metabolism. The effect of lactate in the medium on the nicotinamide adenine dinucleotide+ (NAD+):NADH ratio of the cytoplasm at different stages of development was also examined. The kinetics of the enzyme LDH were assessed at the zygote and blastocyst stages, and the regulation of LDH by the NAD+:NADH ratio was determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryos were obtained from F1 hybrid (C57BL/6 x CBA/Ca) females that were superovulated with 5 IU of eCG (Folligon; Intervet, Lyppard, Australia) followed 48 h later by 5 IU of hCG (Chorulon; Intervet). Immediately after the second injection, females were placed with males of the same strain. Mating was indicated by the presence of a vaginal plug the following morning.
Media Composition
Media used in this study were based on modified Mouse Tubal Fluid medium [12] in which 20 mM NaHCO3 was replaced with 20 mM Hepes at pH 7.4 (H-mMTF). Media, prepared weekly from stock solutions, had the following composition: 103.4 mM NaCl, 4.79 mM KCl, 1.19 mM MgSO4, 1.19 mM KH2PO4, 1.71 mM CaCl2, 5.0 mM NaHCO3, 20 mM Hepes, 0.37 mM sodium pyruvate, 4.79 mM L-sodium lactate, 3.4 mM glucose, 0.10 mM phenol red, 0.06 g/L penicillin, 0.05 g/L streptomycin, supplemented with 4 g/L BSA. Media with increased lactate concentrations had reduced NaCl concentrations to maintain osmolarity. All salts were of high purity (AnalaR brand; BDH, Poole, Dorset, UK); carbohydrates, antibiotics, and Hepes were of embryo-tested grade (Sigma Chemical Co., St. Louis, MO); and BSA was purchased from Bayer Diagnostics (Miles Pentex Crystalline, lot 97; Kankakee, IL).
Collection of Embryos
Zygotes were collected at 21 h post-hCG and denuded by a 1-min incubation with 1 mg/ml hyaluronidase (bovine testes; Sigma). Two-cell embryos were flushed from the oviduct at 46 h post-hCG; 8-cell embryos were flushed from the uterotubal junction at 64 h post-hCG; and blastocysts were flushed from the uterus at 88 h post-hCG. Embryos were washed twice in H-mMTF, and either their metabolism or LDH activity was assessed.
Determination of Pyruvate and Glucose Uptakes
Pyruvate and glucose uptakes were assessed in individual embryos using quantitative microfluorescence [5,6,13,14]. Assays were based on conventional methods of enzymatic analysis employing the pyridine nucleotides NAD(P)H in coupled reactions. Individual embryos were placed in a 25-nl drop of medium H-mMTF modified to contain 0.5 mM glucose, 0.5 mM pyruvate, and lactate concentrations of either 0, 5, 10, 20, or 40 mM. Embryos were incubated at 37°C for up to 2 h. Serial 1-nl samples were taken at 15-min intervals, and glucose and pyruvate concentrations were determined. Linear rates of glucose and pyruvate uptakes were determined for individual embryos and expressed as pmol/embryo per hour. Pyruvate and glucose assays were not affected by the presence of 40 mM lactate.
Analysis of Pyruvate and Lactate Oxidation
Pyruvate and lactate oxidation was assessed by incubation with radiolabeled substrates in a microcentrifuge tube [15]. Pyruvate oxidation by single embryos was determined by incubation with mMTF (with 0.5 mM glucose and no Hepes), [2-14C]pyruvate (0.5 mM; 0.085 mCi/ml) in the presence of 0–40 mM lactate concentrations. Lactate oxidation by individual embryos was determined by incubation in mMTF with 0.5 mM pyruvate and [U-14C]lactate at increasing concentrations (5–40 mM). The amount of labeled lactate in the medium was constant (0.746 mM; 0.117 mCi/ml). The increase in lactate concentration in the medium was achieved by increasing the concentration of unlabeled lactate.
Individual embryos were incubated in 3-µl drops of medium containing the appropriate labeled substrate on the lid of a microcentrifuge vial. A volume of 1.5 ml of 25 mM NaHCO3 pregassed with 5% CO2 in air was added to each vial. The air space between the trap of NaHCO3 and the embryo on the lid was filled with 5% CO2 in air, and the vial was sealed. Embryos were incubated for 3 h at 37°C. Controls were included that contained a 3-µl drop of medium on the lid without an embryo in order to control for spontaneous breakdown of the label. Total counts were determined by the addition of a 3-µl drop of medium straight into the 1.5 ml NaHCO3. After 3 h the vials were opened, and 1 ml of the NaHCO3 was removed and placed into a scintillation vial containing 200 µl of 0.1 M NaOH. Vials were then sealed and stored at 4°C overnight. The following morning, 10 ml of scintillation fluid was added to each vial. The vials were vortexed and counted for 4 min each. Oxidation of labeled substrate was determined from the recovery of CO2 in the vials corrected for the recovery efficiency, which had been previously determined by incubating 3 µl of [14C]sodium bicarbonate on the lid of a vial containing 1.5 ml of NaHCO3. Samples (1 ml) were taken at 30-min intervals for 5 h where the recovery efficiency reached plateau by 3 h [15].
Enzyme Extraction
Individual embryos were washed twice in saline with 4 g/L BSA and placed in 500 nl of enzyme extraction buffer containing 100 mM K2HPO4, 30 mM KF, 1 mM EDTA, 5 mM mercaptoethanol, 2 g/L BSA, and 0.5 g/L PMSF at pH 7.5 [11]. Extraction buffer containing embryos was taken up in a glass capillary tube, sealed in a plastic straw, and plunged into liquid nitrogen. Individual embryos were stored for up to a week at -80°C. Immediately prior to analysis, the extracted embryo was thawed and expelled under oil onto a siliconized glass slide, where it was kept at 4°C until analysis was completed (around 10 min). This extraction was used to assess enzyme activity as described by Martin et al. [11]. It is not necessary to purify the enzyme for assessment of kinetics, as the extraction and assay conditions themselves result in a 2.5 x 106 dilution in the assay mixture. It is not feasible that endogenous regulators would still be active at this dilution.
Determination of LDH Activity
LDH activity was determined using quantitative microfluorescence based on the following reaction: pyruvate + NADH + H+ 
lactate + NAD+. The composition of the reaction buffer was 50 mM K2HPO4, 0.8 mM KH2PO4, 0.63 mM pyruvate, 0.116 mM NaHCO3, and 0.057 mM NADH. Reactions were performed at 37°C, and all reagents were at the concentrations required for assessment of maximal activity [16].
For the assay of each embryo, the enzyme extraction buffer containing the embryo was thawed and expelled under mineral oil onto a siliconized slide, and a 10-nl drop was removed and placed under oil onto a second slide. The initial sample was maintained at 4°C. A 70-nl drop of the reagents was placed onto a siliconized slide and warmed to 37°C, and its fluorescence was quantitated in the presence of UV light. The 10-nl drop of sample was warmed to 37°C; then 3 nl was removed and added to the reagent drop. The change in fluorescence was determined at 5- to 10-sec intervals until a plateau was reached (1–2 min). The change in fluorescence was calculated by a series of standard curves run each day. A control drop of cocktail without sample was warmed to 37°C, and the fluorescence was determined at 5-sec intervals to ascertain the amount of photo-oxidation of NADH by exposure to UV light. A further control drop of sample and reagents in the absence of substrate (pyruvate) was also used to control for nonspecific oxidation. Enzyme activity was determined from a plot of NADH oxidized over time for each embryo. A tangent to the initial linear rate of oxidation of NADH was drawn to determine velocity (Vi). The activity of the enzyme was then calculated from the gradient and has been expressed as nmol NADH oxidized/embryo per hour. The exact amount of enzyme can be calculated for each embryo, as the extraction is in a known volume.
The Km and Vmax of LDH for pyruvate in the zygote and blastocyst were determined in the presence of increasing pyruvate concentrations: 0, 0.04, 0.08, 0.16, 0.32, 0.63, and 1.26 mM (0.63 mM was the concentration present in the assays to determine maximal activity). In each case the buffer solutions for the assay were prepared individually to contain the required pyruvate concentration. The activity of LDH was determined over the range of pyruvate concentrations for each individual embryo. As LDH activity obeys Michaelis-Menten kinetics, initially a plot of reaction velocity (V) as a function of substrate concentration (S) was determined for each embryo. This was then converted to a Lineweaver-Burk plot, and the Vmax and Km were determined for the enzyme of each embryo.
Autofluorescence
The relative redox potential (NAD+:NADH ratio) of the cytoplasm of cells was estimated by determining autofluorescence [17]. In this study the relative redox potential of the cytoplasm was estimated for mouse embryos at the zygote (27 h post-hCG after cumulus cells have dispersed), 2-cell, and morula stage. Embryos were incubated in medium containing 0.5 mM pyruvate and increasing lactate concentrations for 45 min prior to analysis. The relative autofluorescence of the cells was assessed by exposing embryos to UV light (500 µsec), and the fluorescence from a fixed area within the exposed embryo was quantitated using a photometer and photomultiplier attachment. Values are expressed as a scale of arbitrary units where 850 units represents maximum fluorescence.
Statistical Analysis
Data for pyruvate uptake, oxidation, and autofluorescence were found to be normally distributed and were examined using ANOVA. Differences in metabolism between stages of development or concentrations were determined using Duncan's Multiple Range test. Analysis of LDH activity in blastocysts and zygotes was also found to be normally distributed, and differences were therefore determined using Student's t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pyruvate uptake by zygotes and 2-cell embryos was found to be around 4 pmol/embryo per hour at lactate concentrations up to 20 mM (Fig. 1a). Increasing the lactate concentration to 40 mM significantly reduced pyruvate uptake by both zygotes and 2-cell embryos (Fig. 1a). Pyruvate uptake by 8-cell-stage embryos was found to be constant at around 4 pmol/embryo per hour at lactate concentrations between 0 and 10 mM. Increasing the lactate concentration in the medium to 20 mM or above significantly reduced pyruvate uptake of the 8-cell embryos (Fig. 1a). In contrast to the earlier stages, pyruvate uptake at the blastocyst stage was significantly reduced at lactate concentrations of 5 mM. Increasing the lactate concentration to 20 mM resulted in further reductions in pyruvate uptake. Further increasing the lactate concentration to 40 mM did not further reduce pyruvate uptake at the blastocyst stage (Fig. 1a). The concentration of lactate in the culture medium therefore significantly affected the pattern of pyruvate uptake by the developing mouse embryo. In the presence of 20 mM lactate, pyruvate uptake was initially high at the zygote stage, with pyruvate uptake decreasing at the blastocyst stage to less than 1 pmol/embryo per hour. In contrast, in either the absence of lactate or the presence of physiological levels of lactate (5 mM) [13], pyruvate uptake was initially high (around 4 pmol/embryo per hour) in the cleavage-stage embryo and remained high to the blastocyst stage. In contrast to its marked effect on pyruvate uptake, lactate had no effect on glucose uptake at any of the concentrations studied (Fig. 1b).
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Effect of Lactate Concentration on the Oxidation of Pyruvate and Lactate
The effect of increasing the lactate concentration in the medium on pyruvate and lactate oxidation was assessed at the zygote and blastocyst stages. At the zygote stage there was an inverse relationship between decreasing pyruvate oxidation and increasing lactate concentration in the medium (r = 0.94; P < 0.05) (Fig. 2a). Calculation of the percentage of pyruvate taken up by the zygote that was oxidized to CO2 revealed that the percentage of pyruvate oxidized was also significantly reduced by increasing lactate concentration in the medium (Fig. 2b).
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At the blastocyst stage, pyruvate oxidation was also significantly reduced in the presence of an increasing lactate concentration (r = 0.97, P < 0.05). However, in contrast to observations in the zygote, the percentage of pyruvate taken up and oxidized by blastocysts increased linearly with increasing lactate concentration in the medium (r = 0.97; P < 0.05) (Fig. 3). At a lactate concentration of 20 mM, 60% of the pyruvate taken up by the blastocyst was oxidized as compared to 35% in the absence of lactate (P < 0.01).
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At the zygote stage, lactate oxidation was similar at concentrations of 5 and 10 mM. However, at 20 mM, lactate oxidation was significantly increased (Fig. 4). At the blastocyst stage, lactate oxidation significantly increased linearly with concentrations from 5 to 20 mM (Fig. 4).
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Effect of Lactate Concentration on LDH Activity
The activity of LDH for the conversion of pyruvate to lactate was determined at both the zygote and blastocyst stages. The activity of LDH isolated from zygotes was significantly higher than that present in the blastocyst (P < 0.01) (Table 1). There was a significant reduction in the activity of LDH measured in the presence of 20 mM lactate isolated from both zygotes and blastocysts (P < 0.01) (Table 1). This represented a reduction in LDH activity of around 40% in the presence of 20 mM lactate at both stages of development.
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Examination of the kinetics of isolated LDH from both zygotes and blastocysts revealed that the Km values of LDH for pyruvate were similar at the zygote and blastocyst stages, indicating that the isozymes were the same (Table 2). However, the Vmax of LDH isolated from zygotes was significantly higher than that in the blastocyst (P < 0.05), confirming that the activity of LDH was higher in the zygote (Table 2).
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Changing the NAD+:NADH ratio in the enzyme assay significantly altered the activity of isolated LDH in the blastocyst. Only one stage of development was tested here as the isozymes have been shown to be the same in the two stages of development. Increasing the concentration of NAD+ in the incubation buffer from 0 mM for the measurement of maximal activity to 15.0 mM significantly reduced the activity of isolated LDH (P < 0.05) (Table 3).
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Autofluorescence of Embryos
The autofluorescence of embryos at the zygote, 2-cell, and morula stage was determined in order to estimate the relative values of the NAD+:NADH ratio, as autofluorescence is indicative of the inverse of the NAD+:NADH ratio [18]. Increasing the lactate concentration in the medium linearly increased the autofluorescence of zygotes (r = 0.96; P < 0.05). However, at the 2-cell and morula stages, increasing the lactate concentration in the medium did not alter the autofluorescence (Fig. 5). At lactate concentrations of 10 mM or above, the autofluorescence of the zygote was significantly higher than in the later stages (P < 0.01) (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been accepted for many years that the preimplantation mouse embryo has an initial preference for pyruvate at the cleavage stages [1], with glucose uptake remaining low. Two studies have shown that a switch then occurs in nutrient uptake by the blastocyst stage such that pyruvate uptake declines and glucose uptake increases [5,6]; both of these studies were performed in the presence of 23.3 mM lactate. However, Gardner and Leese [19] observed that when pyruvate was present as the sole energy substrate, the documented decrease in pyruvate uptake at the blastocyst stage no longer occurred. The data presented in the present study demonstrate that this reported switch in carbohydrate preference by the preimplantation mouse embryo is dependent on the incubation conditions used for the metabolic assessment. In the presence of high concentrations of lactate (around 20 mM), a significant reduction in pyruvate uptake was observed after the 8-cell stage as previously described. However, in the presence of no lactate or a lactate concentration of less than 10 mM (physiological concentration of lactate is 4.79 mM in mouse oviduct fluid [13]), the uptake of pyruvate remains high throughout development. Therefore, at the blastocyst stage in the presence of a physiological lactate concentration, pyruvate and glucose uptakes were both approximately 5 pmol/embryo per hour. Similar uptake studies on the in vivo-developed sheep embryo in the absence of lactate showed that pyruvate uptake at the cleavage stages was initially high while glucose uptake was low. After compaction, the uptake of glucose increased while pyruvate uptake remained high and further increased at the blastocyst stage [20]. Similarly, studies on the nutrient preferences of in vitro-produced human preimplantation embryos demonstrated that the human embryo continues to take up pyruvate throughout development to the blastocyst stage [21,22]. These studies on pyruvate uptake by the human embryo were also performed in medium either devoid of lactate [22] or containing low lactate concentrations [21]. Therefore the nutrient preferences observed in mouse embryos in this study are similar to that reported in other species. The previously reported decrease in pyruvate uptake by the mouse blastocyst appears to be an artifact of the high concentration of lactate in the medium. This highlights that culture conditions can have a significant effect on the energy metabolism of the preimplantation embryo. This metabolic adaptation by embryos in culture has been demonstrated previously [19,23]. However, it is also known that perturbations in normal embryo metabolism come at a cost of developmental competence [24].
Lactate in the incubation medium not only altered pyruvate uptake but also altered the metabolic fate of the pyruvate. Increasing the lactate concentration up to 20 mM significantly reduced pyruvate oxidation at both the zygote and blastocyst stages. Conversely, increasing the lactate concentration in the medium significantly increased lactate oxidation at both stages of development. This pattern of oxidative metabolism has previously been reported for the 2-cell mouse embryo [7,8], indicating substantial interaction between these two substrates in embryo metabolism. While lactate oxidation increased at the zygote stage when the lactate concentration was increased in the medium, pyruvate oxidation always remained higher than lactate oxidation. However, at the blastocyst stage the reverse was true, with lactate oxidation exceeding pyruvate oxidation at lactate concentrations of 10 and 20 mM. This indicates substantial differences in the abilities of these two stages of embryo development to regulate oxidative metabolism.
In this study, measurement of both pyruvate uptake and oxidation by embryos enabled the interaction between uptake and oxidative metabolism to be determined. Expression of pyruvate oxidation as a percentage of pyruvate uptake revealed that there were striking differences in pyruvate metabolism between the zygote and blastocyst in response to increasing lactate concentrations. For the zygote, increasing the lactate concentration decreased the percentage of pyruvate taken up by the embryo that was oxidized by the tricarboxylic acid cycle. The remaining pyruvate taken up by the embryo was presumably converted to lactate. In contrast to findings for the zygote, the majority of the pyruvate taken up by the blastocyst (68%) in the presence of 20 mM lactate was oxidized by the tricarboxylic acid cycle. Presumably only 32% was converted to lactate. Therefore, the zygote and blastocyst appear to have different capacities to regulate pyruvate metabolism. This difference in regulation of metabolism in response to increasing lactate concentrations may reflect the differing ability of the zygote and blastocyst to utilize lactate as an energy source. Mouse zygote division to the 2-cell stage is supported only by pyruvate or phosphoenolpyruvate [1], while from the 2-cell stage, development can be supported by both pyruvate and lactate [3]. This inability of the zygote to cleave when lactate is the sole energy substrate is seen despite the observation that the zygote can oxidize lactate from the medium. The similar decrease in pyruvate uptake with increasing lactate concentration observed at both the zygote and blastocyst stages can be attributed to competition for the carboxylic acid transporter on the plasma membrane. The higher the lactate concentration, the less pyruvate is able to compete for the transporter. However, once pyruvate is inside the embryo, its fate is determined by cytosolic factors, which appear to differ between the zygote and blastocyst stages. One possible explanation for the different abilities of the zygote and later stages to regulate pyruvate metabolism may be related to differences in the kinetics of the enzyme LDH that catalyses the near-to-equilibrium reaction of pyruvate to lactate with the concomitant conversion of NADH to NAD+. The activity of LDH in the zygote and blastocyst, which differed in their response to exogenous lactate, was therefore determined. Although LDH activity was found to be higher in the zygote compared to the blastocyst, the enzyme activity present at both stages was significantly higher than that required to account for maximal pyruvate utilization. LDH activity has previously been reported to be highest at the zygote, decreasing throughout development to the blastocyst stage [9]. The levels of LDH reported in this study are higher than those previously reported [9,11]. This apparent discrepancy can be explained by the incubation conditions used for LDH measurement. In this study, LDH activity was assessed at 37°C, and activity has been expressed as linear initial velocity. Previous studies employed endpoint determinations after several minutes, which tends to reduce the calculated activity.
LDH isolated from the zygote and the blastocyst were found to be identical in their response to 20 mM lactate, showing a 40% decrease in conversion of pyruvate to lactate at both stages of development. The Km of the enzymes at both stages of development were also found to be the same, indicating that the LDH present at both stages is the same isozyme. The kinetic data presented here confirm previous studies that have identified the isozyme present in mouse preimplantation embryos as LDH-1 through gel separation [9,25–28]. The LDH in the embryo switches to isozyme LDH-5 at the time of implantation [25,29] after the stages used in this study. As the activity of the LDH enzyme of the zygote and blastocyst behaved similarly in isolation in vitro, it appears that the differential activity of the embryos in response to lactate must be due to the action of specific effectors acting on this enzyme in situ.
Increasing the lactate concentration in the medium resulted in a significant decrease in the NAD+:NADH ratio (or increase in autofluorescence) of the zygote. The ratio of NAD+:NADH of 2-cell and morula-stage embryos appears not to be affected by increasing lactate concentrations, as there were no changes in autofluorescence. Cellular utilization of lactate as an oxidative energy substrate requires that NAD+ must be regenerated for further flux to proceed. Two mechanisms commonly used for the regeneration of NAD+ are the conversion of pyruvate to lactate by LDH or by an indirect method involving mitochondrial shuttles such as the malate/aspartate shuttle that transport protons into the mitochondria and regenerates NAD+ in the cytosol [18]. The effect of altering the NAD+:NADH ratio on isolated LDH indicated that increasing the level of NAD+ in the assay resulted in reduced activity of LDH conversion of pyruvate to lactate. It is plausible that the inability of the zygote to utilize lactate as an energy source is due to exhaustion of the NAD+ pools within the zygote resulting from the increased conversion of lactate to pyruvate. As the measurements of lactate oxidation in this study and previous studies [7,8,30] have been endpoint determinations after a 3-h incubation and linear rates of oxidation were not determined, the reduced rates of lactate oxidation in the zygote may actually reflect initial activity similar to pyruvate oxidation that declines or stops due to exhaustion of NAD+ pools within the embryo.
In conclusion, this study has demonstrated that pyruvate metabolism in the zygote and blastocyst is regulated differently in the presence of increasing lactate in the medium. In the presence of lactate the percentage of pyruvate that is oxidized decreases in the zygote, while in the blastocyst the percentage of pyruvate oxidation increases, despite the fact that total oxidation of pyruvate decreases at both stages. Importantly, this work confirms that the mouse blastocyst has a high oxidative capacity when developed in vivo. The high oxidative turnover by the blastocyst coincides with more developed mitochondria [31]. This study highlights the need to measure both nutrient uptake and oxidation before conclusions are made as to the regulation of embryo metabolism. This difference in regulation of pyruvate metabolism was not due to differences in LDH. Although the Vmax of LDH activity was higher in the zygote, the Km and inhibition of activity caused by lactate were similar in the zygote and blastocyst. Therefore the differential effect of lactate on pyruvate metabolism appears to be due to in situ regulation of LDH activity, plausibly by differences in redox potential of the cytosol, which may be due to a reduced capacity of reducing equivalent shuttles in the mitochondria at the zygote.
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FOOTNOTES |
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1 M.L. was the recipient of a Dora Lush Biomedical Studentship from the Australian National Health and Medical Research Council. D.K.G. was the recipient of a Fellowship from the Australian Research Council. 
2 Correspondence: Michelle Lane, Colorado Center for Reproductive Medicine, 799 East Hampden Ave., Suite 300, Englewood, CO 80110. FAX: 303 788 4438; mlane{at}colocrm.com
Accepted: August 18, 1999.
Received: May 6, 1999.
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