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AJP-Endo Articles in PresS. Published on March 27, 2002 as DOI 10.1152/ajpendo.00046.2002 1 Metabolic changes in the glucose-induced apoptotic blastocyst suggest alterations in mitochondrial physiology Maggie M-Y. Chi1, Amanda Hoehn1, and Kelle H. Moley1,2 Departments of 1Obstetrics and Gynecology and 2Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 Running title: Metabolic changes in embryos Address for correspondence: K. H. Moley, Department of OB/GYN, Washington University School of Medicine, 4911 Barnes-Jewish Hospital Plaza, 2nd Floor Maternity, St. Louis, MO 63110; Tel (314)362-1765; FAX (314)747-4150; email: [email protected] Copyright 2002 by the American Physiological Society. 2 Abstract Mammalian preimplantation embryos experience a critical switch from an oxidative to a predominantly glycolytic metabolism. In this study, the change in nutrient metabolism, between the 2-cell and blastocyst stage, was followed by measuring single embryo concentrations of tricarboxylic acid (TCA) cycle and glycolytic metabolites using microfluorometric enzymatic cycling assays. Having established the normal values, further changes that occur as a result of the induction of apoptosis by exposure to high glucose conditions were examined. From a 2-cell to a blastocyst stage, the embryos experienced an increase in TCA metabolites and a dramatic increase in fructose-1,6-bisphosphate. The high TCA metabolites may result from accumulation of substrate due to a slowing of TCA cycle metabolism as glycolysis predominates. Embryos exposed to elevated glucose conditions experienced significantly lower FBP, suggesting decreased glycolysis, significantly higher pyruvate, suggesting increased pyruvate uptake by the embryos in response to decreased glycolysis, and increased TCA metabolites, suggesting an inability to oxidize the pyruvate and a slowing of the TCA cycle. We speculate that the glycolytic changes lead to dysfunction of the outer mitochondrial membrane that results in the abnormal TCA metabolite pattern and triggers the apoptotic event. Key words: Tri-carboxylic Acid cycle, glycolysis, pre-implantation embryo, programmed cell death 3 Glucose transport and metabolism are critical for mammalian blastocyst formation and further development(8, 20). At this stage, the switch occurs from oxidation of pyruvate via the tricarboxylic acid cycle (TCA) to the use of glucose as the main substrate via glycolysis (7, 14). As a result, the blastocyst exhibits extreme sensitivity to glucose deprivation. We have previously shown that any decrease in glucose transport, basal or insulin-stimulated, results in enhanced apoptosis at this stage, which manifests later in pregnancy as a malformation or miscarriage (3, 4, 21, 22). This decrease in blastocyst glucose transport and resulting apoptosis occur in conditions of maternal hyperglycemia and hyperinsulinemia. The blastocyst stage marks a new peak in cellular proliferation and growth as the first epithelial layer, the trophectoderm, is formed. These changes create new biosynthetic demands on the embryo. Maintenance of a high rate of glycolysis is thought to be important for providing a “dynamic buffer” of metabolic intermediates for the biosynthesis of macromolecules (23). For example, glucose-6-phosphate is used in the formation of ribose-5-phosphate required for DNA and RNA synthesis. Another reason for the increased glucose demand may be that increasing amounts of glucose are converted to lactate at the blastocyst stage in humans and rodents (14). In these species in particular, the embryo resides in the uterus for a relatively short time before implantation and the switch to an anaerobic metabolism is in response to the lack of adequate vascularization and oxygenation at the implantation sites or decidual zones. The only source of ATP for the embryo at this point would be conversion of glucose to pyruvate and lactate via glycolysis. 4 Recent studies have shown that cell death caused by reduced availability of glucose, as seen with growth factor withdrawal, is initiated by mitochondrial changes that result in cytochrome c release(10, 27). Overexpression of GLUT1 can prevent this onset of apoptosis(27) and the regulation of outer mitochondrial membrane integrity via the voltage dependent anion channel (VDAC) appears to depend on cellular metabolic changes associated with glycolysis(26). In this study, we attempted to track the changes in nutrient metabolism in the blastocyst by measuring single embryo concentrations of TCA metabolites and glycolytic metabolites using microfluorometric enzymatic cycling assays. We postulate that perhaps some of the metabolic alterations experienced by the blastocyst exposed to hyperglycemia may be responsible for perturbations in mitochondrial physiology and thus may trigger an apoptotic cascade. Materials and Methods Embryo collection and culturing. Embryos were recovered as described previously (21) from superovulated female mice (B6 X SJL F1, Jackson Laboratories). In vivo retrieved embryos were then flushed from the oviducts 48, 72 and 96 hours later corresponding to 2-cell, morula and blastocyst stages respectively. In vitro cultured embryos were flushed at a 2-cell stage and cultured in KSOM mouse embryo culture media(Specialty Media, NJ, USA) containing either 0.2 mM D-glucose, the control concentration of glucose, or 5.6 or 50 mM D-glucose as the test conditions. The embryos were cultured at 37oC in 5% CO2, 5% O2, and 90% N2. 5 Embryo extraction for metabolite assays. At each stage, embryos were washed in BSA-free media for 1 min and then quick frozen on a glass slide by dipping in cold isopentane equilibrated with liquid N2. After freeze-drying overnight in a vacuum at –35oC, the embryos were extracted in nanoliter volumes under oil as previously described (21). Citrate, α-ketoglutarate, aspartate, glycerol-3-phosphate, and ATP were measured in alkaline extracted embryos. These embryos were extracted in 1ul 0.1 N NaOH at room temperature for 20’, 0.5 ul of the extract was heated to 80oC for 20’, and a 0.2 ul mixture of 0.2N HCl and 0.1M Tris HCl pH 6.8 were added. Malate, fumarate and glutamate were measured in acid extracted embryos. These embryos were extracted in 1 ul 0.1 N NaOH at room temperature for 20’, 0.5 ul of the extract was added to 0.1ul of 0.6 N HCl and heated to 80oC for 20’, and finally the extract was neutralized with 0.1 ul of 0.2 M Tris Base. Both treated extracts were stored at –700C. Metabolite microanalytic assays. Separate assays were developed for each metabolite measured and designed to link to reactions requiring NADH or NADPH (Table 1 and 2, step 1 and 2). The NADPH/NADH generated is then enzymatically amplified in a cycling reaction (Table 1, steps 3 and 4) and a byproduct of the amplification step is measured in a fluorometric assay (Table 1, step 5). All metabolites except for fructose-1,6-bisphosphate (FBP) are expressed as millimoles/kg wet weight based on the wet weight of 160 pg per embryo. FBP levels were extremely low in individual embryos and are expressed as micromoles/kg wet weight. Absolute concentrations of metabolites can be calculated in picomoles by multiplying by 0.16. ATP was used as a marker of viability and if any embryos had abnormally low levels, the entire set of 6 experiments was discarded. Previous studies have shown that cells with low ATP undergo necrosis rather then apoptosis, since apoptosis requires energy(6, 15). ATP levels are placed at the outset of each table or figure to demonstrate the equality of the embryos tested. Assay conditions: All assays for a particular metabolite in a given experiment were made at the same time and conducted at ambient temperatures. Metabolites were measured according to the protocols of Table 1 and reagents of Table 2. Standards were carried through the entire procedure. Most of the enzymes were purchased in a suspension of ammonia sulfate, spun down and reconstituted with 20 mM imidazole HCl pH 7.0 and 0.02% BSA. All experiments were completed a minimum of three times. For each metabolite measurement under each condition, at least 16 individual embryos were used. The NADP+ cycling reagent, used only for the ATP assay, contains 100 mM Imidazole HCl, pH 7.0, 7.5 mM a-ketoglutarate, 5 mM glucose-6phosphate, 25 mM NH4Ac, 0.02%BSA, 100 uM ADP, 100 ug/ml beef liver glutamate dehydrogenase and 10ug/ml Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. This enzyme ratio gives approximately 100,000 fold amplification as detailed in Table 1 for ATP. The indicator reaction for the NADP+ cycling step, or step 5 in Table 1, involves adding10ul of the reaction to 1 ml of the indicator, 6-phosphogluconate reagent, containing 50 mM imidazole HAc, pH 7.0, 1 mM EDTA, 30 mM NH4Ac, 5 mM MgCl2, 100 uM NADP+ and 2 ug/ml yeast 6-phosphogluconate dehydrogenase. The NAD+ cycling reagent, which is used for all other metabolite assays in Table 1, contains 100 mM Tris HCl, pH 8.1, 2 mM βmercaptoethanol, 2 mM oxaloacetate, 300 mM ethanol, 0.02% BSA, 15 ug/ml alcohol 7 dehydrogenase and 1.5 ug/ml heart malate dehydrogenase. This enzyme ratio gives about 15,000 fold amplification overnight at room temperature and this is was is used to measure Citrate and Pyruvate. This ratio was altered accordingly to achieve the number of cycles detailed in Table 1. The indicator reaction for the NAD+ cycling step, or step 5 in Table 1, involves adding 10 ul of the reaction to 1 ml of the indicator, malate reagent, containing 20 mM 2-amino-2methylpropanol HCl, pH 9.9, 10 mM L-glutamate, 200 uM NAD+, 5ug/ml malate dehydrogenase 2 ug/ml glutamic-oxalacetic transaminase. Statistical analysis. Data are expressed as means and standard errors. Differences in metabolites between 2-cell and blastocyst stage embryos recovered either in vivo or in vitro were analyzed using Students’ t-test. FBP and pyruvate measurements were the exception. Due to the large number of embryos required for the measurement of these two metabolites, the 2-cell embryos, in vivo blastocyst and in vitro blastocyst assays were grouped together and the results analyzed using ANOVA with Bonferroni/Dunn as a post hoc test. Difference in metabolites from embryos cultured in different concentrations of glucose were analyzed using ANOVA with Bonferroni/Dunn as post hoc analysis. Differences were considered significant at P < 0.05. StatView 4.5 (Abacus Concepts, Inc., Berkeley, CA, USA) was used for statistical analyses. Results Progression from a 2-cell to blastocyst stage in vivo or in vitro results in accumulation of TCA cycle metabolites and a decrease in glycolytic metabolites. The majority of TCA cycle 8 substrates demonstrated a significant increase in concentration as the embryo developed from a 2-cell to blastocyst stage embryo (See Table 3 and Figure 1). This increase comparing 2-cell to blastocyst occurred both in embryos cultured in vitro and in embryos obtained directly in vivo. Similarly, the FBP increased significantly whereas glycerol-3-phosphate, a glycolytic intermediate, dropped during the same period in both conditions. This may reflect the embryo’s adaption to an anoxic peri-implantation existence as well as the embryo’s switch at a blastocyst stage to a predominantly glycolytic metabolism. The low level of FBP in early cleavage stage embryos agrees with previous studies(1). Blastocysts exposed to moderate glucose concentrations (5.6mM) as compared to normal glucose conditions (0.2mM) experience decreased TCA cycle metabolites and increased glycolytic metabolites. Except for citrate and malate, the TCA cycle metabolites in embryos cultured in 5.6 mM glucose were only slightly lower than those in embryos cultured in a normal glucose concentration of 0.2mM. In addition, only glycerol-3-phosphate levels were significantly higher in the blastocysts in 5.6 mM glucose (See Table 4 and Figure 2) and pyruvate and FBP were only slightly higher. Since the prior 2-cell to a blastocyst studies suggested that a slowing of flux via the TCA cycle corresponds to an increase in TCA metabolites and decrease glycerol-6-phosphate levels, these results with 5.6 mM glucose comparing to 0.2 mM glucose suggest the opposite. The mildly elevated glucose concentration appears to be causing increased flux through the TCA cycle with lowering of the levels and 9 perhaps a saturation of the glycolytic pathway with an increase in one of the byproducts used to make glycolipids, glycerol-3-phosphate. Blastocysts exposed to high glucose concentrations (50mM) as compared to normal glucose concentrations (0.2mM) experience increased TCA cycle metabolites, increased pyruvate and decreased glycolytic metabolites. Blastocysts cultured in 50 mM glucose as compared to 0.2 mM glucose had predominantly higher TCA cycle metabolites, significantly higher pyruvate and glycerol-6-phosphate levels and significantly lower FBP levels (See Table 4 and Figure 2). The discrepancy between citrate and pyruvate levels suggests strongly that a block to pyruvate oxidation via the TCA cycle exists in these embryos. Moreover, significantly decreased FBP levels suggest that glycolysis is also blocked, perhaps due to the low free glucose levels that are known to be present in these embryos exposed to high glucose conditions(21). The elevated pyruvate levels may occur due to increased pyruvate uptake that occurs in response to glucose deprivation(7, 8). Discussion Progression from a 2-cell to a blastocyst stage was associated with a significant increase in TCA cycle metabolites, a dramatic increase in FBP and a decrease in glycerol-3-phosphate as seen in this analysis. These results give a profile of a normal transition from a metabolism based on pyruvate oxidation via TCA cycle to a metabolism based on glucose metabolism via glycolysis. TCA cycle metabolites may accumulate due to blastocyst reliance on glycolytic 10 metabolism, slowing of TCA cycle and accumulation of substrates. Less than 1% of glucose consumed at a blastocyst stage is oxidized via the TCA cycle (5) and this finding of increased TCA cycle metabolites corresponds with previous reports using techniques similar to those used in this study (1). Comparing blastocysts cultured in 0.2mM glucose to 5.6mM glucose demonstrates the effect of a moderate increase in glucose on the metabolic products. The decrease in TCA cycle metabolites suggests increased flux through this pathway with increased substrate utilization. Regulation of TCA cycle flux depends in part on increased availability of pyruvate and NAD+. Increasing glucose availability by incubating in 5.6mM glucose would increase pyruvate production and decrease NADH levels as increased pyruvate is converted to lactate. Moreover, increased FBP levels would also stimulate TCA cycle flux and perhaps slow glycolysis. Previous studies have shown that glucose consumption rate is saturated at 0.29 mM but that a further 10% increase is obtained at 3 mM which is similar to the 5.6 mM used in this study (5). All of our findings are consistent with a slight but measurable increase in glucose consumption. Importantly, none of these metabolic changes at this moderate glucose concentration induce apoptosis in the embryo as shown in previous studies (3, 22, 24). In contrast, maternal diabetes or in vitro hyperglycemia, at 50mM glucose, does lead to BAX-dependent apoptosis in the mouse embryo (13, 22). Our previous studies show that this apoptotic event is triggered by decreased GLUT1 expression and glucose transport at the blastocyst stage(3, 21). This study demonstrates that this drop in transport leads to a decrease in 11 glycolysis resulting in lower FBP levels in the individual blastocysts. Other studies have linked inhibition of glycolysis to initiation of an apoptotic cascade (18). Likewise, overexpression of glucose transporters prevent hypoxia-induced programmed cell death (16). Glucose metabolism and glucose uptake also exhibit a protective effect against apoptosis induced by growth factor withdrawal in different cell types (2, 12, 19). We postulate that the decrease in glycolysis in these blastocysts, as demonstrated by decreased FBP levels, and the embryos’ attempts to compensate by increasing pyruvate uptake lead to severe alteration in mitochondrial physiology that result in the triggering of the apoptotic cascade. As shown in the growth factor withdrawal model (27), a depletion of glycolytic byproducts leads to decreased electron transport substrates and a resulting decrease in outer mitochondrial membrane potential (9) due to formation of an electron gradient and closure of the voltage dependent anion channel (VDAC). VDAC is a large conductance channel that when open is the major pathway for metabolite transport across the outer mitochondrial membrane (28). Closure of this channel occurs under conditions of altered intracellular NADH and pyruvate resulting from glycolytic changes (17, 29) and has been shown to trigger apoptosis in growth factor withdrawal models due to hyperpolarization, loss of outer mitochondrial integrity and cytochrome c release (25, 26). Previous data show that VDAC closure leads to severe perturbation in mitochondrial physiology, leading to lack of mitochondrial availability of malate and ADP, slowing down of the TCA cycle and NAD+/NADH shuttles, and accumulation of TCA cycle metabolites as seen 12 in this study (11). This lack of pyruvate oxidation would also support the elevated pyruvate levels. Although the citrate and α-ketoglutarate levels are lower in this report as opposed to all the other TCA cycle components, these two metabolites can readily be converted to glutamate and glutamine and may serve as alternative energy sources under these stress conditions. Due to the anticipated problems with the mitochondrial matrix, the NADH shuttle would not be functioning and thus may result in the increased glycerol-3-phosphate levels. Most of the changes in TCA cycle metabolites seen here can be explained by perturbations in mitochondrial physiology due to a closure of VDAC. In summary, hyperglycemia, by causing a decrease in glucose transport, results in a decrease in glycolysis and a decrease in FBP in the mouse blastocyst as measured in this study. This depletion of glycolytic substrates in combination with the embryo’s attempts to increase pyruvate uptake as reflected here, may be responsible for loss of integrity of the outer mitochondrial membrane as seen in other cell types. An inability of the mitochondria to successfully complete pyruvate oxidation would explain the increase in TCA cycle metabolites seen in these embryos. Recent studies provide strong evidence to support the mitochondria as the site of apoptosis initiation in response to growth factor withdrawal(25-27). These apoptotic effects are believed to be due to metabolic alterations linked to changes in glycolysis, as seen here with the blastocysts undergoing alterations in response to high glucose conditions. Data from this study suggest that hyperglycemia-induced apoptosis in the mouse blastocyst may also 13 involve metabolic alterations leading to problems with outer mitochondrial membrane permeability. 14 ACKNOWLEDGEMENTS This work was supported by grants to K. H. Moley from the National Institutes Child Health and Development (RO1 HD-38061; RO1 HD/DK 40390) and a Research Grant from the American Diabetes Association. 15 REFERENCES 1. Barbehenn, E. K., R. G. Wales, and O. H. Lowry. Measurement of metabolites in single preimplantation embryos: a new means to study metabolic control in early embryos. Journal of Embryology and Experimental Morphology : 29-46, 1978. 2. Berridge, M. V., A. S. Tan, K. D. McCoy, M. Kansara, and F. Rudert. CD95 (Fas/Apo1)-induced apoptosis results in loss of glucose transporter function. Journal of Immunolgy 156: 4092-4099, 1996. 3. Chi, M. M., J. Pingsterhaus, M. Carayannopoulos, and K. H. Moley. Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst. J Biol Chem 275: 40252-7., 2000. 4. Chi, M. M., A. L. Schlein, and K. H. Moley. High insulin-like growth factor 1 (IGF-1) and insulin concentrations trigger apoptosis in the mouse blastocyst via down-regulation of the IGF-1 receptor. Endocrinology 141: 4784-92., 2000. 5. Dufrasnes, E., I. Vanderheyden, D. Robin, J. Delcourt, S. Pampfer, and R. De Hertogh. Glucose and pyruvate metabolism in preimplantation blastocysts from normal and diabetic rats. J Reprod Fertil 98: 169-77., 1993. 6. Eguchi, Y., S. Shimizu, and Y. Tsujimoto. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57: 1835-40., 1997. 7. Gardner, D. K., and H. J. Leese. Non-invasive measurement of nutrient uptake by single cultured pre- implantation mouse embryos. Hum Reprod 1: 25-7, 1986. 8. Gardner, D. K., and H. J. Leese. The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos. Development 104: 423-9, 1988. 9. Hackenbrock, C. R. Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J Cell Biol 30: 269-97., 1966. 10. Harris, M. H., and C. B. Thompson. The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability. Cell Death Differ 7: 1182-91., 2000. 11. Hodge, T., and M. Colombini. Regulation of metabolite flux through voltage-gating of VDAC channels. J Membr Biol 157: 271-9., 1997. 12. Kan, O., S. A. Baldwin, and A. D. Whetton. Apoptosis is regulated by the rate of glucose transport in an interleukin 3 dependent cell line. Journal of Experimental Medicine 180: 917921, 1994. 13. Keim, A. L., M. M. Chi, and K. H. Moley. Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53. Mol Reprod Dev 60: 214-24., 2001. 14. Leese, H. J., and A. M. Barton. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil 72: 9-13, 1984. 16 15. Leist, M., B. Single, A. F. Castoldi, S. Kuhnle, and P. Nicotera. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 185: 1481-6., 1997. 16. Lin, A., J. Weinberg, R. Malhotra, S. Merritt, L. Holzman, and F. Brosius. GLUT1 reduces hypoxia-induced apoptosis and JNK pathway activation. Am. J. Phys. Endocrinol. Metab. 278: E958-E966, 2000. 17. Liu, M. Y., and M. Colombini. Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC. Biochim Biophys Acta 1098: 255-60., 1992. 18. Malhotra, R., and F. C. Brosius, 3rd. Glucose uptake and glycolysis reduce hypoxiainduced apoptosis in cultured neonatal rat cardiac myocytes. J Biol Chem 274: 12567-75, 1999. 19. Malhotra, R., Z. Lin, C. Vincenz, and F. C. Brosius, 3rd. Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose. Am J Physiol Cell Physiol 281: C1596-603., 2001. 20. Martin, K. L., and H. J. Leese. Role of glucose in mouse preimplantation embryo development. Mol Reprod Dev 40: 436-43, 1995. 21. Moley, K. H., M. Chi, and M. Mueckler. Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. American Journal of Physiology 275: E38E47, 1998. 22. Moley, K. H., M. M.-Y. Chi, C. M. Knudson, S. J. Korsmeyer, and M. M. Mueckler. Hyperglycemia induces apoptosis in preimplantation embryos via cell death effector pathways. Nature Medicine 12: 1421-1424, 1998. 23. Newsholme, P., and E. A. Newsholme. Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture. Biochem J 261: 211-218, 1989. 24. Pampfer, S., I. Vanderheyden, J. E. McCracken, J. Vesela, and R. De Hertogh. Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro. Development 124: 4827-36., 1997. 25. Vander Heiden, M. G., N. S. Chandel, X. X. Li, P. T. Schumacker, M. Colombini, and C. B. Thompson. Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc Natl Acad Sci U S A 97: 4666-71., 2000. 26. Vander Heiden, M. G., X. X. Li, E. Gottleib, R. B. Hill, C. B. Thompson, and M. Colombini. Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. J Biol Chem 276: 19414-9., 2001. 27. Vander Heiden, M. G., D. R. Plas, J. C. Rathmell, C. J. Fox, M. H. Harris, and C. B. Thompson. Growth factors can influence cell growth and survival through effects on glucose metabolism. Mol Cell Biol 21: 5899-912., 2001. 17 28. Zalman, L. S., H. Nikaido, and Y. Kagawa. Mitochondrial outer membrane contains a protein producing nonspecific diffusion channels. J Biol Chem 255: 1771-4., 1980. 29. Zizi, M., M. Forte, E. Blachly-Dyson, and M. Colombini. NADH regulates the gating of VDAC, the mitochondrial outer membrane channel. J Biol Chem 269: 1614-6., 1994. 18 FIGURE LEGENDS: Figure 1. Percent change in metabolites comparing 2-cell embryo levels to blastocyst levels in embryos cultured in vitro or in vivo. Values have been normalized to those at the 2-cell stage. The dark vertical line separates TCA cycle metabolites and glycolytic pathway metabolites. CIT, citrate; AKG, alpha-ketoglutarate; MAL, malate; FUM, fumarate; ASP, aspartate; GLU, glutamate; GOP, glycerol-3-phosphate; PYR, pyruvate; FBP, fructose-1,6-bisphosphate. Figure 2. Percent change in metabolites comparing embryos cultured in 0.2 mM D-glucose compared to embryos cultured in 5.6 mM D-glucose or 50 mM D-glucose. 19 Metabolite Extract Step1 Specific Reagent 0.1µl 2X RT 20’ Step 2 Stop Reaction 0.1µl 0.2N NaOH 80oC 20’ ATP 0.1µl Alk Cit 0.1µl Alk 0.1µl 2X RT 30’ 0.1µl 0.15 N HCl RT 10’ Mal 0.1µl Acid 0.1µl 2X RT 30’ 0.1µl 0.2N NaOH 80oC 20’ Fum 0.1µl Acid 0.1µl 2X RT 30’ 0.1µl 0.2N NaOH 80oC 20’ αKG 0.1µl Alk 0.1µl 2X RT 30’ 0.1µl 0.3N HCl Rt 10’ Glut 0.1µl Acid 0.1µl 2X RT 30’ 0.1µl 0.15N NaOH 80oC 20’ GOP 0.1µl Alk 0.1µl 2X RT 30’ 0.1µl 0.18N NaOH 80oC 20’ Pyr 0.1 µl Acid 0.1µl 2X RT 10’ 0.1µl 0.15N HCl 80oC 20’ FBP 0.1 µl Acid 0.1µl 2X RT 20’ 0.1µl 0.25N NaOH 80oC 20’ Step 3 Step 4 Step 5 Standards 0.3µl+10µl NADP CyR RT ON 100,000X 0.3µl +10µl NAD CyR RT ON 15,000X 0.3µl +10µl NAD CyR RT ON 200,000X 0.3µl+10µl NAD CyR+fumarase RT ON 200,000X 0.3µl+NAD CyR RT ON 60,000X 0.3µl+10µl NAD CyR RT ON 30,000X 0.3µl+10µl NAD CyR RT ON 200,000X 0.3µl+10µl NAD CyR RT ON 15,000X 0.3µl+10µl NAD CyR RT ON 200,000X 1µl 1N NaOH 80oC 30’ 10µl to1 ml 6PG Rgt 0.2-0.4µM 1µl 1N NaOH 80oC 20’ 10µl to1ml Malate Rgt 0.25-.50 1-2 µM 1µl 1N NaOH 80oC 20’ 10µl to 1ml Malate Rgt 0.075-0.15 µM 1µl 1N NaOH 80oC 20’ 10µl to 1 ml Malate Rgt 0.075-0.15 µM 1µl 1N NaOH 80oC 20’ 1µl 1N NaOH 80oC 20’ 1 µl 1N NaOH 80oC 20’ 1 µl 1N NaOH 80oC 20’ 1 µl 1N NaOH 80oC 20’ 10µl to 1 ml Malate Rgt 0.175-0.35 µM 10µl to 1 ml Malate Rgt 1.5-3.0µM 10µl to 1 ml Malate Rgt 0.05-.1µM 10µl to 1 ml Malate Rgt 1.28-2.56 µM 10µl to 1 ml Malate Rgt 0.036-0.072 µM Acid, acid extract; Alk, alkaline extract; CyR, cycling reagent; 60,000X, 60,000 fold amplifications; 6PG, 6phosphogluconate; RT, room temperature (22-25oC). TABLE 1. ASSAY PROTOCOLS. Metabolite Buffer pH NADP/NAD Substrates Enzymes 60 mM Tris HCl 8.1 100 µm NADP+ 1 mM DTT 2 mM MgCl2 200 µM glucose 4 µg/mg hexokinase 2.5 µg/ml G-6-phosphate 60 mM Tris 7.4 20 mM NADH 400 µM ZnCl2 20 µg/ml Citrate lyase 2.5 µg/ml MDH 100 mM Imidazole 6.6 25 µm NADH 50 mM NH4Ac 20 µg/ml GDH 100 mM 2-AMP 9.9 100 µM NAD 20 mM glutamate 10 µg/ml MDH 5 µg/ml GOT Fumarate Same Same Same Same Same + 40 µg/ml fumarase Aspartate 100 mM Imidazole 6.7 20 µM NADH 100uM α-KG 20 µg/ml GOT 1 µg/ml MDH Glycerol-3phosphate 100 mM 2-AMP 8.8 200 µM NAD 4 mM β-mercaptoethanol, 2 mM Na2HAsO4 20 µg/ml TPI 100 µg/ml GAPDH 20 µg/ml GOPDH Glutamate 100 mM Tris acetate 8.4 400 µM NAD 200 µM ADP 1 mM H2O2 150 µg/ml GDH 100 mM Imidazole HCl 100 mM Imidazole HCl 6.6 20 µM NADH 6.4 80 µM NAD ATP Citrate α-Ketoglutarate Malate Pyruvate Fructose-1,6bisphosphate 2 µg/ml LDH 2 mM EDTA 4 mM β-mercaptoethanol, 2 mM Na2HAsO4 5 µg/ml Aldolase 5 µg/ml TPI 100 µg/ml GADPH MDH, malate dehydrogenase; GDH, glutamate dehydrogenase; GOT, glutamic-oxalacetic transaminase; 2-AMP, 2-amino-2-methylpropanol HCl; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GOPDH, glycerol-3-phosphate dehydrogenase. TABLE 2. ASSAY REACTIONS ATP Citrate aKG Fum Mal Asp Glut GOP Pyr FBP In vitro 2.650+.08a 1.425+0.05c 1.147+.07e .011+.007c .066+.013c .717+.06c 7.13+.22c 2.853+.116c - - In vivo 2.974+.04b 2.177+0.05d 1.501+.08d .05+.005d .086+.006d 1.47+.05d 8.52+.15d 3.08+.079d .303+.006f .207+.073f In vitro 2.420+.05a 6.342+.77c 1.092+.08e .189+.016c .851+.04c 4.63+.19c 12.42+.25c .485+.016c .558+.067f 10.6+0.4f In vivo 1.955+.03b 9.409+.53d 1.316+.06d .215+.02d .811+.06d 3.95+.10d 13.2+.39d .522+.021d .528+.034f 2.764+.36f 2-cell Blastocyst a,b,e = NS c,d = p<0.005 for metabolite between 2-cell and blastocyst in in vitro or in vivo category respectively. f = p<0.01 for metabolite between 2-cell and blastocyst by ANOVA with Bonferroni/Dunn post hoc test. TABLE 3. METABOLITE LEVELS IN 2-CELL AND BLASTOCYST STAGE EMBRYOS OBTAINED EITHER IN VIVO OR AFTER IN VITRO CULTURE IN 0.2mM D-GLUCOSE IN KSOM. All measurements are in mmoles/kg wet weight/embryo except for FBP which is umoles/kg wet weight/embryo. For all measurements except pyruvate and FBP, at least 20 individual blastocysts were used for each metabolite measurement. At least 20 sets of two 2-cell embryos each were used for the metabolite measurements. For pyruvate, twenty 2-cell embryos and 32 blastocysts were measured. For FBP, thirty-six 2-cell embryos were used and 30 blastocysts were measured. ATP Citrate αKG Fumarate Malate Aspartate Glutamate GOP Pyruvate FBP 0.2 mM Glc 3.316+.09 13.61+1.1 2.081+.14 0.185+.04 0.909+.10 2.927+.144 19.91+.94 .254+.01 .290+.015 12.5+2.1 5.6mM Glc 3.42+.16a 7.15+0.94b 1.905+.086a 0.175+.03a .664+.10c 2.825+.17a 17.83+1.4a .379+.013d .327+.02a 14.9+2.5a 50mM Glc 3.086+.13a 9.13+.71b 2.042+.17a .261+.025a 1.148+.088c 4.427+.20d 25.26+.99b .380+.022d .484+.05c 9.2+1.1c a=NS vs 0.2 mM; b=p<0.01 vs 0.2mm; c=p<0.05 vs 0.2 mM; d=p<0.001 vs 0.2 mM. TABLE 4. METABOLITE LEVELS IN EMBRYOS CULTURED IN VITRO FROM A 2-CELL TO BLASTOCYST STAGE IN DIFFERENT CONCENTRATIONS OF D-GLUCOSE IN KSOM. All measurements are in mmoles/kg wet weight/embryo except for FBP which is umoles/kg wet weight/embryo. At least 20 individual embryos were used for each metabolite measurement except pyruvate and FBP. For pyruvate, 2 blastocysts were used for each of 5 different assays. For FBP, 3 blastocysts were used for each of 5 different assays. CHANGE IN METABOLITES FROM 2-CELL TO BLASTOCYST IN VIVO AND IN VITRO 10000 1000 In Vivo % OF CONTROL In Vitro 100 10 ATP CIT AKG FUM MAL ASP GLU Figure 1 GOP PYR FBP CHANGE IN METABOLITES COMPARING 0.2 mM TO 5.6 mM OR 50 mM D-GLUCOSE 175 150 125 5.6 mM % OF CONTROL 50 mM 100 75 50 ATP CIT AKG FUM MAL ASP GLU GOP PYR FBP Figure 2