Download Metabolic changes in the glucose-induced apoptotic blastocyst

AJP-Endo Articles in PresS. Published on March 27, 2002 as DOI 10.1152/ajpendo.00046.2002
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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.
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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involve metabolic alterations leading to problems with outer mitochondrial membrane
permeability.
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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.
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Hodge, T., and M. Colombini. Regulation of metabolite flux through voltage-gating of
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Keim, A. L., M. M. Chi, and K. H. Moley. Hyperglycemia-induced apoptotic cell death
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Leese, H. J., and A. M. Barton. Pyruvate and glucose uptake by mouse ova and
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Leist, M., B. Single, A. F. Castoldi, S. Kuhnle, and P. Nicotera. Intracellular adenosine
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Lin, A., J. Weinberg, R. Malhotra, S. Merritt, L. Holzman, and F. Brosius. GLUT1
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Liu, M. Y., and M. Colombini. Regulation of mitochondrial respiration by controlling the
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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.
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Malhotra, R., Z. Lin, C. Vincenz, and F. C. Brosius, 3rd. Hypoxia induces apoptosis via
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Pampfer, S., I. Vanderheyden, J. E. McCracken, J. Vesela, and R. De Hertogh. Increased
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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.
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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