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First published online 21 December 2006
doi: 10.1242/dev.02744
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1 Laboratoire de Biologie du Développement UMR 7009 CNRS/Paris VI,
Observatoire, Station Zoologique, Villefranche sur Mer, 06230 France.
2 Department of Physiology, University College London, Gower Street, London WC1E
6BT, UK.
* Author for correspondence (e-mail: remi.dumollard{at}obs-vlfr.fr)
Accepted 14 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Mouse, Oocyte, Autofluorescence, NAD(P)H, Oxido-reduction, Mitochondria
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), but the development of a chemically defined culture
medium supporting development from fertilisation until blastocyst stage took
decades of research (Summers and Biggers,
2003). Today, even though empirically defined in vitro culture
conditions allow development until the blastocyst stage, the regulation of
intermediary metabolism in the mammalian early embryo is still poorly
understood.
Numerous biochemical studies investigating the substrate requirements of
mammalian embryos suggest that they possess a very specific intermediary
metabolism, which is strictly regulated during early development
(Biggers et al., 1967;
Summers and Biggers, 2003;
Johnson et al., 2003;
Gardner et al., 2002;
Leese, 2002). It is long known
that pyruvate supports development from fertilisation, whereas lactate
supports development from the two-cell stage on and glucose can support early
development only after compaction (Leese,
1995; Leese,
2002). While such a pattern of substrate utilisation supports
sufficient ATP production and biosynthetic pathways in the early embryo,
neither the reasons nor the mechanisms for this highly regulated pattern of
substrate utilisation have been elucidated. Intermediary metabolism also
produces the electron donors NADH and NADPH, which, besides being used for
energetic and biosynthetic metabolism, sets the intracellular redox potential
(or redox state) of the embryo and may thereby have an impact on early
development.
The pyridine nucleotides NADH and NADPH and the thiol tripeptide
glutathione (
-glutamylcysteinylglycine, GSH) form the vast majority of
the reducing equivalents available in the cell. GSH is the major antioxidant
system in the cell (Dickinson and Forman,
2002), while NADH and NADPH (NAD(P)H) are direct antioxidant
molecules (Kirsch and De Groot,
2001). However, whether NAD(P)H has an oxidant or antioxidant
action in the cell depends on the specific enzymatic content of that cell. The
metabolism of lactate, pyruvate and glucose can potentially provide NAD(P)H
and ATP in the cytosol as well as inside the mitochondria
(Fig. 1). However, even though
the impact of these substrates on ATP production is now known
(Biggers et al., 1967;
Houghton et al., 1996;
Leese, 2002;
Johnson et al., 2003), their
impact on the cytosolic and the mitochondrial redox potential remain largely
unexplored. Moreover, biochemical characterisation of the enzymatic activities
present in the early embryo suggests that each of these substrates may be
metabolised by multiple pathways (Cetica et
al., 2002; Comizzoli et al.,
2003; Johnson et al.,
2003; Biggers et al.,
1967; Barbehenn et al.,
1974; Leese, 2002)
(Fig. 1). However, due to the
paucity of studies of the intracellular redox metabolism in live oocytes and
embryos (Lane and Gardner,
2000), the metabolic pathways operating during mammalian early
development to regulate intracellular NAD(P)H levels are mostly unknown.
Oxidative stress, mediated by reactive oxygen species (ROS), results in an
imbalance of the intracellular redox potential towards an oxidised potential
(Balaban et al., 2005).
Oxidative stress has been found to be associated with impaired early
development and fragmented embryos
(Johnson and Nasr-Esfahani,
1994; Yang et al.,
1998); it can induce apoptosis of the oocyte and early embryo
(Liu and Keefe, 2000;
Liu et al., 2000) and is
associated with maternal aging and postovulatory aging of the egg
(Fissore et al., 2002;
Tarin, 1996). Understanding
how mammalian intermediary metabolism regulates the intracellular redox
potential (and hence antioxidant defence) is thus of fundamental
importance.
Finally, the role of redox potential during early development has been
studied only under the perspective of oxidative stress, but the role of the
redox potential per se is still unknown. Recent studies have revealed that
NAD(P)H and its oxidised counterpart NAD(P)+ as well as GSH are
direct modulators of numerous enzymes
(Dickinson and Forman, 2002;
Imai et al., 2000;
Zhang et al., 2002;
Ghezzi, 2005;
Nutt et al., 2005;
Liu et al., 2005). As some of
these enzymes are expressed in oocytes and early embryos
(McBurney et al., 2003;
Hildebrand and Soriano, 2002;
Nutt et al., 2005), mammalian
early development might well be regulated by the intracellular redox potential
itself (for a review, see Dumollard et
al., 2006b). Herein lies another reason to characterise the redox
metabolism operating in the oocyte and embryo and its impact on
development.
We have monitored intracellular NAD(P)H and GSH levels together with the mitochondrial redox state in single mouse oocytes and embryos to study the regulation of redox potential by exogenous substrates. We have also used a pharmacological approach to characterise which metabolic pathways regulate the intracellular redox potential. Finally the effect of manipulations of the redox potential on preimplantation development has been assessed.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)]
containing 1 mg/ml BSA by rupture of the oviduct with a 27 gauge needle. When
it was necessary to remove the cumulus cells, hyaluronidase (150 i.u.
ml-1, Sigma) was added to the H-KSOM/AA. Cumulus-free oocytes were
collected and washed in H-KSOM/AA three times and placed in a drop of the same
medium under mineral oil (Dow Corning, UK). Germinal vesicle (GV) oocytes were recovered by direct dissection of ovaries removed from mice primed with 7 i.u. PMSG 48 hours before. GV oocytes were then cultured in H-KSOM/AA until imaging (typically 30 minutes to 5 hours of culture).
In our laboratory, mouse embryos are routinely cultured in KSOM/AA medium
containing 0.2 mmol/l glucose, 0.2 mmol/l pyruvate and 10 mmol/l lactate. Such
KSOM/AA medium supports development from fertilisation to blastocyst
(Biggers et al., 2000)
(Fig. 8). For embryo culture,
one-cell (pronuclei stage) embryos were collected from hormone-primed mice
27-28 hours post-hCG. They were washed twice in 120 µl drops of fresh
H-KSOM/AA. They were then washed three times in 120 µl of the prospective
KSOM/AA media (Biggers et al.,
2000) in which they were to be cultured, and finally transferred
in groups of 15-30 to pre-equilibrated (5% CO2 in 95% air,
37°C) 60 µl drops of the relevant KSOM/AA media under mineral oil (Dow
Corning, UK) and placed in a water jacketed 37°C incubator (Kendro, UK).
All embryos were cultured for 5 days until the stage of development was
scored.
Subcellular analysis of autofluorescence imaging
The autofluorescence of mouse oocytes and embryos was imaged by confocal or
epifluorescence microscopy essentially as previously described
(Duchen et al., 2003;
Dumollard et al., 2003;
Dumollard et al., 2004;
Dumollard et al., 2006b). Blue
autofluorescence - emitted by the pyridine nucleotides NADH and NADPH in their
reduced form - was excited with UV light (360 or 351 nm on the CLSM) and
emission was collected using a 470 nm longpass or a 435-485 nm bandpass filter
(for CLSM). NAD(P)H autofluorescence is located both in mitochondria and in
the cytosol (Dumollard et al.,
2004; Dumollard et al.,
2006b) (Fig.
1).
The fluorescence of oxidised flavoproteins (FAD++) was excited
using the 458 nm line of an argon laser or with a 440-490 nm bandpass filter,
whereas emitted fluorescence was collected through either a 520 nm longpass
filter or a 505-550 nm bandpass filter. The autofluorescence derived from
oxidised flavoproteins (FAD++) is exclusively localised into the
mitochondria (Dumollard et al.,
2004; Dumollard et al.,
2006b) (Fig. 1).
The organisation of the GV oocyte, which presents a large nucleus (the GV)
surrounded by mitochondria (Dumollard et
al., 2006a; Dumollard et al.,
2006b) (see FAD++ image in
Fig. 2B and
Fig. 3A), allows recording of a
pure nuclear signal (i.e. devoid of mitochondria, dotted line in
Fig. 3A) used to estimate the
cytosolic redox potential and recording of a mitochondrial redox potential by
monitoring changes of fluorescence in a small perinuclear region of interest
(ROI) enriched in mitochondria (dark blue line plotted in
Fig. 3A).
Quantitative analysis of the images obtained was done using the Metamorph (Universal Imaging, USA) and the Zeiss LSM 510 software.
Measurement of intracellular GSH
After the autofluorescence imaging, the increase in MCB fluorescence (exc:
360 nm; em: 470 nm longpass) of oocytes incubated in 12.5 µmol/l MCB was
continuously imaged for 40 to 90 minutes until a plateau was observed (for
details, see Keelan et al.,
2001). In order to compare experiments done on different days, the
level of GSH fluorescence is expressed as a percentage of the fluorescence
emitted from a control embryo (untreated M2 oocyte or GV oocyte) imaged
simultaneously. For treatment with inhibitors, oocytes and embryos were
cultured in the relevant drug for 10 minutes to 5 hours before being imaged in
a drop of control H-KSOM/AA.
Chemicals
D(+)Glucose, 2-deoxyglucose, DL-Lactic acid, pyruvate, oxamate,
oxalomalate, dehydroisoandrosterone (DHEA), FCCP, CN-, rotenone, CIN, dimethyl
malate, BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] and TEMPO
(2,2,6,6-tetramethylpiperidine-1-oxy) are all from Sigma, UK. During
autofluorescence imaging experiments these chemicals were added as 10x
solution of their final concentration (indicated under the graphs).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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These observations are consistent with the finding that glycolysis is
blocked in mouse oocytes (Barbehenn et al.,
1974). Published data suggest that glucose might be metabolised
via the pentose phosphate pathway [PPP, which generates NADPH
(Fig. 1)
(Downs et al., 1998;
Urner and Sakkas, 2005;
Comizzoli et al., 2003)]. To
test this possibility, we imaged mouse oocytes incubated in complete KSOM/AA
medium and applied the PPP inhibitor DHEA
(Nutt et al., 2005). DHEA
provoked an increase in global NAD(P)H mirrored by a decrease in
FAD++ autofluorescence, but cytosolic NAD(P)H (dashed blue line in
Fig. 2B) remained unchanged
(Fig. 2B). This observation
suggests that DHEA unexpectedly inhibits mitochondrial respiration in mouse
oocytes [as has been observed in some preparations
(Correa et al., 2003)] and
suggests that the PPP does not supply NADPH in unfertilised mouse oocytes.
This result was confirmed by inhibiting PPP with another unrelated inhibitor
(6-aminonicotinamide) that did not change NAD(P)H or FAD++
autofluorescence at all (n=9, data not shown). Therefore it seems
that glucose metabolism does not impact the intracellular redox potential in
mouse oocytes.
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The role of lactate dehydrogenase (LDH) in the response to lactate was
explored using oxamate, a specific competitive inhibitor of LDH
(Wilkinson and Walter, 1972).
Fig. 2C shows that NAD(P)H
increased after lactate addition and the subsequent addition of oxamate
decreased NAD(P)H autofluorescence very sharply
(Fig. 2C). Increasing further
lactate in the medium in the presence of oxamate had no effect on the NAD(P)H
signals. Thus, lactate directly reduces the cytosolic redox potential (i.e.
increases cytosolic NADH) via the action of LDH.
To confirm the observed effects of pyruvate on the cytosolic redox potential we blocked LDH (with oxamate, Fig. 3B) or mitochondrial respiration (Fig. 4A) before adding pyruvate. Oxamate prevented the pyruvate-induced decrease in global NAD(P)H without preventing the reduction of FAD++, whereas inhibiting mitochondrial respiration had no effect on the response to pyruvate (Fig. 4A). In addition, mitochondrial NAD(P)H increased upon pyruvate addition when LDH was blocked, further establishing that exogenous pyruvate stimulates mitochondrial NAD(P)H production (Fig. 3B compared with Fig. 3A). These observations demonstrate that pyruvate and lactate are metabolised in the cytosol by LDH to set the cytosolic redox potential and that exogenous pyruvate (but, surprisingly, not lactate-derived pyruvate) is also metabolised in the mitochondria.
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-cyano-4-hydroxy cinnamate (CIN)
(Del Prete et al., 2004).
Application of CIN (0.5 mmol/l) to mature mouse oocytes incubated in complete
KSOM/AA decreased global NAD(P)H autofluorescence and increased
FAD++ autofluorescence (Fig.
4B). Subsequent addition of pyruvate failed to generate the
expected change in FAD++, whereas NAD(P)H autofluorescence still
decreased, consistent with pyruvate entry into the cytosol but not into
mitochondria (Fig. 4B). The decrease in NAD(P)H autofluorescence induced by CIN could be either due to accumulation of pyruvate in the cytosol, which then oxidises NADH via LDH, or to inhibition of the mitochondrial Krebs cycle (see Fig. 1). To discriminate between these, LDH was inhibited by oxamate before addition of CIN (Fig. 4C). This prevented the decrease in NAD(P)H caused by CIN while FAD++ was still oxidised. Subsequent addition of pyruvate no longer changed either NAD(P)H or FAD++ signals. These data together suggest that, in the mature mouse oocyte, exogenous pyruvate may be imported directly into mitochondria to fuel the Krebs cycle, but may also be metabolised by LDH in the cytosol.
Alternate sources of NAD(P)H operating in the mouse oocyte
The observations described so far suggest that the main roles of metabolic
substrates present in culture media are to set the cytosolic
NADH:NAD+ ratio (via LDH). However, as alternate sources of NAD(P)H
could contribute to the regulation of intracellular redox potential, we
investigated further the sources of NAD(P)H in mouse oocytes
(Fig. 5). Malate can be
metabolised by malic enzyme (ME, cytosolic) and by malate dehydrogenase (MDH,
cytosolic and mitochondrial) to produce NADPH and NADH respectively
(Fig. 1)
(Stryer, 1970). Addition of
the permeant malate analogue dimethyl malate (Me2 -malate) provoked
a consistent decrease in FAD++ autofluorescence without
significantly affecting global NAD(P)H autofluorescence
(Fig. 5A). This observation
suggests that Me2-malate is mostly metabolised in mitochondria and
that cytosolic MDH and ME are poorly active in mouse oocytes
(Fig. 5A). It also contradicts
somehow the recent finding that cytosolic MDH is expressed in mouse oocytes
(Lane and Gardner, 2005).
As neither the PPP (Fig. 2)
nor ME (Fig. 5A) supply NADPH
in mouse oocytes, we hypothesised that cytosolic NADP-ICDH - an important
provider of cytosolic NADPH in some somatic cells
(Mallet and Sun, 2003;
McDonald et al., 2005) - could serve this task. Inhibition of NADP-ICDH by
oxalomalate (Yang and Park,
2003), applied to mouse oocytes incubated in complete KSOM/AA
(Fig. 5B,C) also provoked a
decrease in global and cytosolic NAD(P)H autofluorescence without affecting
FAD++ autofluorescence (FCCP was added at the end of the experiment
to completely oxidise the mitochondrial pools). This suggests that cytosolic
NADPH levels are maintained by oxidation of isocitrate by cytosolic NADP-ICDH
(Fig. 1).
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)
and oxalomalate should affect the supply of cytosolic NADPH only
(Yang and Park, 2003), the
sequential addition of these two inhibitors are expected to have additive
effects on NAD(P)H autofluorescence. Oxamate was added before oxalomalate to
further test this hypothesis (Fig.
5C). Here again, inhibiting NADP-ICDH had no effect on NAD(P)H
autofluorescence once LDH had been inhibited. Together, these observations
indicate that, somehow, cytosolic NADH and NADPH pools are in equilibrium in
the mouse oocyte.
Changes in NAD(P)H and in GSH during maturation and after fertilisation
An important of role for NADPH in cells is to fuel the reduction of the
thiol-containing glutathione by glutathione reductase (GR)
(Fig. 7) and the glutaredoxin
(Grx) system (Holmgren et al.,
2005). We thus imaged intracellular NAD(P)H, FAD++ and
GSH in the same oocyte to study how NAD(P)H regulates the levels of GSH in the
mouse embryo (Fig. 6).
Intracellular reduced glutathione (GSH) can be measured by imaging the
fluorescent adduct formed with monochlorobimane (MCB) [see Keelan et al.
(Keelan et al., 2001) for
details on MCB]. First, to validate our GSH imaging technique and to assess
how oxidising GSH impacts autofluorescence levels, we inhibited GSH reduction
by GR (with BCNU) (Gardiner and Reed,
1995a) or oxidised GSH directly [by a short incubation in diamide
(Kosower and Kosower, 1995;
Akella and Harris, 1999)] and
measured autofluorescence and GSH levels
(Fig. 6A).
Fig. 6A shows, as expected,
that inhibiting GR significantly decreased GSH levels (to 23.1±2.9% of
untreated oocytes, P<0.0003, n=28 oocytes), whereas
oxidising GSH with diamide decreased GSH levels to 30.9±4.4% of
untreated controls (n=10 oocytes, P<0.0004,
Fig. 6A). However, BCNU and
diamide have strikingly dissimilar effects on the autofluorescence signal:
BCNU slightly but significantly increased NAD(P)H (107.1±0.8% of
untreated controls, P<0.019, n=28 oocytes) without
affecting mitochondrial FAD++
(Fig. 6A). By contrast, diamide
significantly oxidised both NAD(P)H (observed as a decrease of fluorescence,
74.8±5.8%, P<0.012, n=10 oocytes) and
FAD++ (observed as an increase of fluorescence, 118.0±2.2%,
P<0.004, n=10 oocytes). The acute application of diamide
(15 minutes incubation) used in our protocol mostly oxidises GSH, and to a
lesser extent protein thiols, but not NAD(P)H directly
(Kosower and Kosower, 1995).
Our observations therefore suggest that, in mature mouse oocytes, NADPH is
used by GR to maintain GSH levels.
Then we determined the relationship between NAD(P)H, FAD++ and
GSH during development by measuring them in oocytes before maturation (GV
oocytes) and after maturation (M2 oocytes) as well as in two-cell embryos
(Fig. 6B).
Fig. 6B shows that
FAD++ autofluorescence did not vary during the developmental stages
considered, whereas NAD(P)H was constant during maturation but decreased
significantly between fertilisation and the two-cell stage (to
65.9±0.9% of mature oocytes, P<0.012, n=12
oocytes). By contrast, GSH increased during maturation (GSH levels before
maturation being 64.4±2.4% of M2 oocytes, P<0.002,
n=25 oocytes) and decreased after fertilisation (to 65.5±2.3%
of M2 oocyte, P<0.013, n=12 oocytes), as found earlier in
mammals (for a review, see Luberda,
2005). The concomitant decrease in NAD(P)H and GSH occurring after
fertilisation suggests that GSH production is dependent on NADPH levels during
this stage of development. On the contrary, the increase in GSH levels
observed during maturation was not accompanied by an increase in NAD(P)H
autofluorescence, suggesting that this increase in GSH during maturation does
not rely on increased NADPH. These findings are reminiscent of the different
GSH metabolisms of mouse oocytes before and after fertilisation (see
Gardiner and Reed, 1995a;
Gardiner and Reed, 1995b).
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Surprisingly, we found that increasing pyruvate in the medium was
detrimental for development, as a significantly lower percentage of
blastocysts was consistently obtained (23.8±3.5%, n=87
embryos, four replicates) compared with control conditions (66.5±1.4%,
n=95 embryos, four replicates, P<0.015). Furthermore, the
inhibition of development by 1 mmol/l pyruvate could be rescued by adding the
membrane-permeant antioxidant TEMPO [48.9±2.6% of blastocysts,
n=94 embryos, four replicates versus 23.8±3.5% with high
pyruvate only, P<0.005 (Keelan
et al., 2001)]. This observation demonstrates that such a
concentration of pyruvate is not toxic by itself, as has been reported earlier
(Wales and Whittingham, 1971),
and indicates that an oxidant action of pyruvate inhibits development.
Decreasing the cytosolic NADH:NAD+ ratio with oxamate or the cytosolic NADPH:NADP+ ratio with oxalomalate both inhibited development significantly [oxamate: 23.3±4.9% (n=83, four replicates) versus 65.5±5.1% (n=92 embryos, four replicates), P<0.009; oxalomalate: 5±2.5% (n=83 embryos, four replicates) versus 65.5±5.1% (n=69 embryos, four replicates), P<0.002]. These observations indicate that, in the mouse embryo, both cytosolic NADH and NAD+ and NADPH and NADP+ must be maintained to ensure proper early development.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Dumollard et al.,
2004; Dumollard et al.,
2006b) coupled with a pharmacological approach to decipher the
mechanisms maintaining intracellular redox potential. Finally, we intervened
to manipulate redox potential in order to examine its impact on early
embryonic development. Our observations reveal that an efficient enzymatic system regulates the levels of NADH and NAD+, NADPH and NADP+ and GSH and GSSG in mouse oocytes. This study also shows that the cytosolic and mitochondrial metabolism of exogenous substrates defines a redox potential that supports development to the blastocyst stage.
Intracellular metabolism of glucose in mouse oocytes
Our observations suggest that glucose is poorly metabolised in the mouse
oocyte as neither the acute addition of glucose nor the specific inhibition of
the pentose phosphate pathway (with 6-AN and DHEA) or of glucose metabolism
(with 2-deoxyglucose) affect cytosolic NAD(P)H levels. This is in accord with
previous observations that glycolysis is inhibited in mouse early embryos
(Barbehenn et al., 1974;
Leese, 2002;
Johnson et al., 2003).
However, several studies suggested that the pentose phosphate pathway (PPP)
may be active in maturing oocytes and zygotes and may regulate oocyte
maturation and early development (Cetica et
al., 2002; Downs et al.,
1998; Urner and Sakkas,
2005; Comizzoli et al.,
2003). Our observations show that the PPP plays a minor role, if
any, in the supply of NADPH in the unfertilised oocyte. Such a finding
emphasises that the early mouse embryo has a metabolism that is quite distinct
from that of mature somatic cells (Jain et
al., 2003; Tian et al.,
1999; Tian et al.,
1998) or Xenopus oocytes
(Nutt et al., 2005), in which
the PPP is a major provider of NADPH. Stimulation of the PPP after sperm entry
has been described in the mouse (Urner and
Sakkas, 2005); thus, PPP might have a more significant role in the
regulation of NADPH levels in fertilised oocytes, a hypothesis we are now
investigating.
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) (Fig.
7). Oxamate alone also promoted oxidation of cytosolic NAD(P)H in
oocytes incubated in complete KSOM/AA, further demonstrating a major role for
LDH in supplying cytosolic NADH in the resting oocyte.
Surprisingly, lactate-derived pyruvate seems to be minimally metabolised by
the mitochondria, as mitochondrial autofluorescence was never affected by
lactate addition, even after a delay. This suggests the presence of discrete
pools of pyruvate inside the oocyte: one from the bathing medium, which is
rapidly metabolised by the mitochondria (see below), while a second pool
derived from lactate is poorly used by the mitochondria. Such intracellular
compartmentation of pyruvate pools has been described in neuronal and glial
cells (Cruz et al., 2001;
Zwingmann et al., 2001).
Strikingly, in astrocytes, pyruvate derived from cytosolic alanine is poorly
transported into mitochondria but is preferentially converted to lactate,
which is exported from the cell (Zwingmann
et al., 2001). It seems plausible that lactate-derived pyruvate
may be preferentially converted in the cytosol to alanine by alanine
aminotransferase (ALT) (Fig.
7), because: (1) preferential incorporation of carbon from lactate
into alanine has been measured in mouse embryos
(Quinn and Wales, 1973); (2)
bovine oocytes contain strong ALT activity
(Cetica et al., 2003); and (3)
bovine and porcine embryos produce large amounts of alanine
(Gopichandran and Leese, 2003;
Humpherson et al., 2005).
However, further studies are required to confirm such a hypothesis.
Finally, it is known that lactate alone is not able to support development
from fertilisation, but only from the two-cell stage
(Biggers et al., 1967;
Johnson et al., 2003;
Summers and Biggers, 2003).
Our data showing that lactate-derived pyruvate does not support mitochondrial
oxidative phosphorylation in the oocyte could explain why lactate is unable to
support development from fertilisation.
Regulation of the intracellular NAD(P)H by pyruvate metabolism
Our data clearly demonstrate that pyruvate promotes oxidation of cytosolic
NAD(P)H and reduction of mitochondrial NAD(P)+ and
FAD++. Thus, as in most cell types, pyruvate is, in the mouse
oocyte, a cytosolic oxidant but a mitochondrial reductant. These effects of
pyruvate on mitochondrial redox state are consistent with its central role in
fuelling oxidative phosphorylation and ATP production in the mouse oocyte
(Fig. 7)
(Leese, 2002;
Johnson et al., 2003;
Dumollard et al., 2004).
Two metabolic pathways might explain the cytosolic oxidation by pyruvate;
pyruvate can be metabolised by LDH and ME to consume NADH and NADPH,
respectively (Fig. 1)
(Stryer, 1970). Our
observations suggest that the reduction of pyruvate to lactate by LDH is the
major pathway present in oocytes, as the oxidant effects of pyruvate were
independent of mitochondrial activity and were completely blocked by
inhibition of LDH. Further, we found no evidence for cytosolic ME activity, as
addition of a permeant malate analogue had no effect on NAD(P)H levels while
causing a reduction of mitochondrial FAD++.
Our data suggest that a delicate balance is maintained between cytosolic and mitochondrial metabolism of pyruvate, such that any disruption of that balance strongly impacts on the redox state. Indeed, when uptake of pyruvate into mitochondria was blocked, a sharp oxidation of cytosolic NADH was observed. This poise of cytosolic pyruvate set by pyruvate in the medium and its transport into mitochondria is crucial for optimising energy balance and the cytosolic NADH:NAD+ ratio.
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-ketoglutarate (-KG) in the cytosol.
Such isocitrate originates from citrate synthesised solely by the Krebs cycle
before being exported from the mitochondria [a process called anaplerosis
(MacDonald et al., 2005)
(Fig. 7)]. Therefore the
observation that NADP-ICDH activity maintains cytosolic NADPH levels suggests
that mitochondrial pyruvate metabolism produces citrate in the oocyte. The
earlier finding that mouse zygotes contain high levels of intracellular
citrate supplied by the metabolism of exogenous pyruvate
(Barbehenn et al., 1974)
supports such a hypothesis. This pathway plays a primordial role in providing
cytosolic NADPH in several somatic cell types
(Mallet and Sun, 2003;
Yang and Park, 2003;
MacDonald et al., 2005). Given
the lack of activity of the PPP in mouse oocytes, this pathway may represent a
vital supply of cytosolic NADPH in the mouse oocyte and early embryo, but
further experiments are needed to confirm such a hypothesis.
Together our observations show that both cytosolic and mitochondrial
metabolism of pyruvate regulates cytosolic NADPH and NADH levels via cytosolic
LDH and cytosolic NADP-ICDH. The diverse metabolism of pyruvate in the mouse
oocyte has a dual impact on the intracellular oxidant load, as NADPH has an
antioxidant action (see below) but mitochondrial NADH may promote
mitochondrial ROS production (Fig.
7) (Balaban et al.,
2005; Xu and Finkel,
2002).
GSH metabolism in mouse oocytes and embryos
A major role for intracellular NADPH is to fuel regeneration of GSH via the
glutaredoxin-glutathione reductase (Grx-GR) system and the thioredoxin system
(MacDonald et al., 2005;
Holmgren et al., 2005). By
measuring autofluorescence and GSH levels in the same oocyte, we were able to
determine the relationships between NAD(P)H and GSH during the first stages of
development. Our observations demonstrate that, in mouse oocytes,
intracellular NADPH is consumed to regenerate GSH by the Grx-GR system, as
direct oxidation of GSH by diamide provoked a concomitant oxidation of
NAD(P)H, whereas GR inhibition that depleted GSH levels increased NAD(P)H.
Measuring NAD(P)H and GSH before maturation, before fertilisation and after
the first cleavage revealed that GSH increases during maturation and then
decreases during development, as described previously (for a review, see
Luberda, 2005). However,
NAD(P)H levels were constant during maturation and decreased after
fertilisation. The finding that the increase in GSH observed during maturation
is not accompanied by an increase in NAD(P)H levels suggests that the
production of GSH during maturation depends modestly on NADPH availability. By
contrast, the decrease in GSH observed after fertilisation was accompanied by
a concomitant decrease in NAD(P)H levels, suggesting that GSH production is
limited by NADPH availability. These two hypotheses are consistent with the
earlier findings that GSH production relies crucially on ATP-dependent de novo
synthesis during oocyte maturation, whereas de novo synthesis of GSH is
switched off after fertilisation (Gardiner
and Reed, 1995a; Luberda,
2005). By contrast, NADPH-dependent regeneration of GSH by the
GR-Grx system operates during both maturation and early development
(Gardiner and Reed, 1995b;
Luberda, 2005). Thus, NADPH
levels may be more crucial for the maintenance of GSH levels during early
development than during oocyte maturation.
Together, our observations show that NADPH availability might set the GSH levels in the early embryo, thereby fuelling the antioxidant defence of the embryo, and suggest that early development is associated with an increase in oxidative stress that overpowers antioxidant defences, resulting in a decrease in both NADPH:NADP+ and GSH:GSSG ratios.
Regulation of early development by the redox potential
The detrimental effect of oxidative stress and the protection exerted by
GSH on early development are now well established
(Johnson and Nasr-Esfahani,
1994; Gardner et al.,
2002; Guerin et al.,
2001; Leese, 2002;
Luberda, 2005). However, the
impact on development of the redox potential - set in the mouse zygote mainly
by pyruvate and lactate metabolism - is more difficult to infer due to its
dual action on oxidative stress. The observation that high pyruvate (1 mmol/l)
in the culture medium has a detrimental impact on development that can be
reversed by an antioxidant shows that pyruvate metabolism causes damaging
oxidative stress in the mouse embryo. The oxidant action of pyruvate on the
embryo is illustrated by the fact that, despite the generation of NADPH from
pyruvate via anaplerosis and NADP-ICDH, increasing pyruvate results in a net
oxidation of cytosolic NAD(P)H and a net increase in mitochondrial NADH, which
increases mitochondrial ROS production
(Fig. 7). Therefore, even
though pyruvate can detoxify ROS by directly scavenging hydrogen peroxide
(Constantopoulos and Barranger,
1984), such antioxidant action of pyruvate is surpassed by the
complex metabolism of pyruvate in the oocyte, which increases the
intracellular oxidant load.
The main impact of cytosolic NADPH on oxidative stress is to maintain
antioxidant defence, as it is mainly used to regenerate GSH in the mouse
oocyte. It is important to note that mammalian oocytes do not appear to
possess NAD(P)H oxidase activity as alternative generators of ROS and
H2O2 (Geiszt and
Leto, 2004). The role of NADH on oxidative stress in mouse oocytes
is less straightforward, as NADH is a direct antioxidant
(Kirsch and De Groot, 2001)
but its oxidation by the respiratory chain generates ROS
(Xu and Finkel, 2002;
Balaban et al., 2005)
(Fig. 7). Such an oxidant
action of pyruvate-derived NADH in the mitochondria promotes cell senescence
in human diploid fibroblasts (Xu and
Finkel, 2002). Similarly, in the mouse oocyte, an oxidant action
of pyruvate in the mitochondria may overwhelm the antioxidant defence of the
embryo, thereby impairing development.
Maintenance of cytosolic NADH and NADPH levels by LDH and NADP-ICDH seems crucial for early development of the mouse, even when mitochondrial respiration is low (i.e. when pyruvate is low), as inhibiting each enzyme drastically impaired development. As depleting NADH also depletes NADPH (Fig. 5), it is not possible to differentiate between their respective roles. Further studies are necessary to determine whether these treatments impair early development by increasing the oxidant load experienced by the embryos.
In conclusion, our study describes for the first time how metabolism of exogenous substrates regulates the intracellular redox potential in the mouse oocyte and how such a tightly regulated redox potential supports efficient early development. This work explains the roles played by metabolites present in culture media and will inform future progress in the physiological characterisation of mammalian early development.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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indicates the line of
the scan in the graphs shown below each image. The line-scan analysis reveals
subcellular differences in the distribution of the changing NAD(P)H signal to
the manipulations. Time indicated in minutes. (B) Effect of pyruvate on
global FAD++ (large white ROI, green trace) and NAD(P)H (large
white ROI, light blue trace) and on mitochondrial NAD(P)H (small blue ROI,
dark blue trace) autofluorescence in an MII oocyte incubated in complete
H-KSOM/AA containing 5 mmol/l oxamate (n=11 oocytes). Pyruvate and
rotenone are added as indicated. The x-axes indicate time in
minutes.
