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376
THE JOURNAL
S. DOWNSOF EXPERIMENTAL ZOOLOGY 282:376–384 (1998)
Precursors of the Purine Backbone Augment the
Inhibitory Action of Hypoxanthine and Dibutyryl
cAMP on Mouse Oocyte Maturation
STEPHEN M. DOWNS
Biology Department, Marquette University, Milwaukee, Wisconsin 53233
ABSTRACT
In this study we have tested the hypothesis that precursors of the purine base
backbone—glutamine, glycine, aspartic acid, and formate—promote meiotic arrest when included
in medium containing established meiotic inhibitors and that this occurs in glucose-dependent
fashion. An initial experiment established that in medium supplemented with 4 mM hypoxanthine and containing no purine precursors, very little meiotic arrest was maintained in cumulus
cell-enclosed oocytes after 17–18 hr (90% germinal vesicle breakdown; GVB). Increasing concentrations of glucose reduced the maturation percentage such that only 57% had matured at 0.55
mM. The addition of 2 mM glutamine (Gln) alone reduced the maturation percentage in the absence of glucose (70% GVB), and the further addition of glucose revealed an additive inhibitory
effect between these two supplements. Dose response experiments with Gln, glycine (Gly), aspartic acid and formate showed that in medium supplemented with hypoxanthine, very little inhibitory action was observed in the absence of glucose but that upon addition of this hexose, a dramatic
decrease in maturation percentage was observed in the Gln and Gly groups. Results of experiments using combinations of precursors showed that when Gln and Gly were added together,
greater augmentation of meiotic arrest maintained by either hypoxanthine or dibutyryl cAMP was
achieved in the presence of glucose than with either amino acid alone. The addition of purine
precursors significantly increased the extent of purine nucleotide production by oocyte-cumulus
cell complexes, and this was accentuated by glucose. It is concluded that the presence of purine
precursors can augment the meiosis-arresting action of established meiotic inhibitors in glucosedependent fashion, and that this is due, at least in part, to their incorporation into purine nucleotides via the de novo synthetic pathway. J. Exp. Zool. 282:376–384, 1998. © 1998 Wiley-Liss, Inc.
It has been known for many years that mammalian oocytes, when removed from their intrafollicular environment and placed in a suitable
culture medium, will undergo spontaneous germinal vesicle breakdown in vitro (Pincus and Enzmann, ’35). Numerous studies have reported on
possible physiological compounds present within
developing ovarian follicles that arrest the oocyte
in the germinal vesicle stage, and two of the more
intensely investigated agents are cyclic adenosine monophosphate (cAMP) and purines (see
Tsafriri and Dekel, ’94; Downs, ’95b for recent reviews). Although a number of different compounds
have been shown to suppress spontaneous oocyte
maturation in vitro, the mechanism by which such
meiotic arrest is brought about or the culture conditions that enable this arrest have not usually
been well delineated.
Previous work has shown that the action of meiotic inhibitors can be blocked by treatment of oocytes with purine metabolic perturbants, thereby
© 1998 WILEY-LISS, INC.
implicating the purine metabolic pathways as contributing to meiotic arrest. Purine-maintained meiotic arrest in vitro was reversed by inhibitors of IMP
dehydrogenase, the first enzyme in a two-step pathway from IMP to guanosine monophosphate (Downs
et al, ’86; Eppig, ’91), or by inhibitors of de novo
synthesis (Downs, ’93). These same inhibitors also
abrogated the intrafollicular block to maturation
following intraperitoneal injection to hormonally
primed mice (Downs and Eppig, ’87). It is important to note that the action of the purine metabolic
perturbants was not simply through interrupting
the metabolism of purine meiotic inhibitors such
as hypoxanthine. This is evident from the finding
that, in addition to inducing oocyte maturation in
Grant sponsor: NIH; Grant number: 25291.
*Correspondence to: Stephen M. Downs, Biology Department,
Marquette University, Wehr Life Sciences Building, Room 109, P.O.
Box 1881, Milwaukee, WI 53201-1881.
Received 8 January 1998; Accepted 1 April 1998
PURINE PRECURSORS AND MEIOTIC ARREST
hypoxanthine-arrested oocytes, the IMP dehyrogenase inhibitor, mycophenolic acid, reversed the
meiotic arrest maintained in vitro by the cAMP
analog, dbcAMP, or the phosphodiesterase inhibitors, IBMX or Ro-201724 (Downs, ’93). Hence, an
active de novo purine synthetic pathway is essential, and acts cooperatively with meiotic inhibitors, to achieve optimal meiotic arrest.
The type of culture medium employed and/or
the supplements contained therein can have a profound influence on the ability of established meiotic inhibitors to block oocyte maturation. For
example, hypoxanthine was ineffective in maintaining meiotic arrest in M16 medium, a simple
formulation that lacks vitamins or amino acids;
however, the addition of glutamine significantly
lowered the frequency of maturation to a level
comparable to that seen in more complex media,
including MEM (Downs and Mastropolo, ’97). In
addition, manipulation of potential energy sources
such as glucose, pyruvate and glutamine radically
altered the meiotic response of oocytes in dbcAMPor hypoxanthine-supplemented medium (Fagbohun
and Downs, ’92; Downs and Mastropolo, ’94). Pyruvate exerted a stimulatory influence on maturation,
while glucose and glutamine suppressed germinal
vesicle breakdown. Further evaluation showed that
glycolytic generation of ATP from glucose significantly augmented the inhibitory action of hypoxanthine on oocyte maturation and was dependent
upon gap junctional communication between the
oocyte and cumulus cells (Downs, ’95a). These observations serve to establish that the maturationarresting action of putative meiotic inhibitors
depends upon a multiplicity of factors, not the least
of which is the makeup of the culture medium.
Although glycolytic processing of glucose has
been shown to contribute to the inhibitory action
of hypoxanthine on oocyte maturation, this hexose can also be metabolized through the pentose
phosphate pathway to generate phosphoribosylpyrophosphate (PRPP). PRPP, required for both
the de novo purine synthesis and purine salvage
pathways, could then participate in the production of purine nucleotides that feed into meiosisarresting pathways. Also required for de novo
purine synthesis are small molecular precursors
for the purine backbone, as shown in Fig. 1, that
include glutamine, glycine, formate, and aspartic
acid. The presence of such precursors along with
glucose in the culture medium could affect the level
of purine nucleotides synthesized by oocyte-cumulus cell complexes and thereby modify the action of
putative meiotic inhibitors.
377
Fig. 1. Origins of the purine backbone. Shown are the
sources for the carbon and nitrogen atoms in the purine backbone in a purine nucleoside monophosphate molecule. Glycine and aspartic acid enter the de novo synthetic pathway
once, while glutamine and formate are required twice. CO2
or bicarbonate is also required (for C6), but their effects have
not been addressed in this study.
The present study was undertaken to examine
the effects of these purine backbone precursors
on: (1) the inhibitory action of hypoxanthine and
dbcAMP on the maturation of cumulus cell-enclosed oocytes; and (2) de novo purine nucleotide
synthesis. We report that glutamine and glycine
are particularly effective in increasing both meiotic arrest and the generation of purine nucleotides in culture, and this effect is accentuated
by the presence of glucose.
MATERIALS AND METHODS
Oocyte isolation and culture conditions
C57BL/6J X SJL/J F1 female mice, 20–23 days
old, were used for all experiments. Mice were
primed with 5 I.U. equine chorionic gonadotropin
and 48 hr later were killed and their ovaries were
removed and placed in culture medium. The antral follicles were punctured with sterile needles
and released oocyte-cumulus cell complexes were
pooled and washed through 2–3 changes of medium prior to allocation to the various treatment
groups. Cumulus cell-enclosed oocytes were cultured 17–18 hr in 1 ml medium in capped plastic
Falcon tubes (2058) that were gassed with a humidified mixture of 5% O2, 5% CO2, and 90% N2
and placed in a 37°C water bath. The culture medium used was Eagle’s minimum essential medium supplemented with 0.23 mM pyruvate, 3 mg/
ml lyophilized crystallized bovine serum albumin
378
S. DOWNS
(BSA; ICN ImmunoBiologicals, Lisle, IL), penicillin, and streptomycin sulfate.
Purine de novo synthesis assay
Complexes were cultured 3 hr in medium containing 10 µCi 14C-formate (56.6 mCi/mmol; ICN
Pharmaceuticals, Inc., Irvine, CA) and then extracts were assayed as previously described
(Downs, ’93). Ninety complexes were used per
treatment group per assay.
Chemicals
All culture medium components, hypoxanthine,
and dbcAMP were obtained from Sigma Chem.
Co. (St. Louis, MO).
Oocyte assessment and statistical analysis
At the end of culture, cumulus cells were removed from complexes by repeated pipeting with
a pasteur pipet. Viable oocytes, as determined
morphologically by observation under a stereomicroscope, were then assessed for germinal vesicle
breakdown. In all treatment groups, viability was
maintained above 93%, which is considered normal (Downs and Mastropolo, ’97). Oocyte maturation data are presented as the mean percentage
of germinal vesicle breakdown ±SEM. Maturation
frequencies were subjected to arcsin transformation and data were analyzed by ANOVA followed
by Duncan’s multiple range test. Nontransformed
data for the de novo purine synthesis assay were
analyzed by the same test. A P value less than
0.05 was considered significant.
inhibitory effect of hypoxanthine, with the maximal effect reached at 0.055–5.5 mM (57–67% GVB;
Fig. 2). The addition of glutamine also proved inhibitory, with the maturation percentage reduced
from 90% to 70% in the absence of glucose. The
effects of glucose and glutamine were additive, as
parallel inhibition curves were obtained upon combining the two compounds.
The subsequent dose-response experiments with
purine precursors utilized glucose at a concentration of 5.5 mM, since this concentration is routinely used in most culture media and was in the
range that produced maximum inhibition. Cumulus cell-enclosed oocytes were cultured 17–18 hr
in hypoxanthine-supplemented medium in the
presence or absence of glucose plus increasing concentrations of precursors at concentrations from
0.1 to 2 mM.
In the absence of glucose, glutamine had a marginal inhibitory impact on oocyte maturation in
hypoxanthine-treated oocytes, as only the decrease
at 2 mM (from 97 to 84% germinal vesicle breakdown) was significant (Fig. 3A). The maturation
frequency was lowered upon addition of glucose
and further decreased by glutamine in dose-dependent fashion; the highest concentration of
glutamine (2 mM) producing a 31% reduction in
maturation.
RESULTS
Figure 1 shows the contribution of purine precursor molecules to the 9-member purine backbone. One molecule of aspartic acid and glycine is
required, the former contributing N1 and the latter C4, C5, and N7. Two molecules each of formate and glutamine are also needed, formate
donating C2 and C8 and glutamine donating N3
and N9. C6 is derived from CO2 or bicarbonate,
and the effect of these potential precursors was
not tested in the present study. An initial glucose
dose-response experiment was carried out to analyze the effect of glucose (0.0055–5.5 mM) on the
hypoxanthine-maintained meiotic arrest of cumulus cell-enclosed oocytes during 17–18 hr of culture,
in the absence or presence of 2 mM glutamine. In
the glutamine- and glucose-free group, 90% of the
oocytes underwent germinal vesicle breakdown
(GVB) in the presence of 4 mM hypoxanthine. In
the absence of glutamine, glucose augmented the
Fig. 2. Dose-response effect of glucose on hypoxanthinemaintained meiotic arrest. Cumulus cell-enclosed oocytes were
cultured 17–18 hr in medium containing 4 mM hypoxanthine
plus or minus 2 mM glutamine. To these media were added
increasing concentrations of glucose (0.0055, 0.055, 0.55, and
5.5 mM). At the end of culture, cumulus cells were removed
and oocytes assessed for germinal vesicle breakdown (GVB).
Groups with a common letter are not significantly different.
PURINE PRECURSORS AND MEIOTIC ARREST
379
Fig. 3. Dose-response effect of purine precursors on hypoxanthine-maintained meiotic arrest. Cumulus cell-enclosed
oocytes were cultured 17–18 hr in medium containing 4 mM
hypoxanthine plus or minus 5.5 mM glucose, and each of the
four precursors was added in increasing concentrations (0.1,
0.3, 1, and 2 mM). At the end of culture, cumulus cells were
removed and oocytes assessed for germinal vesicle breakdown
(GVB). Groups with a common letter are not significantly
different.
Glycine had no inhibitory action on oocyte maturation in the absence of glucose (Fig. 3B). Nevertheless, considerable inhibition was achieved in
the presence of glucose, with the two higher concentrations providing the greatest effect (a 39–
44% reduction in maturation compared to +glucose
control). Therefore, this inhibition was primarily
glucose-dependent.
Aspartic acid had no inhibitory effect on oocyte
maturation whether in glucose-free or glucose-containing medium (Fig. 3C). Formate, likewise, had
little influence, although there was a small nega-
tive effect at 0.1 mM in the presence of glucose
(Fig. 3D).
The next series of experiments was carried out
to test the effects of combinations of precursors
on oocyte maturation, both in hypoxanthine- and
dbcAMP-arrested oocytes. The amino acids were
tested at a concentration of 2 mM, and formate
was tested at 0.1 mM, since this concentation produced a slight inhibition in the above experiment.
These agents were tested by themselves, or were
tested in pairs of amino acids, three amino acids
together, or three amino acids plus formate.
380
S. DOWNS
As shown in Fig. 4A, slight inhibition was observed in the hypoxanthine-treated glucose-free
groups containing glutamine or glycine, but these
effects were not significant. The inhibitory effects
were more profound in the presence of glucose. In
this particular experiment (Fig. 4B), aspartate and
formate had little effect, while glutamine and glycine were inhibitory, with the greatest inhibition
occurring in those groups that contained both
amino acids.
The effects of the amino acid precursors were
next tested on dbcAMP-arrested oocytes to determine if their actions were generalized and not just
restricted to hypoxanthine arresting conditions.
Cumulus cell-enclosed oocytes were cultured 17–
18 hr in medium containing 300 µM dbcAMP plus
Gln, Gly, or Asp alone or in combination. Formate
was not tested since it displayed no inhibitory action in earlier experiments. Similar to the results
in hypoxanthine-treated oocytes, Gln and Gly
proved inhibitory, with no appreciable effect of Asp
(Fig. 5). Again, the greatest inhibition was achieved
when Gln and Gly were added together.
Because the compounds tested in this study are
precursors for the purine backbone, it was important to determine how they affect de novo purine
nucleotide synthesis. To this end, oocyte-cumulus
cell complexes were cultured 3 hr in either the
absence or presence of glucose, with different combinations of Gln, Gly, and Asp tested at 2 mM.
Radiolabeled formate was used to assay de novo
synthesis, and so its effects were not assessed on
purine nucleotide production. When all three
amino acids were added to glucose-free medium,
a nearly 300% increase in purine production occurred (Fig. 6). The addition of glucose alone increased the level of purine nucleotides by 42%.
Compared to the +glucose group, Asp had no effect,
while synthesis was increased 80% and 490% by
Gly and Gln, respectively. Purine synthesis was the
highest in the presence of glucose plus all three
amino acids (705 and 1,044% increases over the
–glucose and +glucose control groups, respectively).
Although the level of nucleotide production was
increased nearly 300% when Gln, Gly, and Asp
were added to glucose-free medium, these amino
acids did not significantly augment the inhibitory
action of hypoxanthine after 17–18 hr of culture
(Fig. 4A). Therefore, a kinetics experiment was
performed to determine whether a transient arrest mediated by these amino acids occurred in
the absence of glucose. Cumulus cell-enclosed oocytes were cultured for varying periods from 3 to
15 hr in glucose-free medium containing: (1) no
Fig. 4. Effects of combinations of purine precursors on hypoxanthine-maintained meiotic arrest. Cumulus cell-enclosed
oocytes were cultured 17–18 hr in medium containing 4 mM
hypoxanthine plus (right panel) or minus (left panel) 5.5 mM
glucose. Purine precursors were added alone or in combination, the amino acids at a concentration of 2 mM and for-
mate at a concentration of 0.1 mM. At the end of culture,
cumulus cells were removed and oocytes assesed for germinal vesicle breakdown (GVB). 3 AA, 3 amino acids (Gln, Gly,
and Asp); 3 AA+Form, 3 AA plus formate. Groups with a common letter are not significantly different.
PURINE PRECURSORS AND MEIOTIC ARREST
Fig. 5. Effects of combinations of purine precursors on
dbcAMP-maintained meiotic arrest. Cumulus cell-enclosed
oocytes were cultured 17–18 hr in medium containing 250
µM dbcAMP plus 5.5 mM glucose. Purine precursors were
added alone or in combination, each at a concentration of 2
mM. At the end of culture, cumulus cells were removed and
oocytes assessed for germinal vesicle breakdown (GVB). 3 AA,
3 amino acids (Gln, Gly, and Asp). Groups with a common
letter are not significantly different.
Fig. 6. Effects of glucose and purine precursors on purine
de novo synthesis. Oocyte-cumulus complexes were cultured
3 hr in glucose-free medium or medium containing 5.5 mM
glucose (controls), plus individual amino acids or the three
amino acids together (3 AA). All media contained 14C-formate,
and at the conclusion of culture extracts were assayed for
purine nucleotide production as described in Materials and
Methods. Groups with a common letter are not significantly
different.
381
additional supplements; (2) 4 mM hypoxanthine;
or (3) hypoxanthine plus Gln, Gly, and Asp at 2
mM. These data are shown in Fig. 7. After 3 hr
essentially all of the –glucose control oocytes had
resumed maturation (98%), while only 44 and 23%
of the hypoxanthine and hypoxanthine plus amino
acids groups had matured, respectively. Throughout the remainder of the culture period, the maturation frequency in the hypoxanthine group
continued to rise and by 15 hr had reached 98%.
On the other hand, meiotic arrest was maintained
by the addition of amino acids through the first 9
hr of culture, but then had risen by 15 hr to 55%.
Thus, although the effects of amino acids were
not significant after 17–18 hr in glucose-free medium, they produced a potent, but temporary, augmentation of hypoxanthine-maintained meiotic
arrest consistent with their effect on nucleotide
production.
DISCUSSION
This study has demonstrated that when added
to culture medium, precursors of the purine backbone augment or facilitate the meiosis-arresting
action of meiotic inhibitors such as hypoxanthine
and dbcAMP and increase the de novo production
of purine nucleotides. Glutamine and glycine were
Fig. 7. Effect of purine precursors on the kinetics of oocyte maturation. Cumulus cell-enclosed oocytes were cultured
for varying periods from 3 to 15 hr in glucose-free medium
containing 4 mM hypoxanthine (HX) plus or minus three
amino acids (Gln, Gly, and Asp, each at 2 mM). A single control group lacking hypoxanthine or amino acids was cultured
for 3 hr. At the end of culture, cumulus cells were removed
and oocytes assessed for germinal vesicle breakdown (GVB).
382
S. DOWNS
considerably more effective than aspartic acid or
formate in suppressing germinal vesicle breakdown and had a stimulatory effect on purine
nucleotide production. In addition, the inclusion
of glucose increased both the inhibition of oocyte
maturation and production of purine nucleotides by
these precursors, and these effects likely resulted,
at least in part, from increased PRPP made available by glucose metabolism through the pentose
phosphate pathway. Nevertheless, some of the effects of these precursors on oocyte maturation may
be unrelated to their action on purine synthesis.
Dose-response experiments with purine precursors in hypoxanthine-supplemented medium revealed no consistent inhibitory effect with formate
or aspartic acid, but significant inhibition was observed with glutamine and glycine. The reason
for the lack of effect with formate or aspartic acid
is not known, but could be related to poor uptake
or high endogenous pools already present within
the complex at the time of isolation. Although glycine exhibited strong inhibitory potency, significant suppression of germinal vesicle breakdown
only occurred in the presence of glucose. Glutamine showed glucose-independent inhibition at
the highest concentration tested (2 mM), although
there was some variability in reproducibility (compare Figs. 2 and 3A with Fig. 4A), and this effect
appeared to be additive with that of glucose, as
exemplified by the parallel maturation curves in
Fig. 2. However, at lower concentrations, glutamine exhibited glucose-dependent inhibition; for
example, a concentration of 1 mM had no effect
on maturation in the absence of glucose, but in
its presence reduced the maturation frequency by
38% in glucose-containing medium. Thus, under
the appropriate conditions, a synergistic interaction can be demonstrated between glucose and
glutamine or glycine.
The combination experiments demonstrated
that, in hypoxanthine-containing medium in the
absence of glucose, cultures containing both glycine and glutamine were no more inhibitory than
those containing either amino acid alone. However, in glucose-supplemented medium, the most
inhibitory culture conditions were those that included both glycine and glutamine. In dbcAMPcontaining medium, cultures containing these two
amino acids were again the most inhibitory, but
no more so than in medium containing glycine
alone. These results serve to illustrate further the
cooperative interactions between glucose and purine precursors in augmenting the maturationsuppressing action of meiotic inhibitors.
Previous studies showed that in the absence of
stimulatory ligands, physiological levels of glucose
promoted meiotic arrest when putative meiotic inhibitors were present in the culture medium
(Fagbohun and Downs, ’92; Downs and Mastropolo, ’94). At least part of this glucose effect is
attributable to glycolytic generation of ATP, since
iodoacetate eliminated both the block to spontaneous oocyte maturation and the stimulation of
ATP production that resulted from adding glucose
to hypoxanthine-containing MEM (Downs and
Mastropolo, ’94; Downs, ’95a). But glucose may
also contribute an inhibitory activity through its
ability to influence purine metabolism. As shown
herein, selected amino acids can significantly augment the ability of hypoxanthine and dbcAMP to
maintain meiotic arrest, but optimal inhibition
depends on the presence of glucose. It is proposed
that the inhibitory action of these amino acids is
mediated, at least in part, by their entering the
purine de novo synthetic pathway as precursors
for the purine backbone, since coincident with increased meiotic arrest, the combination of glycine,
glutamine, and aspartic acid significantly promoted the incorporation of 14C-formate into purine nucleotides.
That the degree of both meiotic arrest and purine nucleotide synthesis in response to precursors was increased upon glucose addition suggests
that the hexose contributes to meiotic arrest by
its metabolism through the oxidative arm of the
pentose phosphate pathway to form PRPP, the
starting compound required for de novo purine
synthesis. In support of this idea, glucose stimulated an increase in PRPP during a 6-hr culture
period (Downs et al, ’98) and incubation of oocytecumulus cell complexes with radiolabeled glucose
demonstrated metabolism through the pentose
phosphate pathway (Downs and Utecht, unpublished data). Thus, the oxidative arm of the pentose phosphate pathway is active in oocyte-cumulus
cell complexes and likely metabolizes glucose to
PRPP that feeds into the purine de novo synthetic
pathway along with amino acid precursors to generate inhibitory nucleotides. Nevertheless, we cannot discount the possibility that ATP produced by
glycolytic metabolism also contributes to the inhibitory action of glucose on oocyte maturation
under these experimental conditions.
Some apparent discrepancies exist between the
level of purine nucleotide production and the degree of meiotic arrest in cumulus cell-enclosed oocytes exposed to different precursor combinations.
For example, in the absence of glucose, the com-
PURINE PRECURSORS AND MEIOTIC ARREST
bination of three amino acids produced nearly a
300% increase in nucleotide production while exerting no significant inhibitory effect on oocyte
maturation, yet the glucose alone or glucose-plusglycine groups produced less nucleotide but suppressed meiotic maturation. These differences can
be reconciled if one considers the experimental
protocols involved. The nucleotide assays were
performed on complexes cultured only 3 hr, but
the maturation experiments used culture periods
of 17–18 hr. It was therefore possible that the increase in nucleotide production in response to the
three amino acids in glucose-free medium was a
result of endogenous glucose and/or PRPP present
at the time of isolation that allowed an initial,
yet temporary, augmentation of nucleotide production. Consistent with this idea was the finding
that the addition of glutamine, glycine, and aspartic acid to hypoxanthine-treated oocytes in the
absence of glucose caused a significant attenuation of the maturation kinetics (Fig. 7). The transient nature of this arrest may be due to eventual
depletion of endogenous glucose and its metabolites, since meiotic arrest was maintained when
the medium contained glucose. It is therefore concluded that the greater degree of meiotic inhibition achieved in the glucose and glucose plus
glycine groups is due to sustained levels of nucleotides made possible by the continued presence
of glucose.
A second inconsistency exists between the level
of nucleotide generated in glucose-containing medium in response to glycine versus glutamine and
the actions of these amino acids alone on oocyte
maturation. Glycine produced a 68% increase in
nucleotide production, but this was dwarfed by
the 458% increase stimulated by glutamine. Nevertheless, glycine always exhibited comparable or
greater inhibition of oocyte maturation than
glutamine. Glutamine may stimulate purine
nucleotide synthesis to a greater extent due to its
incorporation at two different sites along the de
novo pathway compared to only one for glycine,
but if a simple direct relationship exists between
nucleotide levels and meiotic arrest, one would
also expect greater inhibition of oocyte maturation with glutamine. It is possible that production of nucleotides beyond a certain level does not
translate into further inhibitory action, or perhaps
upstream events such as PRPP production are affected differently (Boss, ’84). Alternatively, actions
of glutamine or glycine apart from their role in
purine nucleotide production could provide an in-
383
fluence that overrides or augments the purinemediated inhibition.
The synergistic interaction between glucose and
purine precursors is proposed to be due to a collaborative effort generating increased purine
nucleotides. Although not tested in the present
study, it is logical to consider that the resulting
guanyl and adenyl nucleotides each contribute to
meiotic arrest by interacting with the cyclic AMPgenerating adenylate cyclase system, since this
cyclic nucleotide is known to augment the inhibitory actions of purines and follicular fluid fractions in dramatic fashion (Downs and Eppig, ’84;
Downs et al, ’85). The synergistic inhibitory effect on oocyte maturation that results when these
medium supplements are combined with hypoxanthine may therefore be due to increased generation of cAMP through the action of glucose and
purine precursors combined with suppression of
cAMP phosphodiesterase activity by hypoxanthine
(Eppig et al., ’85; Downs et al., ’89).
In summary, hypoxanthine and dbcAMP exhibited a limited ability to maintain meiotic arrest
in cumulus cell-enclosed mouse oocytes when medium lacked glucose or precursors of the purine
base backbone. When added alone to inhibitorsupplemented medium, glucose, glutamine, or glycine had a limited inhibitory effect on oocyte
maturation, but when glucose and amino acids
were added in combination a synergistic inhibition was achieved. Formate and aspartic acid were
ineffective. In general, the degree of inhibition correlated with levels of purine nucleotide produced
by the oocyte-cumulus cell complexes. These results therefore serve to illustrate how medium
components—in particular, glucose and purine
precursors—can profoundly influence the ability
of established meiotic inhibitors to maintain meiotic arrest in vitro.
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