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. 282:376384, 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. LITERATURE CITED Boss, G.R. (1984) Decreased phosphoribosylpyrophosphate as the basis for decreased purine synthesis during amino acid starvation of human lymphoblasts. J. Biol. Chem., 259:2936– 2941. Downs, S.M. (1993) Purine control of mouse oocyte maturation: Evidence that nonmetabolized hypoxanthine maintains meiotic arrest. Mol. Reprod. Dev., 35:82–94. Downs, S.M. (1995a) The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte. Dev. Biol., 167:502–512. Downs, S.M. (1995b) Ovulation 2: Control of the resumption of meiotic maturation in mammalian oocytes. In: Gametes— The Oocyte. J.G. Grudzinskas and J.L. Yovich, eds. Cambridge University Press, Cambridge, pp. 150–192. Downs, S.M, D.L. Coleman, P.F. Ward-Bailey, and J.J. Eppig (1985) Hypoxanthine is the principal inhibitor of murine 384 S. DOWNS oocyte maturation in a low molecular weight fraction of porcine follicular fluid. PNAS, 82:454–458. Downs, S.M., D.L. Coleman, and J.J. Eppig (1986) Maintenance of murine oocyte meiotic arrest: uptake and metabolism of hypoxanthine and adenosine by cumulus cell-enclosed and denuded oocytes. Dev. Biol., 117:174–183. Downs, S.M., S.A.J. Daniel, E.A. Bornslaeger, P.C. Hoppe, and J.J. Eppig (1989) Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity. Gamete Res., 23:323–334. Downs, S.M., and J.J. Eppig (1984) Cyclic adenosine monophosphate and ovarian follicular fluid act synergistically to inhibit mouse oocyte maturation. Endocrinology, 114:418–427. Downs, S.M., and J.J. Eppig (1987) Induction of mouse oocyte maturation in vivo by perturbants of purine metabolism. Biol. Reprod., 36:431–437. Downs, S.M., P.G. Humpherson, and H.J. Leese (1998) Meiotic induction in cumulus cell-enclosed oocytes: involvement of the pentose phosphate pathway. Biol. Reprod., 58:1084–1094. Downs, S.M., and A.M. Mastropolo (1994) The participation of energy substrates in the control of meiotic maturation in murine oocytes. Dev. Biol., 162:154–168. Downs, S.M., and A.M. Mastropolo (1997) Culture conditions affect meiotic regulation in cumulus cell-enclosed mouse oocytes. Mol. Reprod. Dev., 46:551–566. Eppig, J.J. (1991) Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles. Biol. Reprod., 45:824–830. Eppig, J.J., P.F. Ward-Bailey, and D.L. Coleman (1985) Hypoxanthine and adenosine in murine ovarian follicular fluid: Concentrations and activity in maintaining oocyte meiotic arrest. Biol. Reprod., 33:1041–1049. Fagbohun, C.F., and S.M. Downs (1992) Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes. J. Reprod. Fert., 96:681–697. Pincus, G., and E.V. Enzmann (1935) The comparative behavior of mammalian eggs in vitro and in vivo. J. Exp. Med., 62:665–675. Tsafriri, A. and N. Dekel (1994) Molecular mechanisms in ovulation. In: Molecular Biology of the Female Reproductive System. J.K. Findlay, ed. Academic Press, San Diego, pp. 207–258.