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a Biology Department, Marquette University, Milwaukee, Wisconsin 53201-1881
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a series of papers detailing the influence of energy substrates on oocyte maturation, we have established that FSH induction of meiotic maturation, but not spontaneous maturation, requires the presence of glucose [4–6]. We have postulated that FSH-stimulated uptake and metabolism of glucose by the cumulus cells contributes to the production of a positive stimulus that triggers germinal vesicle breakdown (GVB) in the oocyte. Although the majority of glucose taken up in response to gonadotropin is metabolized to lactate, the glycolytic pathway does not appear to be vital for meiotic induction [6]. Nor is the oxidation of glucose-derived pyruvate important in mediating the positive meiotic response [7].
A logical alternative route for glucose metabolism is the pentose phosphate pathway. The oxidative arm of this pathway links glucose utilization and purine metabolism via conversion of the end product, ribose-5-phosphate, to phosphoribosylpyrophosphate (PRPP), a compound required for the purine de novo and salvage pathways. This is an important consideration since the de novo purine synthetic pathway is involved in meiotic induction [8]. A recent study provided evidence that the pentose phosphate pathway does, indeed, participate in meiotic induction [9]. For example, in the absence of a hormonal stimulus, activators of this pathway induced GVB when meiotic arrest was maintained with hypoxanthine or isobutylmethylxanthine. In addition, agents that modify the redox state of nicotinamide dinucleotide phosphate (NADP[H]) or inhibit enzymes of the pentose phosphate pathway prevented FSH-induced maturation. Furthermore, meiotic induction was associated with elevated levels of PRPP, an important metabolic bridge between the pentose phosphate and purine biosynthetic pathways.
While the latter study supported the idea that the pentose phosphate pathway is important in mediating meiotic induction, much of the evidence linking these two processes was indirect. Hence, the present study was undertaken to more directly assess the route of glucose metabolism in cumulus-oocyte complexes during hormone-stimulated maturation using [1-14C]-, [6-14C]-, and [5-3H]glucose as substrate. The results support earlier conclusions that increased pentose phosphate pathway activity in the somatic compartment is associated with meiotic induction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C57BL/6J x SJL/J F1 female mice, 20–23 days old, were used for all experiments. Mice were primed with 5 IU eCG and 48 h later were killed; their ovaries were placed in culture medium. Antral follicles were pierced with sterile needles, and the released oocyte-cumulus cell complexes (OCC) were washed free of contaminating ovarian tissue and transferred to the appropriate culture groups. For some experiments, primed mice received an injection of 5 IU hCG, and 2.5 h later OCC were obtained for metabolic assessment. Denuded oocytes (DO) were obtained by repeated pipetting of OCC with a Pasteur pipette.
The culture medium used was Eagle's minimum essential medium (MEM) containing 50 µg/ml streptomycin sulfate, 75 µg/ml penicillin G, and 3 mg/ml lyophilized crystallized BSA (ICN ImmunoBiologicals, Lisle, IL). Bicarbonate was reduced to 5 mM, and the medium was buffered with 25 mM Hepes, pH 7.2. Glucose and pyruvate concentrations were varied according to the experiment.
For the glucose metabolism experiments, 10 DO or six OCC were cultured in a 5-µl hanging drop suspended from the cap of a 1.5-ml microfuge tube. The tube contained 1.45 ml 0.1 N NaOH that served as a trap to collect metabolically released 3H2O (via glycolysis) or 14CO2 (via tricarboxylic acid [TCA] cycle or pentose phosphate pathway) when radiolabeled glucose was included in the culture medium. The metabolic routes for release of these products by the various glucose analogues used is shown in Figure 1. For oocyte maturation experiments, five DO or three OCC were cultured in a 5-µl hanging drop suspended from the cap of a 1.5-ml microfuge tube containing NaOH and then assessed for GVB.
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Oocyte Maturation and Metabolic Measurements
For assessment of oocyte maturation, DO or OCC were transferred at the end of culture to a Petri dish and examined under a stereomicroscope for initiation of meiotic maturation, as manifested by GVB.
An initial experiment established the efficacy of the microfuge culture system in collecting the gaseous byproducts of glucose metabolism. Hanging drops containing [14C]bicarbonate or 3H2O were incubated for 10-min intervals up to 60 min. The amount of label in the trap was then measured and converted to a percentage of the total amount added to the hanging drop.
For metabolic analyses, all cultures contained 0.75 µCi of the appropriate glucose analogue: [5-3H]glucose (12.8 mCi/mmol); [1-14C]glucose (55.0 mCi/mmol); or [6-14C]glucose (56.0 mCi/mmol). Cold glucose was added to bring the final glucose concentration to 4 mM. For each treatment group, blanks were included in which identical tubes were prepared and incubated minus tissue. The amount of substrate metabolized was calculated by subtracting the mean blank radioactivity from the sample radioactivity, dividing this value by the total radioactivity of labeled substrate added, and multiplying by the glucose dilution factor.
Chemicals
MEM amino acids were purchased from Gibco BRL (Grand Island, NY). All other culture medium components were obtained from Sigma Chemical Co. (St. Louis, MO). Radiolabeled glucose analogues were purchased from Amersham Life Science Inc. (Arlington Heights, IL).
Statistics
Oocyte maturation data were subjected to arcsin transformation prior to statistical analysis, while nontransformed metabolism data were analyzed. For comparison of three or more groups, data were examined by ANOVA followed by Duncan's multiple range test. For paired comparisons, Student's t-test was utilized. A p value < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For analysis of spontaneous maturation, oocytes were cultured in inhibitor-free medium for up to 4 h and GVB was assessed at hourly intervals. As shown in Figure 2A, comparable kinetics of maturation were displayed by the two groups of oocytes, with greater than 95% GVB after 4 h.
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We next tested the ability of FSH to stimulate meiotic resumption in hypoxanthine-arrested oocytes. OCC were cultured in medium containing 4 mM hypoxanthine, in the presence or absence of FSH, for up to 18 h. With culture in hypoxanthine alone, meiotic arrest was effectively maintained, with only a small increase in the maturation percentage from 8% to 31% between 3 and 18 h (Fig. 2B). Consistent with previous reports [2, 10, 11], the addition of FSH produced an inhibitory action during the initial 9 h of incubation, followed by stimulation, such that 80% of the oocytes had resumed maturation by 18 h. These results therefore establish that the culture system used for the glucose metabolism experiments supports the expected maturation response.
The success of the metabolic experiments depends on the efficient collection of 14CO2 and 3H2O produced during the culture period. To test the effectiveness of this system, a preliminary experiment was carried out in which [14C]bicarbonate or 3H2O was added to the hanging drop and equilibration with the NaOH trap was monitored for up to 1 h. These results are shown in Figure 3. The radiolabeled molecules equilibrated rapidly with the trap, as 98.3% of 14CO2 and 96.7% of 3H2O were collected within the 1-h incubation period.
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The first metabolic experiments examined the kinetics of glucose metabolism when OCC were cultured in medium containing 4 mM hypoxanthine plus or minus FSH. OCC were cultured for up to 6 h, and samples were collected every 1.5 h. Figure 4 shows the results for glucose labeled at the C-1 and C-6 positions. Very little glucose is metabolized through both glycolysis and the TCA cycle, as labeled CO2 from [6-14C]glucose was very low in hypoxanthine-containing medium (0.5 pmol/complex after 6 h). In addition, FSH had no effect on 14CO2 generation from this compound. On the other hand, considerably more 14CO2 was retrieved from FSH-free cultures that utilized [1-14C]glucose (1.9 pmol/complex after 6 h), and FSH stimulated an increase in the oxidation of this compound (increases to 1.56- to 2.2-fold between 3 and 6 h of incubation). Thus, oxidative metabolism of glucose through the pentose phosphate pathway, but not the tricarboxylic acid cycle, was stimulated by FSH.
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Metabolism of [5-3H]glucose was greater by about two orders of magnitude, with 104 pmol H2O produced per complex after 6 h in FSH-free medium (Fig. 5). Also, FSH stimulated increases to 2.4- to 3.4-fold in glycolytic activity between 3 and 6 h of incubation.
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In the next two experiments, OCC were exposed either to an activator of the pentose phosphate pathway or to a high concentration of pyruvate, and metabolism of the three radiolabeled glucose compounds was measured after 4.5 h. These values are shown in Table 1 (experiments 2 and 3).
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In experiment 2, we employed a stimulator of the pentose phosphate pathway, pyrroline carboxylate, that has been shown to trigger meiotic resumption in hypoxanthine- and isobutylmethylxanthine-arrested mouse oocytes [9]. This agent had no effect on metabolism of [6-14C]glucose but stimulated oxidation of [1-14C]glucose to over 2-fold, thereby indicating stimulation of the pentose phosphate pathway. A modest increase (25%) in [5-3H]glucose metabolism was also observed.
In the third experiment, OCC were cultured in inhibitor-free medium containing 4 mM glucose in the presence or absence of 1 mM pyruvate to determine whether this latter energy substrate would alter glucose utilization. We reasoned that high pyruvate levels might promote meiotic maturation by an increased flux of glucose through the pentose phosphate pathway. However, the relative metabolism of the three glucose compounds in pyruvate-free medium was similar to that observed in the hypoxanthine-containing cultures. In addition, the inclusion of pyruvate resulted in significant reductions in metabolism of all three glucose compounds.
To determine how OCC responded metabolically to a meiosis-inducing stimulus in vivo, eCG-primed mice received an ovulatory injection of hCG, and 2.5 h later OCC were isolated and tested for glucose metabolism during a 4.5-h culture period. We have established that at this time post-hCG, 80–85% of oocytes isolated from antral follicles have resumed meiotic maturation. Results are presented in Table 2. Human CG stimulated both the oxidation of [1-14C]glucose and glycolytic metabolism of [5-3H]>glucose, and by an identical margin (to 2.2-fold). No significant effect on the oxidation of [6-14C]glucose was observed. These data show that the metabolic response of complexes to an in vivo stimulus mimics their response in vitro to FSH and pyrroline carboxylate.
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The last two experiments were carried out to ascertain how DO metabolize glucose and how pyruvate and pyrroline carboxylate affect such metabolism during a 4.5-h culture period. Although little glucose is thought to be consumed directly by the oocyte [12], significant utilization was detected in these experiments, but to a lesser extent than in complexes. In the absence of pyruvate, 2.53 pmol glucose per oocyte was processed through glycolysis (Table 3) compared to 126.6 pmol per complex (Table 1), constituting 2% of the total glycolytic activity in complexes. This value was not affected by the addition of 1 mM pyruvate (2.23 pmol/oocyte). Oocyte oxidation of glucose was much lower, with 0.18 and 0.05 pmol CO2 per oocyte generated from [1-14C]glucose and [6-14C]glucose, respectively (Table 3), representing 8% and 9.6% of glucose oxidized by complexes under identical conditions. Pyruvate did not affect the metabolism of [1-14C]glucose but rendered CO2 production from [6-14C]glucose undetectable.
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In the second DO experiment, in the absence of pyrroline carboxylate, relative levels of [1-14C]glucose and [5-3H]glucose metabolism were identical to those of the first experiment; but the presence of pyruvate (0.23 mM) again eliminated detectable [6-14C]glucose oxidation (Table 3). The ratio of metabolism of [1-14C]glucose and [5-3H]glucose by the oocyte versus the complex (8.6% and 1.9%, respectively) was identical to that in experiment 1 but was reduced to zero in the case of [6-14C]glucose. Upon addition of pyrroline carboxylate, glycolytic metabolism was reduced (from 1.68 to 0.51 pmol/oocyte). Although a small amount of [6-14C]glucose was oxidized (0.06 pmol/oocyte), no significant increase in [1-14C]glucose oxidation was observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Meiotic Induction and the Pentose Phosphate Pathway in OCC
FSH increased the activity of the pentose phosphate pathway in hypoxanthine-treated complexes. Although [1-14C]glucose can produce 14CO2 from either the pentose phosphate pathway or the glycolysis/TCA cycle, the failure of FSH to increase the oxidation of [6-14C]glucose indicates that the effect of this gonadotropin on [1-14C]glucose metabolism is attributable principally to an augmented flux through the pentose phosphate pathway. As demonstrated in previous studies [6, 13, 14], OCC displayed a tremendous capacity for glycolytic metabolism, and stimulation with FSH enhanced the activity of this pathway to 1.8- to 2.7-fold. Assuming that 14CO2 generation from [1-14C]glucose reflects total glucose oxidation, glycolytic activity is approximately two orders of magnitude greater, particularly in FSH-stimulated cultures. Indeed, measurement of pyruvate, glucose, and lactate in spent medium has shown that most of the pyruvate produced by the OCC via glycolysis is further converted to lactate instead of being oxidized through the TCA cycle [6, 7]. Nevertheless, consumption of glucose by this route does not mediate the meiosis-inducing action of FSH, since blocking glycolysis does not affect meiotic resumption [6]. Rather, evidence indicates that the pentose phosphate pathway is a critical metabolic route for glucose in FSH-induced maturation [9]. Although the absolute increase in pentose phosphate pathway activity by OCC in response to FSH is much less than the increase in glycolysis (1 pmol compared to 156 pmol per complex), it still represents a significant augmentation of the metabolic flux through this pathway. These results are thus consistent with a role for the pentose phosphate pathway in meiotic induction.
A variety of agents are known to oxidize NADPH to NADP and thereby stimulate the pentose phosphate pathway [15–20]. In a previous study, NADPH-oxidizing agents were shown to stimulate meiotic resumption in hypoxanthine- or isobutylmethylxanthine-arrested oocytes, but only indirect evidence was obtained for activation of the pentose phosphate pathway [9]. It was therefore important to implement the present culture system to test the effect of one of these agents, pyrroline carboxylate, on glucose metabolism. Similar to the results using FSH, pyrroline carboxylate produced an increase to 2.2-fold in [1-14C]glucose oxidation without any significant change in [6-14C]glucose oxidation, again indicating a preferential effect on the oxidative arm of the pentose phosphate pathway. O'Fallon and Wright [21] also reported increased [1-14C]glucose metabolism by mouse preimplantation embryos in response to another activator of the pentose phosphate pathway, phenazine ethosulfate, with no effect on [6-14C]glucose metabolism. These data further demonstrate a direct relationship between increased pentose phosphate pathway activity and the resumption of meiotic maturation in mouse oocytes. Additionally, if one evaluates the [5-3H]/[1-14C] metabolic ratio (Table 1), it is apparent that a relative shift toward less glycolytic metabolism occurred in response to pyrroline carboxylate whereas an increase in this ratio occurred in response to FSH. Thus, although the two agents affect relative metabolic flux through these two pathways differently, they each augment the activity of the pentose phosphate pathway, coincident with induction of GVB.
It was important to establish whether OCC receiving a meiosis-inducing stimulus in vivo responded in a manner similar to those induced in vitro by FSH or pyrroline carboxylate. When primed mice received hCG to stimulate meiotic resumption in vivo, the OCC showed an increase to 2.2-fold in both [1-14C]- and [5-3H]glucose metabolism compared to unstimulated OCC, with no effect on [6-14C]glucose metabolism. These data are, indeed, consistent with those from FSH- or pyrroline carboxylate-treated OCC and suggest that a similar metabolic response results from meiosis-inducing stimuli whether OCC are treated in vitro or in vivo. Unlike the response of OCC in vitro to FSH, OCC stimulated in vivo with hCG did not exhibit a relative shift toward increased glycolytic activity compared to that of the pentose phosphate pathway, perhaps signifying subtle differences in metabolic control between the in vitro and in vivo meiotic induction protocols.
The means by which increased activity in these two pathways is brought about by hormonal stimulation is not known, but almost certainly results, at least in part, from augmented glucose phosphorylation. A translation-dependent increase in hexokinase activity occurs in mouse complexes in response to gonadotropin [6] that would provide more glucose-6-phosphate as substrate for either pathway. Consistent with this idea is the finding that 2-deoxyglucose, which is not metabolized beyond the initial phosphorylation step, blocked meiotic induction by either FSH [6] or pyrroline carboxylate [9]. It is not clear whether a change in glucose phosphorylation alone is sufficient to account for the increase in glucose consumption or whether specific pathway enzymes are also activated. However, we were unable to demonstrate increases in several critical enzymes of these pathways in OCC in response to FSH [6].
Millimolar levels of pyruvate can stimulate oocyte maturation in the absence of glucose [5], although oxidation via the TCA cycle is apparently not the mechanism [7]. It was therefore of interest to determine whether the addition of 1 mM pyruvate to pyruvate-free medium could alter glucose metabolism and produce a preferential shift toward the pentose phosphate pathway, a consequence consistent with meiotic induction [9]. However, a significant decrease in metabolism was observed with all three glucose analogues. The reason for this response is not known with certainty but probably represents negative feedback of pyruvate on glycolysis and an ultimate reduction in hexokinase activity. Consistent with this scheme is the observation that pyruvate reduces overall glucose consumption by the OCC ([7]; data herein). Thus, it does not appear that high pyruvate levels stimulate oocyte maturation by increasing glucose flux through the pentose phosphate pathway.
Glucose Metabolism by DO
Experiments carried out more than 30 years ago established that the DO has a compromised ability to metabolize glucose in comparison to the cumulus cell-enclosed oocyte [12]. Glucose is poorly utilized by the oocyte as an energy source and must first be converted to pyruvate by the follicle cells to ensure long-term viability of the oocyte and successful meiotic maturation [4, 5, 12, 22–24]. In support of this, oocytes from numerous species exhibit preferential oxidation of pyruvate as compared to glucose, though measurable metabolism of glucose has been observed [25–29]. There are exceptions, as Zuelke and Brackett [14] were unable to obtain evidence for glycolysis in denuded bovine oocytes, and two groups reported an inability of denuded mouse oocytes to incorporate 2-deoxyglucose [30, 31].
In the current study, mouse oocytes demonstrated an ability to metabolize glucose, though at a much reduced rate when compared to activity in the intact OCC. Glycolytic flux was only 2% as high and metabolism of [1-14C]glucose 8% as high, while oxidation of [6-14C]glucose was undetectable or negligible. If the oocyte occupies approximately 16% of the volume of the OCC, as indicated by the oocyte-cumulus cell coupling index [32], these values are below what would be expected if the oocyte is capable of comparable metabolic activity. Our values for glucose oxidation by mouse oocytes are similar, but somewhat lower, than that obtained by Brinster [26] (0.145 pmol/oocyte per hour). The difference may be attributable to the use of [U-14C]glucose by Brinster or to differing media (MEM/BSA here versus Krebs-Ringer bicarbonate/15% calf serum used by Brinster).
Several possibilities may explain the inability of the oocyte (and early embryo) to efficiently utilize glucose, such as a deficiency in glucose transport [33, 34], low or unmeasurable hexokinase activity [6, 25, 31, 35–38], or a block in phosphofructokinase activity [39]. Despite its limited utilization by the DO, glucose can influence meiotic maturation in the absence of cumulus cells, as evidenced by findings that iodoacetate partially reversed the meiosis-arresting action of hypoxanthine on DO [40]. The fact that the baseline [5-3H]/[1-14C] ratio in complexes ranges from 51.4 to 62.2, while that in DO is 12.9–14.1, indicates that relatively less glucose is metabolized through the Embden-Meyerhoff pathway by DO than by complexes and is consistent with previous studies demonstrating active pentose shunt enzymes in oocytes and unfertilized ova [36, 41–43]. Compromised glucose transport or low hexokinase activity is probably not the primary reason for this difference, because either condition would be expected to affect glycolysis and the pentose phosphate pathway equally and have little effect on the ratio. A block in glycolysis, presumably at the phosphofructokinase step [34], would be consistent with the present observations.
Pyrroline carboxylate significantly reduced glycolytic flux of glucose in DO but had no effect on [1-14C]glucose metabolism. The lack of stimulation of the pentose phosphate pathway was unexpected, since we have shown that this agent induced meiotic maturation in hypoxanthine-arrested DO [9]. However, this failure to increase [1-14C]glucose oxidation may be due to the limited ability of the oocyte to take up glucose or to the shorter incubation period (4.5 h) used for the assay in this study compared to the longer 17–18 h period used to test the effects of pyrroline carboxylate on oocyte maturation [9].
It is important to emphasize that the DO is an artifactual entity that never exists during normal folliculogenesis and that the somatic cumulus cells associated with the oocyte constitute the dominant influence on the metabolism of energy substrates by the complex (cf. [44]). Hence, the metabolic behavior of the isolated oocyte may not be physiologically relevant. Work in progress has revealed that while the DO oxidizes exogenous pyruvate at over twice the rate at which it consumes glucose, the OCC metabolizes glucose at a rate nearly 60-fold greater than its oxidation of exogenous pyruvate. These observations suggest that, although the oocyte has the ability to metabolize glucose, it may actually process negligible amounts of the hexose when coupled to follicle cells due to the greater efficiency of the somatic tissue in carrying out this function. Metabolic intermediates or end products could then be transferred directly to the oocyte through the gap junctional pathway. Indeed, Saito et al. [31] have presented evidence that 2-deoxyglucose-6-phosphate produced in the cumulus cells can accumulate in the oocyte.
In summary, glucose metabolism has been demonstrated in DO and OCC under a variety of conditions. We have confirmed a high capacity of the complex for glycolytic metabolism and activation of this pathway by FSH. In addition, conditions that trigger meiotic maturation also bring about increased flux through the pentose phosphate pathway, supporting the proposal that these two phenomena are causally related. Although the oocyte can take up and metabolize glucose, this is primarily the role of follicle cells that subsequently transmit metabolites and/or other regulatory molecules to the oocyte and thereby influence its metabolic and meiotic status.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Stephen M. Downs, Biology Department, Marquette University, Wehr Life Sciences Building, PO Box 1881, Milwaukee, WI 53201-1881. FAX: 414 288 7357; downss{at}vms.csd.mu.edu
Accepted: January 19, 1999.
Received: October 2, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-pyrroline-5-carboxylic acid in human fibroblasts. Biochem Biophys Res Commun 1979; 87:363–370.[CrossRef][Medline]This article has been cited by other articles:
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R. Roberts, J. Stark, A. Iatropoulou, D. L. Becker, S. Franks, and K. Hardy Energy Substrate Metabolism of Mouse Cumulus-Oocyte Complexes: Response to Follicle-Stimulating Hormone Is Mediated by the Phosphatidylinositol 3-Kinase Pathway and Is Associated with Oocyte Maturation Biol Reprod, July 1, 2004; 71(1): 199 - 209. [Abstract] [Full Text] [PDF]
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S. A. Colton, P. G. Humpherson, H. J. Leese, and S. M. Downs Physiological Changes in Oocyte-Cumulus Cell Complexes from Diabetic Mice that Potentially Influence Meiotic Regulation Biol Reprod, September 1, 2003; 69(3): 761 - 770. [Abstract] [Full Text] [PDF]
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S. A. Colton, G. M. Pieper, and S. M. Downs Altered Meiotic Regulation in Oocytes from Diabetic Mice Biol Reprod, July 1, 2002; 67(1): 220 - 231. [Abstract] [Full Text] [PDF]
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Y. G. Chung, M. R.W. Mann, M. S. Bartolomei, and K. E. Latham Nuclear-Cytoplasmic "Tug of War" During Cloning: Effects of Somatic Cell Nuclei on Culture Medium Preferences of Preimplantation Cloned Mouse Embryos Biol Reprod, April 1, 2002; 66(4): 1178 - 1184. [Abstract] [Full Text] [PDF]
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