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Gamete Biology |
Biology Department,3 Marquette University, Milwaukee, Wisconsin 53202
Department of Biology,4 University of York, Y01 5YW, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, gametogenesis, meiosis, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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300 mg glucose/dl blood before eCG priming), the kinetics of spontaneous oocyte maturation were accelerated when compared to controls, and the response of cumulus cell-enclosed oocytes (CEO) to gonadotropin stimulation was suppressed both in vitro and in vivo [13]. That the effects of streptozotocin were caused by the loss of insulin and/or subsequent hyperglycemia was shown by the results of exogenous insulin treatment, in which blood glucose levels were lowered to physiological levels and normal meiotic regulation and ovulation were restored [13]. Previous work has demonstrated that altering the concentrations of energy substrates such as glucose and pyruvate can profoundly influence the process of meiotic resumption [14, 15]. Glucose is required for hormone-induced maturation [16], but under the appropriate culture conditions, elevated levels of glucose have been shown to suppress maturation [14, 17, 18]. Although cumulus cells are very active glycolytically [19–21], this pathway does not appear to be vital for meiotic induction [22]; however, it may play a role in spontaneous maturation [18]. Additionally, the oxidation of glycolytically derived pyruvate is not important in mediating the positive signal in meiotic induction [23, 24].
Alternatively, glucose can be metabolized through the pentose phosphate pathway (PPP) that produces ribose-5-phosphate, which is converted to phosphoribosylpyrophosphate (PRPP), a necessary precursor for de novo purine synthesis (Fig. 1). Metabolic studies have implicated both the PPP and the de novo purine synthetic pathway in hormone-induced meiotic maturation [25–27]. Because hormone-induced maturation is compromised in diabetic animals, it is possible that these metabolic pathways are also adversely affected. De novo purine synthesis leads to the production of GTP and ATP, which are involved in the action of adenylate cyclase and, hence, cAMP production (Fig. 1). This cyclic nucleotide mediates the action of gonadotropins on granulosa cells, and a lesion in this transduction system might explain the reduced meiotic response to FSH in CEO from diabetic mice. Thus, changes in glucose metabolism may be linked to cAMP generation in the cumulus cell compartment in response to gonadotropin stimulation, and aberrations at any point in this pathway could be instrumental in restricting the meiotic response in diabetic animals.
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Suppression of hormone-induced maturation is not the only meiotic lesion manifested in diabetic mice. We also observed a reduction in the transient meiosis-arresting action of FSH in CEO and an acceleration of spontaneous maturation kinetics [13]. Taken together, such effects appear to be contradictory, but if one considers that cumulus cells provide both inhibitory and stimulatory signals to the oocyte [17], then the above changes in meiosis are consistent with a loss of cell-cell communication between germ cell and somatic compartments. Thus, depending on the culture conditions, a loss in metabolic coupling could decrease the transfer of inhibitory or stimulatory signals and, thereby, either promote or suppress meiotic progression, respectively.
As shown in Figure 1, we propose that glucose is involved in a series of metabolic pathways that collectively contribute to hormonal induction of meiotic maturation. Altered function at any point could result in a compromised ability to respond to gonadotropin stimulation with germinal vesicle breakdown (GVB). The present study was undertaken to investigate how type I diabetes affects these physiological parameters in oocyte-cumulus cell complexes (OCC), with special emphasis on those processes thought to be involved in meiotic regulation. We demonstrate decreased intercellular communication and defects in glucose, purine, and cAMP metabolism in complexes from diabetic mice, and we propose that each of these conditions could contribute to the altered meiotic behavior.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature C57BL/6J x SJL/J F1 mice (age, 20–24 days) were used for all experiments. Diabetic and age-matched control mice received 5 IU of eCG by i.p. injection, and 48 h later, the ovaries were removed and placed in a dish containing 2.5 ml of culture medium. Oocytes were isolated by puncturing the antral follicles with sterile needles, then washed through several changes of medium and transferred to the appropriate test medium. For maturation experiments, incubation was carried out in 1 ml of medium in plastic culture tubes that were gassed with a humidified mixture of 5% O2, 5% CO2, and 90% N2 and then placed in a 37°C water bath for the culture period. The culture medium used for all experiments, except when otherwise noted, was Eagle minimum essential medium (MEM) supplemented with 0.23 mM pyruvate, 3 mg/ml of BSA (ICN Immunobiologicals, Lisle, IL), 50 µg/ml of streptomycin sulfate, and 75 µg/ml of penicillin. For assessment of maturation, cumulus cells were removed and the oocytes examined under a stereomicroscope for GVB, an indicator of meiotic resumption.
Generation of Diabetic Mice
To establish type I diabetes, female mice (age, 20–23 days; weight, 11 g) received a single injection of streptozotocin (dissolved in sodium citrate buffer; Sigma, St. Louis, MO), which destroys the ß-cells in the pancreas and blocks production of insulin. Control mice received an equal volume of the buffer. Three days postinjection, a tail-blood sample was measured for glucose concentrations via a commercial glucometer, and mice with glucose levels of 300 mg/dl or greater were selected as diabetic and received a priming injection of eCG.
Measurement of Glucose Metabolism
For the glucose metabolism experiments, six OCC were cultured in a 5-µl drop of Hepes-buffered MEM suspended from the cap of a 1.5-ml microfuge tube for 3 h in a 37°C incubator. The tube contained 1.47 ml of 0.1 N NaOH that served as a trap to collect metabolically released 3H2O (via glycolysis) or 14CO2 (via the tricarboxylic acid cycle [TCA] or PPP) when radiolabeled glucose was added to the culture medium [27]. For each experiment, 0.75 µCi of one of the following analogs was used: [5-3H]glucose (12.8 mCi/mmol), [1-14C]glucose (56 mCi/mmol), or [6-14C]glucose (56 mCi/mmol; Amersham, Piscataway, NJ). Unlabeled glucose was added to bring the final glucose concentration to 4 mM. For each treatment group, blanks were assayed in which identical tubes were prepared without a tissue sample. At the end of the culture period, the NaOH trap was transferred to a scintillation vial containing scintillation fluid, and radioactivity was determined by scintillation spectroscopy. The amount of substrate metabolized was determined by subtracting the mean blank radioactivity from the sample radioactivity, dividing this value by the total radioactivity of the labeled substrate added, and then multiplying by the glucose dilution factor [27].
Measurement of Pyruvate Oxidation
For each metabolic measurement, six OCC or 10 denuded oocytes were cultured in a 5-µl hanging drop suspended from the cap of a 1.5-ml microfuge tube as described above. Cultures contained 0.044 µCi [2-14C]pyruvate (17.5 mCi/mmol; New England Nuclear, Boston, MA), and the final pyruvate concentration was 0.5 mM. For each group, blanks were included in which identical tubes were prepared and incubated without a tissue sample. The amount of substrate metabolized was calculated in a fashion similar to that described for glucose metabolism [27].
Energy Substrate Assay
For the determination of energy substrate consumption or production, 10 OCC were cultured in 8-µl microdrops of MEM under oil for 18 h. At the conclusion of culture, 6 µl of medium were removed and placed in a Cobas Mira assay tube (Roche Products Ltd., Welwyn Garden City, UK). The oocytes were scored for maturation. Each medium sample was diluted with 144 µl of water and placed in a freezer at -80°C until assayed. Medium samples were assayed for pyruvate, glucose, and lactate concentrations using a Cobas Mira automated analyzer as previously described [28]. These substrate experiments were performed four times, with three to four measurements per treatment group per experiment.
De Novo Purine Synthesis
Freshly isolated OCC from control and diabetic mice were cultured for 1 h in 1 ml of MEM/BSA containing 10 µCi [14C]glycine (112.7 mCi/mmol; New England Nuclear) with or without FSH and then assayed for de novo purine synthesis as previously described [29]. Seventy-five complexes were used for each determination.
ATP Assay
The ATP levels were determined for freshly isolated OCC. For each group, 25 OCC were assayed as previously described [14]. Luminescence was measured using a Model 6900 luminometer (Optical Technology Devices, Inc., Elmsford, NY). A standard curve was prepared from a set of ATP standards that had been extracted in a fashion identical to that of the tissue samples for each experiment.
cAMP Assay
The cAMP levels were determined using an indirect cAMP immunoassay kit (Sigma). The OCC were freshly isolated or cultured for 3 h in the absence or presence of FSH. Twenty-five OCC were assayed for the freshly isolated groups and the non-FSH treated groups; five OCC were assessed for the FSH-treated groups. Measurements of cAMP in the complexes were determined via a microtiter plate reader, comparing those values to a cAMP standard curve.
Oocyte-Cumulus Cell Coupling
The OCC from control or diabetic mice were incubated in MEM/BSA containing 10 µCi [3H]hypoxanthine (24 Ci/mmol; Amersham) or [3H]uridine (32 Ci/mmol; Amersham) for 3 h at 37°C. At the end of the culture period, the complexes were divided into two groups. One group was denuded of the cumulus cells by repeated pipetting, and the other group was left intact for determination of the total incorporation of the radiolabel by the complex. Both groups were then washed through three changes of Dulbecco PBS (Sigma) supplemented with 3 mg/ml of BSA to remove excess radiolabel. Fifteen OCC or denuded oocytes were then transferred to separate scintillation minivials; an equal amount of buffer from the last wash dish served as a blank. The oocytes were solubilized with 1 N NaOH and neutralized with 1 N HCl; radioactivity was determined by liquid scintillation spectroscopy. The extent of oocyte-cumulus cell coupling was measured as the percentage of radiolabel found in the oocytes relative to the amount of label in the intact complexes.
Uptake of Metabolites
To measure uptake of [3H]hypoxanthine and [3H]uridine by the oocyte, CEO were isolated from control and diabetic mice. The complexes were then denuded by mouth pipetting, and the oocytes were cultured in 1 ml of MEM/BSA containing 10 µCi [3H]hypoxanthine or [3H]uridine for 3 h at 37°C. At the end of culture, the oocytes were washed through three changes of PBS/BSA; 45 oocytes were collected and placed into a scintillation vial. Blanks were obtained for each group from the last wash dish. The oocytes were solubilized with 1 N NaOH and then neutralized with 1 N HCl. Each group was then analyzed by liquid scintillation spectroscopy. Uptake was determined by dividing the amount of radiolabel present by the number of oocytes in the group.
Hypoxanthine Phosphoribosyl Transferase Assay
Denuded oocytes were obtained from control or diabetic mice. Oocytes were washed four times with PBS containing polyvinylpyrrolidone and then frozen at -80°C in a small volume of the same solution (5 denuded oocytes/ml) until assayed as previously described [30].
Statistical Analysis
Each oocyte maturation experiment was conducted at least three times, with 23–56 oocytes per treatment group. Data from oocyte maturation experiments are reported as the mean percentage GVB ± SEM. Maturation frequencies were subjected to arcsine transformation and analyzed statistically by ANOVA followed by the Duncan multiple-range test. Nontransformed data from the glucose metabolism, pyruvate oxidation, energy substrate, de novo purine synthesis, hypoxanthine phosphoribosyl transferase (HPRT), ATP, and cAMP assays are presented as the mean ± SEM and were analyzed by the same statistical tests. A P value of less than 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Earlier work from our lab indicated that glucose is required for hormone-induced meiotic maturation [16] and that increased transit of glucose through the PPP is involved in the meiotic induction process [27, 31]. Because FSH-induced maturation was compromised in oocytes isolated from diabetic mice [13], it was important to determine how the diabetic condition affects glucose metabolism. Toward this end, a hanging-drop culture system and differentially radiolabeled glucose were utilized. [5-3H]Glucose was used to test for glycolytic activity. In the absence of FSH, complexes from control mice metabolized 124.3 pmol/complex through the glycolytic pathway. In complexes from control mice, FSH stimulated glycolysis by 1.9-fold and 2.5-fold in the absence and presence, respectively, of hypoxanthine (Fig. 2A). [5-3H]Glucose metabolism in complexes from diabetic mice was no different from that of controls in any of the treatment groups.
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To determine whether activity through the PPP is altered because of the diabetic condition, [1-14C]glucose and [6-14C]glucose were used. In control complexes, when [1-14C]glucose was used as a substrate, FSH had no effect in the absence of hypoxanthine, but the presence of the purine base stimulated CO2 generation from 2.0 to 5.7 pmol/complex (Fig. 2B). Complexes from diabetic mice exhibited the same pattern, but the increased metabolism in response to FSH in hypoxanthine-supplemented medium was significantly less than that observed in controls. The FSH did not stimulate the release of 14CO2 when complexes from control mice were cultured with [6-14C]glucose, whether in the absence or the presence of hypoxanthine (Fig. 2C). These results show that the TCA cycle was not stimulated by FSH. Again, complexes from diabetic mice did not deviate from this metabolic pattern. Because FSH had no effect on [6-14C]glucose metabolism, any changes in the metabolism of [1-14C]glucose can be attributed to changes in the activity of the PPP. Thus, the results indicate that FSH stimulates the PPP in hypoxanthine-treated complexes, although less effectively in those from diabetic mice.
To assess the effects of diabetes on pyruvate metabolism, oxidation of pyruvate by both the OCC and the denuded oocyte was assayed using the hanging-drop method. Consistent with a recent study [24], denuded oocytes from both control and diabetic mice oxidized significantly more pyruvate (more than 3-fold more) than intact complexes. Moreover, no effect of diabetes or FSH was observed on pyruvate oxidation by complexes in either group (data not presented). These results indicate that no significant difference is found in pyruvate metabolism in either the intact complex or the denuded oocyte from diabetic mice when compared to controls.
To further examine the relationship between diabetes and energy substrate dynamics in OCC, glucose consumption and pyruvate and lactate production were measured following 18-h cultures in microdrops. The OCC were cultured in 8-µl drops of control medium (normal substrate composition, 0.23 mM pyruvate and 5.5 mM glucose) under oil that was supplemented with 300 µM dbcAMP with or without FSH. The pattern of oocyte maturation was similar to that observed in oocytes cultured in 1 ml of medium in tubes: FSH effectively stimulated maturation in control oocytes (a 72% increase in GVB) but was less effective in oocytes from diabetic mice (a 38% increase; data not presented). Despite suppressing meiotic induction, the diabetic condition had no significant impact on these energy substrate dynamics (Fig. 3). The OCC consumed approximately 1 nmol of glucose in the absence of FSH, and this value doubled on addition of FSH. Consumption of glucose was associated with an accumulation of lactate and pyruvate in the medium, and in both treatment groups, a significant increase in lactate production was also stimulated by FSH.
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De Novo Purine Synthesis
The glucose metabolism assay demonstrated that significantly less glucose was metabolized through the PPP in OCC from diabetic mice compared to controls. Because the end product of the PPP, ribose-5-phosphate, is converted to PRPP, which is the starting substrate for de novo purine synthesis, it was important to determine whether this latter pathway was also compromised in OCC from diabetic mice. We therefore measured purine nucleotide synthesis in OCC from control and diabetic mice in the absence and presence of FSH.
When OCC from control mice were treated with FSH, a 2-fold increase was observed in purine nucleotide production (Fig. 4). In the absence of FSH, OCC from diabetic mice exhibited a significantly higher amount of de novo purine synthesis when compared to OCC from control mice. These data suggest that the additional glucose caused by the diabetic condition may result in an increase in de novo purine synthesis. Nevertheless, although FSH stimulated an increase in de novo purine synthesis by complexes from diabetic mice, it was significantly less that that seen in FSH-treated OCC from control mice (Fig. 4).
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Influence of Diabetes on Epidermal Growth Factor and Phorbol Ester-Induced Maturation
Diabetes suppresses both hCG-induced maturation in vivo and FSH-induced maturation in vitro [13]. To determine if this compromised response is unique for gonadotropins, induction of GVB in arrested CEO by epidermal growth factor (EGF) or the phorbol ester, phorbol 12-myristate 12-acetate (PMA), was tested. In each of these experiments, an FSH-stimulated group was included as a positive control. When CEO from control mice were cultured 17–18 h in 300 µM dbcAMP alone, 18% of oocytes underwent maturation, and this increased to 95% and 97% on addition of FSH and EGF, respectively (Fig. 5A). Whereas the diabetic condition suppressed FSH-induced oocyte maturation by 23%, no effect of diabetes was observed on EGF-induced meiotic maturation.
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To test the effects of PMA on oocyte maturation, CEO from control and diabetic mice were cultured 17–18 h in 50 µM isobutylmethylxanthine (IBMX) in the absence or presence of FSH or PMA. The IBMX was chosen to maintain meiotic arrest because a previous study showed limited stimulation of GVB by PMA in dbcAMP-arrested CEO [32]. In control CEO, 10% maturation was observed in the absence of ligand, which increased to 75% and 87% on addition of FSH and PMA, respectively (Fig. 5B). Very little stimulation of maturation occurred in response to FSH in CEO from diabetic mice (a 17% increase), but PMA was able to stimulate a 53% increase in maturation. Although meiotic maturation in PMA-treated CEO from diabetic mice was significantly less than that in PMA-treated control CEO, the decrease in meiosis-inducing capability was considerably less than that observed in FSH-treated CEO.
These results demonstrate that when compared to meiotic resumption induced by FSH, maturation triggered by EGF or PMA is less sensitive to the diabetic condition. Taken together, the data from these experiments suggest that the inability of CEO from diabetic mice to respond to stimulatory ligands is not a generalized response but, rather, is the result of a specific lesion in the FSH-induction pathway.
cAMP and ATP Levels and the Meiotic Response to cAMP Pulsing
Both the short-term meiotic arrest and longer-term meiotic induction that occur in response to FSH treatment are compromised in CEO from diabetic mice [13]. Because the principle mediator of gonadotropin action on granulosa cells is thought to be cAMP, it was important to examine whether FSH-stimulated cAMP synthesis was compromised in complexes from diabetic mice. When levels of cAMP were compared in freshly isolated complexes, no difference was observed between control and diabetic mice (1 fmol/complex) (Table 1). After 3 h of culture in 4 mM hypoxanthine, the level of cAMP was unchanged in both groups. However, whereas OCC from control mice cultured for 3 h in the presence of FSH demonstrated an 11-fold increase in cAMP concentration, FSH stimulated only a 4.5-fold increase in OCC from diabetic mice. No difference was observed in the size of complexes (reflecting the number of cumulus cells) between control and diabetic mice that could account for this difference. These data therefore show that OCC from diabetic mice have a reduced ability to synthesize cAMP in response to FSH stimulation.
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Because ATP serves as a substrate for adenylate cyclase-catalyzed cAMP production, one possible explanation for the lower level of cAMP in FSH-treated OCC from diabetic mice was a lower level of ATP in OCC at the time of FSH stimulation. To test this idea, freshly isolated complexes from eCG-primed control and diabetic mice were assayed for ATP. As shown in Table 1, the ATP level in complexes from diabetic mice was only 75% of that in complexes from control mice.
Although OCC from diabetic mice have a reduced ability to synthesize cAMP in response to FSH treatment, it was important to determine whether their responsiveness to elevated cAMP was also altered. Previous work has shown that pulsing CEO for 3 h with high levels of cAMP can induce maturation in meiotically arrested oocytes [33, 34]. We therefore compared the effect of cAMP pulsing in control and diabetic CEO groups. The CEO from control and diabetic mice were cultured for 3 h in medium containing 300 µM dbcAMP in the absence or presence of either 8-bromo-cAMP (8-Br-cAMP) or 8-thiomethyl-cAMP (8-SMe-cAMP) at a concentration of 1 mM. Oocytes were then washed in inhibitor-free medium and returned to dbcAMP-supplemented medium for 17–18 h and assessed for maturation. When oocytes from control mice were exposed to dbcAMP alone, 20% of oocytes resumed maturation (Fig. 6). Following exposure to 8-SMe-cAMP or 8-Br-cAMP, the maturation frequency increased to approximately 90% (an increase of 70%). When oocytes from diabetic mice were cultured in dbcAMP alone, 15% of the oocytes resumed maturation, and treatment with 8-SMe-cAMP or 8-Br-cAMP was less effective, producing an increase in maturation of only 43%–44%. These data indicate that not only do the complexes from diabetic mice have a reduced ability to synthesize cAMP following FSH treatment, but their response to a transient cAMP increase is also compromised.
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Oocyte-Cumulus Cell Coupling
We have shown that CEO from diabetic mice exhibit both accelerated spontaneous maturation kinetics and restricted hormone-induced maturation in vitro [13]. Because the metabolic coupling pathway between the oocyte and cumulus cells provides an avenue through which both inhibitory and stimulatory signals may pass to the oocyte, a loss in communication between these two cells types might explain, at least in part, these alterations in meiosis. We therefore compared the metabolic coupling in freshly isolated OCC from control and diabetic mice. When [3H]hypoxanthine was used as the labeling agent, the coupling percentage in OCC from both control and diabetic mice was identical (13%) (Fig. 7). However, when the experiment was repeated using [3H]uridine, the coupling percentage in OCC from diabetic mice was significantly lower than that in control OCC (8% vs. 12%) (Fig. 7).
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Because determination of the coupling percentage assumes negligible uptake by the oocyte of the labeling agent, it was possible that the diabetic condition altered oocyte permeability to these agents. To determine if direct uptake of radiolabel by oocytes was affected by the diabetic state, denuded oocytes from control and diabetic mice were cultured for 3 h in medium containing [3H]hypoxanthine or [3H]uridine. The results are presented in Figure 8. Only a negligible amount of [3H]uridine was taken up by oocytes from control and diabetic mice, and no significant difference was observed between the two groups. Uptake of hypoxanthine by control oocytes was also negligible; however, oocytes from diabetic mice took up 3-fold more [3H]hypoxanthine than oocytes from control mice. Such an increase in permeability to hypoxanthine in oocytes from diabetic mice would produce an artificially high coupling percentage and may explain, at least in part, why no difference in coupling was detected between the two groups when hypoxanthine was used as a radiolabeled marker.
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To further examine this, the average radioactive counts taken up by denuded oocytes were subtracted from the average counts per minute for CEO from both control and diabetic mice to control for the direct uptake of [3H]hypoxanthine by oocytes, and the coupling percentage was again calculated. Under these conditions, the control percentage was reduced only 0.1%, from 13.4% to 13.3%, whereas the coupling percentage was reduced from 13.6% to 13% in complexes from diabetic mice, a 0.6% decrease. Although not as dramatic a reduction in the coupling percentage as observed using [3H]uridine, it should be emphasized that it is impossible to know whether direct uptake of hypoxanthine by the oocyte is the same in the denuded state as it is in the cumulus cell-enclosed state. The results of such normalization suggest that differences in oolemma permeability could mask alterations in gap junctional patency, but they also indicate that other factors may contribute to the altered coupling.
One explanation for the reduced uptake of hypoxanthine by oocytes may be an increase in the activity of the salvage enzyme, HPRT, because this enzyme has been shown to regulate hypoxanthine uptake in somatic cells [35–38]. However, assay of HPRT in oocyte extracts showed no difference in activity between the two groups (data not presented).
Simple manipulation of energy substrates has a profound influence on meiotic regulation. For example, eliminating glucose and raising the pyruvate concentration to 1 mM negates the inhibitory action of hypoxanthine and dbcAMP on oocyte maturation, but the addition of a small amount of glucose dramatically lowers the maturation percentage [15, 16]. Evidence suggests that the inhibitory action of glucose under these circumstances is caused by glycolytic production of ATP in the cumulus cells that is shunted to the oocyte through the gap junctional pathway [14, 17]. Therefore, if somatic-germ cell coupling is compromised in OCC from diabetic mice, the response to glucose should be blunted. To test this, CEO from control and diabetic mice were cultured in MEM/BSA supplemented with 4 mM hypoxanthine and 1 mM pyruvate in the absence or presence of 5.5 mM glucose. Without glucose, 95% of the oocytes from control mice underwent maturation, and addition of glucose reduced the number of maturing oocytes by 56% (Fig. 9). When CEO from diabetic mice were cultured under the same conditions, a comparable number of oocytes underwent maturation in the absence of glucose (94%), but glucose reduced the maturation frequency by only 36%. This was a significantly weaker inhibitory response, consistent with poor communication between the two cell types.
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When oocytes are cultured in the presence of hypoxanthine, a higher percentage of maturation is usually observed in denuded oocytes than in the CEO. This is likely caused by the more efficient uptake of hypoxanthine by the cumulus cells and transfer of the purine base or its metabolites to the oocyte through the coupling pathway [39]. We therefore carried out a hypoxanthine dose-response experiment with denuded and CEO to determine whether differences in this differential meiotic arrest existed between control and diabetic mice. If so, we expected the difference to be less pronounced in oocytes from diabetic mice. When oocytes were cultured in increasing concentrations of hypoxanthine, a dose-dependent decrease in maturation was observed in oocytes from both control and diabetic mice (Fig. 10). In control oocytes, significantly fewer CEO than denuded oocytes underwent maturation at 2 and 4 mM hypoxanthine; however, the diabetic condition compressed this difference such that no significant differences in maturation between CEO and denuded oocytes were observed in vitro at any of the hypoxanthine concentrations (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have shown that glucose is required for hormone-induced maturation [16] and that the PPP is the metabolic route for glucose utilization in meiotic induction [27]. Because we observed a lesion in FSH-induced meiotic maturation in CEO from diabetic mice, it was important to assess to what extent FSH is able to stimulate the PPP under diabetic conditions. Indeed, in the presence of hypoxanthine plus FSH, significantly less PPP activity is found in complexes from diabetic mice when compared to those from controls. No defects in glycolysis were detected, which is consistent with previous findings showing that this pathway is not required for meiotic induction [22, 24, 39]. Similarly, pyruvate oxidation was not affected by the diabetic condition. No differences in the pattern of net energy substrate consumption or production were detected in the conditioned medium from microdrop cultures. This was not surprising, however, because most of the glucose consumed by complexes is metabolized through glycolysis.
Because the PPP leads into de novo purine synthesis via the production of ribose-5-phosphate and its conversion to PRPP, it was important to assess de novo purine synthetic activity. The production of purine nucleotides was significantly reduced in FSH-stimulated OCC from diabetic mice when compared to controls, thus indicating that the de novo purine synthetic pathway is also adversely affected by the diabetic condition. Because inhibiting the purine synthesis pathway suppresses meiotic induction [25], the lack of flux through this metabolic pathway may contribute to the reduced meiotic induction observed in diabetic mice.
Meiotic induction can be brought about by a number of different stimulatory ligands. To determine whether the diabetic condition generally affects all meiotic induction pathways, we compared the effects of diabetes on FSH-, EGF-, and PMA-stimulated maturation. FSH acts in granulosa cells by stimulating the conversion of ATP to cAMP through G protein-coupled adenylate cyclase. EGF and PMA act through tyrosine kinase and protein kinase C, respectively, and both are potent inducers of meiotic resumption [32, 33]. In CEO from diabetic mice, no reduction in EGF-induced maturation was observed, and although fewer oocytes underwent maturation in response to PMA, the decrease in meiotic maturation was much less profound than that observed in FSH-treated oocytes. These results demonstrate greater sensitivity of the FSH transduction system to diabetes, and they suggest that the deficit in hormone responsiveness is specific for gonadotropins. It is possible that the slight reduction in meiotic induction in PMA-treated CEO from diabetic mice is caused by cross-talk between the protein kinase C and protein kinase A transduction systems.
FSH produces short-term meiotic arrest and longer-term meiotic induction in isolated CEO, and each of these is mediated by the cumulus cells. In CEO from diabetic mice, both meiotic responses are suppressed. One possible explanation for the lack of response is reduced production of cAMP. Levels of cAMP rise in the mouse OCC in vivo in response to a meiosis-inducing injection of human chorionic gonadotropin and stay elevated while oocytes are resuming maturation [40]. A sustained rise in somatic cell cAMP mediates the meiosis-inducing action of gonadotropins and likely requires the sequential metabolism of glucose through the PPP and de novo purine synthetic pathways (Fig. 1). We propose that deficits in these pathways are instrumental in limiting gonadotropin-stimulated cAMP production, which leads to the subsequent alterations in meiotic maturation. Indeed, a significant reduction in FSH-stimulated cAMP production was manifested by OCC from diabetic mice.
Levels of ATP in freshly isolated OCC from diabetic mice were also significantly lower, which could contribute to the reduced cAMP production and compromised meiotic response to FSH treatment. Additionally, the lowered ATP levels could explain the accelerated spontaneous maturation kinetics reported in our earlier study [13]. Spontaneous maturation is thought to result from a loss of inhibitory input, and we have presented evidence that elevated levels of ATP contribute to the maintenance of meiotic arrest [14, 17]. Moreover, a significant decrease in oocyte ATP levels accompanies spontaneous maturation in vitro [17]. If the freshly isolated OCC from diabetic mice have less inhibitory potential because of a reduced ATP content, then the oocyte may resume meiosis at an accelerated rate when compared to controls.
Although we demonstrated a reduction in cAMP, it was also important to assess the responsiveness of the oocyte to this cyclic nucleotide. Previous work has shown that meiotic maturation can be induced through pulsing the CEO with cAMP analogs that activate type II protein kinase A, such as 8-Br-cAMP and 8-SMe-cAMP [34]. When dbcAMP-arrested oocytes were pulsed with either cAMP analog, meiotic maturation was greatly stimulated; however, the response of CEO from diabetic mice to the same pulsing conditions was reduced by approximately 30%. It was therefore apparent that not only does the diabetic condition compromise cAMP production in response to hormone stimulation, it also limits the responsiveness of the oocyte to increases in cAMP.
Oocyte maturation data [13] suggest that the diabetic condition affects metabolic coupling between the oocyte and the cumulus cells. In the present study, a significant reduction in metabolic coupling was demonstrated in complexes from diabetic mice. Such a condition might affect the transfer of inhibitory factors from the cumulus cells to the oocyte, resulting in an accelerated rate of spontaneous maturation or a reduced FSH-mediated transient arrest as previously reported [13]. The reduced inhibitory effect of glucose on CEO treated with hypoxanthine and 1 mM pyruvate is consistent with this idea. On the other hand, a compromised pathway would also prevent stimulatory agents from passing from the cumulus cells into the oocyte, thus suppressing meiotic induction.
Oocytes from diabetic mice were able to take up significantly more hypoxanthine from the medium than control oocytes, whereas the level of HPRT activity was not different between the two groups. These results indicate that oolemma permeability may be altered. Elevated levels of glucose have been shown to cause an imbalance in the osmolarity of cells, which may cause damage to tissues [41]. A breach of oolemma selectivity could then lead to increased uptake by the oocyte of certain molecules such as hypoxanthine and might explain the increased accumulation of hypoxanthine in oocytes from diabetic mice. It also appears to interfere with the coupling assay when [3H]hypoxanthine is used as the radiolabeled marker and to eliminate the differential inhibition of maturation in denuded and CEO by hypoxanthine.
One inconsistency in the present study is the lack of effect of diabetes on EGF-induced meiotic maturation. If oocyte-cumulus cell coupling is compromised in diabetic mice, and if the coupling pathway is important for meiotic induction, then all meiosis-inducing ligands would be expected to be negatively affected. Indeed, we have shown in CEO from nondiabetic mice that EGF-induced maturation depends on a patent gap junctional pathway (unpublished data). The failure of the diabetic condition to alter EGF-induced maturation is difficult to reconcile, but two possibilities warrant discussion. First, lesions in the coupling pathway may not play as prominent a role in the changes to meiotic induction as do lesions in metabolism. Second, the metabolic lesions may not affect EGF-stimulated complexes as profoundly as they do FSH-stimulated complexes. Because EGF and FSH act by different transduction systems, downstream production of a positive meiotic signal may be differentially affected. A more robust signal may be generated in EGF-treated complexes that is not affected significantly by the small decrease in oocyte-cumulus cell coupling. More experimentation would be required to address these possibilities.
In summary, we have proposed that a series of metabolic pathways, initially involving glucose and culminating in a sustained rise in cAMP levels (Fig. 1), participate in the meiotic induction process. We have shown in the present study that the diabetic condition produces a myriad of physiological changes along this metabolic route that are associated with aberrant regulation of oocyte maturation. Metabolically, the OCC has a reduced flux of glucose through the PPP, which likely contributes to the reduced de novo purine synthesis and hormone-stimulated cAMP production. Results also indicate that altered responsiveness to cAMP as well as changes in metabolic coupling and oolemma permeability may adversely affect meiotic regulation. These data therefore reflect a pleiotropic response to diabetes in which an additive effect of these different defects may lead to miscues in meiotic regulation.
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FOOTNOTES |
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2 Correspondence: Stephen M. Downs, Biology Department, Marquette University, P.O. Box 1881, Milwaukee, WI 53202-1881. stephen.downs{at}marquette.edu
Received: 20 November 2002.
First decision: 6 December 2002.
Accepted: 15 April 2003.
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