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a Clinic of Sterility, Department of Obstetrics and Gynecology, University Hospital of Geneva, 1211 Geneva 14, Switzerland
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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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While glucose metabolism has been measured independently in spermatozoa, oocytes, and early embryos [5, 8,9], it has not been investigated during sperm entry into oocytes. Swezey and Epel [10] have demonstrated in the sea urchin that PPP activity plays an important role shortly after sperm penetration into the oocyte. They have shown that the NADPH produced by the PPP activity is used for the generation of H2O2 postfertilization, which in turn is implicated in modifications to the fertilization membrane. In light of these data, PPP activity may also be important for events related to fertilization in mammals.
The main purpose of this study was therefore to characterize glucose metabolism, by assessing glycolytic and PPP activities, during sperm fusion and decondensation into zona-free mouse oocytes. The metabolic activities of the gametes during the fertilization process have also been compared with the metabolism of parthenogenetically activated mouse oocytes. The characterization and attribution of the metabolic roles of each gamete during sperm fusion and decondensation into the oocyte will provide us with a greater understanding of the process of fertilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The basic culture medium used in all experiments was M16 [11] containing 23.3 mM lactate, 0.33 mM pyruvate, and 5.56 mM glucose, and supplemented with 15 mg/ml type V BSA. Cytochalasin B and phloretin were dissolved in dimethyl sulfoxide (DMSO) as a stock solution (1000x concentrated) and stored at -20°C. Dilutions in culture medium were made before use. All chemicals were obtained from Sigma Pharmaceuticals (Buchs, Switzerland).
Oocyte Preparation
Oocytes were obtained from 3- to 4-wk-old female B6D2F1 mice (IFFA-CREDO, L'Arbresle, France), which received injections of 5 IU eCG (Folligon; Veterinaria, Zürich, Switzerland) followed 48 h later by 5 IU hCG (Choluron, Veterinaria). Fourteen to 16 h after administration of hCG, oocytes surrounded by their cumulus cells were collected. After cumulus digestion by 0.2 mg/ml hyaluronidase (Sigma), zonae pellucidae were mechanically removed as previously described [5].
Sperm Preparation
Spermatozoa were obtained from the cauda epididymidis and the vas deferens of 10- to 16-wk-old OF1 male mice (BRL, Füllinsdorf, Switzerland). In all experiments, spermatozoa were incubated in 200 µl of M16 under oil (light white mineral oil; Sigma) at 37°C for 3 h to achieve capacitation and to maximize the proportion of acrosome-reacted sperm [12]. The concentration of motile sperm at the end of capacitation was 20–40 x 106 cells/ml.
For insemination of zona-free oocytes, suspensions of capacitated sperm were diluted in M16 medium to obtain a final concentration of 0.1–0.5 x 106 motile sperm/ml. For experiments performed in the absence of Ca2+, sperm suspensions were washed and diluted in Ca2+-free M16. Insemination droplets of 50 µl were prepared from these suspensions and covered with oil.
Gamete Fusion Assay
To measure glucose metabolism in fusing gametes, zona-free oocytes were incubated for 15 min in insemination drops under oil for sperm binding, washed quickly to remove excess sperm, and transferred into 3-µl incubation droplets placed on the lids of Eppendorf (Hamburg, Germany) tubes. After the lids were fitted on the Eppendorf tubes, the oocytes with bound sperm were incubated for 1 h at 37°C to allow sperm entry and decondensation, and metabolic measurements. To prevent gamete fusion, sperm binding as well as the following incubation were performed in Ca2+-free medium. When glucose uptake inhibitors were tested, they were added together with radiolabeled glucose, after sperm binding to the oolemma. For assessment of sperm binding to the oolemma, and sperm entry and decondensation, the oocytes were fixed in formaldehyde and stained with Hoechst 33342 (Sigma) as previously described [13].
Oocyte Activation
Cumulus-free oocytes were incubated for 3 min in M16 containing 7% ethanol, washed quickly in M16, and transferred into 3-µl incubation droplets placed on the lids of Eppendorf tubes for metabolic measurements. After 1 h of incubation, the oocytes were collected, fixed, and stained with Hoechst 33342 to determine whether the resumption of meiosis had occurred.
Metabolic Measurements
The metabolism of glucose was measured in ovulated metaphase II oocytes (MII oocytes), in activated oocytes, and in fusing gametes (during sperm penetration and decondensation in zona-free oocytes) essentially as described by Rieger et al. [14] in cattle embryos. Measurements were performed in M16, a bicarbonate-buffered medium containing pyruvate and lactate that supports fertilization. The final glucose concentration was 1 mM (unlabeled plus labeled glucose).
Glucose metabolism via glycolysis was measured by collecting 3H2O released from [5-3H]glucose (13.6 Ci/mmol; Amersham Rahn, Zurich, Switzerland). Tracer quantities of [5-3H]glucose (250 µCi/ml; about 20 µM) were added to M16 medium supplemented with 1 mM of unlabeled glucose. Glucose metabolism via the PPP was assessed by measuring the 14CO2 produced from [1-14C]glucose (55 mCi/mmol; Amersham). [1-14C]glucose was added to glucose-free M16 at a final concentration of 1 mM (55 µCi/ml). In addition, some experiments were performed with [6-14C]glucose (55 mCi/mmol; Amersham) to evaluate the oxidation of labeled glucose by the Krebs cycle in MII oocytes.
Measurements were performed in a closed incubation system, essentially as described by Rieger et al. [14]. Gametes were incubated with radiolabeled glucose in 3-µl droplets and placed on the inner side of the lids of Eppendorf tubes. Tubes were filled with 1.5 ml of 25 mM NaHCO3, the function of which was to trap 3H2O and 14CO2 released from [5-3H]glucose and [1-14C]glucose, respectively. Both M16 medium and NaHCO3 were equilibrated with 5% CO2 in air at 37°C before measurements.
To measure glucose metabolism in MII oocytes, activated oocytes, or fusing gametes, the following steps were taken. Groups of 10 oocytes were transferred in a minimal amount of medium into the 3-µl incubation droplets just deposited on the lid. As soon as the gametes were transferred, the Eppendorf tubes were closed and incubated at 37°C for 3 h (MII oocytes) or 1 h (activated oocytes, fusing gametes). Sham preparations, containing no gametes but 3-µl droplets of medium supplemented with radiolabeled glucose, were included in each experiment and for each medium tested. They served to determine the levels of passive diffusion and possible spontaneous breakdown of the labeled glucose as well as the background of the radioactivity counter.
At the end of incubation, the NaHCO3 fraction was collected quickly in scintillation vials containing 200 µl of 0.1 M NaOH, which promoted the conversion of dissolved CO2 and bicarbonate into carbonate. After an overnight incubation, 10 ml of scintillation cocktail (Lumagel; Lumac, Groningen, the Netherlands) was added to the vials and the radioactivity counted. The mean counts per minute of the sham preparations was subtracted from the counts per minute obtained for each batch of oocytes. The difference obtained, which was representative of the metabolism of the oocytes, was divided by the total counts per minute of labeled glucose and multiplied by the total quantity of glucose present in the 3-µl droplet. Although the metabolism of [1-14C]glucose by MII oocytes gave low levels of counts per minute, they repeatedly exceeded the counts per minute of the sham preparations by at least 50% after a 3-h incubation. In the cases in which the count-per-minute values obtained after oocyte incubation were not different from the values of the sham preparations, the 14CO2 production was considered undetectable.
Statistics
ANOVA followed by Scheffé's test for multiple comparisons was used to compare the numbers of bound sperm, the numbers of decondensed sperm, and glucose metabolism in the different media. The percentages were compared using the same method but after arc sine transformation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The metabolism of glucose through both glycolysis and the PPP was measured in ovulated MII oocytes with [5-3H]glucose and [1-14C]glucose, respectively (Table 1). As reported for spermatozoa [15], the glycolysis rate obtained in bicarbonate-buffered medium was higher than that measured previously in Hepes-buffered medium, which was 132 ± 52 fmol/oocyte per h [5]. The levels of glucose metabolized through the PPP were low compared to those metabolized through glycolysis and represented 2.4% of total glucose metabolism as measured from the [5-3H]glucose metabolism. The incubation of MII oocytes with [6-14C]glucose gave count-per-minute values that were not different from the values of the sham preparations, indicating that no detectable levels of 14CO2 were generated with this radiolabel (data not shown). The absence of calcium did not influence metabolic activity through either pathway.
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Glucose Metabolism during Gamete Fusion andSperm Decondensation
The metabolism of glucose was investigated during the early events of fertilization, and both glycolysis and PPP activity were measured during the first hour following sperm binding to zona-free oocytes. During this period, sperm entered the oocytes and their chromatin decondensed whereas the oocytes resumed meiosis to the telophase II stage. Parallel experiments were performed in which gamete fusion, but not sperm binding to the oolemma, was prevented by removal of Ca2+ [16].
As shown in Figure 1A, the metabolism of [5-3H]glucose was significantly higher in fertilized (+Ca2+) compared to unfertilized (-Ca2+) oocytes, indicating a higher rate of glycolysis associated with fertilization. This difference was not due to sperm attached to the oolemma because the numbers of bound sperm were similar in the two groups. Neither was this difference due to the absence of Ca2+ because, in capacitated sperm, we found that the absence of Ca2+ did not induce a decrease in glycolysis rate. In capacitated spermatozoa, we found a glycolysis rate of 10.5 ± 5.0 fmol/sperm per h with Ca2+ and 10.8 ± 5.9 fmol/sperm per h without Ca2+.
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Gamete fusion triggers the activation of the oocyte. To determine whether the elevation in glucose metabolism reported in Figure 1 was part of the activation process, the metabolism of activated oocytes was measured during the first hour following a 3-min exposure to ethanol. Although nearly all eggs had resumed meiosis to telophase II after this treatment, no increase in metabolism was observed (Fig. 1A).
Similar results were obtained after investigation of the metabolism of glucose through the PPP (Fig. 1B). The production of 14CO2 from [1-14C]glucose was not detectable in unfertilized oocytes (-Ca2+) after 1 h of incubation, although we were able to measure PPP activity in MII oocytes after a 3-h incubation (Table 1). It appeared that the basal activity of this pathway was too low to be detected after a 1-h incubation with [1-14C]glucose. In contrast, the metabolism of glucose through the PPP became clearly detectable in fertilized oocytes (+Ca2+). As for the unfertilized oocytes, the metabolism of [1-14C]glucose by parthenogenetically activated oocytes was not detectable.
Inhibition of the PPP during Gamete Fusion andSperm Decondensation
In a previous study [5], we have shown that inhibitors of facilitative glucose transport (cytochalasin B and phloretin) induced an inhibition of glycolysis in MII oocytes. However, PPP activity has not been measured in the presence of these inhibitors. Therefore, the effects of cytochalasin B and phloretin on both glycolysis and PPP activities were tested in zona-free oocytes during sperm entry and decondensation.
Both cytochalasin B and phloretin were able to prevent the increase in glycolysis (Fig. 2A) associated with sperm penetration into zona-free oocytes. In contrast, glucose metabolism through the PPP was inhibited by cytochalasin B but not by phloretin (Fig. 2B). Phloretin was also ineffective when the PPP was enhanced by the electron acceptor brilliant cresyl blue (BCB) [17] (Table 2).
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The inhibition of glycolysis and PPP activities in the presence of these inhibitors did not prevent fusion and sperm decondensation (Fig. 2). When the PPP was enhanced 3- to 4-fold by BCB (Table 2), the percentage of penetrated oocytes and the number of decondensed spermatozoa per oocyte were not different from the control.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to glycolysis [5], the presence of an active PPP in oocytes has already been suggested by studies reporting high levels of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the PPP, in mouse oocytes [18, 19] and the ability of the oocyte to generate 14CO2 from [1-14C]glucose [20, 21]. In this study, we have used the 14CO2 production from [1-14C]glucose to measure PPP activity since no detectable levels of 14CO2 were measured after incubation of MII oocytes with [6-14C]glucose. When there is no detectable production of 14CO2 from [6-14C]glucose, the 4CO2 generated from [1-14C]glucose metabolism is considered to arise exclusively from the PPP [22]. This absence of 14CO2 production from [6-14C]glucose was certainly due to the presence of unlabeled pyruvate and lactate, which greatly decrease the specific activity of radiolabel entering the Krebs cycle and prevent 14CO2 release by this pathway [23]. Brinster [20] reported 14CO2 formation from [6-14C]glucose in mouse MII oocytes in the absence of any energy source. The ratio of the production of 14CO2 from [1-14C]glucose to the production of 3H2O from [5-3H]glucose, which has been used to determine the relative activity of the PPP [17] was low. However, the activity of this pathway would be higher if recycling of ribose 5-phosphate occurred; this could not be evaluated by using [1-14C]glucose [23]. In addition, the presence of CO2 can allow gluconeogenesis to occur by conversion of pyruvate into glucose and therefore decrease the specific activity of [1-14C]glucose entering the PPP and lead to an underestimation of its activity.
An increase in both glycolysis and PPP activity was associated with fertilization. The increase in 14CO2 production from [1-14C]glucose has been interpreted as an increase in PPP but not in Krebs cycle activity since pyruvate and lactate were present in the incubation medium. However, oxidation by the Krebs cycle is probably enhanced as glucose metabolism is increased. An increase in glucose utilization promoted by fertilization has been suggested by studies reporting higher glucose oxidation [20], hexokinase activity [24], and glycolysis rate [25] in fertilized oocytes (zygote or 1-cell stage) than in MII oocytes in the mouse. The present study has shown that this metabolic activation began very early at fertilization, during gamete fusion and/or sperm decondensation, and that the PPP was part of this activation. This up-regulation occurred when the external glucose concentration was constant, implying that changes in enzyme activities and/or in glucose transport were responsible for higher glucose utilization.
The fertilizing sperm appears to be necessary for this metabolic activation since parthenogenetically activated oocytes did not display an increased glucose metabolism. Therefore, the spermatozoa may bring a factor that stimulates oocyte metabolism. Alternatively, it may be envisaged that the enzymes of the spermatozoa, which are incorporated into the ooplasm during fusion, may be activated by an undetermined factor of the oocyte.
The role of this metabolic activation does not appear to be related to successful gamete fusion. The inhibition of glucose uptake by facilitative glucose transport inhibitors (cytochalasin B, phloretin) did not prevent sperm entry into zona-free oocytes [5] even though these inhibitors were able to decrease the activities of glycolysis and the PPP (this study). The stimulation of the PPP by BCB, as measured by an increase in 14CO2 production, did not change the penetration rate. The effects of this PPP stimulation on the production of metabolic intermediates remain speculative, but it has been proposed that PPP stimulation by electron acceptors (PES, P5C) in mouse oocytes could induce an increase in ribose 5-phosphate [26].
This early activation of glucose metabolism may be related to other aspects of fertilization and/or to embryo development. For example, NADPH, which is produced by the PPP, has been shown to play an important role in the sea urchin, whereby fertilization induces an activation of the G6PDH to provide NADPH, which is then used to produce H2O2 via NADPH oxidase. In this species, H2O2 plays a physiological role in the hardening of the fertilization membrane [10]. The existence of an oolemma block to polyspermy in the mouse has been previously described; however, the underlying mechanism is not understood [27,28]. Interestingly, Horvath et al. [29] described the establishment of a plasma membrane block to polyspermy in the mouse that was sperm-dependent, as artificial egg activation did not result in a block response. In our study, an increase in PPP activity was observed only when the oocytes were activated by spermatozoa. The consequent elevation of NADPH may be used to produce H2O2 via a putative NADPH oxidase at fertilization. This mechanism could subsequently establish the oolemma block to polyspermy, similar to that observed in the sea urchin. Alternatively, ribose 5-phosphate, which is also generated by the PPP, may also play an important role as a precursor of nucleotide synthesis, since the first DNA replication cycle occurs during pronucleus migration at the zygote stage [30], and activation of the embryonic genome occurs at the 2-cell stage in the mouse [31].
The significance of our observations during the fertilization process for the developing embryo is as yet unclear and warrants further investigation.
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FOOTNOTES |
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2 Correspondence: Françoise Urner, Laboratoire des Gamètes, Clinique de Stérilité, Hôpital Cantonal, 30 Bd. de la Cluse, 1211 Geneva 14, Switzerland. FAX: 022 382 43 85; francoise.urner{at}hcuge.ch
3 Current address: Denny Sakkas, Reproductive Biology and Genetic Group, Department of Medicine, University of Birmingham and Assisted Conception Unit, Birmingham Women's Hospital, Birmingham, B15 2TG, UK.
Accepted: November 25, 1998.
Received: March 3, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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