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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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To permit gamete fusion, glucose must be metabolized because non-metabolizable glucose analogues (L-glucose, 3-O-methylglucose, 2-deoxyglucose) are unable to support this process [6]. In contrast to the metabolism of oocytes, which does not appear necessary for this event, the metabolism of spermatozoa may be linked to the fusion step. While it is well accepted that glucose metabolism through glycolysis provides energy to spermatozoa [9], the existence and the role of the pentose phosphate pathway (PPP) have not been unequivocally demonstrated in spermatozoa. The main function of the PPP is to generate NADPH, which is required for reductive reactions, and to form ribose 5-phosphate for the synthesis of nucleic acids. The implication of NADPH in certain human sperm functions [10, 11] indicates that the PPP may be involved in successful fertilization.
The main purpose of this study was therefore 1) to demonstrate the presence of a functional PPP in mouse spermatozoa and 2) to determine whether this pathway is implicated in the spermatozoa's acquisition of competence to fuse and enter the oocyte.
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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 [12] 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, phloretin, and iodoacetate were dissolved as stock solutions (1000x concentrated in dimethyl sulfoxide) and stored at -20°C. Dilutions in culture medium were made before use. All chemicals were obtained from Sigma Pharmaceuticals (Buchs, Switzerland). The antibodies anti-GLUT3 and anti-GLUT1 were kindly provided by Dr. G.W. Gould (University of Glasgow, Scotland) and Dr. B. Thorens (University of Lausanne, Switzerland), respectively.
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 we have previously described [6].
Sperm Preparation
Spermatozoa were obtained from the cauda epididymidis and the vas deferens of 10- to 16-wk-old OF1 males (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 [13]. 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. To remove glucose, 20 µl of capacitated sperm was mixed with 4 ml of M16 without glucose and centrifuged at 150 x g for 10 min. The pellet was then resuspended in an appropriate volume of glucose-free medium to give a final concentration of about 0.1 x 106 motile sperm/ml. Insemination droplets of 50 µl were prepared from these suspensions and covered with oil.
For measurements of sperm metabolism, suspensions of capacitated sperm were diluted to the appropriate concentration (100, 500, or 1000 spermatozoa/3 µl) in glucose-free M16 for measurement of the PPP activity or in medium containing 1 mM glucose for glycolysis measurement.
Gamete Fusion Assay
Zona-free oocytes were incubated in insemination drops for 20 min to allow sperm binding to the oolemma. They were then transferred into 25-µl droplets of M16 for 45 min to allow bound sperm to enter the oocytes and to decondense. The oocytes were then washed as described by Wolf and Hamada [14] to remove loosely attached sperm. 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 we have previously described [15].
To inhibit glucose uptake or glycolysis in sperm, cytochalasin B (50 µM), phloretin (0.5 mM), and iodoacetate (10 µM) were added to the insemination drops 60 min before addition of the oocytes. Cytochalasin B was also added 30 and 0 min before gamete mixing. When NADPH was used instead of glucose, it was added to sperm suspensions containing no glucose 1 h or 15 min before addition of the oocytes. These different compounds were present in the culture medium until the end of the experiment.
Metabolic Measurements
Glucose metabolism was measured in capacitated sperm essentially as described for cattle embryos by Rieger et al. [16]. Measurements were made in M16, a bicarbonate-buffered medium containing pyruvate and lactate that supports both sperm capacitation and fertilization.
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 sperm suspensions 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 (55 µCi/ml; 1 mM) was added to sperm suspensions containing no unlabeled glucose. 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.
Measurements were performed in a closed incubation system, essentially as described by Rieger et al. [16]. Spermatozoa (100, 500, 1000) were incubated with radiolabeled glucose in 3-µl droplets placed on the inner side of the lids of Eppendorf (Hamburg, Germany) tubes previously filled with 1.5 ml of 25 mM NaHCO3, whose function 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. As soon as the 3-µl droplets were deposited on the lids, the Eppendorf tubes were closed and incubated at 37°C. Sham preparations, containing no spermatozoa 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) was added to the vials, and the radioactivity was counted.
The mean cpm of the sham preparations was subtracted from the cpm obtained for each batch of spermatozoa. The difference obtained, which was representative of the metabolism of the spermatozoa, was divided by the total cpm 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 spermatozoa gave low cpm, they repeatedly exceeded the cpm of the sham preparations by 50–100% when the numbers of spermatozoa were sufficient. In the cases in which the cpm values obtained after sperm incubation were not different from the values of the sham preparations, the 14CO2 production was considered undetectable.
Immunoblotting
Western analysis was performed using procedures similar to those described for the detection of GLUT in preimplantation embryos by Aghayan et al. [17]. Briefly, 0.25 x 106 spermatozoa or 30 mouse blastocysts were lysed in 10 µl of lysis buffer and stored at-20°C. Before electrophoresis, the samples were diluted in a double-strength solution of Laemmli's sample buffer [18]. Electrophoresis was performed under reducing conditions using a 10% polyacrylamide SDS minigel system (Bio-Rad, Zurich, Switzerland), and the proteins were transferred to the nitro-cellulose membrane with the Bio-Rad mini-transfer system. After transfer, the membrane was washed and prepared, using a slight modification of the procedure described by Aghayan et al. [17]. To detect GLUT1 and GLUT3, respectively, the membrane was incubated in anti-GLUT1 (1:1500 dilution) or anti-GLUT3 (1:1500 dilution) for 1 h at room temperature. Proteins were visualized by using the enhanced chemiluminescence system (ECL; Amersham).
Statistics
ANOVA followed by Scheffe'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 sin transformation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a first series of experiments, identification of the glucose transporter and metabolic studies were undertaken in a population of capacitated sperm. Using Western blot analysis (Fig. 1), we found that a band migrating at 45 kDa was recognized by anti-GLUT3, indicating that this transporter was expressed in mouse spermatozoa. Mouse sperm proteins did not react with anti-GLUT1, but mouse blastocysts, which were used as positive controls, reacted positively to anti-GLUT1.
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The metabolism of glucose through glycolysis and the PPP was measured in sperm after capacitation by using [5-3H]glucose and [1-14C]glucose, respectively. The production of 3H2O and 14CO2 by spermatozoa was first measured as a function of time of incubation and number of spermatozoa. 3H2O (Fig. 2A) and 14CO2 (Fig. 2B) increased linearly with time, but the best time courses were obtained with 1000 spermatozoa. It appears that the levels of glucose metabolized were high through glycolysis and were considerably lower through the PPP: the ratio of the production of 14CO2 from [1-14C]glucose to the production of 3H2O from [5-3H]glucose ranged from 0.5 to 0.7%. The incubation of spermatozoa with [6-14C]glucose lead to cpm values that were not different from the values of the sham preparations, indicating that no detectable levels of 14CO2 were produced with this radiolabel (data not shown).
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Glucose metabolism was measured in the presence of phloretin and cytochalasin B (Fig. 3), which are inhibitors of facilitative glucose transport [19]. Blocking glucose uptake by 50 µM cytochalasin B induced an inhibition of both glycolysis and PPP activity by 36% and 46%, respectively. The PPP activity was diminished by 80% in the presence of 200 µM cytochalasin B (data not shown). In contrast, 0.5 mM phloretin, which induced a glycolysis inhibition of 80%, was unable to induce a decrease in the PPP activity. Iodoacetate was a strong inhibitor of sperm glycolysis at 10 µM.
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Involvement of the PPP in theFusion Capacity of Spermatozoa
To determine whether the glucose metabolism of the sperm was involved in gamete fusion, the fusing capacity of the sperm was assessed after it had been depressed for 1 h with glucose uptake or glycolysis inhibitors prior to insemination (Table 1). When 0.5 mM phloretin and 10 µM iodoacetate were used to inhibit glycolysis but not PPP activity, the percentage of penetration and the number of decondensed sperm per oocyte were similar to those of the control. When both glycolysis and the PPP activities were decreased by using 50 µM cytochalasin B, the fusing capacity of sperm was significantly reduced. A 1-h exposure of sperm to cytochalasin B was required to induce an inhibition of fertilization; when the preincubation time was reduced to 30 min or 0 min, spermatozoa fused with the oocytes as in the control medium.
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In the next series of experiments, NADPH, the main product generated by the PPP, was substituted for glucose. Spermatozoa were capacitated in glucose-containing medium, washed to remove glucose, and incubated in glucose-free medium for 1 h to eliminate their ability to fuse with the oocytes. Glucose (5.5 mM) or NADPH (5 mM) was then added to these sperm suspensions before insemination of the zona-free oocytes and was continuously present in the medium during sperm penetration and decondensation. Table 2 shows that the fertilizing capacity of the mouse sperm was rapidly restored by the addition of NADPH or glucose. However, longer incubation (1 h) of the sperm in the presence of NADPH tended to diminish its beneficial effect, and a 2-h preincubation did not result in successful gamete fusion (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of a PPP in spermatozoa has been investigated in some species, but its existence has not been unequivocally demonstrated. Activities of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the PPP, have been reported in spermatozoa in humans [20, 21] and mice [22], but not in bulls [23]. In addition, the transcription of a retroposed gene, encoding for a functional G6PDH and not linked to the X chromosome, has been evidenced in postmeiotic spermatogenic cells in the mouse [24]. The ability of spermatozoa to metabolize labeled glucose through the PPP has been reported in humans [25, 26] and rabbits [27], but not in mice [1], rams, or bulls [28]. Although species differences may exist, the techniques used to assess the activity of the PPP are questionable. From studies performed in rams, bulls [29], and mice [1], an absence of the PPP in spermatozoa was concluded because the yields of 14CO2 from [1-14C]glucose and [6-14C]glucose were not significantly different and gave ratios of about one. However, evaluation of the PPP by using the ratios of 14CO2 produced from [1-14C]glucose and [6-14C]glucose is not appropriate, and the absence of a difference in yields of 14CO2 does not prove that the PPP is absent or inactive [30–32].
14CO2 can be released from [1-14C]glucose by metabolism through the PPP or in the Krebs cycle after glycolytic metabolism to [3-14C]pyruvate. In the presence of unlabeled pyruvate and lactate, which greatly decrease the specific activity of radiolabel entering the Krebs cycle, the 14CO2 released by the Krebs cycle is necessarily negligible [33]. Under our experimental conditions (medium containing pyruvate and lactate), the oxidation of labeled glucose through the Krebs cycle was prevented since no detectable levels of 14CO2 were measured after incubation with [6-14C]glucose. Therefore, the 14CO2 measured after incubation of spermatozoa with [1-14C]glucose originated exclusively from the PPP [32] and was indicative of a functional PPP. The ratio of the 14CO2 produced from [1-14C]glucose to the 3H2O produced from [5-3H]glucose, which has been used to determine the relative activity of the PPP [34], was very low. The activity of this pathway would be higher if recycling of ribose 5-phosphate occurred; however, this could not be evaluated by using [1-14C]glucose [33].
As in rats and humans [35, 36], we have shown the presence of the high-affinity glucose transporter GLUT3 [37] in mouse sperm, indicating the immense need of sperm for glucose. Interestingly, the presence of cytochalasin B but not phloretin inhibited the PPP activity, although both are able to bind to facilitative glucose transporters (GLUT1, GLUT3) and to inhibit glucose uptake by sperm in different species [38, 39]. Cytochalasin B has been shown to bind to GLUT3 in human testis [36], to inhibit glucose transport [40], and to decrease the glycolytic rate in human sperm [41]. The differential response of the PPP to cytochalasin B and phloretin was surprising since we expected that metabolism through both glycolysis and the PPP would be decreased after glucose uptake inhibition. Although the lack of inhibition of the PPP in the presence of phloretin has not been explained, it may be supposed that the cellular effect of phloretin was not limited to glucose uptake inhibition. For example, phloretin has been shown to enhance chick myoblast fusion by activating a calcium-dependent potassium channel [42]. In our study, phloretin may activate the PPP by an unknown mechanism, leading to an increased generation of 14CO2 from small quantities of [1-14C]glucose entering the gametes despite transport inhibition.
As demonstrated previously, glucose must be metabolized to exert its positive effect on fusion [6]. Although glycolysis is important for sperm functions such as hyperactivated motility, this metabolic pathway does not appear to be responsible for successful gamete fusion since its inhibition by phloretin or by iodoacetate (inhibitor of the glycolytic enzyme glyceraldehyde phosphate dehydrogenase [43]) in capacitated spermatozoa before insemination of zona-free oocytes and during gamete interaction did not prevent fusion. The reduced penetration into zona-free oocytes observed when spermatozoa were pretreated for 1 h with cytochalasin B, which was shown to diminish the PPP activity, suggests that this pathway is necessary to render the sperm fusogenic. In contrast, the lack of inhibition when 1) the oocytes were pretreated with cytochalasin B [6] and 2) this inhibitor was present during gamete interaction indicates that the PPP activity is not essential either for the fusibility of the oocyte nor for the fusion step per se. Niemierko and Komar [44] have also shown that pretreatment of sperm with cytochalasin B before insemination drastically decreased the fertilization rate (73.3% vs. 15.6%) when using cumulus-enclosed zona intact mouse eggs. The inhibitory effect of cytochalasin B has been interpreted as the result of its inhibition of glucose uptake and the consequent decrease in PPP activity. The influence of a cytoskeletal destabilization of the spermatozoa by a long exposure to cytochalasin B cannot be totally excluded. Maro et al. [45], however, have shown that in vitro fertilization of mouse cumulus-enclosed oocytes was not affected by cytochalasin D, which is a more efficient inhibitor of the cytoskeleton than cytochalasin B and does not interfere with glucose transport across the plasma membrane [46]. The results of Maro et al. [45], points 1 and 2 above, and the observation that spermatozoa pretreated for 30 min with cytochalasin B did not alter penetration rate into the oocytes indicate that a cytoskeletal effect was most likely not responsible for our observation. In the event that cytochalasin B does not acting via destabilization of cytoskeletal elements, we can speculate that its role as an inhibitor of glucose uptake and PPP activity was responsible.
In this context, the observation that exogenously added NADPH can support gamete fusion in the absence of glucose is particularly relevant to the implication of the PPP in the fusing capacity of the sperm. Exogenous addition of NADPH to human spermatozoa has been shown to induce reactive oxygen species (ROS) and tyrosine protein phosphorylation [10, 47]. Although NADPH has limited membrane permeability, the diffusion of a small proportion of the molecules through the plasma membrane has been suggested [11]. An intracellular effect of exogenously added NADPH would be consistent with intracellular production of NADPH by the PPP. In the case of an extracellular effect of NADPH, it may be speculated that the reduction of NADP to NADPH by glucose 6-phosphate dehydrogenase and 6-phospho-gluconate dehydrogenase takes place at the membrane level and that NADPH is released at the external face of the plasma membrane.
The NADPH produced via the PPP appears to work at two levels that are finely balanced. Overactivity of this pathway may damage the sperm, whereas underactivity may render the sperm unable to fertilize. Excessive G6PDH activity has been found to be associated with defective sperm function in humans [48], suggesting that an appropriate regulation of the PPP is important for the fertilizing capacity of sperm. NADPH could activate the glutathione (GSH)-dependent scavenger system to decrease the levels of ROS; however, this system is probably not efficient in human spermatozoa since they contain very low quantities of GSH peroxidase [49]. In the mouse, despite a significant amount of both GSH and GSH peroxidase [50], the efficiency of this scavenger system is abolished in case of oxidative stress [49]. Alternatively, NADPH has been postulated to be a cofactor for a putative NADPH oxidase that would be responsible for the production of ROS in human sperm [11]. One of these ROS, H2O2, exerts a physiological function in controlling the levels of protein tyrosine phosphorylation [10, 51]. In the mouse, the phosphorylation of tyrosine residues is important for sperm functions such as capacitation [52] and acrosome reaction [53]. In addition, glucose has been shown to inhibit tyrosine phosphorylation during capacitation of bovine sperm [54]. In mouse spermatozoa, glucose may exert indirect control of protein tyrosine phosphorylation of the sperm, after its metabolism through the PPP and the concomitant production of NADPH.
In conclusion, it appears that the beneficial effect of glucose on gamete fusion is probably mediated by its metabolism through the PPP of the spermatozoa, which we have shown to be functional. The ability of capacitated sperm to fuse with the oocyte is lost after removal of glucose or PPP inhibition by cytochalasin B and is restored by the addition of glucose or NADPH. This suggests the existence of an event in spermatozoa that is linked to gamete fusion and controlled by glucose via the production of NADPH, although the key functions that depend on NADPH remain to be defined.
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ACKNOWLEDGMENTS |
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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: Reproductive Biology and Genetic Group, Department of Medicine, University of Birmingham and Assisted Conception Unit, Birmingham Women's Hospital, Birmingham B15 2TG, United Kingdom.
Accepted: October 23, 1998.
Received: March 3, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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