BIOLOGY OF REPRODUCTION 60, 733–739 (1999)
A Possible Role for the Pentose Phosphate Pathway of Spermatozoa in
Gamete Fusion in the Mouse 1
Françoise Urner2 and Denny Sakkas3
Clinic of Sterility, Department of Obstetrics and Gynecology, University Hospital of Geneva,
1211 Geneva 14, Switzerland
ABSTRACT
Glucose metabolism is essential for successful gamete fusion
in the mouse. Although the metabolic activity of the oocyte does
not appear to play a significant role in the fusion step, the metabolic role of the spermatozoon is not known. The aim of this
study was therefore to characterize the role of glucose metabolism in mouse spermatozoa. Initially, the high-affinity glucose
transporter GLUT3 was identified in mouse sperm. In characterizing the glucose metabolism of mouse sperm, we have
shown 1) that mouse epididymal spermatozoa have a functional
pentose phosphate pathway (PPP), implying that they produce
NADPH, which is required for reducing reactions, and ribose 5phosphate, which is required for nucleic acid synthesis; and 2)
that sperm are able to fuse with the oocyte when NADPH is
substituted for glucose, suggesting that sperm need to produce
NADPH via the PPP in order to be able to achieve fertilization.
The existence of an NADPH-regulated event that influences the
ability of the sperm to fuse with the oocyte is envisaged.
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 5phosphate 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.
INTRODUCTION
MATERIALS AND METHODS
The fertilization process requires glucose in a number of
species (mouse [1, 2]; rat [3]; human [4]). In the mouse,
the analysis of the sequence of events leading to fertilization in vitro has indicated that glucose is essential for the
penetration of the zona pellucida [5] and for the fusion of
the gametes [6]. The failure to penetrate the zona pellucida
in the absence of glucose has been attributed to the altered
hyperactivated motility of the sperm [2, 7]. In contrast to
hyperactivated motility, sperm capacitation and the acrosome reaction are necessary steps for the spermatozoa to
fuse with the oocytes [8]. Although the influence of glucose
on the acrosome reaction in the mouse is uncertain [2, 5],
spermatozoa that have acrosome-reacted spontaneously in
the presence of glucose lose their ability to fuse with the
oolemma after the removal of this hexose [6]. This suggests
that, in addition to the acrosome reaction, a glucose-regulated event influences the ability of the spermatozoon to
fuse with the oocyte. Despite the abundant studies on gametes and fertilization, the precise role of glucose in specific
gamete functions and, in particular, in those involved in
sperm-oocyte fusion, have not yet been identified.
To permit gamete fusion, glucose must be metabolized
because non-metabolizable glucose analogues (L-glucose,
3-O-methylglucose, 2-deoxyglucose) are unable to support
Chemicals and Media
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 (10003 concentrated in
dimethyl sulfoxide) and stored at 2208C. 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].
Accepted October 23, 1998.
Received March 3, 1998.
1
Supported by the Fonds National Suisse de la Recherche Scientifique
(32–45435.95).
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; e-mail: [email protected]
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.
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 ml of M16 under oil (light white
mineral oil; Sigma) at 378C for 3 h to achieve capacitation
and to maximize the proportion of acrosome-reacted sperm
733
734
URNER AND SAKKAS
FIG. 1. GLUT3 expression in mouse sperm. Western blot analysis of
GLUT1 (left) and GLUT3 (right) was performed in mouse spermatozoa
(0.25 3 106/lane). As a positive control, mouse blastocysts (30/lane) were
probed for the presence of GLUT1.
[13]. The concentration of motile sperm at the end of capacitation was 20–40 3 106 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 3 106 motile sperm /ml.
To remove glucose, 20 ml of capacitated sperm was mixed
with 4 ml of M16 without glucose and centrifuged at 150
3 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 3 106 motile sperm/ml. Insemination droplets of 50 ml 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 ml) in glucosefree 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-ml 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 mM), phloretin (0.5 mM), and iodoacetate
(10 mM) 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-buff-
ered 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 mCi/ml; about 20 mM) 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 mCi/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-ml 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 [114C]glucose, respectively. Both M16 medium and NaHCO
3
were equilibrated with 5% CO2 in air at 378C before measurements. As soon as the 3-ml droplets were deposited on
the lids, the Eppendorf tubes were closed and incubated at
378C. Sham preparations, containing no spermatozoa but 3ml 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 ml 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-ml 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
3 106 spermatozoa or 30 mouse blastocysts were lysed in
10 ml of lysis buffer and stored at2208C. 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 nitrocellulose membrane with the Bio-Rad mini-transfer system.
After transfer, the membrane was washed and prepared, us-
GLUCOSE METABOLISM DURING GAMETE FUSION
735
FIG. 2. Time courses of glucose metabolism through A) glycolysis and B) the PPP in capacitated sperm. Glucose metabolism was measured in 100,
500, or 1000 capacitated spermatozoa for 1, 2, or 3 h. Each value represents the mean (6 SD) of triplicate measurements performed in 2 different
males.
ing 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.
RESULTS
Glucose Metabolism of Capacitated Spermatozoa
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.
The metabolism of glucose through glycolysis and the
PPP was measured in sperm after capacitation by using [53H]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
FIG. 3. Inhibition of the metabolism of glucose through A) glycolysis and B) the PPP in capacitated spermatozoa. Glucose metabolism was measured
in 1000 capacitated spermatozoa for 3 h, in the absence or presence of the different inhibitors. Each bar represents the mean levels of metabolized
glucose, expressed as a percentage of the control (6 SD), the latter being fixed at 100%. Controls were normalized to 100% because of their high
variability (6 32%). Triplicate measurements were performed in 4 different males. CytoB, cytochalasin B; IA, iodoacetate. *Significantly different from
the control ( p , 0.05).
736
URNER AND SAKKAS
TABLE 1. Fertilizing ability of sperm pretreated with inhibitors of glucose uptake or glycolysis before insemination of zona-free oocytes.
Treatment
Sperm preincubation time
(min)
No. of
oocytes
No. of
replicates
—
0
30
60
60
60
163
58
65
118
55
64
12
4
4
8
4
5
Control
Cytochalasin B
Phloretin
Iodoacetate
a
b
No. of bound
sperm per oocytea
9.0
1.9
1.5
6.3
6.8
11.9
6
6
6
6
6
6
2.9
1.7b
0.5b
4.9
3.7
3.3
No. of decondensed
sperm per penetrated
oocytea
1.3
1.8
1.5
1.1
2.0
1.5
6
6
6
6
6
6
0.3
0.6
0.5
0.2
0.9
0.5
Penetration rate
(%)a
93
81
76
28.7
100
78
6
6
6
6
6
6
6
29
21
10b
0
29
Mean 6 SD.
Significantly different from the control (p , 0.05).
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).
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 mM 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 mM 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 mM.
Involvement of the PPP in the
Fusion 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 mM
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 mM 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.
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 glucosefree 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).
DISCUSSION
In this study, we have presented data that further characterize the glucose metabolism of mouse spermatozoa and
emphasize its importance in fertilization. We have shown
1) that mouse epididymal spermatozoa have a functional
PPP, and 2) that NADPH can substitute for glucose in gamete fusion. These results strongly indicate that sperm need
to generate NADPH via the PPP in order to achieve fertilization.
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
14CO produced from [1-14C]glucose and [6-14C]glucose is
2
not appropriate, and the absence of a difference in yields
TABLE 2. Restoration of the fertilizing ability of sperm by NADPH and glucose after preincubation in glucose-free medium.
Treatmenta
No glucose
NADPH
Glucose
a
b
c
Sperm preincubation time
(min)
No. of
oocytes
No. of
replicates
60
15
60
60
78
77
71
73
5
5
5
5
No. of bound
sperm per oocyteb
16.3
8.7
7.6
8.0
6
6
6
6
6.4
3.8
2.0
3.6
No. of decondensed
sperm per penetrated
oocyteb
1.0
1.5
1.2
1.9
6
6
6
6
All sperm suspensions were incubated for 1 h in glucose-free medium before the addition of NADPH or glucose.
Mean 6 SD.
Significantly different from the control without glucose (p , 0.05).
0
0.7
0.4
0.9
Penetration rate
(%)b
7
78
53
94
6
6
6
6
4
33c
38
5c
GLUCOSE METABOLISM DURING GAMETE FUSION
of 14CO2 does not prove that the PPP is absent or inactive
[30–32].
14CO can be released from [1-14C]glucose by metabo2
lism 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
14CO released by the Krebs cycle is necessarily negligible
2
[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 [614C]glucose. Therefore, the 14CO measured after incuba2
tion 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
737
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
738
URNER AND SAKKAS
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.
ACKNOWLEDGMENTS
23.
24.
25.
26.
We gratefully acknowledge the assistance of Dr. Greet Leppens-Luisier,
and we would like to thank Prof. John Aitken for comments.
27.
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