From the Center for Research on Reproduction and Women's Health, Biomedical Research Building II/III, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6142
Received for publication, July 13, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Spermatozoa are highly polarized cells with
specific metabolic pathways compartmentalized in different regions.
Previously, we hypothesized that glycolysis is organized in the fibrous
sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a
recent report suggested that glucose is not essential for murine sperm
capacitation, we demonstrated that glucose (but not lactate or
pyruvate) was necessary and sufficient to support the protein tyrosine
phosphorylation events associated with capacitation. The effect of
glucose on this signaling pathway was downstream of cAMP, and appeared
to arise indirectly as a consequence of metabolism as opposed to a
direct signaling effect. Moreover, the phosphorylation events were not
affected by uncouplers of oxidative respiration, inhibitors of electron
transfer, or by a lack of substrates for oxidative respiration in the
medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either
at the level of substrate availability or by downstream enzymes. These
data support the hypothesis that ATP specifically produced by a
compartmentalized glycolytic pathway in the principal piece of the
flagellum, as opposed to ATP generated by mitochondria in the
mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system
for the study of integration of compartmentalized metabolic and
signaling pathways.
Mammalian spermatozoa are highly differentiated cells that
display extreme polarization of cellular architecture and function. For
example, the sperm head has evolved to interact with the egg's extracellular matrix and plasma membrane, and contains the paternal genetic material, whereas the sperm flagellum acts to provide motility
for these cells. In regard to this polarization of function, sperm have
two major constraints. First, they have little cytoplasm, and therefore
have a reduced ability to translocate metabolic intermediates or
substrates from one region to another. In addition, they are
transcriptionally inactive, and therefore cannot make new proteins in
response to changing needs. To overcome these constraints, we, along
with others, have hypothesized that sperm possess compartmentalized
metabolic and signaling pathways in specific regions of the cell poised
to function in a localized fashion (1-4).
The most obvious example of metabolic compartmentalization in
spermatozoa is that of oxidative respiration. This pathway is restricted to the mid-piece of the flagellum, because mitochondria are
located solely in this region. Oxidative respiration provides the most
efficient generation of ATP, yet the major sites of ATP consumption in
sperm are the dynein ATPases found associated with the axoneme
throughout the entire length of the flagellum (5). In some species such
as the sea urchin, a phosphorylcreatine shuttle has been observed to
transfer high-energy phosphate from the mitochondria to the principal
piece (6). However, in most mammalian species, this system either is
poorly developed or is absent entirely (see Kaldis et al.
(7) for a review). How then do the principal piece and end-piece of the
flagellum meet their energy needs?
We, along with others, have hypothesized that glycolysis is
compartmentalized in a cytoskeletal element in the principal piece of
the flagellum known as the "fibrous sheath"
(FS).1 This structure would
then be able to provide localized ATP production to the dynein ATPases
along the entire length of the principal piece of the flagellum (3, 4,
8). This theory was first suggested following observations that the
enzymes of glycolysis downstream from aldolase are all associated with
a cytoskeletal structure of the rabbit sperm flagellum (2). It was
later demonstrated that one of these enzymes,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localizes to the FS
in several mammalian species (3, 9). Concurrently, a germ cell-specific
isoform of type 1 hexokinase (HK1-SC), the first enzyme in glycolysis,
was also found to target to the FS (4, 8). This localization
demonstrated for the first time that the proximal portion of the
glycolytic pathway was also associated with the FS.
In addition to tethering enzymes of glycolysis, the FS can also act as
a scaffold for signaling molecules involved in the control of motility.
A major component of the FS has been identified as an
A-kinase anchoring
protein (AKAP) (10). This protein, AKAP4, has been shown to
bind protein kinase A (10), which plays a role in many of the
phosphorylation events postulated to govern motility (11). Therefore,
glycolysis organized in the FS could be used not only to provide
motility by generating ATP for the dynein ATPases, but also to help
regulate motility by providing ATP to protein kinases (4).
Changes in sperm motility occur not only within the epididymis as sperm
acquire motility, but also within the female reproductive tract as
sperm mature functionally and become competent to fertilize an egg.
This process, known as "capacitation," is associated with the
activation of a novel signal transduction cascade that includes the
efflux of cholesterol from membranes, alterations in membrane phospholipid content, alterations in intracellular calcium and bicarbonate ions, alterations in membrane potential, and elevations in
cAMP that ultimately result in the downstream phosphorylation of a
number of proteins on tyrosine residues (see Kopf et al. (12) for a review). It has long been recognized that capacitated sperm
display increased energy demands (13), presumably to effect these
changes in sperm signaling and function. However, the relationship between the signaling events associated with capacitation and changes
in sperm energy metabolism is poorly understood, despite the
co-compartmentalization of these pathways in regions such as the
FS.
Several studies using different model systems have been published on
the role of glucose in sperm capacitation and fertilization. In some
species (e.g. the mouse and human), glucose is necessary for
capacitation, the onset of hyperactivated motility, and acrosomal exocytosis (14-17). Contrary to the stimulatory role seen in the mouse
and human, glucose inhibits capacitation in the bull (18) and guinea
pig (19). These species differences might be related to the fact that
glucose can have other effects on sperm unrelated to the generation of
ATP. For example, glycolysis might affect sperm function by regulating
intracellular pH (18). Alternatively, glucose might function as a
signaling molecule by acting on regulatory enzymes such as cyclic
nucleotide phosphodiesterases (20). Finally, glucose might be used in
alternative metabolic pathways such as the pentose phosphate pathway
(PPP).
A possible role for the PPP in capacitation/fertilization is pertinent
to a discussion of metabolic compartmentalization in sperm in that we
have hypothesized that this pathway might be localized to the mid-piece
and/or sperm head (4). Although GAPDH is found in the FS, it and most
other glycolytic enzymes have not been found elsewhere in the sperm.
Yet the same germ cell-specific HK found in the FS, HK1-SC, is also
found in the mid-piece and head (4). The presence of HK in compartments lacking GAPDH suggests that HK might be supplying glucose 6-phosphate (Glu-6-P) to a pathway other than glycolysis, such as the PPP, in these
regions. In the first step unique to this pathway, glucose-6-phosphate dehydrogenase (G6PDH) produces an alternative type of energy, reducing
power in the form of NADPH. Of interest, reducing power produced from
glucose metabolism has been implicated in several steps of
fertilization including fusion of the sperm with the egg and sperm head
decondensation (21-23).
A prime candidate to regulate glucose metabolism in sperm is HK1-SC.
HK1 enzymatic activity is altered during male germ cell development in
the rat (24), and during epididymal maturation in the bull (25).
Because HK functions to generate Glu-6-P, and this intermediate can be
used by both glycolysis and the PPP, HK1-SC is in an ideal position to
regulate two pathways of glucose metabolism. Such regulation during
sperm capacitation might occur at several levels. For example, enzyme
activities might vary absolutely, or shift in subcellular or
biochemical localization (e.g. shift from a particulate to a
soluble phase). Alternatively, substrate availability might be altered,
or pathway inhibition might be regulated by the consumption/removal of
end products.
The highly differentiated structure of sperm, the co-localization of
metabolic and signaling pathways to specific structures, and the
importance of these pathways in sperm function, make these cells a
potentially valuable model for studying the cross-talk between
metabolic and signaling pathways. In the present report, we have
therefore investigated the functional relationships between metabolic
and signaling pathways during murine sperm capacitation.
Reagents and Media--
All reagents were purchased from Sigma,
unless otherwise noted. A polyclonal antiserum against HK type 2 ( Collection and Capacitation of Spermatozoa--
Cauda epididymal
sperm were collected from retired breeder CD1 mice (Charles River,
Wilmington, MA) by a swim-out procedure in 2 ml of ModW at 37 °C.
Epididymal tissue was removed, and the sperm washed at 100 × g for 1 min in a clinical centrifuge to remove any gross
tissue debris. The sperm were resuspended in a final volume of 3-8 ml
of ModW, and then centrifuged at 500 × g for 8 min in
a round-bottomed tube. The resultant "fluffy" pellet was counted,
assessed for motility, and diluted for use. In all cases, large-bore
plastic transfer pipettes or large-orifice pipette tips were used to
minimize damage to the sperm membranes.
After collection and washing, sperm were incubated under
noncapacitating (ModW medium alone) or capacitating conditions (ModW with 10 mM NaHCO3, and either 3 mM
2-hydroxypropyl- Electrophoresis and Immunoblotting--
Protein extracts from
spermatozoa were prepared by boiling the cells in sample buffer with
Spectrophotometric Analysis of HK1-SC and G6PDH
Activity--
Spermatozoa were incubated for 1 h at 37 °C
under noncapacitating or capacitating conditions as described above.
After incubation, they were split into 4 treatment groups for different
methods of solubilization or permeabilization, with each sample
ultimately being resuspended in a final volume of 800 µl of either
ModW or ModW + 10 mM NaHCO3 depending upon the
capacitation status. Pellet and supernatant fractions were obtained
for each treatment group.
The first group was solubilized with 0.5% Triton X-100 in ModW (± NaHCO3) for 15 min at room temperature with occasional hand vortexing. The sperm were centrifuged for 2 min at 5000 × g, and the supernatant collected. The pellet was resuspended
and centrifuged again to remove any trace amounts of supernatant, and
then resuspended. The second group was referred to as "intact"
sperm, because they were merely washed by centrifugation and split into
supernatant and pellet fractions. These sperm were not truly intact,
because centrifugation has been shown to induce sublethal membrane
damage in human (32), and
murine2 sperm. In the third
group, sperm were concentrated by centrifugation at 3000 × g for 1 min and then resuspended in 790 µl of
reagent-grade water (Milli-Q, Millipore water purification system).
These "hypotonically-treated" sperm were incubated at room
temperature for 15 min with occasional hand vortexing, prior to
separation into pellet and supernatant fractions. The last group was
permeabilized with streptolysin-O (SLO) (33) (Murex Diagnostics
Limited, Dartford, United Kingdom) in ModW (± NaHCO3) at a
final concentration of 0.6 IU/106 sperm. These
"SLO-treated" sperm were incubated at 37 °C for 15 min. Again,
supernatant and pellet fractions were obtained by centrifugation.
After treatment, all of the resuspended pellet and supernatant
fractions were loaded into individual cuvettes for spectrophotometric determination of hexokinase (HK) activity. HK activity was measured by
means of a coupled enzyme reaction pathway that led to the reduction of
NADP+ to NADPH. The rate of this reduction was measured as
a change in absorbance at 365 and 395 nm using an American Instrument
Co. (AMINCO) dual wavelength spectrophotometer (Travenol Laboratories, Inc., Silver Springs, MD). The coupled reactions were as follows: glucose + ATP
Measurements of endogenous G6PDH activity were performed in a manner
similar to the above methods, except that only a single reaction, not a
coupled reaction pathway, was utilized. Exogenous NADP+ and
varying concentrations of Glu-6-P were added to the sperm extracts
(Triton X-100 treatment was utilized to provide maximal solubilization)
and reduction of the NADP+ was quantified. Unlike earlier
measurements of HK activity, additional glucose, CP, and CPK were
excluded to reduce de novo generation of Glu-6-P within the
cuvette. The Km and Vmax with
respect to Glu-6-P for G6PDH were deduced by quantifying G6PDH activity at various concentrations of Glu-6-P, and fitting the data to a
hyperbolic function.
Glucose Was Critical for Protein Tyrosine Phosphorylation Events
Associated with Capacitation--
Despite an extensive literature on
sperm metabolism, the basic question of which metabolic substrates are
required to support the maturational process of capacitation remains a
matter of controversy. Species-specific differences have further
complicated the issue, but even within the murine model system there is
some disagreement over the role of glucose. To determine potential
roles for glucose in supporting capacitation, we first determined
whether glucose was required for protein tyrosine phosphorylation
events associated with capacitation. Epididymal sperm were washed free
of glucose and then incubated under conditions that support
capacitation, with the inclusion of glucose, lactate, and pyruvate,
alone or in combination (Fig. 1). Using
these substrates at concentrations typically present in media
supporting capacitation, only glucose was found to be both necessary
and sufficient to support the full pattern of protein tyrosine
phosphorylation associated with capacitation (34, 35).
We next investigated the concentration of glucose required to support
this signaling pathway by incubating sperm with varying concentrations
of glucose (Fig. 2). Two different
methods of capacitation were employed to test whether glucose was
acting either upstream in the signaling pathway at the level of
cholesterol efflux (incubation with 2-OH-
These results suggested an absolute requirement for glucose in
supporting the protein tyrosine phosphorylation signaling pathway associated with capacitation. Yet they did not indicate whether the
effect of glucose was by a direct action on a signaling molecule, or
whether the effect was mediated by some product of glucose metabolism.
To resolve this distinction, sperm were incubated under capacitating
conditions in the absence of glucose, but in the presence of various
concentrations of a nonmetabolizable glucose analog, 2-DG (Fig.
3). This analog is phosphorylated by HK,
but will not enter glycolysis or the PPP. If 2-DG stimulated tyrosine phosphorylation to the same degree as glucose, then the effect on
signaling would result from cross-talk between the signaling pathway
leading to protein tyrosine phosphorylation and 1) the transporters
bringing 2-DG into the sperm, 2) HK which phosphorylates the 2-DG, or
3) the 2-DG itself. Any of these possibilities would suggest that
glucose might directly regulate signaling leading to protein tyrosine
phosphorylation. If the 2-DG did not stimulate the increase in protein
tyrosine phosphorylation, then the effect of glucose on signaling would
most likely result from some product or intermediate of glucose
metabolism.
Spermatozoa incubated with increasing concentrations of 2-DG did not
show an increase in protein tyrosine phosphorylation (Fig. 3),
suggesting that the effect of glucose on protein tyrosine phosphorylation in murine sperm probably resulted from a product of
glucose metabolism. Moreover, with concentrations of 2-DG >10 µM, a decrease in protein tyrosine phosphorylation was
observed, even below that seen in sperm incubated in the absence of
2-DG. This finding suggested that 2-DG could function as a
"phosphorylation sink," and that the phosphorylation events
associated with capacitation were not terminal. If these events had
been terminal, there would not have been a decrease in protein tyrosine
phosphorylation below that seen in the uncapacitated state. Thus the
protein substrates appeared to be alternately phosphorylated and
dephosphorylated during capacitation.
The failure of lactate and/or pyruvate to support signaling events
associated with capacitation suggested a minimal role for ATP produced
by the oxidation of these compounds in this cascade. However, these
experiments did not completely rule out input from oxidative
respiration, as ATP may also be derived from other endogenous sources
such as fatty acid oxidation. To eliminate any ATP production from the
mitochondria, sperm were incubated with uncouplers of oxidative
phosphorylation and inhibitors of electron transfer, alone and in
combination (Fig. 4). Regardless of the
presence of these compounds, the pattern of protein tyrosine
phosphorylation was not decreased so long as glucose was present in the
medium. These experiments were also performed using a ModW medium
lacking lactate and pyruvate (with 2.4 mM CaCl2
added to compensate for the removal of the hemi-calcium salt). Results
were identical to those shown in Fig. 4 (data not shown).
Quantitation of Sperm HK and G6PDH Activities during
Capacitation--
These data confirmed a critical role for ATP derived
from glycolysis as opposed to oxidative respiration to support
signaling events associated with capacitation. A prime candidate for
regulating glucose metabolism in sperm is HK, because of its upstream
position in glycolysis and the PPP. To investigate whether HK activity was regulated during capacitation in murine sperm, the first step was
to ensure that only one type of HK was present. The germ cell-specific HK found in murine spermatozoa has been identified as HK1-SC, and has
been localized to the FS of the flagellum, as well as the mitochondria
and the membranes of the sperm head (4). No evidence for the somatic
HK1 has been found in these cells (4). However, other HK family members
also exist, and the first studies to document the existence of a germ
cell-specific HK suggested that HK2 might be found associated with male
reproductive tissues (36). To rule out the possibility that HK2 might
also be found in spermatozoa, thereby complicating the interpretation
of data, sperm proteins were probed with an antiserum against HK2 (Fig. 5). No evidence for HK2 was found in
mature murine spermatozoa, although a positive control (cardiac tissue)
demonstrated the efficacy of the antiserum.
HK activity during capacitation could be regulated in two ways. The
first would be an absolute increase in overall activity, and the second
would be a biochemical shift in activity, for example, from an
insoluble form to a soluble form, as occurs in somatic cells as HK can
be bound to mitochondrial porins or may be found in the cytosol. To
investigate these possibilities, we incubated sperm under
noncapacitating or capacitating conditions and then subjected them to a
variety of treatments resulting in different degrees of
permeabilization or solubilization (Fig.
6). In no test group was overall HK
activity altered during the process of capacitation. Moreover, HK was
not seen to shift in regard to its solubility characteristics with
capacitation. These findings were true whether sperm were capacitated
with 2-OH-
Although HK enzymatic activity appeared to be equal in noncapacitated
and capacitated sperm, these activities might represent the action of
different amounts of protein in the supernatant and pellet fractions.
For example, half the amount of an enzyme that was twice as active
would give the same overall activity as twice the amount of enzyme that
was half as active. To rule out this possibility, amounts of HK protein
in the supernatant and pellet fractions from the different treatment
groups were quantified by immunoblotting and found not to vary in three
replicate experiments (data not shown). In addition, treatment groups
that were not subjected to solubilization with Triton X-100 had this detergent added after initial measurements were recorded, and then
additional measurements of HK activity were taken. Triton X-100
provided maximal solubilization and revealed approximately equivalent
amounts of HK activity in the appropriate fractions (data not shown).
These data suggested that HK activity neither was regulated with
capacitation, nor underwent a shift in solubility characteristics.
Finally, the Km with respect to glucose and
Vmax for HK1-SC were determined by
spectrophotometric analysis at 24 °C utilizing supernatant fractions
from sperm treated with Triton X-100. The Km was
equal to 35.4 ± 5.6 µM (S.D.) (n = 6) and the Vmax was equal to 3.93 ± 0.54 nmol/min/106 sperm (S.D.) (n = 6). Our
measurement of the Km is similar to the estimation
made by Katzen (36) of 100 µM for HK1 in spermatozoa, and
is very close to his quantification of 30 µM for the
Km of brain HK1. The high affinity and rate of HK
activity in sperm underscores the physiological importance of this
enzyme in the function of these cells.
Although exogenous G6PDH was added in excess in the previous
experiments as part of the coupled reaction pathways, endogenous G6PDH
represented the first enzyme specific to the PPP in the sperm. This
alternative pathway of glucose metabolism has been suggested to play a
role in murine fertilization by generating NADPH. The enzymatic
activity of endogenous G6PDH with respect to Glu-6-P was assessed
spectrophotometrically at 24 °C utilizing supernatant fractions from
noncapacitated sperm treated with Triton X-100. The
Km was equal to 5.4 ± 4.8 µM
(S.D.) (n = 3) and the Vmax was
equal to 0.069 ± 0.011 nmol/min/106 sperm (S.D.)
(n = 3). These values were obtained from noncapacitated sperm that were incubated and solubilized with Triton X-100 in the
absence of glucose. Analysis of G6PDH in sperm incubated under capacitating conditions did not reveal remarkable differences versus noncapacitated
sperm.3 However, strict
values for G6PDH activity in capacitated sperm are problematic because
capacitation requires the presence of glucose in the medium.
Determination of exact kinetic values for G6PDH was impossible given
the unknown intracellular concentration of Glu-6-P in the sperm, which
resulted from their incubation condition.
ATP Produced by Glycolysis Was Required for Signaling Events during
Murine Sperm Capacitation--
We have demonstrated that of the three
metabolic substrates (glucose, lactate, and pyruvate) commonly included
in media designed to support capacitation, only glucose was necessary
and sufficient to support the protein tyrosine phosphorylation
signaling events associated with capacitation. Moreover, glucose
concentrations as low as 10-100 µM were able to support
the same level of tyrosine phosphorylation as higher concentrations of
this sugar. Glucose could exert this effect on the signaling pathways
associated with capacitation in one of two ways. The first would be a
direct effect on a signaling molecule such as an adenylyl cyclase or a
cyclic nucleotide phosphodiesterase, whereas an alternative possibility would be an indirect effect through some intermediate or end product of
glucose metabolism. Regardless, the fact that the response to glucose
was identical whether capacitation was induced at the level of
cholesterol efflux, or at the level of cAMP, suggests that the effect
of glucose was downstream to the rise in cAMP associated with capacitation.
Recently, Redkar and Olds-Clarke (37) suggested that in the absence of
glucose, the binding of sperm to the plasma membrane of zona
pellucida-free eggs was significantly retarded, but still occurred. In
contrast to previous reports suggesting the importance of glucose in
sperm function (14-16), these authors inferred that glucose was not
required for stages of capacitation leading up to and including
acrosomal exocytosis (37). One methodological difference that might
explain the disparity between the results is that in the present study
sperm were washed free of glucose, whereas Redkar and Olds-Clarke (37)
utilized a swim-out method of sperm collection without any washes.
While presumably designed to avoid potential sublethal membrane damage
induced by centrifugation (32), their methodology did not ensure a
glucose-free condition; based on their methods the medium may have
actually contained glucose at concentrations
Incubation of sperm with the nonmetabolizable glucose analog, 2-DG, did
not support the pattern of protein tyrosine phosphorylation observed
with glucose. While it could be argued that 2-DG might not elicit an
identical signaling response as glucose, this possibility is unlikely
given the fact that this analog is brought into the cell through the
same transporters and with the same affinity as glucose (38). Rather,
these data suggest that the role of glucose in supporting protein
tyrosine phosphorylation was to produce a specific metabolite, or an
end product of metabolism such as ATP or NADPH. The phosphorylation
sink effect noted with higher concentrations of 2-DG also speaks
toward the importance of glucose metabolism in supporting these dynamic
post-translational modifications.
Intuitively, the most straightforward connection between glucose
metabolism and phosphorylation events is the production of ATP and its
subsequent utilization by protein kinases. To confirm that in sperm,
ATP produced by glycolysis is not equivalent to ATP produced by
oxidative respiration, we incubated sperm with uncouplers of oxidative
respiration. These compounds allow respiration to occur, but do not
allow the concomitant production of ATP (26, 29). Whether sperm were
incubated with uncouplers of oxidative respiration or inhibitors of
electron transfer, we observed no significant decreases in patterns of
protein tyrosine phosphorylation, suggesting that glycolysis provides
the ATP for these signaling events.
Interestingly, spermatozoa remained motile in the presence of
uncouplers of oxidative respiration, but had significant decreases in
percent motility when uncouplers and electron transfer inhibitors were
added together.5 Although
surprising, there is precedence in canine sperm for the maintenance of
motility in the presence of uncouplers of respiration (1). These
observations suggest in sperm that, unlike uncouplers which enhance
flux through glycolysis, inhibitors of electron transfer feedback
negatively on glycolysis, perhaps through the build-up of intracellular
lactate. In addition, it has been suggested that inhibitors such as
antimycin A might have effects on sperm motility distinct from
inhibiting electron transfer (1). Although the spermatozoa remained
visibly motile in the presence of the uncouplers 1799 and
pentachlorophenol, it remains unclear whether the mitochondria have
some input into the pattern or frequency of flagellar motility.
Differentiating the input from the various compartmentalized metabolic
pathways in sperm motility will form the basis of future investigations.
The phosphorylation of individual proteins on tyrosine residues has
been correlated with the onset of hyperactivated motility in hamster
sperm (39). The link between signaling events and changes in motility
is further strengthened by the fact that most all of the
tyrosine-phosphorylated proteins identified thus far localize to the
sperm flagellum (39, 40). Taken together, these findings support the
hypothesis offered by Travis et al. (4), that a
glycolytic pathway organized in the FS would be positioned not only to
supply ATP to the flagellar ATPases, but also to supply ATP to the
protein kinases believed to be involved in the regulation of
capacitation and motility. These results suggest a functional
relationship between the glycolytic and signaling pathways co-localized
to the FS.
Hexokinase Activity Was Unchanged during Murine Sperm
Capacitation--
Given the importance of glycolysis in sperm
function, and the need to regulate changes in energy production as
sperm mature, it becomes important to investigate potential regulators
of sperm glucose metabolism. HK generates Glu-6-P from glucose,
supplying this substrate to both glycolysis and the PPP. Because both
of these pathways have been suggested to be of importance in sperm function, and because HK activity is regulated at different stages of
male germ cell development (24, 25), we investigated whether HK
activity was regulated with capacitation. A potential problem with
several earlier studies on enzymatic activities in sperm is that these
activities were only tested in soluble extracts or homogenates (41,
42). Given the cytoskeletal attachment of the glycolytic enzymes and
the close apposition of the overlying plasma membrane, this methodology
might produce nonphysiologic results as the enzymes are removed from
the influence of potential regulators. Therefore, we quantified HK
activity in noncapacitated and capacitated sperm that were
permeabilized or solubilized to varying degrees.
Despite these precautions, no change in HK activity or solubility was
noted with capacitation. The value for the Km of
HK1-SC of 30 µM was similar to the estimates made by
Katzen (36) for both the germ cell-specific and brain HK1. The HK
activity detected was high, and would provide rapid phosphorylation of glucose as it was taken into the cell. This reaction prevents loss of
the Glu-6-P from the cell by means of the negative charge on the sugar
phosphate group.
The Vmax for G6PDH, the first enzyme specific to
the PPP, suggests an important role for this enzyme in murine sperm.
Standardization of units to 106 cells allows a direct
comparison of Vmax for G6PDH in human and murine
sperm. The Vmax for G6PDH in human spermatozoa,
0.012 ± 0.002 nmol/min/106 cells (43), is several
times lower than our calculation of Vmax for
G6PDH in murine sperm, 0.069 ± 0.011 nmol/min/106
cells. Most likely, this difference resulted from increased activity or
increased protein abundance in the mouse. If the G6PDH is functioning in the sperm head, this difference in activity might reflect the fact
that the murine sperm head is larger than the human sperm head.
Regardless, these values corroborate the suggested important role for
this alternative pathway of glucose metabolism in murine sperm function
(21-23).
The highly polarized structure and function of mammalian sperm dictates
that these cells compartmentalize specific metabolic and signaling
pathways to regions where they are needed. These pathways must be
integrated to support normal cellular function. This inter-relationship
underscores the fact that in these cells, metabolic pathways often
regarded as "housekeeping" in nature cannot be dissociated from
very specific changes in maturation and function. We propose to extend
the use of spermatozoa as a model system not only to study
compartmentalized metabolic pathways, but also to study the
relationship between signaling and metabolic pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HK2) was generously provided by Dr. John Wilson (Michigan State
University). Anti-phosphotyrosine antibodies (
-PY, clone 4G10) were
purchased from Upstate Biotechnology (Lake Placid, NY), and used at a
1:10,000 dilution for immunoblots. A polyclonal antiserum against the
germ cell-specific domain of HK1-SC (
-qcs) had been generated
previously and was used as described by Travis et al. (4).
Compound 1799, an uncoupler of oxidative respiration, was generously
provided by Dr. Peter Heytler (E. I. duPont Nemours & Co.,
Wilmington, DE) (26). A modified Whitten's medium (ModW) (22 mM HEPES, 1.2 mM MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic
acid, 4.8 mM lactic acid hemi-calcium salt, pH 7.3) was
used for most assays involving mature spermatozoa, except where noted.
This medium was designed to support sperm functions yet not interfere
with spectrophotometric assays.
-cyclodextrin (2-OH-
-CD) or 1.0 mM
dibutyryl-cAMP and 0.1 mM IBMX) for 1 h in a 37 °C
water bath at a final concentration of 2 × 106
sperm/300 µl. In place of bovine serum albumin, 2-OH-
-CD was used
as a cholesterol acceptor so as to use a completely defined medium
(27). Depending upon the experiment, the sperm were incubated with
varying concentrations of glucose, lactate, pyruvate, and 2-deoxyglucose (2-DG) as metabolic substrates. Uncouplers of oxidative respiration and inhibitors of electron transfer were used at or above
concentrations used in previously published experiments with
spermatozoa (28-30). For experiments in which protein tyrosine phosphorylation was assessed, sperm were concentrated by centrifugation at 10,000 × g and then washed in 1 ml of ModW
containing 0.2 mM Na3VO4 to inhibit
phosphatase activity during extraction.
-mercaptoethanol (31), and then centrifuging at 10,000 × g to yield a supernatant fraction. Cardiac tissue was
obtained from male CD1 mice, minced with dissecting scissors, and
homogenized in a Teflon/glass homogenizer 20 times on ice. Sample
buffer was added to the extract, which was sonicated on ice prior to
boiling and centrifugation to yield a soluble fraction. Proteins were
separated under reducing conditions by SDS-polyacrylamide gel
electrophoresis using 10% polyacrylamide gels (31), and transferred to
Immobilon-P membranes (Millipore, Bedford, MA). Blocking,
immunoblotting, and detection of the immunoreactive proteins by
chemiluminescence (Renaissance, PerkinElmer Life Sciences, Boston, MA)
were performed as described in Travis et al. (4).
Glu-6-P + ADP and Glu-6-P + NADP+
NADPH + 6-phospho-glucono-
-lactone, with the first reaction catalyzed by endogenous HK, and the second catalyzed by exogenous G6PDH. These reactions were carried out using an ATP-regenerating system of creatine phosphate (CP) and creatine phosphokinase (CPK). Exogenous reagents (with accompanying final concentrations in parentheses) were added in the following order: glucose (5 mM), CP (20 mM), CPK (20 IU/ml),
NADP+ (0.1 mM), G6PDH (7 IU/ml), and ATP (1 mM). Measurements of activity were determined from slopes
taken from within the linear range, and were recorded using two
different gain settings to provide optimal resolution. After recording
enzyme activity, the samples not treated with Triton X-100 had this
detergent added to a final concentration of 0.5%, to effect complete
solubilization of the HK and obtain a maximal reading from each sample.
This procedure was used as an internal control for equal loading of
extracts into the cuvettes. In addition, this step demonstrated whether identical amounts of HK were either solubilized or retained in the
particulate fraction under noncapacitating and capacitating conditions.
The Km and Vmax with respect
to glucose for the germ cell-specific HK1-SC were deduced by
quantifying HK activity at various concentrations of glucose, and
fitting the data to a hyperbolic function.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of glucose, lactate, and pyruvate on
protein tyrosine phosphorylation during murine sperm capacitation.
Spermatozoa were incubated under capacitating conditions (10 mM NaHCO3 and 3 mM 2-OH- -CD), in
the presence of lactate (4.8 mM) (L), pyruvate
(1 mM) (P), and glucose (5.5 mM)
(G), alone or in combination. Proteins were then extracted,
separated by electrophoresis, transferred to Immobilon P membranes, and
immunoblotted with anti-phosphotyrosine (
-PY). A control
("
"), with no metabolic substrate, revealed a minimal pattern of
protein tyrosine phosphorylation. The one phosphorylated band
visualized in this lane represented HK1-SC, which is constitutively
phosphorylated on these residues in mature sperm (44), and can thereby
function as a loading control. All immunoblots presented in this paper
were performed with sperm extracts made from CD-1 males, and are
representative examples of experiments that were performed a minimum of
three times with similar results.
-CD) (27), or downstream at
the level of the second messenger cAMP (incubation with the
cell-permeable dibutyryl-cAMP and IBMX) (35). Regardless of the method
of capacitation, a glucose concentration between 10 and 100 µM was sufficient to support the same degree of protein
tyrosine phosphorylation as that seen with 5 mM glucose in
ModW. Similar findings were observed using sperm from both CD-1 and
B6SJLF1/J males, showing that the phenomenon was not strain-specific
(data not shown).
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Fig. 2.
Effects of varying concentrations of glucose
on patterns of protein tyrosine phosphorylation during murine sperm
capacitation. Murine spermatozoa were incubated either under
noncapacitating conditions ( ), or two different conditions that would
support capacitation (10 mM NaHCO3 and 3 mM 2-OH-
-CD; or 10 mM NaHCO3,
1.0 mM dibutyryl-cAMP, and 0.1 mM IBMX), with
varying concentrations of glucose. Extracts were made from the sperm
under reducing conditions, separated by electrophoresis, transferred to
a membrane, and immunoblotted with anti-phosphotyrosine antibodies
(
-PY). The results obtained with this experiment were
identical, whether sperm were obtained from CD-1 or B6SJLF1/J
males.
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Fig. 3.
Effects of varying concentrations of 2-DG on
patterns of protein tyrosine phosphorylation during murine sperm
capacitation. Spermatozoa were incubated either under
noncapacitating conditions ( ), or capacitating conditions (10 mM NaHCO3 and 3 mM 2-OH-
-CD),
with varying concentrations of 2-DG. Protein extracts were made from
the sperm under reducing conditions, separated by electrophoresis,
transferred to a membrane, and immunoblotted with
-PY
(
-PY).
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Fig. 4.
Effects of uncouplers of oxidative
respiration and inhibitors of electron transfer on patterns of protein
tyrosine phosphorylation during murine sperm capacitation.
Spermatozoa were incubated either under noncapacitating conditions
("N") or capacitating conditions ("C")
(10 mM NaHCO3 and 3 mM
2-OH- -CD), with: A, no other reagents added;
B, 20 µM 1799 and 20 µM
pentachlorophenol; C, 4 µM antimycin A and 2 µg/µl oligomycin; D, 20 µM 1799, 20 µM pentachlorophenol, 4 µM antimycin A, and
2 µg/µl oligomycin. Control samples with 0.5% (v/v) dimethyl
sulfoxide and 0.5% (v/v) dimethyl formamide (used to solubilize the
uncouplers and inhibitors), showed no difference from the experimental
samples (data not shown). Protein extracts were made from the sperm
under reducing conditions, separated by electrophoresis, transferred to
a membrane, and immunoblotted with
-PY.
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Fig. 5.
Immunoblotting of murine spermatozoa and
cardiac tissue with antisera to hexokinase type 2 and the germ
cell-specific domain of HK1-SC. Extracts (50 µg each) of murine
spermatozoa (S), and murine cardiac tissue (H)
were separated by electrophoresis, transferred to a membrane, and
immunoblotted with -HK2 ("
-HK2") or the
-germ
cell-specific domain of HK1-SC (
-gcs).
-CD, or with dibutyryl-cAMP and IBMX (Fig. 6).
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Fig. 6.
Hexokinase enzyme activity in noncapacitated
and capacitated sperm prepared under a variety of extraction
conditions. HK enzyme activity was not observed to vary in a
statistically significant fashion whether sperm were incubated under
noncapacitating conditions (white bars), in the presence of
3 mM 2-OH- -CD and 10 mM NaHCO3
(diagonally hatched bars), or 1 mM
dibutyryl-cAMP, 0.1 mM IBMX, and 10 mM
NaHCO3 (black bars). This result was true for
both supernatant (S) and particulate (P)
fractions, whether the sperm were extracted with Triton X-100
(TX-100), washed with ModW (intact), incubated in
a hypotonic medium (hypotonic), or permeabilized with SLO
(SLO). Note that the extremely low values for the hypotonic
supernatants resulted as both an artifact of their preparation (they
were centrifuged to remove the original medium, thereby removing HK as
revealed in the "intact" supernatants) as well as the resistance of
murine sperm to hypotonic treatment (45). One-way analysis of variance
was employed to evaluate differences between values, with
p < 0.05 used as the criterion for significance.
Error bars denote S.E. of the mean. The JMP (version 3)
statistical package (JMP Statistical Visualization Software, SAS
Institute, Inc., Carey, NC) was used for these analyses.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 µM. In
light of our findings, even a concentration as low as this might have a
significant impact on signaling events associated with capacitation. In
the present study, care was taken to minimize membrane damage by the
use of large-bore transfer pipettes, low-speed centrifugations in
swinging-bucket rotors, and round-bottomed tubes. The ultimate
assessment of membrane damage in sperm is testing fertilizing ability,
and this end point was not affected by these
methods.4
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HD-33052 (to A. J. T., T. M., G. S. K., and S. B. M.), T32-HD-07305-15 (to A. J. T.), 1RO1-RR00188-01 (to A. J. T.), PO1-HD-06274 (to B. H. J., B. T. S., G. S. K., and S. B. M.), and RR-07065 (to D. M. D.), and by the Bourat Foundation (C. J. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 215-573-4782;
Fax: 215-573-7627; E-mail: smoss@mail.med.upenn.edu.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M006217200
2 A. J. Travis, S. B. Moss, and G. S. Kopf, unpublished observations.
3 A. J. Travis, B. H. Jones, D. M. Dess, B. T. Storey, S. B. Moss, and G. S. Kopf, unpublished observations.
4 A. J. Travis, C. J. Jorgez, S. B. Moss, G. S. Kopf, and C. J. Williams, unpublished observations.
5 A. J. Travis, B. H. Jones, D. M. Dess, S. B. Moss, and G. S. Kopf, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
FS, fibrous sheath;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HK1, type 1 hexokinase;
AKAP, A-kinase anchoring protein;
PPP, pentose phosphate
pathway;
Glu-6-P, glucose 6-phosphate;
G6PDH, glucose-6-phosphate
dehydrogenase;
-HK2, antiserum against type 2 hexokinase;
-PY, antiserum against phosphotyrosine residues;
ModW, modified Whitten's
medium;
2-OH-
-CD, 2-hydroxypropyl-
-cyclodextrin;
2-DG, 2-deoxyglucose;
SLO, streptolysin-O;
CP, creatine phosphate;
CPK, creatine phosphokinase;
IBMX, isobutylmethylxanthine.
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REFERENCES |
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