BIOLOGY OF REPRODUCTION (2016) 94(4):96, 1–9
Published online before print 16 March 2016.
DOI 10.1095/biolreprod.115.137752
Energy Utilization for Survival and Fertilization—Parsimonious Quiescent Sperm Turn
Extravagant on Motility Activation in Rat1
Lokesh Kumar,3 Santosh K. Yadav,3 Bhavana Kushwaha,3 Aastha Pandey,3 Vikas Sharma,3 Vikas Verma,3
Jagdamba P. Maikhuri,3 Singh Rajender,3 Vishnu L. Sharma,4 and Gopal Gupta2,3
3
4
Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India
Division of Medicinal & Process Chemistry, CSIR-Central Drug Research Institute, Lucknow, India
Quiescent sperm survive in cauda epididymis for long periods
of time under extreme crowding conditions and with a very
limited energy substrate, while after ejaculation, motile sperm
live for a much shorter period with an unlimited energy resource
and without crowding. Thus, the energy metabolism in relation
to the energy requirement of the two may be quite different. A
simple physiological technique was evolved to collect viable
quiescent sperm from rat cauda epididymis to compare its
energy metabolism with motile sperm. Quiescent sperm
exhibited 40%–60% higher activities of mitochondrial electron
transport chain complexes I–IV and ATP synthase in comparison
to motile sperm and accumulated Ca2+ in the midpiece
mitochondria to enhance oxidative phosphorylation (OxPhos).
In contrast, motile sperm displayed up to 75% higher activities
of key glycolytic enzymes and secreted more than two times the
lactate than quiescent sperm. Quiescent sperm phosphorylated
AMPK and MAPK-p38, while motile sperm phosphorylated AKT
and MAPK/ERK. Glycolytic inhibitor iodoacetamide prevented
motility activation of quiescent rat sperm and inhibited
conception in rabbits more effectively than OxPhos uncoupler
2,4-dinitrophenol. Apparently, quiescent sperm employ the most
energy efficient OxPhos to survive for extended periods of time
under extreme conditions of nutrition and crowding. However,
on motility initiation, sperm switch predominantly to glycolysis
to cater to their high- and quick-energy requirement of much
shorter periods. This study also presents a proof of concept for
targeting sperm energy metabolism for contraception.
epididymis, gamete biology, metabolism, sperm, sperm motility
and transport
INTRODUCTION
The sperm are specialized cells produced in the testis by
spermatogenesis and pass sequentially through the epididymis
for maturation in a tightly regulated manner [1]. In most
mammals, including rat and human, completely mature sperm
1
Supported by research fellowships from the University Grants
Commission (L.K., V.S.), the Council of Scientific and Industrial
Research (S.K.Y.), and the Indian Council of Medical Research (B.K.,
A.P.), New Delhi, India. This study was supported by the CSIR Network
Project ‘‘PROGRAM’’ (BSC0101). CDRI Communication Number 9200.
2
Correspondence: E-mail: [email protected]
Received: 12 December 2015.
First decision: 12 January 2016.
Accepted: 8 March 2016.
Ó 2016 by the Society for the Study of Reproduction, Inc. This article is
available under a Creative Commons License 4.0 (Attribution-NonCommercial), as described at http://creativecommons.org/licenses/by-nc/
4.0
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
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with the full potential to exhibit vigorous motility are stored
quiescently in the cauda epididymis for periods exceeding 30
days to conserve energy. Sperm motility initiation is coincident
with ejaculation, when epididymal semen is diluted with
seminal plasma containing a special recipe of proteins/
enzymes, buffering agents, redox regulators, and energy
substrates. Cauda epididymis provides a special milieu to
ensure a maximal survival period of potentially motile
quiescent spermatozoa. Quiescence is nature’s unique process
of keeping the sperm viable for relatively long periods of time
with their vital functions retained [2] and ensures the supply of
millions of vigorously motile sperm during ejaculation for
fertilization. Various factors released in cauda epididymis and
some intrinsic features may be responsible for keeping sperm
quiescent [3]. During passage of testicular semen from the
caput to cauda epididymis, ;90% of water is absorbed, which
results in a large number of sperm being stored and crowded
tightly in the smallest feasible space [4]. On the contrary,
during ejaculation, a variety of fluids rich in energy substrate
(fructose) are added to dilute the epididymal semen very
significantly. Motility initiation takes place at ejaculation in the
female reproductive tract, where sperm survive for just a few
days. Thus, highly crowded quiescent sperm with a very
limited energy resource survive much longer than the
uncrowded, highly motile sperm with an unlimited energy
resource, and therefore the former may have a very different
energy metabolism than the latter.
The mammalian sperm is fully equipped to carry out both
glycolysis and oxidative phosphorylation (OxPhos) to produce
ATP [5]. Mitochondrial OxPhos in the sperm midpiece can
produce ATP much more efficiently than glycolysis occurring
along the entire sperm tail. However, there is doubt that
sufficient ATP can diffuse from the sperm midpiece to the
distal segments of the sperm tail for vigorous motility [6].
Recent studies indicate that sperm may rely on glycolysis for
generating the required energy for motility [7]. This view is
supported by the fact that sperm motility is sustained in
medium containing OxPhos inhibitors antimycin-A and
rotenone [8] and that OxPhos uncoupler carbonyl cyanide 3chlorophenylhydrazone (CCCP) could reduce sperm mitochondrial membrane potential without affecting sperm motility/
velocity [9]. On the other hand, in the absence of glucose,
mouse sperm could sustain motility in pyruvate-containing
medium but not in the presence of CCCP [1]. Most of the
published literature highlights the importance of glycolysis in
sperm motility [10, 11], but the subtle role of OxPhos cannot
be ruled out [5]. Energy metabolism of motile sperm is well
studied [1], but our knowledge of quiescent sperm metabolism
is very modest, including the changes associated with motility
activation. A major hurdle has been the unavailability of
quiescent sperm since it tends to gain motility on isolation in
any isotonic medium; nevertheless, limited success was
ABSTRACT
KUMAR ET AL.
[16]. Reaction mixture (200 ll) contained potassium phosphate buffer (25 mM,
pH 7.5), oxidized cytochrome c (75 lM), sodium azide (40 mM), EDTA (100
lM), Tween-20 (0.025% v/v), and sample (10–30 lg protein). The reaction
was started by adding decylubiquinol (100 lM), mixing rapidly, and measuring
the increase in absorbance at 550 nm for 1–2 min.
Complex IV (cytochrome c oxidase, EC 1.9.3.1). Complex IV was
assayed by monitoring the oxidation of cytochrome c as described by Spinazzi
et al. [16] with slight modification. The final reaction mixture (200 ll)
contained potassium phosphate buffer (25 mM, pH 7.0) and reduced
cytochrome c (50 lM). The reaction was initiated by addition of reduced
cytochrome c. Oxidation of cytochrome c was monitored spectrophotometrically at 550 nm for 5 min.
achieved by Nakamura et al. [12]. A serious review of the
literature on this subject indicates that sperm motility initiation
is basically a biochemical event involving several signaling and
metabolic enzymes, with minor effects of physicochemical
parameters, such as viscosity and pH [3]. As enzymes undergo
reversible inactivation at low temperatures, we isolated the
quiescent caudal sperm in a medium at 08C–28C to prevent
them from gaining motility. This first study reports the energy
metabolism of quiescent sperm in comparison to motile sperm
in rat. A proof of concept has also been presented to indicate
the vulnerability of sperm energy metabolism to chemical
interference for contraception.
Citrate Synthase Assay
MATERIALS AND METHODS
Citrate synthase, the mitochondrial matrix enzyme, was assayed according
to Spinazzi et al. [16] and was used to normalize the results of the assays of
respiratory chain enzymes. The final reaction assay mixture (200 ll) contained
Tris (100 mM, pH 8.0), Triton X-100 (0.1%), DTNB (100 lM), Ac-CoA (300
lM), and 10 ll of sample. Reaction was initiated by adding oxaloacetic acid
(500 lM), and the increase in absorbance at 412 nm was monitored for 3 min.
A unit of activity is defined as lM/min/mg protein.
Chemicals and Reagents
Hanks Balanced Salt Solution (HBSS) and all other chemicals/reagents
were from Sigma Aldrich unless stated otherwise. The lactate estimation kit
was purchased from Cayman Chemicals, and the calcium detection kit was
purchased from Enzo Life Sciences.
ATP Synthase Assay
Sperm Isolation and Sample Preparation
Qualitative Analysis of Calcium Content
Qualitative analysis of calcium content in quiescent and motile rat sperm
was made by labeling with the fluorometric calcium detector using a calcium
detection assay kit (Enzo Life Sciences). Sperm samples were incubated with
calcium detector for 30 min at 378C as per the instructions provided in the kit
manual. Thereafter, sperm samples were washed with dilution buffer and
pelleted at 700 3 g at 258C and resuspended in dilution buffer. A small drop of
diluted sample was taken on a clean glass slide, covered with a cover glass, and
imaged using a Nikon Eclipse 80i microscope equipped with epifluorescence
illumination. Exposure time was the same for all the samples.
Estimation of Rate-Limiting Glycolytic Enzyme Activities
Enzyme activities of hexokinase (EC 2.7.1.1), phosphofructokinase (EC
2.7.1.11), and pyruvate kinase (EC 2.7.1.40), which catalyze irreversible, ratelimiting kinase (phosphorylation) reactions in glycolysis, were measured by
coupled enzyme assays in a spectrophotometer using KC Junior software (BioTek). Washed sperm were incubated in RIPA buffer (with 13 protease and
phosphatase inhibitor cocktail) at 48C for 2–3 h and then sonicated on ice.
Sonicated samples were centrifuged at 14 000 rpm for 20 min at 48C, and the
supernatant was used for the enzyme assays. Hexokinase (HK) activity was
assayed according to Nakamura et al. [12] in a reaction mixture (150 ll)
containing 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 4 mM EDTA, 1.0 unit/ml
G6PDH, 10 mM glucose, 0.6 mM b-NADP, and 0.1% Triton X-100. The
reaction was initiated by adding 4 mM ATP, and NADPH production was
measured at 340 nm by spectrophotometry. Phosphofructokinase (PFK) activity
was assayed in a final reaction volume of 150 ll containing 100 mM Tris-HCl
(pH 7.5), 7 mM MgCl2, 0.3 mM NADH, 1 mM phosphoenolpyruvate, 2 mM
ATP, 0.5 M KCl, 2 mM fructose-6-phosphate, 1.65 U LDH, and 2 U pyruvate
kinase [18]. Pyruvate kinase (PK) was assayed according to Bergmeyer [19] in
a reaction volume of 150 ll containing 39 mM potassium phosphate (pH 7.9),
0.58 mM phosphoenolpyruvate, 0.11 mM b-NADH, 6.8 mM MgCl2, 1.5 mM
ADP, and 10 units LDH. About 15–30 lg of sperm protein were used for all
glycolytic enzyme assays, and the quantification of protein was done by
Bradford assay. A unit of activity is defined as lM/min/mg protein.
Activity of Respiratory Complexes
Quiescent and motile sperm samples were pelleted and incubated in lysis
buffer (Tris 50 mM with 0.01% n-dodecyl-b maltoside) for 2 h at 48C and
sonicated on ice (25% amplitude, four pulses of 10 sec). After sonication,
samples were centrifuged at 14 000 rpm at 48C for 20 min, and the supernatants
were used for enzyme assays. Protein was estimated by Bradford reagent. The
assays were performed using 15–30 lg of protein per enzyme reaction.
Activities of all complexes were estimated by a spectrophotometer using KC
Junior software (Bio-Tek). A unit of activity is defined as nM/min/mg protein.
Complex I (NADH-ubiquinone oxidoreductase, EC 1.6.5.3). Complex I
was estimated by monitoring oxidation of NADH using the ubiquinone analog
decylubiquinone as an electron acceptor by following the method of Tatarkova
et al. [15] with slight modification. The final assay reaction mixture (200 ll)
contained 35 mM KH2PO4 (pH 7.2), 5 mM MgCl2, 40 mM sodium azide, 5 lM
antimycin-A, 60 lM decylubiquinone, and 0.1 mM NADH. NADH oxidation
was followed spectrophotometrically at 340 nm.
Complex II (succinate-ubiquinone oxidoreductase, EC 1.3.5.1).
Complex II was assayed by following the conversion of succinate to fumarate
through the reduction of an artificial electron acceptor dichlorophenolindophenol (DCPIP) using the method of Spinazzi et al. [16] with slight modification.
The final reaction mixture (200 ll) contained 25 mM KH2PO4, 20 mM
succinate, 40 mM sodium azide, 80 lM DCPIP, and 0.1% BSA, pH 7.5.
Reaction was initiated by adding 50 lM decylubiquinone and the enzyme
reaction was followed for 3 min at 600 nm in a spectrophotometer.
Complex III (ubiquinol-cytochrome c reductase, EC 1.10.2.2).
Complex III assay was adapted from the modified protocol of Spinazzi et al.
Estimation of Extracellular Lactate/ECAR (Extracellular
Acidification Rate)
Glycolytic flux was measured by estimating extracellular lactate (the end
product of glycolysis) using an ECAR estimation kit (Cayman Chemicals). An
equal number of sperm cells of both (quiescent and motile) samples in HBSS
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Activity of ATP synthase was measured in the direction of ATP hydrolysis
(ATPase activity), using spectrophotometry [17] with some modification. Final
reaction mixture for this assay (150 ll) contained 50 mM Tris-HCl, 20 mM
KCl, 2.5 mM MgCl2, 1 mM ATP, and 0.5 mM EGTA, pH 7.4, with 160 lM
NADH, 2.5 units of pyruvate kinase, and 2.5 units of lactate dehydrogenase.
The linear reaction was followed at 340 nm at 378C. A unit of activity is
defined as lM/min/mg protein.
Quiescent and motile sperm samples were isolated from the cauda epididymis
of Sprague Dawley rats. Adult rats (aged 16–20 wk) were procured from the
Laboratory Animal Division of CSIR-Central Drug Research Institute and were
autopsied in accordance with the guidelines and protocols approved by the
Institutional Ethics Committee for the Use of Laboratory Animals of CSIRCDRI, Lucknow, India. For the quiescent sperm sample, left epididymides were
dissected out, wrapped in aluminum foil, and put in ice for 10–15 min.
Thereafter, several small incisions were made in the cauda and immersed in
sterile, ice-cold HBSS (maintained in ice at 08C–28C) and shaken briskly. The
contamination of caudal tissue in sperm samples, if any, was negligible. The
sperm did not gain any motility. It has been reported that cooling motile rat sperm
to 48C significantly reduced motility without affecting acrosomal membrane
integrity [13]. Similarly, motile mouse sperm cooled gently to 08C and then
rewarmed were comparable to those maintained at 228C [14]. In the present
study, the epididymides (wrapped in aluminum foil) were precooled in ice to cool
the sperm gently before isolation in ice-cold medium. In parallel, the right
epididymides were kept in HBSS at 378C for 10–15 min and then for another 5
min after making small incisions in the cauda and gentle shaking. The sperm here
gained vigorous motility. Both quiescent and motile sperm were viable as tested
by Syber-14 fluorescent dye (Invitrogen). Except for measurement of the
extracellular lactate secretion, both quiescent and motile samples were washed
with sterile PBS twice at 28C–48C and pelleted at 700 3 g for 5 min in a
refrigerated centrifuge, resuspended in PBS, and used for further experiments.
QUIESCENT SPERM ENERGY METABOLISM
3 ml of saline) per rabbit, and the animals were hand mated once with fertile
males. Control rabbits received 3 ml of saline intravaginally as a vehicle.
Mating was confirmed by the presence of sperm in vaginal smears. The animals
were allowed to complete gestation (30–35 days), and the number of pups
delivered was counted and recorded. This study was approved by the
Institutional Animal Ethics Committee of CSIR-Central Drug Research
Institute, Lucknow, India.
medium was incubated for 30–60 min at 378C. Thereafter, the samples were
centrifuged at 10 000 rpm for 10 min, and the supernatant was used for lactate
estimation according to the kit protocol.
Expression of Hypoxia Inducible Factor in Sperm
Qualitative analysis and localization of hypoxia inducible factor (HIF) was
done in quiescent and motile sperm by fluorimetric assay. Sperm were placed
on slide (coated with 0.1% poly-L-lysine) fixed with 4% paraformaldehyde for
15–20 min and permeabilized with 0.25% Triton X-100. After blocking
nonspecific sites with 1.0% BSA for 1 h at room temperature, samples were
incubated with HIF-1a antibody at a dilution of 1:200 overnight at 48C and then
washed with Tris-buffered saline (TBS) with 0.1% Tween-20. Washed samples
were incubated with Alexafluor (AF546; Invitrogen) secondary antibody at
room temperature for 1.0 h in the dark. After washing with TBS, a small drop
of antifade reagent with DAPI (Invitrogen) was placed on the sample and
covered with a cover glass for imaging on a Nikon 80i microscope equipped
with epifluorescence illumination. Exposure time was the same for all samples.
Statistical Analysis
All experiments were performed in triplicates and repeated three times.
Results have been expressed as mean values and were analyzed by one-way
analysis of variance using the Graph Pad Prism software (version 3.0). P-values
, 0.05 were considered significant.
RESULTS
Mitochondrial Complexes Exhibit Significantly Higher
Activity in Quiescent Sperm
Western Blot Analysis
Sperm Motility Initiation in the Presence of Glycolytic and
OxPhos Inhibitors
The motility parameters on activation of quiescent sperm (% motility, %
progressive motility [% PM], average path velocity [VAP], amplitude of lateral
head displacement [ALH], and beat cross frequency [BCF]) in the presence of
glycolytic and OxPhos inhibitors were studied using the Computer Assisted
Sperm Analyzer; (CASA; Hamilton Thorne IVOS, version 12.3, Hamilton
Thorne Inc.). The following CASA settings were used to acquire data: analyzer
setup: STANDARD RAT; frames acquired: 30 frames; frame rate: 60 Hz;
minimum contrast: 80; minimum cell size: 7 pixels; minimum static contrast:
15; straightness threshold:80.0%; VAP cutoff: 20.0 lm/sec; progressive
minimum VAP: 50.0 lm/sec; VSL cutoff: 30.0 lm/sec; cell size: 25 pixels;
cell intensity: 80; static head size: 0.71–8.82; static head intensity: 0.14–1.84;
static elongation: 0–63; magnification: 0.83; video frequency: 60; LED
illumination intensity: 2224; IDENT illumination intensity: 3000; and
temperature set: 37.58C. Glycolytic inhibitors 2-deoxyglucose (2DOG;
hexokinase inhibitor), N-ethyl oxamate (EO; lactate dehydrogenase inhibitor),
iodoacetamide (IAA; glyceraldehyde 3-phosphohate dehydrogenase inhibitor),
and OxPhos inhibitors oxaloacetic acid (OAA; OxPhos complex II inhibitor)
and 2,4-dinitrophenol (2-DNP; OxPhos uncoupler) were used. Stock solutions
of inhibitors were diluted to 100 mM and thereafter serially double diluted to
50, 25, 12.5, 6.25, 3.125, and 1.56 mM concentrations in HBSS. Quiescent
caudal sperm obtained directly from cauda epididymis were diluted with each
of these concentrations at 378C starting from 100 mM, and movement of sperm
(if any) was recorded. 2DOG solution was prepared both in HBSS and in
physiological saline since the inhibition by 2DOG is reversed by the presence
of glucose in HBSS. The motility data acquired by CASA at the highest
concentration of the inhibitor at which quiescent sperm could initiate motility
are reported in Figure 4A.
Quiescent Sperm Exhibit High ATP Synthase Activity
ATP synthase traps the free energy released during reflux of
protons into the mitochondrial matrix by synthesizing ATP
molecules. The activity of ATP synthase in quiescent sperm
was approximately 2.5 times higher at 4.64 units against 1.81
units in motile sperm (Fig. 1B).
Exclusive Localization of Intracellular Calcium in the
Midpiece of Sperm Cells
Fertility of Rabbits Instilled Vaginally with Glycolytic or
OxPhos Inhibitor Before Mating
Calcium was exclusively localized in the midpiece of sperm
cells, and the level of fluorescence was markedly higher in
quiescent sperm than in motile sperm (Fig. 1C). The head and
tail regions were almost completely devoid of fluorescence.
The most active inhibitors of glycolysis (IAA) and OxPhos (2,4DNP) were
instilled intravaginally in coeval female rabbits separately at a dose of 50 mg (in
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The respiratory chain consists of four protein complexes: I,
II, III, and IV. Complexes I, III, and IV are proton (H þ) pumps
that form an electrochemical (H þ) gradient across the inner
mitochondrial membrane. Complex II is not a proton pump but
delivers additional electrons to ubiquinone via flavin adenine
dinucleotide (FAD). Respiratory complex I (NADH-ubiquinone oxidoreductase, EC 1.6.5.3), removes two electrons from
NADH and transfers them to a lipid-soluble carrier ubiquinone
(Q), while respiratory complex II (succinate-ubiquinone
oxidoreductase, EC 1.3.5.1) delivers additional electrons from
succinate to the ubiquinone pool via FAD. Further, respiratory
complex III (ubiquinol- [QH2-] cytochrome c reductase, EC
1.10.2.2) removes two electrons from QH2 and transfers them
to two molecules of cytochrome c and a water-soluble electron
carrier, and finally respiratory complex IV (cytochrome c
oxidase, EC 1.9.3.1) removes four electrons from four
molecules of cytochrome c and transfers them to molecular
oxygen to produce two molecules of water. Complexes I and
III pump four protons out of the inner mitochondrial matrix,
while complex IV utilizes four protons from the inner
mitochondrial matrix, resulting in a proton gradient across
the inner mitochondrial membrane. The reflux of protons back
into the mitochondrial matrix releases a large amount of free
energy that is coupled to ATP synthase and drives the synthesis
of ATP molecules. The activity of mitochondrial complexes I–
IV after normalization to the steady activity of citrate synthase
in mitochondrial matrix (i.e. per unit of citrate synthase
activity) in quiescent sperm was estimated to be 110.3, 61.11,
191.76, and 40.74 units, respectively, whereas in motile sperm
it was 55.97, 28.74, 119.66, 16.68 units, respectively. Thus, on
motility initiation, sperm exhibited just about 40%–60% of the
OxPhos activity seen in quiescent sperm (Fig. 1A). Maximum
change in activity was seen in the case of complex IV.
Quiescent and motile sperm samples were lysed in RIPA buffer (with 13
protease and phosphatase inhibitor cocktail), incubated for 2 h at 48C, and
sonicated on ice. After sonication, cells were centrifuged at 14 000 rpm for 20
min, and the protein concentration of supernatant was determined by the BCA
(bicinchoninic acid) protein estimation kit (Thermo Fisher Scientific). An equal
amount of protein was loaded in each lane and resolved on 12% SDS gels by
PAGE, and protein bands were transferred onto a PVDF microporous
membrane (Immobilon-P; EMD Millipore). Membranes were blocked with
5% milk protein in TBS containing 0.1% Tween to prevent nonspecific binding
and then incubated overnight in a cold room at 48C with primary antibodies for
HIF, 1:3000 (Sigma); p-AMPK, 1:2500; AMPK, 1:3000; pAKT, 1:2000; AKT,
1:3000; p-ERK, 1:2500; ERK, 1:3000 (Cell Signaling Technology); and
dilutions in TBS with 1% BSA. Thereafter, the membranes were washed four
times with 13 TBS for 10 min each and incubated with horseradish peroxidase–
conjugated secondary antibodies (Sigma Aldrich) at room temperature at
1:6000 dilution in TBS with 1% BSA. The blots were washed and developed
with the Enhanced Chemiluminescence Kit (EMD Millipore) and imaged on an
ImageQuant LAS 4000 (GE Healthcare).
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FIG. 1. Oxidative phosphorylation in quiescent and motile sperm. A) The activities of mitochondrial electron transport chain complexes I–IV in
quiescent and motile sperm. Normalized specific activities of complexes (nM/min/mg protein) were obtained by dividing with the steady activity of
mitochondrial matrix enzyme citrate synthase, and the values are expressed as a ratio of the activities in quiescent sperm (taken as 100 units). B) Specific
activities of ATP synthase in quiescent and motile sperm (lM/min/mg protein). Significant differences from quiescent indicated as *P , 0.05, **P , 0.01,
and ***P , 0.001. C) Fluorescent, bright-field and merged images of quiescent (Q) and motile (M) sperm labeled with fluorescent Ca2þ detector fluoforte
dye. Note the specific labeling of the midpiece region. Original magnification 3600.
to fructose-1,6-bisphosphate. The mammalian sperm PFK is
allosterically inhibited by ATP, citrate, and H þ and activated
by AMP and fructose-2,6-bisphosphate [22]. PK catalyzes the
final phosphorylation reaction in glycolysis and converts
phophoenolpyruvate to pyruvate with phosphorylation of
ADP to ATP. Mammalian sperm PK is similar to muscle PK
and is activated by mono- and divalent cations and inhibited by
phenylalanine [23]. A sperm-specific PK (PK-S) has been
reported in boar sperm along with normal PK [24], with similar
catalytic properties. In rat quiescent sperm, HK, PFK, and PK
exhibited activities of 9.4, 6.0, and 4.8 units, respectively,
Motile Sperm Exhibit High Activity of Rate-Limiting
Glycolytic Enzymes
HK, PFK, and PK catalyze rate-limiting, phosphorylation
reactions in glycolysis. HK phosphorylates glucose to glucose
6-phosphate and prevents its diffusion out of the cell, as the cell
membrane is impermeable to phosphorylated sugars. It is the
principal control point in glycolysis [20] and is limited by the
supply of glucose. In mouse sperm, this step is catalyzed
almost exclusively by spermatogenic cell-specific hexokinase
HK1S [21]. PFK is the second point for control of glycolytic
flux and catalyzes the phosphorylation of fructose-6-phosphate
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whereas in motile sperm these activities were 13.1, 9.8, and 8.4
units, respectively. Thus, motile sperm had approximately
40%–75% higher glycolytic activity as compared to quiescent
sperm (Fig. 2A).
Lactate Production Is Higher in Motile Sperm
Quiescent sperm produced ;853 nM lactate/106 cells/h,
which was just 39% of that produced by motile sperm (;2174
FIG. 3. Energy-sensing (AMPK) and cell-signaling (MAPK/p38, AKT, MAPK/ERK) proteins in quiescent and motile sperm. Representative Western blots
(upper panel) and statistical data of three experiments (lower panel) for phospho-AMPK (A), phospho-p38 (B), phospho-AKT (C), and phospho-ERK (D) in
quiescent (Q) and motile (M) sperm. Significant differences from quiescent indicated as *P , 0.05, **P , 0.01, and ***P , 0.001.
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FIG. 2. Glycolysis in quiescent and motile sperm. A) Specific activities (lM/min/mg protein) of rate-limiting glycolytic enzymes hexokinase (HK),
phosphofructokinase (PFK), and pyruvate kinase (PK) in motile sperm expressed as a ratio of the activities in quiescent sperm (taken as 100 units). B)
Lactate secretion by quiescent and motile sperm (lM/106 sperm/h). C) HIF-1a levels in quiescent (Q) and motile (M) sperm. Significant differences from
quiescent indicated as *P , 0.05, **P , 0.01, and ***P , 0.001. D) Immunofluorescent labeling of HIF-1 (red) and DNA (DAPI, blue) of quiescent (Q)
and motile (M) sperm. Note specific localization of HIF-1 in the principal piece (inset). Original magnification 3400. BF, bright-field.
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FIG. 4. Inhibition of sperm motility initiation and fertility by glycolytic and OxPhos inhibitors. A) Motility initiation in the presence of different
concentrations of glycolytic inhibitors—2-deoxyglucose (2DOG), N-ethyl oxamate (EO), and iodoacetamide (IAA)—and OxPhos inhibitors—oxaloacetate
(OAA) and 2,4-dinitrophenol (2,4DNP) in HBSS—in vitro. B) Percent motility, percent progressive motility (% PM), average path velocity (VAP), amplitude
of lateral head displacement (ALH), and beat cross frequency (BCF) of sperm initiating motility in the presence of the highest concentration of inhibitor
supporting motility initiation (mean 6 SEM of three experiments). C) In vivo evaluation of the most active glycolytic (IAA) and OxPhos (2,4DNP) inhibitors
as vaginal contraceptives in rabbits.
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TABLE 1. Glycolytic and OxPhos inhibitors used in the study and their actions and mechanisms.
Inhibitor
Action
Mechanism
2-deoxyglucose (2-DOG)
Reversible inhibitor of HK
N-ethyl oxamate (EO)
Reversible inhibitor of lactate dehydrogenase
Iodoacetamide (IAA)
Irreversible inhibitor of GAPDH
Oxaloacetate (OAA)
Competitive inhibitor of succinate ubiquinone
oxidoreductase (respiratory complex II)
2,4-dinitrophenol (2,4DNP)
Mitochondrial uncoupler (uncouples electron transport
chain with ATP synthesis)
OAA was the weakest inhibitor of sperm motility activation.
On the other hand, quiescent sperm failed to initiate any kind of
motility in the presence of 50 mM 2,4DNP (OxPhos uncoupler)
(Fig. 4A). Nevertheless, motility could be observed in
quiescent sperm in a medium containing 3.125 mM IAA, 50
mM 2DOG or EO, and 25 mM 2,4DNP. While most of the
motility parameters, such as % motility, % PM, VAP, and
ALH, were reduced in proportion to the concentration and type
of the inhibitor, BCF increased in the case of glycolytic
inhibitors and OAA and decreased in the case of 2,4DNP.
When the most potent inhibitor of rat sperm motility initiation
(IAA; glycolytic/GAPDH inhibitor) at 3.125 mM was
combined with 50 mM OAA, the overall reduction in motility
parameters was more pronounced as compared with IAA alone
at 3.125 mM, with a major fall in progressive motility.
Similarly, an improvement in the immobilizing effects of IAA
(at 3.125 mM) was also observed in the presence of 25 mM
2,4DNP with a noticeable drop in BCF (Fig. 4B).
Higher Expression of HIF-1a in Motile Sperm
The expression signal (fluorescent red) of HIF was observed
to be very prominent in motile sperm, while this signal in
quiescent sperm was comparatively quite weak (Fig. 2, C and
D). Interestingly, the high HIF fluorescence was localized
almost exclusively to the principal piece region of the sperm
with negligible fluorescence in the midpiece and head regions
(Fig. 2D).
Increased Phosphorylation of AMPK and p38 in Quiescent
Sperm and of MAPK/ERK and AKT in Motile Sperm
MAPK/ERK and p38 cascades play crucial roles in cell
proliferation and apoptosis [25]. Despite sperm being fully
differentiated, cells lacking transcriptional activity and the
presence and activation of these cascades have been detected in
spermatozoa, and specific inhibitors of MAPK/ERK and p38
cascades reduce and stimulate sperm motility, respectively
[26]. Similarly, inhibition of PI3K/AKT cell signaling in sperm
using wortmannin reduces the number of motile, progressively
motile, and hyperactivated sperm [27]. On the other hand,
AMPK is a cell energy sensor that is shown to play an essential
role in boar sperm motility and viability [28]. The expression
levels of AMPK, MAPK/ERK and p38, and AKT were similar
in motile and quiescent sperm. However, the expression of
active phospho-AMPK and phospho-p38 were higher in
quiescent sperm with weak signals in motile sperm (Fig. 3,
A and B). On the other hand, phospho-ERK and phospho-AKT
signals were higher in motile sperm than in quiescent sperm
(Fig. 3, C and D).
Fertility Inhibition in Rabbits Treated Intravaginally with
Glycolytic and OxPhos Inhibitors
None of the five mated female rabbits that received 50 mg
of glycolytic inhibitor IAA intravaginally before mating
delivered any pups, displaying a pregnancy rate and fertility
rate of 0%. On the other hand, four out of five mated females
treated with 50 mg of 2,4DNP intravaginally before mating
delivered 16 (two, six, three, and five) pups, displaying a
pregnancy rate of 80% and fertility rate of 45% compared with
control animals (Fig. 4C).
DISCUSSION
Quiescent sperm remain virtually ‘‘frozen’’ at scrotal
temperature to maintain viability for periods exceeding 30
days in rats. However, quiescence is different from cryopreservation, where sperm are actually frozen with a complete
metabolic halt. At scrotal temperature, the metabolic enzymes
are fully active in quiescent sperm and cater to their survival
needs during storage and to their motility initiation during
ejaculation. The quiescent sperm survive in a dynamic balance
with the caudal lumen environment and are prone to death and
dissolution with adverse changes in epididymal physiology
[30]. Fully mature sperm held completely immotile in the
cauda epididymis are ready to gain motility in any physiological medium provided or ejaculated [12]. For vigorous motility,
Inhibition of Quiescent Sperm Motility Initiation in the
Presence of Glycolytic and OxPhos Inhibitors
The inhibitors of glycolysis and OxPhos used in the present
study and their primary action and mechanisms are detailed in
Table 1. Quiescent sperm failed to initiate motility in 6.25 mM
IAA (GAPDH inhibitor), 100 mM 2DOG, and 100 mM EO
(HK and LDH inhibitors, respectively). However, quiescent
sperm initiated motility at 100 mM OAA (OxPhos complex II
inhibitor), but there was a major drop in progressive motility.
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nM lactate/106 cells/h), which revealed an approximately 2.5
times higher glycolytic flux in motile sperm than in quiescent
sperm (Fig. 2B).
2-DOG is a glucose analog with the hydroxyl group at
carbon 2 replaced by hydrogen. It competitively
inhibits the HK reaction and prevents further
progress of glycolysis.
EO is a pyruvate analog that competitively binds with
LDH and prevents LDH action.
IAA forms a thio-ether bond with the cysteine residue
at the active site of GAPDH, which is essential for
activity, thus preventing its reaction with the
physiological substrate glyceraldehyde-3-phosphate.
OAA forms a stable complex with succinate
ubiquinone oxidoreductase (succinate
dehydrogenase), transforming it to an inactive form
[29].
Dissipates the proton gradient across the mitochondrial
membrane and collapses the electron motive force
generated by the respiratory complexes.
KUMAR ET AL.
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with energy-metabolizing enzymes greatly removed from
equilibrium. This would keep the cells geared up for very
active energy metabolism when substrates become available
during motility initiation at ejaculation. Similarly, HIF-1a is an
oxygen-sensing transcription factor that accumulates in the
nucleus and mitochondria [40] in response to low-oxygen
tension and/or increased concentration of lactate/pyruvate [41].
The increased concentration of lactate in motile sperm due to
active glycolysis could stabilize HIF-1a, but its unique
localization exclusively in the sperm principal piece was
surprising. This first report of HIF-1a in rat sperm may suggest
its direct role in sensing and maintaining active glycolysis in
transcriptionally inactive sperm, but this aspect needs detailed
investigation. Phospho-AKT is well known to stimulate
glycolysis through the activation by phosphorylation of
hexokinase [42] and phosphofructokinase-2 (PFK-2), which
catalyzes the production of fructose-2,6-bisphosphate, a potent
allosteric activator of glycolytic PFK-1 [43]. Increased
phosphorylation of AKT would favor glycolysis in rat sperm
after motility initiation.
Among glycolytic inhibitors, iodoacetamide (IAA; glyceraldehyde phosphate dehydrogenase inhibitor) was most
effective in blocking sperm motility initiation and maintaining
the quiescence of caudal sperm at 6.25 mM (in HBSS), while
the same result was achieved with 50 mM of OxPhos
uncoupler 2-dinitrophenol (2DNP in HBSS). Thus, it is
apparent that while glycolysis may be the preferred pathway
after motility initiation, OxPhos may have a supportive role. It
would be pertinent to mention here that GAPDH/ sperm have
profound defects in motility with sluggish movement and no
forward progression, making males infertile [11]. The ATP
levels of these sperm have been reported to be ;90% lower
than wild-type sperm without any change in oxygen consumption [11]. Similarly, the glycolytic inhibitors 2-DOG (hexokinase inhibitor in saline) and EO (LDH inhibitor in HBSS) were
more effective than oxaloacetate (mitochondrial complex II
inhibitor in HBSS). Since 2-DOG is a reversible inhibitor of
hexokinase, its efficacy was diminished in the presence of
glucose (contained in HBSS). Another important observation
was that the beat cross frequency of rat sperm apparently
increased by glycolytic inhibitors and reduced by the 2,4DNP.
Perhaps OxPhos and glycolysis dually fuel the axoneme in the
proximal region, where it passes through the midpiece
surrounded by a sheath of closely packed mitochondria to
initiate the whiplash movement. On the other hand, glycolysis
alone seems to generate the force of whiplash movement in the
distal axoneme passing through the principal piece for
generating the propulsion required for forward motility.
Consequently, paralyzing the principal piece by glycolytic
inhibitors dissipated the available OxPhos energy in the
midpiece and increased BCF. On the other hand, uncoupling
of OxPhos in the midpiece by 2,4DNP could reduce the force
for initiating whiplash, resulting in lowered BCF. However,
OAA, being the weakest OxPhos inhibitor, did not behave like
2,4DNP. Nevertheless, in rabbits, blocking glycolysis of
ejaculated sperm in female (vagina) by placing 50 mg of
IAA just before mating completely blocked pregnancy, while
an equal amount of 2,4DNP could suppress the pregnancy rate
by only 20% and the fertility rate by ;50%.
Thus, the present study for the first time reports the status of
energy metabolism of quiescent sperm stored in the cauda
epididymis and highlights some of the crucial metabolic events
that occur in mammalian sperm during motility activation. The
dual role of sperm midpiece mitochondria is indicated in
sustaining sperm longevity during storage in the cauda
epididymis before ejaculation through optimal utilization of
sperm require an ample amount of ATP, which could be
supplied by either OxPhos or Glycolysis [6]; however, the
crucial metabolic changes and the selection of ATP-producing
machinery during motility initiation are not well studied.
Previous attempts to obtain quiescent sperm used physical
methods, such as the micropuncture technique [31], retrograde
perfusion [32], or capillary centrifugation in a MicroHematocrit centrifuge [12]. However, these methods provided
short-term quiescence with the apprehension of activating
sperm motility on dilution in any isotonic medium required for
washing and further processing of samples for biochemical
estimations. We have for the first time used a simple, reliable
physiological method of arresting the motility activation of
caudal sperm by isolating and further processing sperm
samples at low (08C–28C) temperature without freezing. This
method provides the required quiescent sperm samples with a
fully intact cellular structure for processing and estimating
various biochemical parameters, as low temperature prevents
motility activation during dilution in isotonic media.
The results of the present study indicate that for surviving
long storage periods in extremely crowded conditions and with
very limited energy resources, quiescent sperm adopt the most
efficient method of energy metabolism, that is, oxidative
phosphorylation, to meet their low but prolonged energy
requirements during rest. However, during motility activation,
the sperm switch over to predominantly glycolytic energy
metabolism to cater to their quick-energy requirement [6]. This
is somewhat analogous to the skeletal muscle cells, which
actively glycolyze during brisk physical activity but switch
over to OxPhos during rest. In fact, it has been demonstrated
for muscle cells that during low metabolic activity (low ATP
demand), OxPhos is more efficient than glycolysis, while when
ATP demands are high, glycolysis with concomitant lactate
secretion becomes highly efficient and the preferred pathway
for energy [33]. In earlier studies, a switch from glycolysis to
OxPhos has been reported during meiosis in rat testicular germ
cells [34]. Mammalian sperm have a neatly demarcated
midpiece with mitochondria arranged in a sheath beneath the
plasma membrane. The role of mitochondria in predominantly
glycolytic motile sperm has often been questioned [11],
especially with respect to its remote location from the motility
appendage and the efficacy of energy transfer [5]. Nevertheless, high levels of mitochondrial calcium in the midpiece of
quiescent sperm (rather than in motile sperm) as seen in the
present study can accelerate oxidative phosphorylation [35] to
enable the mitochondria to play a crucial role in survival of
sperm during their storage in the cauda epididymis under
crowding stress by providing the required energy through
efficient utilization of available substrates. The stress-activated
p38-MAPK, which is reported to be associated with low
motility in the case of human sperm [36], was phosphorylated
in the quiescent sperm of rat in the present study. In fact,
activation of MAP kinase/ERK1/2 stimulates and p38 inhibits
forward and hyperactivated motility, respectively, in human
sperm [26]. We also demonstrate markedly increased ERK1/2
phosphorylation in motile rat sperm, which in other cell types
is positively correlated with glucose consumption, lactate
production, and increased glycolytic flux but not with OxPhos
[37]. On the other hand, AMP-activated protein kinase
(AMPK) is a crucial cellular energy sensor (ATP:AMP ratio)
and promotes ATP production when cellular energy status is
low through the activation of catabolic pathways of energy
metabolism [38], including upregulation of mitochondrial
function [39]. However, the most important information about
quiescent sperm derived from the activation (phosphorylation)
of AMPK is that they survive under low, energy-starved status
QUIESCENT SPERM ENERGY METABOLISM
REFERENCES
1. Mukai C, Okuno M. Glycolysis plays a major role for adenosine
triphosphate supplementation in mouse sperm flagellar movement. Biol
Reprod 2004; 71:540–547.
2. Moore HD, Samayawardhena LA, Brewis IA. Sperm maturation in vitro:
co-culture of spermatozoa and epididymal epithelium. J Reprod Fertil
Suppl 1998; 53:23–31.
3. Aitken RJ. Possible redox regulation of sperm motility activation. J Androl
2000; 21:491–496.
4. Cornwall GA. New insights into epididymal biology and function. Hum
Reprod Update 2009; 15:213–227.
5. Takei GL, Miyashiro D, Mukai C, Okuno M. Glycolysis plays an
important role in energy transfer from the base to the distal end of the
flagellum in mouse sperm. J Exp Biol 2014; 217:1876–1886.
6. Ford WCL Glycolysis and sperm motility: does a spoonful of sugar help
the flagellum go round? Hum Reprod Update 2006; 12:269–274.
7. Kim J, Copley SD. Why metabolic enzymes are essential or nonessential
for growth of Escherichia coli K12 on glucose. Biochemistry 2007; 46:
12501–12511.
8. Krzyzosiak J, Molan P, Vishwanath R. Measurements of bovine sperm
velocities under true anaerobic and aerobic conditions. Anim Reprod Sci
1999; 55:163–173.
9. Nascimento JM, Shi LZ, Tam J, Chandsawangbhuwana C, Durant B,
Botvinick EL, Berns MW. Comparison of glycolysis and oxidative
phosphorylation as energy sources for mammalian sperm motility, using
the combination of fluorescence imaging, laser tweezers, and real-time
automated tracking and trapping. J Cell Physiol 2008; 217:745–751.
10. Williams AC, Ford WC. The role of glucose in supporting motility and
capacitation in human spermatozoa. J Androl 2001; 22:680–695.
11. Miki K, Qu W, Goulding EH, Willis WD, Bunch DO, Strader LF,
Perreault SD, Eddy EM, O’Brien DA. Glyceraldehyde 3-phosphate
dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for
sperm motility and male fertility. Proc Natl Acad Sci U S A 2004; 101:
16501–16506.
12. Nakamura N, Miranda-Vizuete A, Miki K, Mori C, Eddy EM. Cleavage of
disulfide bonds in mouse spermatogenic cell-specific type 1 hexokinase
isozyme is associated with increased hexokinase activity and initiation of
sperm motility. Biol Reprod 2008; 79:537–545.
13. Varisli O, Uguz C, Agca C, Agca Y. Effect of chilling on the motility and
acrosomal integrity of rat sperm in the presence of various extenders. J Am
Assoc Lab Anim Sci 2009; 48:499–505.
14. Tao J, Du J, Kleinhans FW. The effect of collection temperature, cooling
rate and warming rate on chilling injury and cryopreservation of mouse
spermatozoa. J Reprod Fertil 1995; 104:231–236.
15. Tatarkova Z, Kuku S, Racay P, Lehotsky J, Dobrota D, Mistuna D, Kaplan
P. Effects of aging on activities of mitochondrial electron transport chain
complexes and oxidative damage in rat heart. Physiol Res 2011; 60:
281–289.
16. Spinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C. Assessment of
mitochondrial respiratory chain enzymatic activities on tissues and
cultured cells. Nat Protoc 2012; 7:1235–1246.
17. Rosing J, Harris DA, Kemp A, Slater EC. Nucleotide binding properties of
native and cold-treated mitochondria. Biochim Biophys Acta 1975; 376:
13–26.
18. Pickl A, Johnsen U, Schönheit P. Fructose degradation in the
Haloarchaeon Haloferax volcanii involves a bacterial type phosphoenolpyruvate-dependent phosphotransferase system, fructose-1-phosphate
kinase, and class II fructose-1,6-bisphosphate aldolase. J Bacteriol 2012;
194:3088–3097.
19. Bergmeyer HU, Gawehn, K, Grassl M. Methods of Enzymatic Analysis,
vol. 1, 2nd ed. New York: Academic Press; 1974:509–510.
20. Wilson JE. Brain hexokinase, the prototype ambiquitous enzyme. Curr
Top Cell Regul 1980; 16:1–54.
9
Article 96
Downloaded from www.biolreprod.org.
21. Nakamura N, Shibata H, O’Brien DA, Mori C, Eddy EM. Spermatogenic
cell-specific type 1 hexokinase is the predominant hexokinase in sperm.
Mol Reprod Dev 2008; 75:632–640.
22. Kamp G, Schmidt H, Stypa H, Feiden S, Mahling C, Wegener G.
Regulatory properties of 6-phosphofructokinase and control of glycolysis
in boar spermatozoa. Reproduction 2007; 133:29–40.
23. Storey BT, Kayne FJ. Properties of pyruvate kinase and flagellar ATPase
in rabbit spermatozoa: relation to metabolic strategy of the sperm cell. J
Exp Zool 1980; 211:361–367.
24. Feiden S, Stypa H, Wolfrum U, Wegener G, Kamp G. A novel pyruvate
kinase (PK-S) from boar spermatozoa is localized at the fibrous sheath and
the acrosome. Reproduction 2007; 134:81–95.
25. Pearson G, Robinson F, Beers GT, Xu BE, Karandikar M, Berman K,
Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation
and physiological functions. Endocr Rev 2001; 22:153–183.
26. Almog T, Lazar S, Reiss N, Etkovitz N, Milch E, Rahamim N, Dobkin
BM, Rotem R, Kalina M, Ramon J, Raziel A, Breitbart H, et al.
Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK
as regulators of human sperm motility and acrosome reaction and as
predictors of poor spermatozoan quality. J Biol Chem 2008; 23:
14479–14489.
27. Sagare-Patil V, Vernekar M, Galvankar M, Modi D. Progesterone utilizes
the PI3K-AKT pathway in human spermatozoa to regulate motility and
hyperactivation but not acrosome reaction. Mol Cell Endocrinol 2013;
374:82–91.
28. Hurtado de Llera A, Martin-Hidalgo D, Gil MC, Garcia-Marin LJ,
Bragado MJ. New insights into transduction pathways that regulate boar
sperm function. Theriogenology 2016; 85:12–20.
29. Wojtczak L, Wojtczak AB, Ernster L. The inhibition of succinate
dehydrogenase by oxalacetate. Biochim Biophys Acta 1969; 191:10–21.
30. Jones R. Sperm survival versus degradation in the mammalian epididymis:
a hypothesis. Biol Reprod 2004; 71:1405–1411.
31. Turner TT, Howards SS. Factors involved in the initiation of sperm
motility. Biol Reprod 1978; 18:571–578.
32. Turner TT, Giles RD. The effects of carnitine, glycerylphosphorylcholine,
caffeine and egg yolk on the motility of rat epididymal spermatozoa.
Gamete Res 1981; 4:283–295.
33. Vazquez A, Oltvai ZN. Molecular crowding defines a common origin for
the Warburg effect in proliferating cells and the lactate threshold in muscle
physiology. PLoS ONE 2011; 6:e19538.
34. Bajpai M, Gupta G, Setty BS. Changes in carbohydrate metabolism of
testicular germ cells during meiosis in the rat. Eur J Endocrinol 1998; 138:
322–327.
35. Ivannikov MV, Macleod GT. Mitochondrial free Ca2þ levels and their
effects on energy metabolism in Drosophila motor nerve terminals.
Biophys J 2013; 104:2353–2361.
36. Ding YQ, Jiang H, Wang CL. ERK and P38MAPK expressions and
human sperm motility. Zhonghua Nan Ke Xue 2011; 17:809–812.
37. Través PG, de Atauri P, Marı́n S, Pimentel-Santillana M, Rodrı́guezPrados JC, Marı́n de Mas I, Selivanov VA, Martı́n-Sanz P, Boscá L,
Cascante M. Relevance of the MEK/ERK signaling pathway in the
metabolism of activated macrophages: a metabolomic approach. J
Immunol 2012; 188:1402–1410.
38. Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor
that maintains energy homeostasis. Nat Rev Mol Cell Biol 2012; 22:
251–262.
39. Hardie DG. AMP-activated protein kinase: an energy sensor that regulates
all aspects of cell function. Genes Dev 2011; 25:1895–1908.
40. Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 2006;
70:1469–1480.
41. Lu H, Forbes RA, Verma A. Hypoxia-inducible factor 1 activation by
aerobic glycolysis implicates the Warburg effect in carcinogenesis. J Biol
Chem 2002; 28:23111–23115.
42. Miyamoto S, Murphy AN, Brown JH. Akt mediates mitochondrial
protection in cardiomyocytes through phosphorylation of mitochondrial
hexokinase-II. Cell Death Differ 2008; 15:521–529.
43. Deprez J, Vertommen D, Alessi DR, Hue L, Rider MH. Phosphorylation
and activation of heart 6-phosphofructo-2-kinase by protein kinase B and
other protein kinases of the insulin signaling cascades. J Biol Chem 1997;
11:17269–17275.
the energy substrates and, in addition, fueling the proximal
axoneme along with glycolysis to generate the whiplash action.
Apparently, glycolytic inhibitors may be more effective in
paralyzing sperm for contraception than OxPhos uncouplers
due to the major role of the glycolysis in sperm motility
postejaculation.