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Localisation and function of glucose transporter GLUT1
in chicken (Gallus gallus domesticus) spermatozoa:
relationship between ATP production pathways and
flagellar motility
Rangga Setiawan
A
,Chathura Priyadarshana
A
,Atsushi Tajima
B
,
Alexander J. Travis
C
and Atsushi Asano
B
,
D
A
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305-8572, Japan.
B
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-8572, Japan.
C
Baker Institute for Animal Health, Cornell University, Hungerford Hill Road, Ithaca, NY 14853,
USA.
D
Corresponding author. Email: asano.atsushi.ft@u.tsukuba.ac.jp
Abstract. Glucose plays an important role in sperm flagellar motility and fertility via glycolysis and oxidative
phosphorylation, although the primary mechanisms for ATP generation vary between species. The glucose transporter
1 (GLUT1) is a high-affinity isoform and a major glucose transporter in mammalian spermatozoa. However, in avian
spermatozoa, the glucose metabolic pathways are poorly characterised. This study demonstrates that GLUT1 plays a major
role in glucose-mediated motility of chicken spermatozoa. Using specific antibodies and ligand, we found that GLUT1
was specifically localised to the midpiece. Sperm motility analysis showed that glucose supported sperm movement during
incubation for 0–80 min. However, this was abolished by the addition of a GLUT1 inhibitor, concomitant with a
substantial decrease in glucose uptake and ATP production, followed by elevated mitochondrial activity in response to
glucose addition. More potent inhibition of ATP production and mitochondrial activity was observed in response to
treatment with uncouplers of oxidative phosphorylation. Because mitochondrial inhibition only reduced a subset of sperm
movements, we investigated the localisation of the glycolytic pathway and showed glyceraldehyde-3-phosphate
dehydrogenase and hexokinase I at the midpiece and principal piece of the flagellum. The results of this study provide
new insights into the mechanisms involved in ATP production pathways in avian spermatozoa.
Additional keywords: sperm motility.
Received 4 July 2019, accepted 30 October 2019, published online 28 January 2020
Introduction
Spermatozoa acquire energy from nutrient molecules present in
extracellular environments for a variety of functions, such as
flagellar motility. ATP is the principal form of energy. It is
generated via glycolysis and oxidative phosphorylation in the
flagellum, which comprises the midpiece and principal piece.
Glucose is the predominant substrate for glycolysis in female
reproductive tract fluids in mice (Gardner and Leese 1990), pigs
(Nichol et al. 1992) and cows (Carlson et al. 1970). However,
the effect of glucose on spermatozoa varies between species. For
example, glucose stimulates capacitation-associated changes,
including hyperactivated motility (Fraser and Quinn 1981) and
activation of signal transduction pathways (Travis et al. 2001).
However, capacitation and successful fertilisation are inhibited
by the presence of glucose in bovine (Parrish et al. 1989) and
guinea pig (Hyne and Edwards 1985) spermatozoa. Birds
maintain unusually high concentrations of glucose in uterine
fluid (van Eck and Vertommen 1984;Dupuy and Blesbois
1996). Although capacitation is not recognised in birds, our
recent study and others have found that glucose stimulates a
signal transduction pathway and flagellar motility in chicken
spermatozoa, resulting in an elevated fertilisation potential
(McLean et al. 1997;Ushiyama et al. 2019). Despite reports of a
close relationship between glucose and sperm motility in some
species, the molecular mechanisms involved in glucose uptake
remain poorly understood in birds.
Facilitative diffusion of glucose into spermatozoa along a
concentration gradient is catalysed by specific carriers. The
CSIRO PUBLISHING
Reproduction, Fertility and Development
https://doi.org/10.1071/RD19240
Journal compilation CSIRO 2020 www.publish.csiro.au/journals/rfd
glucose transporter (GLUT) family comprises 14 glucose trans-
port proteins with high structural similarity and sequence homol-
ogy. Multiple GLUT isoforms, including GLUT1, 2, 3, 4 and 8,
are suggested to mediate glucose transport in spermatozoa
(Burant et al. 1992;Haber et al. 1993;Angulo et al. 1998;
Schurmann et al. 2002). Studies into GLUT expression in
mammalian spermatozoa have established that these GLUT iso-
forms are localised at differential cellular compartments that
require ATP to function (Bucci et al. 2011;Dias et al. 2014). The
different affinities for glucose between isoforms (Devaskar and
Mueckler 1992) suggest specific roles for these isoforms. It has
recently been reported that GLUT3 is present in spermatozoa and
shows differences in localisation between murine and chicken
spermatozoa (Simpson et al. 2008;Ushiyama et al. 2019).
GLUT1 is a major GLUT isoform in mammalian spermatozoa
and is localised to the acrosome region and principal piece of the
flagellum in human, rat and bull spermatozoa (Angulo et al.
1998). In addition, GLUT1 is a low-K
m
, high-affinity GLUT
believed to play a central role in maintaining basal glucose uptake
in many mammalian cells (Devaskar and Mueckler 1992).
Together, these observations imply functional roles of this
isoform in mammalian spermatozoa. However, the function of
GLUT1 in mammalian and avian spermatozoa remains unclear.
This is due, in part, because targeted deletion of GLUT1 results in
embryonic lethality in mice (Heilig et al. 2003).
In spermatozoa, ATP is generated from glucose via glycoly-
sis and oxidative phosphorylation, which occur in different
regions of the cell. Numerous mammalian sperm studies have
demonstrated that oxidative phosphorylation occurs in the
mitochondria, which are highly packed into the midpiece of
the flagellum, whereas glycolysis occurs in the principal piece
that occupies the major part of the flagellum (Mukai and Okuno
2004;Storey 2008). Most of the enzymes necessary for glycol-
ysis, such as hexokinase I and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), possess sperm-specific isoforms that
are primarily associated with the fibrous sheath in the principal
piece due to an N-terminal domain anchor (Bunch et al. 1998;
Travis et al. 1998). Due to its higher efficiency of ATP produc-
tion, oxidative phosphorylation is considered to be the main
provider of ATP for sperm motility, although some studies have
shown that there is wide variation among the primary mechan-
isms for ATP production in mammalian species (Storey 2008).
For example, glycolysis plays a major role in ATP production
for flagellar movement in murine and human spermatozoa
(Williams and Ford 2001;Mukai and Okuno 2004). Conversely,
ATP production is also critical for the maintenance of motility
and fertilisation ability in avian spermatozoa (Wishart and
Palmer 1986;McLean et al. 1997), potentially via a metabolic
signalling pathway (Nguyen et al. 2014). However, the ATP
production pathways along the flagellum are poorly charac-
terised in avian spermatozoa.
The present study demonstrates that GLUT1 is specifically
localised to the midpiece of the flagellum and contributes to
glucose uptake and ATP production via both glycolysis and
oxidative phosphorylation for flagellar motility. Because of
differences in mammalian data and a lack of knowledge of the
subcellular regions involved in glycolysis in avian spermatozoa,
we undertook immunodetection of hexokinase I and GAPDH
and showed that these enzymes were localised to both the
midpiece and principal piece of the flagellum in chicken
spermatozoa. The results of the present study provide new
insight into the glucose metabolic pathways involved in sup-
porting flagellar motility and suggest the involvement of the
midpiece in both glycolysis and oxidative phosphorylation in
avian spermatozoa.
Materials and methods
Reagents and animals
Unless stated otherwise, all chemicals were purchased from
Sigma-Aldrich. Fasentin, a GLUT1 inhibitor (Wood et al. 2008),
and a broad spectrum GLUT inhibitor (GLUTi; glucose trans-
porter inhibitor II) were obtained from Santa Cruz Biotechnol-
ogy. Recombinant GLUT1 ligand tagged with green fluorescent
protein (GFP) was purchased from Metafora Biosystems. 2-(N-
[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]-amino)-2-deoxy-D-glucose
(2-NBDG) and 5,50,6,60-tetrachloro-1,103,30-tetraethylbenzimi-
dazolylcarbocyanine iodide (JC-1) were obtained from Invitro-
gen and Cayman Chemical respectively. The ‘Cellno’ ATP assay
reagent was from TOYO B-Net. Polyclonal antiserum against
chicken GLUT1 was raised in two rabbits at Eurofins Genomics
using the antigenic epitope [Lys
476
–Gln
489
] specific for GLUT1.
Polyclonal and monoclonal antibodies against hexokinase I and
GAPDH were purchased from Proteintech and FUJIFILM Wako
Pure Chemicals respectively.
Semen was collected from fertile male Rhode Island Red
roosters using the dorsal–abdominal massage method (Burrows
and Quinn 1937). Pooled semen from at least four roosters was
washed twice by centrifugation at 1000 gfor 5 min at 258C with
phosphate-buffered saline (PBS) to exclude secretory fluids,
including seminal plasma. All animal work was approved by the
Institutional Animal Care and Use Committee of the University
of Tsukuba (Approval no. 18–349).
Localisation
For immunostaining, spermatozoa were fixed with 4% para-
formaldehyde for 15 min at room temperature, permeabilised in
0.5% Triton X-100 for 1 min and blocked in 10% goat serum for
1 h. Spermatozoa were incubated with anti-GLUT1 (1 : 150),
anti-hexokinase I (1 : 200) or anti-GAPDH (1 : 200) antibodies
at 48C overnight, followed by incubation with either anti-rabbit
or anti-mouse IgG Alexa Fluor 488 (1 : 200) for 1 h at room
temperature. Coverslips were mounted using Vectashield
mounting medium with 40,6-diamidino-2-phenylindole (DAPI;
Vector Laboratories) on glass slides.
Labelling with GLUT1 ligand tagged with GFP (GLUT1–
GFP) was performed by fixing spermatozoa with 1% paraformal-
dehyde, followed by incubation with GLUT1–GFP (1: 25) at 48C
overnight and mounting on glass slides as described above.
Immunoblotting
Spermatozoa were resuspended in PBS containing a protease
inhibitor cocktail (Roche Diagnostics), Dounce homogenised
and total protein measured using a Bradford protein assay.
Samples of 70 mg sperm protein were processed for sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
BReproduction, Fertility and Development R. Setiawan et al.
analysis. Transfer, blocking and immunodetection of GLUT1
were performed as described previously (Asano et al. 2009).
Anti-GLUT1 antibody and anti-rabbit IgG conjugated with
horseradish peroxidase were diluted 1 : 3000 and 1 : 10 000
respectively. Chemiluminescence was used to detect the
immunoreactivity.
Sperm incubation and motility
Spermatozoa (1 10
7
)wereincubatedinN-Tris-
(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)–
NaCl medium (20 mM TES, 150 mM NaCl, pH 7.4) containing
5mMCa
2þ
at 398Cwith0–50mMfasentin,10mMantimycinA
(AA) or 10 mMcarbonylcyanidem-chlorophenyl hydrazine
(CCCP) in the presence or absence of 20mM glucose for 0, 40 or
80 min. AA (a respiratory chain inhibitor) and CCCP (a proton
ionophore) were used to inhibit mitochondrial oxidative phos-
phorylation. Sperm motility profiles were analysed using a sperm
motility analysis system (SMAS; DITECT). At least 200 sper-
matozoa per sample were examined for the following movement
characteristics: motility (%), straight linevelocity (VSL; mms
1
),
curvilinear velocity (VCL; mms
1
), average path velocity (VAP;
mms
1
), linearity (LIN; calculated as VSL/VCL), straightness
(STR; calculated as VSL/VAP), amplitude of lateral head dis-
placement (ALH; mm) and beat cross frequency (BCF; Hz).
Glucose uptake
A glucose uptake assay using a fluorescent glucose analogue
was performed as described previously (Ushiyama et al. 2019).
Briefly, spermatozoa (1 10
7
) were incubated in the presence
or absence of 60 mM 2-NBDG with 0, 30 or 50 mM fasentin in
TES–NaCl solution containing 5 mM Ca
2þ
for 40 min at 398C,
then centrifuged at 1000gfor 5 min at 398C and resuspended in
TES–NaCl. Fluorescence intensity was measured using a Mul-
timode Detector DTX800 (Beckman Coulter) at excitation and
emission wavelengths of 488 and 530 nm respectively. The
subcellular localisation of 2-NBDG was viewed using a Leica
DMI 4000B microscope (Leica Microsystems). Images were
captured using identical exposure times for groups incubated
with different concentrations of fasentin.
Mitochondrial activity
Mitochondrial activity was assessed using JC-1, a lipophilic
fluorescent probe that reversibly changes its fluorescence from
green (monomeric form) to orange (aggregate form) as the mito-
chondrial membrane potential (MMP) increases. Spermatozoa
(2 10
7
)wereincubatedwith0,30or50mM fasentin, 1 mM
GLUTi, 10 mMAAor10mM CCCP in TES–NaCl solution
containing 5 mM Ca
2þ
, with or without 20 mM glucose at 398Cfor
40 min. Then, spermatozoa were centrifuged at 1000gfor 5 min at
room temperature and resuspended in 100 mL TES–NaCl solution
containing 5 mL JC-1. After incubation at 398C for 15 min, samples
were washed twice by centrifugation at 1000gfor 5 min at 258C
and resuspended in Cell-Based Assay Buffer (Cayman Chemical).
The fluorescence intensity in each sample (2 10
6
spermatozoa)
was analysed using a Multimode Detector DTX800 (Beckman
Coulter) with excitation and emission wavelengths set at 535 and
590 nm respectively for the aggregate form and 485 and 530 nm
respectively for the monomeric form. The fluorescence intensity
ratio 590/530 nm was used to evaluate MMP.
ATP quantification
ATP content was quantified using the ‘Cellno’ ATP assay
reagent (TOYO B-Net). Spermatozoa (2 10
7
) were incubated
with 0, 30 or 50 mM fasentin, 1 mM GLUTi, 10 mM AA and
10 mM CCCP in the presence or absence of 20 mM glucose in
TES–NaCl solution containing 5 mM Ca
2þ
at 398C for 40 min.
After washing with TES–NaCl solution, spermatozoa (6 10
4
)
were solubilised in ATP assay reagent at 238C for 10 min, and
the luminescence signal was measured using a Multimode
Detector DTX800 (Beckman Coulter).
Statistical analysis
Multiple comparisons were conducted using one-way analysis
of variance (ANOVA), followed by Tukey’s honestly signifi-
cant difference (HSD), except for the effects of glucose sup-
plementation and fasentin concentration on motility profile,
which were analysed using two-way ANOVA. Differences were
considered significant at two-tailed P,0.05.
Results
Expression and localisation of GLUT1
Immunoblot analysis using chicken spermatozoa showed
detection of GLUT1 at the predicted molecular weight (Fig. 1a).
Immunostaining showed localisation of GLUT1 at the midpiece
region along with the flagellum (Fig. 1b). In addition, specific
localisation of GLUT1 was found at the midpiece when sper-
matozoa were labelled with GLUT1–GFP, a recombinant
GLUT1 ligand. No signals were observed in control spermato-
zoa that were only treated with a fluorescent secondary anti-
body. These results showed that GLUT1 was expressed and
specifically localised at the midpiece of chicken spermatozoa.
Effects of glucose on sperm motility
To examine the effects of glucose on sperm motility, sperma-
tozoa were investigated using SMAS immediately (0 min) or
after 40 and 80 min incubation with or without 20 mM glucose.
The sperm motility parameters at 0 min were as follow: motility,
95.98%; VSL, 27.75 mms
1
; VCL, 110.38 mms
1
; VAP,
44.12 mms
1
; LIN (VSL/VCL), 0.27; STR (VSL/VAP), 0.66;
ALH, 1.73 mm; BCF, 7.91 Hz (Table 1). When spermatozoa
were incubated without glucose supplementation, significant
(P,0.05) time-dependent decreases were observed at 40 and
80 min in motility (83.89% and 76.30% respectively), VSL
(20.08 and 16.77 mms
1
respectively), VCL (75.41 and
70.73 mms
1
respectively), VAP (29.72 and 24.21 mms
1
respectively) and ALH (1.31 and 1.19 mm respectively). No
changes were observed at 40 and 80 min for LIN (0.28 and 0.26
respectively), STR (0.72 and 0.72 respectively) and BCF (8.43
and 7.43 Hz respectively). In contrast, in the presence of 20 mM
glucose, there were no decreases in motility (93.37% and
88.84% at 40 and 80 min respectively) and VAP (38.93 and
37.08 mms
1
at 40 and 80 min respectively) after 40 min incu-
bation, or in VSL (28.04 and 26.13 mms
1
at 40 and 80 min
Sperm GLUT1 and ATP production pathways in poultry Reproduction, Fertility and Development C
respectively) and LIN (0.36 and 0.32 at 40 and 80 min
respectively) after 80 min incubation. Higher motility, VSL and
VAP were detected after 40 min -incubation following glucose
supplementation. Similar to observations without glucose, there
was no change in BCF throughout the incubation period
following glucose supplementation (11.12 and 10.59 Hz at 40
and 80 min respectively). Further, AA, an electron transport
inhibitor, inhibited motility, VSL, VCL, VAP and ALH even
in the presence of glucose, suggesting the involvement of mito-
chondrial oxidation in motility regulation (see Fig. S1, available
as Supplementary Material to this paper). Together, these results
indicate that glucose supports sperm flagellar motility.
Role of GLUT1 in sperm motility
To examine the glucose-dependent role of GLUT1 in supporting
sperm motility, spermatozoa were incubated with 0, 30 or 50 mM
fasentin, a GLUT1 inhibitor, for 40 min in the presence or
absence of 20mM glucose, followed by SMAS. In the absence of
glucose, there were no differences in any parameters among
fasentin concentrations (motility, 75.75–83.00%; VSL, 17.81–
18.72 mms
1
; VCL, 74.18–86.47 mms
1
;VAP,24.27–
27.55 mms
1
; LIN, 0.24–0.25; STR, 0.72–0.74; ALH, 1.36–1.58
mm; BCF, 6.05–7.38 Hz; Table 2).
Consistent with the findings presented in Table 1, incubation
with glucose improved sperm motility (91.81%), VSL
(27.96 mms
1
) and VAP (39.63 mms
1
) in the absence of
fasentin (P,0.05). In contrast, these effects were abolished
by the addition of 30 or 50 mM fasentin (motility, 83.65% or
81.93% respectively; VSL, 18.46 or 18.68 mms
1
respectively;
VAP, 25.55 or 27.51 mms
1
respectively). Furthermore, motil-
ity, VSL, VCL and VAP decreased significantly with increasing
concentrations of fasentin, suggesting GLUT1 inhibition by
Table 1. Changes in sperm motion parameters following incubation for 0–80 min, with or without (control) 20 mM glucose supplementation
Data are expressed as the mean s.e.m. (n¼5). Within rows, different superscript letters indicate significant differences (P,0.05). VSL, straight line
velocity; VCL, curvilinear velocity; VAP, average path velocity; LIN, linearity; STR, straightness; ALH, amplitude of lateral head displacement; BCF, beat
cross frequency
0 min 40 min 80 min
Control Control Glucose Control Glucose
Motility (%) 95.98 0.64
a
83.89 1.93
c
93.37 0.53
ab
76.30 0.81
d
88.84 0.65
bc
VSL (mms
1
) 27.75 0.70
a
20.08 1.16
b
28.04 0.89
a
16.77 0.41
b
26.13 1.01
a
VCL (mms
1
) 110.38 3.40
a
75.41 2.70
cd
87.34 1.82
bc
70.73 3.48
d
93.10 2.43
b
VAP (mms
1
) 44.12 1.25
a
29.72 1.63
c
38.93 0.89
ab
24.21 0.55
c
37.08 1.15
b
LIN (VSL/VCL) 0.27 0.00
ab
0.28 0.02
ab
0.36 0.02
a
0.26 0.02
b
0.32 0.02
ab
STR (VSL/VAP) 0.660.01
b
0.72 0.02
ab
0.76 0.02
a
0.72 0.01
ab
0.76 0.01
a
ALH (mm) 1.73 0.04
a
1.31 0.08
b
1.37 0.03
b
1.19 0.07
b
1.40 0.06
b
BCF (Hz) 7.91 0.15 8.43 1.04 11.12 0.66 7.43 0.81 10.59 0.64
(b)
55-
kDa
100-
70-
35-
SP
(a)
cAB GFP ligand Control
GLUT1
Nucleus
Merged
Fig. 1. Expression and localisation of the glucose transporter GLUT1 in chicken spermatozoa. (a)
Immunoblotting using a chicken GLUT1-specific antibody (cAB) showed the presence of GLUT1 at the
predicted molecular weight. SP, sperm. (b) Spermatozoa were fixed and subjected to immunostaining with cAB
or labelling with GLUT1 ligand fused with green fluorescent protein (GFP ligand). GLUT1 was localised at the
midpiece in addition to the flagellum. No signals were detected in spermatozoa labelled with secondary
antibody used as control. Nuclei were stained using 40,60-diamidino-2-phenylindole (blue). Images are
representative of three independent experiments. Scale bars ¼5mm.
DReproduction, Fertility and Development R. Setiawan et al.
fasentin. These results suggest that GLUT1 plays an important
role in in supporting flagellar motility in chicken spermatozoa.
Glucose uptake assay
To determine the role of GLUT1 in glucose uptake in chicken
spermatozoa, the fluorescent analogue 2-NBDG was used to
visualise spermatozoa incubated with different concentrations
of fasentin; both 30 and 50 mM fasentin reduced glucose uptake
(Fig. 2).
Together with the GLUT1 localisation data, these results
strongly suggest that GLUT1 contributes to glucose uptake in
the midpiece and indicate the involvement of GLUT1 in
mitochondrial activity.
Role of GLUT1 in ATP production
To examine the role of GLUT1 in chicken sperm ATP produc-
tion, cellular ATP content was quantified in spermatozoa
incubated with fasentin, GLUTi, AA or CCCP, with or without
20 mM glucose. Incubation with 20 mM glucose increased the
ATP content (mean increase 80%), whereas fasentin decreased
ATP content in a dose-dependent manner (22–33%; Fig. 3a).
There was no difference in ATP content between the 50 mM
fasentin and no glucose groups, suggesting that GLUT1 plays a
major role in ATP production. GLUTi, AA and CCCP markedly
reduced the ATP content (mean decreases of 93.1%, 88.4% and
82.4% respectively vs glucose alone), but no differences were
observed between them. Considering the specific localisation of
GLUT1 in the midpiece, together with abolishment of glucose-
stimulated motility by fasentin, these results reinforce that
glucose uptake via GLUT1 plays a major role in ATP produc-
tion. When spermatozoa were incubated without glucose, ATP
content was lower in the fasentin, GLUTi, AA and CCCP groups
than in the control with no glucose addition, suggesting that a
small amount of remnant glycosable substrates may be present
in the sperm suspension or cytoplasm before incubation.
To examine the effect of GLUT1 on mitochondrial activity,
MMP was assessed in spermatozoa incubated with fasentin,
GLUTi, AA or CCCP, in the absence or presence of 20 mM
Table 2. Motility parameters of sperm incubated for 40 min in the
presence of fasentin, with or without (control) 20 mM glucose
supplementation
Data are expressed as the mean s.e.m. (n¼5–6). Within rows, different
superscript letters indicate significant differences (P,0.05). *P,0.05
compared with control. VSL, straight line velocity; VCL, curvilinear
velocity; VAP, average path velocity; LIN, linearity; STR, straightness;
ALH, amplitude of lateral head displacement; BCF, beat cross frequency
Fasentin (mM)
03050
Motility (%)
Control 82.96 2.21 75.75 4.57 83.00 3.58
Glucose 91.81 1.14
a
* 83.65 1.17
ab
81.93 2.16
b
VSL (mms
1
)
Control 18.72 1.95 17.98 1.61 17.81 0.91
Glucose 27.96 1.01
a
* 18.46 1.73
b
18.68 3.92
b
VCL (mms
1
)
Control 86.47 7.85 86.16 9.99 74.18 2.34
Glucose 110.37 8.25
a
73.81 7.83
b
77.19 15.08
b
VAP (mms
1
)
Control 27.55 2.75 26.22 2.57 24.27 0.96
Glucose 39.63 1.39
a
* 25.55 2.12
b
27.51 5.09
b
LIN (VSL/VCL)
Control 0.24 0.01 0.25 0.02 0.25 0.01
Glucose 0.28 0.01 0.28 0.02 0.25 0.01
STR (VSL/VAP)
Control 0.72 0.02 0.73 0.02 0.74 0.01
Glucose 0.74 0.02 0.75 0.02 0.71 0.02
ALH (mm)
Control 1.41 0.08 1.58 0.18 1.36 0.04
Glucose 1.65 0.09 1.40 0.18 1.29 0.04
BCF (Hz)
Control 6.78 0.65 7.38 1.08 6.05 0.75
Glucose 6.98 0.33 7.99 1.15 7.89 1.50
(b)
(a)
0
10
20
30
40
50
60
70
03050
Fluorescence intensity
(×103 a.u.)
Fasentin BF Merged
0 µM
30 µM
50 µM
NC
Fasentin (µM)
∗
∗
Fig. 2. Glucose uptake in spermatozoa incubated with 0–50 mM
fasentin. Chicken spermatozoa were incubated with 0–50 mM fasentin
and 60 mM 2-(N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]-amino)-2-deoxy-D-
glucose (2-NBDG) in N-Tris(hydroxymethyl)methyl-2-aminoethane
sulfonic acid (TES)–NaCl with 5 mM CaCl
2
before the glucose uptake assay
using a fluorescent glucoseanalogue. (a) Fasentin treatment inhibited glucose
uptake. Data are expressed as the means.e.m. (n¼5). *P,0.05 compared
with 0 mM fasentin. (b) Representative images showing that signals for
2-NBDG were found exclusively in the midpiece and were diminished with
increasingconcentrations of fasentin. No fluorescent signalswere found in the
negative control (NC). BF, Bright field. Scale bars ¼10 mm.
Sperm GLUT1 and ATP production pathways in poultry Reproduction, Fertility and Development E
glucose. Glucose supplementation markedly increased MMP
(Fig. 3b), whereas MMP was dose-dependently decreased by
fasentin, resulting in no difference in MMP between fasentin-
treated groups with and without glucose. AA and CCCP were
used as negative controls, and these groups had a lower MMP
than the 50 mM fasentin-treated group. Together, these results
demonstrate that GLUT1 plays a major role in the stimulation of
oxidative phosphorylation in response to glucose uptake.
The results of this study indicated that oxidative phosphory-
lation is the predominant source of ATP in chicken spermatozoa.
Therefore, movement was analysed in spermatozoa treated with
uncouplers of mitochondrial oxidative phosphorylation. The
results showed a significant decrease in motility, VSL, VCL
and VAP compared with treatment with glucose alone (Fig. 3c).
Despite the potent inhibition of VSL, VCL and VAP, more than
60% of spermatozoa were still motile. In addition, a marked
difference was seen in the extent of the reduction in response to
mitochondrial oxidative inhibitors between ATP content and
motility, suggesting the involvement of both glycolysis and
oxidative phosphorylation in flagellar motility in chicken
spermatozoa.
Localisation of glycolytic enzymes
Several glycolytic enzymes have been identified in mammalian
spermatozoa that are primarily localised in the principal piece of
the flagellum, suggesting this is the primary site for glycolysis
(Miki et al. 2004). However, expression and localisation of
glucose metabolic pathways has not been demonstrated in avian
spermatozoa; therefore, we aimed to identify the localisation of
hexokinase I and GAPDH, major glycolytic enzymes, in
chicken spermatozoa. Immunoblotting for GAPDH and hexo-
kinase I showed they could be detected at the predicted
molecular weights (Fig. 4a).
GAPDH and hexokinase I were localised to both the mid-
piece and principal piece, along with the flagellum (Fig. 4b). In
addition, hexokinase I showed patch-like localisation at the
sperm head region despite it being largely devoid of GAPDH.
Together, these results suggest the glycolytic pathway occurs in
both the midpiece and principal piece of chicken spermatozoa.
Discussion
Spermatozoa have a high requirement for ATP as an energy
source to maintain flagellar motility. Despite the importance of
glucose transport for ATP production and fertility in chicken
spermatozoa, the regulatory mechanisms involved in glucose-
dependent sperm motility remain poorly understood. We found
that GLUT1 is specifically localised to the midpiece and con-
tributes to flagellar motility by ATP production via glycolysis
and oxidative phosphorylation. The results of this study provide
new insights into sperm function, such as flagellar motility, and
suggest dual roles of the midpiece in the glucose metabolic
pathways in avian spermatozoa.
In the present study, immunoblotting and immunostaining
using a specific antibody revealed GLUT1 in the midpiece. This
was confirmed by labelling spermatozoa with GLUT1–GFP, a
specific ligand tagged with GFP. This recombinant ligand was
originally generated from a binding domain of human T-cell
leukaemia virus to GLUT1, and has been widely used to
determine the expression and localisation of GLUT1 (Manel
et al. 2003;Kinet et al. 2007). Together, the results of the present
ac
b
ac
bc
ac c
ac ac aaaa
0
1
2
3
4
5
6
JC-1 aggregate/monomer
Glu – + – + – + – + – + – +
ATP (mol per 6×104
spermatozoa)
(a)
a
b
d
c
d
ac
fef f
def
f
de
0E+00
1E-08
2E-08
3E-08
4E-08
5E-08
Glu – + – + – + – + +––+
F30 F50 GLUTi AA CCCP
F30 F50 GLUTi AA CCCP
a
b
cc
0
20
40
60
80
100
Motility (%)
a
a
bb
0
20
40
60
80
100
VCL (µm s–1)
a
b
c
c
0
20
40
VAP (µm s–1)
a
b
cc
0
15
30
VSL (µm s–1)
(c)
–Glu
AA
CCCP
–+++
––+–
–– –+ –––+ –– –+ – ––+
––+– ––+– ––+–
–+++ +++ –+++
(b)
Fig. 3. Changes in (a) ATP content, (b) mitochondrial activity and (c)
sperm motility profile in response to various inhibitors. Spermatozoa were
incubated with 30 or 50 mM fasentin (F30 and F50), 1 mM glucose transporter
inhibitor (GLUTi), 10 mM antimycin A (AA) or 10 mM CCCP, with 0 or
20 mM glucose supplementation, for 40 min. (a) ATP was quantified and (b)
mitochondrial activity was measured using 5,50,6,60-tetrachloro-1,103,30-
tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) fluorescence. (a) ATP
content was increased by glucose supplementation, but decreased in the
presence of F30 and F50. GLUTi, AA and CCCP markedly reduced the ATP
content. (b) Mitochondrial activity was also higher following glucose
supplementation, but was inhibited by the addition of F50 and GLUTi.
AA and CCCP reduced mitochondrial activity. (c) The sperm motility
analysis system (SMAS) revealed reduced motility, straight line velocity
(VSL), curvilinear velocity (VCL) and average path velocity (VAP) for
spermatozoa incubated with glucose in combination with either AA or
CCCP compared with glucose alone or no glucose supplementation. Data are
expressed as mean s.e.m. (n¼4, 6 and 5 in (a), (b) and (c) respectively).
Different letters above columns indicate significant differences (P,0.05).
FReproduction, Fertility and Development R. Setiawan et al.
study strongly suggest the specific localisation of GLUT1 at the
midpiece in chicken spermatozoa. A previous localisation study
showed that the midpiece region was largely devoid of GLUT1,
and that GLUT1 was localised to the acrosomal region and
principal piece in several mammalian species (Angulo et al.
1998). This suggests a functional distinction of GLUT1 between
mammalian and avian spermatozoa.
Previous studies in chicken spermatozoa demonstrated the
importance of glucose transport in sperm motility and fertility
(Wishart 1982;McLean et al. 1997). Consistent with this,
SMAS in this study showed that glucose supplementation
supports sperm motility. It was previously shown that several
GLUTs were associated with the flagellar regions in mammalian
spermatozoa (Bucci et al. 2011). In addition, we recently
reported that GLUT3 is localised to the entire flagellum as well
as acrosomal regions in chicken spermatozoa (Ushiyama et al.
2019). However, it remains unclear which GLUT support
flagellar motility. Fasentin binds to a unique site of the intracel-
lular domain of GLUT1, resulting in the inhibition of glucose
uptake (Wood et al. 2008). As a preliminary experiment, we first
attempted to identify effective fasentin concentrations on the
motility profile and found concentration-dependent inhibition
(data not shown). This, combined with the fact that the IC
50
of
fasentin is 68 mM, was why we chose to use concentrations of 30
and 50 mM fasentin in this study, resulting in almost complete
abolition of sperm motility dependent on extracellular glucose.
Similarly, uptake of 2-NBDG decreased following fasentin
treatment. Furthermore, these concentrations showed no cyto-
toxicity, at least in terms of chicken sperm motility, which is in
agreement with a previous study performed using cultured cells
(Wood et al. 2008). Thus, the results suggest that GLUT1 plays a
major role in glucose transport to power flagellar motility.
In mammalian spermatozoa, glucose-mediated acceleration
of flagellar motility depends on ATP production via oxidative
phosphorylation in mitochondria and glycolysis in the principal
piece (Ford 2006). In agreement with this, we found that GLUT1
inhibition abolished the increase in ATP production and mito-
chondrial activity in response to glucose supplementation in
chicken spermatozoa. In addition, more potent inhibition of
ATP production was observed when spermatozoa were treated
with GLUTi. Considering that this inhibitor blocks not only
GLUT1-, but also GLUT3-, GLUT4- and GLUT9-mediated
transport to some extent (Granchi et al. 2014), these results
suggest an important role of GLUT1 in glucose metabolic
pathways as well as the potential involvement of other GLUTs.
Supporting this, we and others previously demonstrated that
GLUT3 is present along the length of the flagellum in chicken
and murine spermatozoa (Simpson et al. 2008;Ushiyama et al.
2019). Furthermore, flagellar localisation of the GLUT9 iso-
form was reported in murine spermatozoa (Kim and Moley
2007), although no information is available regarding avian
spermatozoa. Considering the restricted localisation of chicken
GLUT1 to the midpiece of the flagellum, together with differ-
ences in the affinity of GLUT for glucose (Devaskar and
Mueckler 1992), these findings suggest differential roles of
GLUT in ATP production pathways.
The present study showed that disruption of mitochondrial
ATP production using AA or CCCP, uncouplers of mitochon-
drial oxidative phosphorylation, reduced cytoplasmic ATP and
sperm movement, concomitant with mitochondrial activity.
These findings are consistent with a previous study that showed
a strong correlation between mitochondrial ATP production and
motility in chicken spermatozoa (Froman et al. 1999). Surpris-
ingly, approximately 60% of spermatozoa were still motile after
mitochondrial inhibition in chickens. Studies in mammalian
spermatozoa have shown different roles of ATP produced via
different metabolic pathways (Travis et al. 2001;Mukai and
Okuno 2004). For example, in mice, CCCP treatment had no
effect on motility or ATP levels, but diminished mitochondrial
activity following glucose supplementation (Goodson et al.
2012). Furthermore, studies in guinea pig spermatozoa showed
that glucose stimulates motility and ATP production, even with
inhibition of oxidative phosphorylation, although it retarded the
acrosome reaction inherent with the capacitation process
(Rogers and Yanagimachi 1975;Mu
´jica et al. 1991). Despite
a wealth of knowledge about mammalian spermatozoa, the
localisation and functional roles of the glucose metabolic
pathway remain poorly characterised in avian spermatozoa.
The results of this study showed localisation of GAPDH and
hexokinase I at the midpiece and principal piece of the flagel-
lum, suggesting that the glycolytic pathway operates along the
entire length of the flagellum in chicken spermatozoa. Several
glycolytic enzymes, including GAPDH and hexokinase I, have
GAPDH HK1
GAPDH
37-
100-
75-
50-
kDa
250-
150-
DAPI
Merged
kDa HK1
250-
100-
150-
75-
50-
(a)
(b)
Fig. 4. Expression and localisation of glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) and hexokinase 1 (HK1) in chicken spermatozoa.
(a) GAPDH and HK1 were expressed at the predicted molecular sizes.
(b) GAPDH and HK1 were both localised to the flagellum, with clear
enrichment in the midpiece (green). Faint, dotted distribution of HK1 was
observed in the sperm head, where GAPDH was not present. Nuclei were
stained using 40,60-diamidino-2-phenylindole (DAPI; blue). Images are
representative of five independent experiments. Scale bars ¼5mm.
Sperm GLUT1 and ATP production pathways in poultry Reproduction, Fertility and Development G
sperm-specific isoforms directly or indirectly tethered to the
fibrous sheath present exclusively in the principal piece of the
flagellum (Welch et al. 1992;Westhoff and Kamp 1997),
resulting in the distinction of its localisation from the somatic
isoform. Despite the similarity in the flagellar structure between
mammals and birds, antibodies were not able to distinguish
tissue-specific isoforms due to commonality of the epitope
region between isoforms. Recent studies in bull spermatozoa
using tissue-specific antibodies against GAPDH showed that
somatic GAPDH was localised to the midpiece, but the sperm-
specific isoform was confined to the principal piece (Feiden
et al. 2008). It was also reported that the midpiece contains
hexokinase I isoforms (Travis et al. 1998;Nakamura et al.
2008). These findings suggest that chicken spermatozoa may
possess both somatic and sperm-specific isoforms. Of note, we
found faint patch-like distribution of hexokinase I in the sperm
head, largely devoid of GAPDH. Hexokinase I is also involved
in the pentose phosphate pathway (PPP), a semi-independent
and parallel pathway to glycolysis. Previous studies in mamma-
lian spermatozoa showed that an active PPP is localised to the
midpiece and sperm head, and plays a role in multistage of
fertilisation (Bolton and Linford 1970;Urner and Sakkas 2005).
The results of the present study suggest a future investigation
into the functional nature of PPP in avian spermatozoa.
Although oxidative phosphorylation is more efficient than
glycolysis for ATP production, in most mammalian spermato-
zoa glycolysis is exclusively responsible for supporting
capacitation-associated changes, such as protein tyrosine phos-
phorylation and hyperactivated motility in some species (Travis
et al. 2001;Williams and Ford 2001). Considering that avian
spermatozoa, unlike mammalian spermatozoa, do not require
capacitation for functional changes to acquire the competency to
fertilise, our results provide a foundation for investigations into
ATP production pathways in vertebrates.
In summary, the present study demonstrated that GLUT1 is
specifically localised to the midpiece region in chicken sperma-
tozoa and plays a major role in ATP production and flagellar
motility supported by glucose uptake. Furthermore, the results
suggest that chicken spermatozoa rely on both glycolysis and
oxidative phosphorylation to support flagellar motility. These
findings provide new insights into the glucose metabolic path-
ways in avian spermatozoa, and highlight their distinction from
mammalian spermatozoa.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
This study was supported by Japan Society for the Promotion of Science
(Grants 17KK0150 and 15K07761 to Atsushi Asano) and the Indonesian
Endowment Fund for Education, Republic of Indonesia (to Rangga
Setiawan).
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