ROLE OF GLYCOLYSIS AND RESPIRATION IN SPERM METABOLISM AND

MOTILITY

A thesis submitted to Kent State

University in partial fulfillment of the
requirements for the degree of Master of Science

By
Vinay Pasupuleti
December, 2007

Thesis written by
Vinay Pasupuleti
M.B., B.S., Kasturba Medical College, 2001
M.S., Kent State University, 2007

Approved by

______________________________, Advisor
S. Vijayaraghavan
______________________________, Director, School of Biomedical Sciences
Robert V. Dorman
______________________________, Dean, College of Arts and Sciences
Jerry Feezel

ii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ..v
INTRODUCTION .1
Background ........1
Aims .....13
METHODS ..14
RESULTS ....19
DISCUSSION ..38
REFERENCES.....47

iii

LIST OF FIGURES
Figure 1. Anatomy of spermatozoa....3
Figure 2. Glycolysis .......7
Figure 3. ATP production from glycolysis and respiration8
Figure 4. Sperm ATP and motility in media sustaining glycolysis or respiration ...20
Figure 5. Effect of DOG on sperm ATP and motility in presence of pyruvate and
lactate22
Figure 6. Effect of iodoacetamide on sperm ATP and motility in presence of pyruvate
and lactate...........23, 24
Figure 7. Effect of DOG and iodoacetamide on sperm ATP and motility in presence
of glucose......25
Figure 8. Effect of DOG on sperm ATP and motility in presence of fructose.....27
Figure 9. Western blot of mouse sperm extracts probed with GSK-3 antibody.........28
Figure 10. Aligned GSK-3 peptide sequence of human, rat and mouse......30
Figure 11. Western blot of mouse sperm extracts probed with GSK-3/ antibody.....31
Figure 12. Western blot of bovine sperm extracts probed with GSK-3 antibody...32
Figure 13. Intracellular localization of GSK-3/ in mouse sperm ..34
Figure 14. Intracellular localization of GSK-3/ in bovine sperm ..35
Figure 15. Sperm ATP and motility in presence of GSK-3 inhibitors...37
Figure 16. Schematic of modes of action of DOG and iodoacetamide..46

iv

ACKNOWLEDGEMENTS
The author extends his sincere gratitude to the following individuals:
Dr. Srinivasan Vijayaraghavan, Department of Biological Sciences, Kent State University
for his guidance and support throughout the duration of this endeavor.
Dr. Douglas Kline and Dr. Jennifer L. Marcinkiewicz, my committee members, for their
valuable time and advice.
Pawan Puri, doctoral student in Department of Biological Sciences and Dr. Rumela
Chakrabarti, for their help with the experiments and review of the thesis.

INTRODUCTION

Background
Motility is a characteristic function of most male gametes and this feature enables
the spermatozoa to reach a female gamete for fertilization. The sperm must be highly
motile for an extended period of time under varying conditions. Despite decades of
research, relatively little is known about how various metabolic and biochemical
pathways operate to induce and sustain motility in mature spermatozoa.

Several

intracellular mediators and exogenous substances have been found to stimulate or inhibit
motility in spermatozoa.

A complete understanding of energy utilization and the

mechanism of motility mediators will ultimately lead to the elucidation of this complex
biological process.

Spermatogenesis
Spermatogenesis is the process by which a complex, interdependent population of
germ cells produces spermatozoa. Three major stages can be distinguished:
spermatogoniogenesis, meiosis of spermatocytes and spermiogenesis. Spermatogenesis
occurs within the seminiferous tubules of the testes in intimate association with Sertoli
cells.

Sertoli cells provide nourishment and protection to the developing gametes.

Leydig cells in the interstitial spaces between the tubules secrete testosterone hormone
which is essential to spermatogenesis. During spermatogoniogenesis, germ cells divide

mitotically to form spermatogonia, some of which differentiate and undergo mitotic

division to form primary spermatocytes. The meiotic division of primary spermatocytes
produces secondary spermatocytes which complete the second meiotic division to form
spermatids. Spermatids are haploid, round, cells without flagella that differentiate
morphologically to form mature spermatozoa by a process called spermiogenesis. During
spermiogenesis, spermatids begin to grow a tail, and develop a thickened mid-piece,
where the mitochondria gather around an axoneme. The chromatin undergoes packaging,
becoming highly condensed and transcriptionally inactive. The Golgi apparatus surrounds
the condensed nucleus, becoming the acrosome. Mature spermatozoa are released into the
seminiferous tubule lumen at the completion of spermiogenesis.

A complete

spermatogenetic cycle from spermatogonium to mature spermatozoa requires

approximately 56 days in a mouse and 65 days in humans.

Spermatozoa
The two main components of the mature sperm are the head and flagellum, as
shown in Fig.1. The head contains the nucleus, acrosome and a small amount of
cytoplasm. The flagellum is divided successively into midpiece, principal piece and the
end piece. It contains the central complex of microtubules forming the axoneme,
surrounded in turn by outer dense fibers extending from the neck into the principal piece.
The midpiece contains the mitochondria. The axoneme has the conserved 9+2
structure, consisting of a central doublet of microtubules surrounded by a ring of nine
A/B microtubule doublets [1].

Fig.1. Anatomy of a bovine spermatozoon. A mature spermatozoa consists of a

head containing the acrosome and the nucleus, the mid-piece containing the
mitochondria, the tail and the end-piece.

Spermatozoon maturation
Sperm morphogenesis is accomplished in the testis, but testicular sperm remain
physiologically immature. Once formed within the seminiferous tubules, the immotile
spermatozoa are released into luminal fluid and transported into epididymis, where they
gain the ability to move [2]. Epididymal maturation of spermatozoa is an androgen
dependent process [3]. The testicular spermatozoa are transported passively into the rete

testis and then to the epididymis via the efferent ducts. The efferent ducts absorb most of
the fluid discharged from the testis with the spermatozoa, thus increasing the epididymal
sperm concentration [4]. The epididymis can be divided into three parts: caput, corpus
and cauda. In most mammals, the transit of spermatozoa through the epididymis usually
takes 10-13 days and in humans the estimated transit time is 2-6 days [5]. Generally,
spermatozoa isolated from the caput epididymis are immotile and spermatozoa isolated
from the caudal epididymis show high motility and forward progression [6-8]. To attain
the capacity to fertilize, sperm undergo many maturational changes during its transit in
the epididymal duct [4]. These include changes in plasma membrane lipids, proteins and
glycosylation, alterations in the outer acrosomal membrane and cross-linking of nuclear
protamines and proteins of the outer dense fiber and fibrous sheath. Spermatozoa are
maintained in a low energy consumption state during epididymal storage in the cauda
epididymis, thus conserving energy and favoring long-term survival of the cells [9].
Motility is activated when spermatozoa contact substances in semen upon ejaculation
[10]. Sperm artificially isolated from the caput and caudal epididymis are called caput
sperm and caudal sperm respectively and are used to study changes in motility
parameters and metabolism.
Though caudal spermatozoa are motile they are unable to fertilize the egg.
Spermatozoa need to undergo further maturational changes including capacitaion,
hyperactivation and acrosome reaction before they can fuse with the female gamete.
These changes begin once sperm are deposited into the female reproductive tract.
Capacitation is initiated and possibly already completed in the cervix [11]. During

capacitation there are changes in the sperm plasma membrane, intracellular ions,
metabolism, nucleus and acrosome [12]. Hyperactivation takes place in the oviduct and
helps the spermatozoa to swim in the viscous oviduct fluid [13]. The acrosome reaction
enables spermatozoa to penetrate through the zona pellucida and fuse with the egg
plasma membrane [14].

Mechanics of flagellar motility

Activation of sperm flagellar motility involves activation of both energy
metabolism and the motile apparatus. The flagellar movement is generated by the motor
activities of the axonemal dynein arms working against stable microtubule doublets. The
initiation of the flagellar waveform is dependent on the phosphorylation of the axonemal
dynein [15]. After phosphorylation, the dynein ATPase is activated. The energy released
by the hydrolysis of ATP, converted to force, causes the microtubules to slide past one
another [16, 17]. Dephosphorylation of dynein by the calmodulin-dependent protein
phosphatase

calcineurin

then

reverses

the

process

[18].

The

phosphorylation/dephosphorylation and the corresponding activation and inactivation of

the dynein arms occur in an asynchronous manner around the circumference and along
the length of the axoneme [19]. The axoneme propagates bends in both directions by
regulating the timing and location in which dynein arms are active [1]. The sliding
activity of the central pair of microtubules is modulated by intracellular calcium [20].

Sperm metabolism
The sperm axoneme engine requires a continuous supply of ATP to maintain
motility in the male and female reproductive tract. Sperm ATP requirements change
during epididymal maturation and later in the female reproductive tract when they
undergo capacitation and hyperactivation. Sperm require an adequate and increasing
supply of ATP as they go through these events.
As early as the 1930s, bovine spermatozoa were shown to convert glucose,
fructose or mannose to lactic acid to sustain motility. Studies over the next several
decades led to the conclusion that mammalian sperm can produce energy by anaerobic
glycolysis, by oxidation of the metabolic products of glycolysis or by oxidation of
endogenous substrates [22-24]. There has been a disagreement regarding the relative
importance of these three processes and this confusion might be partly because of
considerable species variation in metabolic patterns [27, 28].
Sperm can use variety of simple sugars such as glucose, fructose and mannose
[27] and have the ability to metabolize glycerol, lactate, pyruvate and acetate by utilizing
them into glycolytic pathway [27]. Glycolysis can occur with a variety of substrates.
Glucose is converted to glyceraldehyde-3-phosphate by using 2 mol of ATP per mol of
glucose. Four mol of ATP are produced when pyruvate is made from glyceraldehyde-3phosphate giving a net production of 2 mol ATP by the oxidation of one mol of
glucose(Fig. 2). Pyruvate enters the TCA cycle where NADH and FADH2 molecules are
produced, oxidation of NADH and FADH2 in the electron transport chain generates the
ATP molecules by oxidative phosphorylation (Fig. 3). Mammalian sperm can also

produce energy by anaerobic glycolysis or by oxidation pyruvate. A unique intra

mitochondrial lactate dehydrogenase X allows the NADH resulting from pyruvate
oxidation to convert pyruvate to lactate [26].

Fig.2. Glycolysis

Fig.3. ATP production from glycolysis and respiration .

There are remarkable species differences in the relative dependence of sperm on

different energy resources. Human spermatozoa survive well anaerobically in the present
of exogenous glycolysable substrate and depend less on the energy of oxidative
respiration [27]. Sperm from guinea-pig and boar are essentially unable to support
motility by anaerobic glycolysis and depend on a much greater degree on oxidative
respiration [28, 29]. Bull and rhesus monkey lie in between these extremes since their

energy requirements can be met by glycolysis as well as by respiration [30]. Bull sperm
velocities were found to be comparable in the aerobic and anaerobic conditions in the
medium containing glucose [27]. Ejaculated ram spermatozoan motility was shown to be
sustained only on mitochondrial oxidation in quercetin (a glycolytic inhibitor) treated
cells [38]. Bull sperm motility parameters were not significantly different in the presence
or absence of Antimycin A and Rotenone (inhibitors of mitochondrial respiration) when
glucose was present in the medium which indicated that glycolysis can support motility
on its own. Medium containing pyruvate but with 2-deoxyglucose (an inhibitor of
glycolysis) could support motility in aerobic conditions suggested that mitochondrial
oxidation can support motility in the absence of glycolysis. Pyruvate has been shown to
yield ATP and maintain motility in the presence of rutamycin and rotenone (inhibitors of
mitochondrial respiration) which implied that pyruvate is metabolised to produce ATP by
a pathway independent of oxidative phosphorylation associated with electron transport
chain [28].
Sperm contain many mitochondria strategically located in the mid-piece where
they can efficiently power the flagellum. Although mitochondrial oxidation is more
efficient than glycolysis for ATP production, some have questioned whether diffusion of
ATP from mid-piece mitochondria could be adequate and rapid enough to fulfill the
energy needs for active sliding in the distal end of long mouse sperm flagella [31].
Glycolysis is likely to be utilized in the distal flagella since the enzymes of glycolysis
such as hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase
[33] are localized to fibrous sheath of various mammalian species [33].

10

A recent study by Mukai and Okuno [34] suggested that glycolysis is important
for mouse sperm motility, and that respiratory substrates (pyruvate) cannot maintain
sperm motility unless glycolysis is also functional. This was apparently supported by the
observation that mouse sperm motility decreased when incubated in media containing
glycolytic inhibitor, 2-deoxyglucose (DOG). DOG is a glucose analog which inhibits
hexokinase in the first step of glycolysis. This inhibition occurred in the presence of
pyruvate. Maintenance of motility in the presence of pyruvate and absence of glucose
was proposed to be due to glycolysis of glucose obtained through gluconeogenesis.
However, no direct evidence for gluconeogenesis was presented. This also raises the
question as to why sperm would use gluconeogenesis, which consumes three times more
ATP than being produced by glycolysis, to form glucose and then use glucose in
glycolysis to generate ATP. DOG gets converted to DOG-6-phosphate by hexokinase.
This study did not take into account the possibility that ATP consumed for
phosphorylation of DOG, might be depleting ATP produced by oxidative
phosphorylation. Another report which showed that glyceraldehyde-3- phosphate
dehydrogenase (GAPDH) knock out mouse sperm had very low ATP and lacked
progressive motility suggested that glycolysis is essential for mouse sperm motility and
fertility [35]. GAPDH knock out mouse would be unable to generate ATP by glycolysis.
Sperm motility in GAPDH knock out mice was measured in medium containing glucose
which leads to accumulation of the glycolytic intermediate, glyceraldehyde-3-phosphate.
However an alternate explanation is that increased concentration of glycolytic
intermediates decrease sperm phosphate levels and thus consume ATP being produced by

11

oxidative phosphorylation [36]. This might be responsible for decreased motility seen in
these sperm. It has been shown that oxidative phosphorylation can support mouse sperm
motility on its own when glycolysis is inhibited by -cholrohydrin, a GAPDH inhibitor
[37].
The focus of this research was to determine whether mitochondrial and glycolytic
pathways individually can sustain ATP production and motility over time or whether both
operating together are needed to maintain motility and ATP levels required by the sperm.

Glycogen Synthase Kinase-3

Glycogen synthase kinase-3 (GSK-3), originally identified as a regulator of
glycogen metabolism [39] is a signaling enzyme involved in insulin [40] and growth
factor function [41, 42]. GSK-3 acts as a downstream regulatory switch that determines
the output of numerous signaling pathways initiated by diverse stimuli [43]. Several
mechanisms play a part in controlling the actions of GSK-3, including its
phosphorylation, complex with other proteins, and its subcellular distribution. These are
used to control and direct the far-reaching influences of GSK-3 on cellular structure,
growth, motility and apoptosis [44]. In mammals there are two isoforms, GSK-3 (51
kDa) and GSK-3 (47 kDa) which are encoded by two independent genes [45]. Studies
show that GSK-3 and its regulating kinases are important signaling enzymes involved in
sperm regulation and specifically the development of sperm motility [46, 47]. GSK-3
along with the upstream signaling proteins, protein kinase B (PKB; also known as cAkt)
and phosphoinositide 3-kinase (PI3-kinase), involved in its phosphorylation, are present

12

in the spermatozoa [48, 49]. In somatic cells, GSK-3 is regulated by serine and tyrosine
phosphorylation [50, 51]. GSK-3 activity is much lower and tyrosine phosphorylation of
GSK-3 is much higher in caudal compared to caput spermatozoa [46, 52, 53].
Stimulation of motility is associated with an increase in GSK-3 tyrosine phosphorylation
while inhibition of motility results in the disappearance of the phosphorylation [53].
Serine phosphorylation of GSK-3 increases significantly in spermatozoa during their
passage through the epididymis [49]. These studies on GSK-3 phosphorylation support
the idea that GSK-3 has an important role in sperm function.
Glycolysis is regulated by availability of substrate, concentration of enzymes
responsible for the rate limiting steps, allosteric regulation of enzymes and covalent
modification of enzymes (e.g. phosphorylation). GSK-3 is one of the potential kinases
responsible for regulation of glycolysis by enzyme phosphorylation and therefore can
have an indirect role in ATP production in sperm. Studying the changes in subcellular
distribution and localization of GSK-3 in caput and caudal sperm as the sperm attains
motility and its role in sperm metabolism and motility would further understanding of the
role of GSK-3 in sperm function.

13

Summary of Aims
The focus of this research was to determine whether mitochondrial and glycolytic
pathways individually can sustain or both operating in tandem are needed to maintain
motility and ATP levels required by the sperm.
Hypothesis #1: Respiration and glycolysis compensate for each other but they do not
have individual obligatory roles in sperm metabolism and motility.
-

Aim #1: Study the role of mitochondrial respiration and glycolysis in

sperm ATP production and sperm motility.

The contribution and relative importance of these two pathways will be assessed by
suspending sperm in media with glycolytic or respiratory inhibitors. ATP levels will be
quantified using lucifearse assay and motility by computer assisted sperm motility
analysis.
Hypothesis #2: GSK-3 has a role in sperm ATP production.
-

Aim #2: Determine distribution and localization of GSK-3 and GSK-3

in bovine and mouse sperm and study the role of GSK-3 in sperm
metabolism.

Western blotting and immunocytochemistry will be used to determine the distribution

and localization of GSK-3/ in sperm. GSK-3 inhibitors will be used to look for
changes in sperm ATP levels and motility.

METHODS
Sperm Extract Preparation
Testes of mature bulls with intact tunica were obtained from a local
slaughterhouse. CD1 strain, wild-type mice were obtained from the animal facility at
Kent State University. Mice were sacrificed by CO2 asphyxiation. Bovine and mouse
caput and caudal spermatozoa were isolated and washed twice in CESD buffer (10mM
Tris-HCl pH 7.2, 100mM NaCl, 40mM KCl, and 5mM MgCl2). Bovine ejaculated
received in milk solution were given three to four washes before they were used in the
experiments. Sperm pellets derived from a suspension of 109 sperm/ml were suspended in
HB+, homogenization buffer (10mM Tris pH 7.2, 1mM EDTA, 1mM EGTA)
supplemented with protease inhibitors (10mM benzamidine, 1mM PMSF, 0.1mM TPCK,
and 5mM -mercaptoethanol), and cells were lysed with three 5-sec bursts of a Biosonic
(Bronwell Scientific, Rochester, NY) sonicator at maximum setting. The sperm sonicate
was centrifuged at 16,000 x g for 15 min. For RIPA+ (50mM Tris HCl pH 8.0, 150mM
NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors) and RIPA+SDS
(RIPA+ with 1% SDS) buffer extracts sperm were suspended in these solutions for 30
minutes, kept on ice and then centrifuged. The supernatant which is the soluble sperm
extract and the pellet which is the insoluble sperm extract were used in the western blot
experiments. Bovine sperm in homogenization buffer were centrifuged at 16,000 x g and
the supernatant (16k extracts) obtained were used as controls in western blot experiments.

14

15

Antibodies
A rabbit polyclonal antibody against the carboxy-terminus domain of GSK-3
was used to identify GSK-3 in mouse sperm extracts by western blotting. This antibody
was produced commercially (Zymed Inc., South San Francisco, CA) with a synthetic
polypeptide with the amino acid sequence WQSTDATPTLTNSS corresponding to the
carboxy-terminus of GSK-3 (Fig. 10).
A

mouse

monoclonal

antibody

against

amino

acid

sequence,

KQLLHGEPNVSYICSRY, a sequence common for both the isoforms of GSK-3 (Fig.

10) was used in western blotting and immunocytochemistry to identify and localize GSK3 and GSK-3. This antibody was purchased from Upstate biotechnology (UBI), Lake
Placid, NY.

Western blot analysis

Eluates or flow-through samples from the various experiments were separated by
SDS-PAGE through 12% polyacrylamide slab gels. After electrophoresis, proteins were
electrophoretically transferred to Immobilon-P, PVDF membrane (Millipore Corp.,
Bedford, MA). Non-specific protein binding sites were blocked with 5% nonfat dry milk
in TTBS (Tris-buffered saline (TBS: 25mM Tris-HCl pH 7.4, 150mM NaCl) containing
0.1% Tween 20).

The blots were incubated with the primary antibody overnight,

shaking, at 4C. After washing twice for 10 min each with TTBS, the blots were
incubated

with

peroxidase-labeled

anti-rabbit

secondary

antibody

(Amersham,

Piscataway, NJ) for 1 h at room temperature. After washing twice for 15 min each and

16

four times for 5 min each in TTBS, the blots were developed with an ECL
chemiluminescence kit (Amersham) and exposed onto Kodak X-OMAT film.

Fluorescence Immunocytochemistry
Caudal spermatozoa were isolated and washed twice as previously described, then
resuspended in PBS. Cells (50-100 l of 1 x 108 cells/ml) were attached to poly-L-lysinecoated coverslips and then fixed in 100% methanol for 5 min at -20C or cells in
suspension were fixed with 4% formaldehyde in PBS for 30 min at 4C, permeabilized
briefly with 0.2% Triton X-100, then attached to poly-L-lysine-coated coverslips. Once
air-dried the attached cells were washed three times with 200 l TTBS, then incubated
overnight with 200 l blocking buffer (2.5% BSA and 5% normal goat serum in TTBS)
in a humidified chamber. GSK-3/ (UBI) antibody was diluted in blocking buffer. The
cells were incubated in 200 l of this diluted (1:2 to 1:200) primary antibody overnight at
4C in a humidified chamber. For the negative control, the primary antibody was omitted
and the cells were incubated in blocking buffer overnight at 4C. The cells were washed
three times with TTBS, then incubated, shielded from light, for 1 h at room temperature
in 200 l goat anti-rabbit or anti-mouse secondary antibody conjugated to Cy3 (Jackson
Laboratories, West Grove, PA) diluted 1:200 in blocking buffer. The cells were washed
five to six times in cold TTBS and air-dried. The coverslips were mounted on slides
using Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA). Cells
were examined by fluorescence and phase-contrast microscopy and images were saved as
24-bit JPEG files.

17

ATP Assay
The amount of ATP contained in mouse sperm was measured by using
ENLITEN rLuciferase/Luciferin Reagent (Promega USA) and 20/20n Luminometer
(Turner Biosystems USA). Principle of the assay: Luciferin in presence of O2 and ATP is
converted to oxyluciferin and emits light. This reaction is catalyzed by luciferase. When
ATP is the limiting component in the luciferase reaction, the intensity of the light emitted
is proportional to the concentration of ATP. After sperm counts were done using a
hemocytometer, sperm were suspended in test solutions and incubated at 37C for 30 min
in a 5% CO2 incubator. The suspension was centrifuged at 600 x g for 5 min and 1%
trichloroacetic acid (TCA) was added to the pellet. This solution was then vortexed and
centrifuged at 16000 x g for 10 min. Ten l of the supernatants for each experiment was
added to 100l of the reagent for the ATP measurement. Relative light unit (RLU)
values thus obtained were plotted on an ATP standard curve whose RLU values were
obtained from 10-fold serial dilutions of the ATP standard (10-6 M to 10-11 M). The
concentration of ATP is reported in nanomoles ATP/109 sperm. Each experiment was
repeated thrice.

Computer Assisted Sperm Motility Analysis

Cauda epididymal sperm were harvested in BSA-fortified (10%) Whittinghams
medium (99.3 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl22H2O, 0.5 mM MgCl26H2O, 0.36
mM NaH2PO4, 25 mM NaHCO3, 100 U/ml penicillin G-K salt, 50 g/ml streptomycin
sulfate, 25 mM sodium lactate, 0.50 mM sodium pyruvate, 5.55 mM glucose, pH 7.4) or

18

Whittinghams media lacking glucose or pyruvate and lactate. Debris and dead sperm
population were reduced by centrifuging the sperm at 1000 rpm for 2 min and after 10
min of "swim up" sperm were collected and incubated in media containing 0.5mM
iodoacetamide, 5mM DOG and 4M antimycin A. Sperm were incubated with each
inhibitor for 30 min. Sperm motility were analyzed by computer assisted sperm analysis
using CEROS sperm analyzer from Hamilton Thorne Biosciences. This procedure uses
the pattern analysis statistical computer program which calculates the percentage of
motile sperm in a population and various motion parameters of which average path
velocity has been shown in the results. Each experiment was repeated thrice.

Statistical Analysis
Values for ATP, path velocity and percentage motility have been expressed as the mean
and standard error of mean (SEM). The mean and SEM were calculated using a statistical
program in Microsoft Excel which calculates descriptive statistics for a set of variables.
The standard errors were compared and means were considered different if SEM did not
overlap.

RESULTS

Aim #1 Study the role of mitochondrial respiration and glycolysis in sperm ATP
production and sperm motility.
Mouse sperm ATP levels and motility can be maintained in medium supporting either
glycolysis or mitochondrial respiration.
Glycolysis and oxidative respiration are the main sources of ATP production in a sperm.
There has been much debate on their contribution and individual ability to maintain
sperm motility. First, to see the relative contribution of glycolysis and oxidative
respiration in maintaining the sperm ATP pool and motility, mouse caudal sperm were
incubated in medium containing 1) glucose along with pyruvate and lactate i.e.
supporting both glycolysis and mitochondrial respiration, 2) only pyruvate and lactate,
supporting only mitochondrial respiration or 3) glucose and Antimycin A, a medium
supporting only glycolysis. Pyruvate is converted to acetyl coA before entering the
Krebs cycle and in the process NAD+ gets converted to NADH. Lactate is converted to
pyruvate by lactate dehydrogenase and in the process converts NADH to NAD+. Using
both lactate and pyruvate as respiratory substrates ensures a continuous supply of NAD+
in sperm. NAD+ is required in the fifth step of glycolysis. Antimycin A is a well
established mitochondrial site III electron transport chain inhibitor [54].

19

20

Fig. 4. Mouse caudal epididymal sperm ATP concentration (A) and sperm
motility (B) in media supporting both glycolysis and respiration (glucose +
pyruvate & lactate) or oxidative respiration (pyruvate & lactate) or glycolysis
(glucose + antimycin A). ATP and motility data are the mean of three
experiments. Error bars represent SEM.

21

Sperm ATP levels were highest when medium supported both glycolysis and
respiration. A decrease in sperm ATP levels were observed when either glycolysis or
mitochondrial respiration was inhibited (Fig. 4A). This decrease was not statistically
significant. No significant differences were observed in sperm motility parameters and
path velocity of sperm suspended in all three above mentioned conditions (Fig. 4B).

The glycolytic inhibitor, DOG decreases mouse sperm ATP levels and motility in a
medium containing respiratory substrates.
In order to clarify the importance of glycolysis, we studied the effect of two
glycolytic inhibitors, 2-deoxyglucose (DOG) and iodoacetamide on mouse sperm ATP
levels and motility. DOG inhibits hexokinase by competition with glucose [55].
Iodoacetamide is an irreversible inhibitor of glyceraldehyde-3-phosphate dehydrogenase.
Mouse caudal sperm were incubated with 5mM DOG in medium containing pyruvate and
lactate without glucose for 30 minutes and sperm motility and ATP levels were
measured. DOG drastically decreased sperm ATP levels (Fig. 5A) and also decreased the
percent of motile sperm and the average path velocity (Fig. 5B). These results are in
agreement with a previous report [34]. As DOG competitively blocks hexokinase and
gets phosphorylated to DOG-6-phosphate, it might be using a substantial amount of ATP
generated from oxidative respiration as it is phosphorylated. To address this possibility
we used iodoacetamide, 0.5mM to inhibit glycolysis in presence of pyruvate and lactate
(no glucose) and measured sperm motility and ATP levels.

22

Fig. 5. Effect of DOG on sperm ATP concentration (A) and sperm

motility (B) in presence of pyruvate and lactate (no glucose). The
concentration of DOG was 5mM. ATP and motility data are the mean of
three experiments. Error bars represent SEM.

23

Iodoacetamide, a potent inhibitor of glycolysis did not decrease mouse sperm ATP and
motility levels in medium supporting only mitochondrial respiration.
Surprisingly, sperm ATP levels were comparable to the control (Fig. 6A) and
sperm motility (Fig. 6B) was maintained after 30 minutes. Our observations and those of
Mukai and Okuno [34] that show a depletion of ATP with DOG treatment suggests that
glycolysis is required to sustain ATP levels and that oxidative phosphorylation in the
presence of pyruvate and lactate cant compensate for a reduction in glycolysis. However,
inhibition of glycolysis with iodoacetamide does not cause a reduction in ATP
concentration indicating that oxidative production of ATP is sufficient to maintain ATP
levels. The reduction of ATP concentration using DOG as a glycolytic inhibitor can be
explained by the depletion of ATP from oxidative phosphorylation as DOG is
phosphorylated.

24

Fig. 6. Effect of iodoacetamide on sperm ATP concentration (A) and

motility (B) in presence of pyruvate and lactate (no glucose). The
concentration of the glycolytic inhibitor, iodoacetamide was 0.5mM. ATP
and motility data are the mean of three experiments. Error bars represent
SEM.

Iodoacetamide but not DOG could decrease sperm ATP levels and motility in medium
containing glucose.
The contrasting results of two glycolytic inhibitors on sperm ATP levels and
motility might be related to their respective mechanism of actions and their effectiveness
under different conditions. To explore the glycolytic inhibition efficacy of the two
glycolytic inhibitors, sperm were incubated in medium containing glucose (no pyruvate
and lactate) in presence of either iodoacetamide or DOG. Sperm were dependent on
glycolysis for generation of respiratory substrates under both the conditions.

25

Fig. 7. Effect of DOG and iodoacetamide on sperm ATP concentration (A) and
sperm motility (B) in presence of glucose (no pyruvate and lactate).

The

concentrations of inhibitors were 5mM for DOG and 0.5mM for iodoacetamide.
ATP and motility data are the mean of three experiments. Error bars represent
SEM.

26

Iodoacetamide effectively blocked glycolysis and hence decreased sperm ATP

levels (Fig. 7A) and motility (Fig. 7B). DOG, being a weak competitive inhibitor of
glycolysis in the presence of glucose, was unable to significantly suppress sperm ATP
production and motility as shown in Fig. 7A and Fig. 7B respectively. It has been shown
[55] that DOG in presence of glucose acts as competitor for the hexokinase enzyme.

Mouse sperm ATP levels and motility were decreased by DOG in medium containing
fructose.
The effect of DOG on sperm ATP and motility was examined in presence of
another glycolytic substrate, fructose in medium. This would give indirect evidence
whether DOG is using up ATP produced from either glycolysis or respiration. Unlike
glucose, fructose bypasses the first step of glycolysis when it enters the pathway thereby
all hexokinase binding sites would be free. DOG significantly decreased sperm ATP
levels and motility parameters as depicted in Fig. 8A and 8B respectively. This is because
of lack of competition for hexokinase enzyme as seen in presence of glucose.

27

Fig. 8. Effect of DOG on sperm ATP concentration (A) and sperm

motility (B) in presence of fructose. The concentration of DOG was
5mM. ATP and motility data are the mean of three experiments. Error
bars represent SEM.

28

Aim #2 - Determine distribution and localization of GSK-3 and GSK-3 in bovine

and mouse sperm and study the role of GSK-3 in sperm metabolism.
GSK3 western blotting
Western blotting analysis was used to determine the presence of GSK-3 in mouse caput
and caudal RIPA sperm extracts. Bovine caput and caudal sperm extracts were used as
positive controls. No bands corresponding to GSK-3 were seen in either supernatant or
pellet of mouse extracts (Fig. 9) using antibody against the C-terminus sequence
WQSTDATPTLTNSS.

Fig. 9. Western blot of mouse caput (cp) and caudal (cd) sperm,
supernatant and pellet extracts probed with GSK-3 antibody.

29

The apparent absence of GSK-3 in mouse sperm extracts was surprising since this
antibody had been used to detect GSK-3 in sperm extracts from different species such as
hamster, sea urchin, elephant etc. Also, this antibody had been raised against the carboxy
terminal domain of GSK-3 which is conserved across several species.
There were no gene and peptide sequence of mouse GSK-3 in NCBI website
(http://www.ncbi.nlm.nih.gov/). Mouse GSK-3 gene and peptide sequences were
computationally derived using the NCBI and www.ensembl.org websites. By using the
known human GSK-3 sequence and the whole mouse genome in Blast search tool we
were able to annotate the mouse GSK-3 sequence. This derived sequence was aligned
with the known human and rat GSK-3 peptide sequence. We found that the conserved
(across different species such as human and rat) carboxy terminus domain
(WQSTDATPTLTNSS) of the GSK-3 against which the Zymed antibody was raised to
be significantly different in mouse (Fig. 10).

30

Human GSK3 MSGGGPSGGGPGGSGRARTSSFAEPGGGGGGGGGGPGGSASGPGGTGGGKASVGAMGGGV

Rat GSK3
MSGGGPSGGGPGGSGRARTSSFAEPGGGGGGGGGGPGGSASGPGGTGGGKASVGAMGGGV
Mouse GSk3 MSGGGPSGGGPGGSGRARTSSFAVARRRRRRWWRRPGGSASGPGGTGGGKASVGAMGGGV
*********************** .
*************************
Human GSK3 GASSSGGGPGGSGGGGSGGPGAGTSFPPPGVKLGRDSGKVTTVVATLGQGPERSQEVAYT
Rat GSK3
GASSSGGGPSGSGGGGSGGPGAGTSFPPPGVKLGRDSGKVTTVVATLGQGPERSQEVAYT
Mouse GSk3 GASSSGGGPSGSGGGGSGGPGAGTSFPPPGVKLGRDSGKVTTVVATVGQGPERSQEVAYT
*********.************************************:*************
Human GSK3 DIKVIGNGSFGVVYQARLAETRELVAIKKVLQDKRFKNRELQIMRKLDHCNIVRLRYFFY
Rat GSK3
DIKVIGNGSFGVVYQARLAETRELVAIKKVLQDKRFKNRELQIMRKLDHCNIVRLRYFFY
Mouse GSk3 DIKVIGNGSFGVVYQARLAETRELVAIKKVLQDKRFKNRELQIMRKLDHCNIVRLRYFFY
************************************************************
Human GSK3 SSGEKKDELYLNLVLEYVPETVYRVARHFTKAKLTIPILYVKVYMYQLFRSLAYIHSQGV
Rat GSK3
SSGEKKDELYLNLVLEYVPETVYRVARHFTKAKLIIPIIYVKVYMYQLFRSLAYIHSQGV
Mouse GSk3 SSGEKKDELYLNLVLEYVPETVYRVARHFTKAKLITPIIYIKVYMYQLFRSLAYIHSQGV
********************************** **:*:*******************
Human GSK3 CHRDIKPQNLLVDPDTAVLKLCDFGSAKQLVRGEPNVSYICSRYYRAPELIFGATDYTSS
Rat GSK3
CHRDIKPQNLLVDPDTAVLKLCDFGSAKQLVRGEPNVSYICSRYYRAPELIFGATDYTSS
Mouse GSk3 CHRDIKPQNLLVDPDTAVLKLCDFGSAKQLVRGEPNVSYICSRYYRAPELIFGATDYTSS
************************************************************
Human GSK3 IDVWSAGCVLAELLLGQPIFPGDSGVDQLVEIIKVLGTPTREQIREMNPNYTEFKFPQIK
Rat GSK3
IDVWSAGCVLAELLLGQPIFPGDSGVDQLVEIIKVLGTPTREQIREMNPNYTEFKFPQIK
Mouse GSk3 IDVWSAGCVLAELLLGQPIFPGDSGVDQLVEIIKVLGTPTREQIREMNPNYTEFKFPQIK
************************************************************
Human GSK3 AHPWTKVFKS-RTPPEAIALCSSLLEYTPSSRLSPLEACAHSFFDELRCLGTQLPNNRPL
Rat GSK3
AHPWTKVFKS-RTPPEAIALCSSLLEYTPSSRLSPLEACAHSFFDELRSLGTQLPNNRPL
Mouse GSk3 AHPWTKVFKSSKTPPEAIALCSSLLEYTPSSRLSPLEACAHSFFDELRRLGAQLPNDRPL
********** :************************************ **:****:***
Human GSK3 PPLFNFSAGELSIQPSLNAILIPPHLRSPAG-----TTTLTPSSQALTETPTSSDWQSTD
Rat GSK3
PPLFNFSPGELSIQPSLNAILIPPHLRSPSG-----PATLTSSSQALTETQTGQDWQAPD
Mouse GSk3 PPLFNFSPGELSIQPSLNAILIPPHLRSPAGPASPLTTSYNPSSQALTEAQTGQDWQPSD
*******.*********************:*
.:: ..*******: *..***..*
Human GSK3 AT-PTLTNSS
Rat GSK3
AT-PTLTNSS
Mouse GSk3 ATTATLASSS
** .**:.**

Fig. 10. Aligned GSK-3 peptide sequence of human, rat and mouse. The stars
indicate a perfect match and the dots indicate the number of mismatches in the peptide
sequences. The domains recognized by the Zymed GSK-3 antibody (highlighted in
green) and the UBI GSK-3/ antibody (highlighted in pink) are shown.

31

Fig. 11. Western blot showing supernatant and pellet extracts of mouse
caput (cp) and caudal (cd) sperm prepared in HB+, RIPA+ and RIPA+SDS
probed with GSK-3/ antibody.

Mouse caudal sperm extracts were prepared in HB+, RIPA+ and RIPA+SDS
buffers. Western blot was done to look for the presence of either GSK-3/ using another
antibody. This antibody from Upstate Biotechnologies was against the polypeptide
sequence KQLLHGEPNVSYICSRY, a region different from the conserved GSK-3
carboxy-terminus domain sequence (Fig. 10). With this antibody western blotting shows
the presence of both GSK-3 and GSK-3 in the mouse caudal sperm extracts which can
be recognized by their different molecular weights: GSK-3, 47 KDa; GSK-3, 51 KDa
(Fig. 11). The figure also shows that differences in extraction of GSK-3/ in different

32

buffers. In HB+ buffer extracts most of the GSK-3/ was in the pellet but with RIPA
and RIPA-SDS buffer, all of the GSK-3/ was present in the supernatant.

Fig.12. Western blot of supernatant and pellet of bovine caput (cp) and caudal
(cd) sperm extracts in HB+ (A), RIPA+SDS (B) and RIPA+ (C) buffers
probed with GSK-3 antibody.

33

Extracts were prepared in different buffers to identify a buffer which can bring all of the
GSK-3 into the supernatant thereby identifying the buffer best suited for GSK-3 activity
studies. HB+ buffer mainly extracts cytoplasmic proteins and RIPA+ buffer which has
both sodium deoxycholate and NP-40, which are detergents, allows extraction of the
membrane proteins too.

GSK-3 Immunocytochemistry
Next immunocytochemistry was used to determine intracellular localization of
GSK-3. This was done to see if there is any change in localization of GSK-3 across caput
and caudal mouse sperm and across caput, caudal and ejaculated bovine sperm. Any
change in GSK-3 localization across caput to caudal to ejaculated sperm would increase
the

probability

of

GSK-3

having

functional

role

in

sperm

motility.

Immunocytochemistry of mouse (Fig. 13) and bovine (Fig. 14) sperm with GSK-3/
antibody shows almost all of the GSK-3 or GSK-3 localized to the post acrosomal
region of the sperm. No change in GSK-3 localization was seen in caput and caudal
mouse sperm and caput, caudal and ejaculated bovine sperm by immunofluorescence.

34

Fig. 13. Intracellular localization of GSK-3/ in mouse caput (A, B) and

caudal (C, D) sperm. Left, indirect immunofluorescence using the GSK-3/
(UBI). Right, fluorescence image of the sperm head with the DNA binding
dye, DAPI (4-6 Diamidino-2-phenylindole). Bar in C represents 40 m.

35

Fig. 14. Intracellular localization of GSK-3/ in bovine caput (A, B), caudal (C,
D) and ejaculated (E, F) sperm. Left panels, bar is 100 m. Right, additional
image at higher magnification, bar is 20 m.

36

Sperm ATP assay and motility with GSK-3 inhibitors

GSK-3, a kinase enzyme, might be in involved in phosphorylating one or more
enzymes of glycolysis and thereby playing an indirect role in ATP production in sperm.
To understand the role of GSK-3 in sperm metabolism we checked sperm ATP levels and
motility parameters with various established GSK-3 inhibitors. Bisindolylmaleimides,
Bis-I and Bis-IX are potent inhibitors of GSK-3 activity. Bis-V has no effect on GSK-3
activity [56, 57]. Lithium potently inhibits GSK-3 but is not a general protein kinase
inhibitor [58]. SB216763 and SB415286 are selective small molecule inhibitors of GSK3 [59]. Bovine caudal sperm were incubated in medium (CESD buffer) containing
glucose in water bath at 37C for 30 min with the above GSK-3 inhibitors. No specific
pattern was observed when ATP levels and motility parameters were compared with the
control (no inhibitors in medium). Sperm incubated with LiCl and Bis-I showed an
apparent increase in ATP and with Bis-V, Bis-IX, SB216763 and SB414286 showed an
apparent decrease in ATP levels. Sperm incubated with LiCl, Bis-I and Bis-IX showed
decreased motility levels whereas with SB216763 and SB415286 increased motility
parameters (Fig. 15). However, this experiment was only done once.

37

Fig. 15. ATP levels (A) and motility and path velocity (B) in bovine
caudal sperm after incubation with various GSK-3 inhibitors: LiCl
(20mM), Bis-I (5M), Bis-V (1M), Bis-IX (2M), SB415286
(12.5M), SB216763 (10M).
experiment.

Results shown are from a single

DISCUSSION
The sperm requires an adequate supply of ATP for motility to complete the task
of fertilization. Glycolysis and mitochondrial oxidation provide ATP. Sperm
mitochondria are strategically located at mid piece to provide ATP to axoneme. Recently
there has been controversy over the relative importance of glycolysis and mitochondrial
oxidation to supply ATP to maintain sperm motility. A recently published paper has
questioned the importance of mitochondrial oxidation in supplying ATP and has
concluded that glycolysis is the main pathway required for sperm ATP production. Miki
et al (2004) suggested that sperm glycolysis is the main pathway to support motility and
that the mitochondria were redundant [35]. This was shown by gene knock out of the
germ cell specific isoform of GAPDH, which selectively blocks glycolysis. Sperm
lacking glyceraldehyde-3-phosphate dehydrogenase had defects in sperm motility and
fertility with no progressive motility. ATP levels in these mice were only 10% of ATP
levels in wild type mice although the mitochondrial oxidation between wild type and null
mice was apparently comparable. As GAPDH null mice did not show motility in the
presence of physiological or even higher concentrations of pyruvate, it was concluded
that the majority of ATP required for sperm motility is supplied by glycolysis.
Supportive evidence of the study of Miki et al, were studies which showed that mice
lacking the testis specific cytochrome C, an essential component of electron transport
chain, have the ability to fertilize eggs [60]. Although fertility is significantly reduced in

38

39

cytochrome C null mice, it has been suggested that glycolysis on its own can provide
enough ATP to sustain motility and perform sperm function. Indirect evidence for the
importance of glycolysis is localization of glycolytic enzymes along the entire length of
flagellum to supply ATP where it is required instead of diffusion from mid-piece
mitochondria [33]. Mukai and Okuno (2004) concluded that glycolysis plays a major
role in ATP production in mouse sperm since sperm motility could not be maintained in
the presence of respiratory substrates when glycolysis was suppressed with DOG, a
glycolytic inhibitor [34].
Although the results obtained from above mentioned studies were interpreted to
emphasize the essential role of glycolysis in sperm motility, it is paradoxical that the
specialized cell such as sperm will depend on only glycolysis when ATP synthesis by
oxidative phosphorylation is fifteen times more efficient than glycolysis. Furthermore,
glycolysis and mitochondrial respiration are interconnected processes and are highly
regulated by feed back pathways, so results of the in vitro experiments in papers
discussed above require very careful interpretation. These questions prompted me to
study the role of glycolysis and mitochondrial respiration in maintaining the sperm ATP
pool and motility. In the present study we tested the hypothesis proposed by Mukai and
Okuno with additional set of experiments to see the importance of mitochondrial ATP in
sustaining motility in mouse model.
First, ATP and motility levels in mouse sperm dependant either exclusively on
glycolysis or mitochondrial respiration were measured. Mouse sperm motility was
sustained by glycolytic ATP pool or mitochondrial ATP independently. This was in

40

agreement with studies conducted in which mouse and bull sperm motility could be
maintained under aerobic and anaerobic conditions [34, 66]. Sperm were motile in the
medium supporting only oxidative phosphorylation. This confirms the previously
published results in varied species of spermatozoa in which mitochondrial ability to
sustain ATP and motility has been shown by incubating sperm with different
mitochondrial inhibitors e.g. oligomycin, Antimycin A, KCN and

also shows the

functionality of sperm mitochondria [25]. Sperm are specialized cell which undergo
spermiogeneis followed by maturation in the epididymis and keep the organelles which
are indispensable for their function. Mitochondria are wrapped around the sperm midpiece suggesting important energy function. This experiment confirms the previous
studies that mouse sperm mitochondria contribute to ATP production and that
mitochondrial ATP can sustain motility.
Recently it has been shown that mouse sperm cannot sustain motility in presence
of pyruvate and lactate if DOG is added to the medium [34]. As the sperm could not
maintain motility in presence of oxidative phosphorylation substrates when glycolysis
was inhibited by DOG, these results were interpreted by Mukai and Okuno as glycolysis
in the principal piece is essential for maintenance of motility. DOG is known inhibitor of
glycolysis. It gets phosphorylated by hexokinase to 2-deoxyglucose 6-phosphate, which
cannot be further metabolized. As DOG gets phosphorylated, it has been speculated that
this phosphorylation might drain out the mitochondrial ATP produced by metabolism of
pyruvate and lactate making the sperm immotile [61]. To test this hypothesis we
incubated mouse sperm with DOG or iodoacetamide in presence of pyruvate and lactate.

41

Iodoacetamide blocks step 6 of glycolysis by inhibiting glyceraldehyde-3-phosphate

dehydrogenase. Mouse sperm incubated with DOG showed significant loss in sperm
ATP and motility. No significant decrease in motility and ATP levels were observed in
mouse sperm incubated in iodoacetamide. These results were in agreement with the
studies done with chlorohydrin which is another GAPDH inhibitor. Contraceptive doses
of

-chlorohydrin or 6-chloro-6deoxyglucose [62, 63] did not decrease oxidative

respiration, and sperm from rats made infertile with 6-chloro-6-deoxyglucose remained
motile with a normal ATP concentration when incubated with pyruvate plus lactate (no
glucose) [64].
DOG is a weak competitive inhibitor of glycolysis in presence of glucose and so
increasing glucose would overcome DOG inhibition. Iodoacetamide acts by inhibiting
step 6 of glycolysis. Mouse sperm were incubated in medium containing glucose with
DOG or iodoacetamide. DOG did not cause significant decrease in sperm ATP levels and
motility in presence of glucose. Glucose undergoing glycolysis due to incomplete
inhibition by DOG is a source of pyruvate and ATP generation. On the other hand, when
iodoacetamide was used, a sharp fall in ATP and motility levels was observed. This
experiment established the inhibitory action of iodoacetamide on sperm glycolysis.
Effect of DOG in presence of fructose was studied. This was done to see whether
DOG reduces ATP levels if another sugar is provided instead of glucose. DOG
significantly reduced the sperm ATP and motility in presence of fructose. This result
might be because of two reasons. Fructose bypasses the first step of glycolyis when it
enters the pathway thereby leaving all hexokinase binding sites for DOG. It has also been

42

shown that DOG has higher affinity to hexokinase than fructose. Km values of
hexokinase for fructose and DOG are 1.6x10-3M and 2.7x10-5M [65]. This could explain
the fall in sperm ATP and motility by DOG in presence of fructose as DOG has higher
affinity to hexokinase than fructose.
Our experiments establish the action of DOG and iodoacetamide under different
conditions and provide sufficient evidence that mouse sperm motility can be maintained
by ATP generated by mitochondrial oxidation. Inhibition of motility by DOG in the
presence of pyruvate and lactate might be due to its ability to use up phosphate by
utilizing ATP generated by oxidative phosphorylation (Fig. 16). Mitochondrial
respiration and glycolysis can compensate for each other but they do not have obligatory
roles in maintaining sperm ATP production and sperm motility.
GSK-3 is a multi-tasking kinase involved in variety of cellular processes e.g.
signal transduction, metabolism, apoptosis and cell cycle regulation etc. The presence of
both isoforms of GSK-3 in sperm and their upstream regulators PKB and PI3 kinase in
sperm have been shown [49]. GSK-3 is regulated by its phosphorylation at tyrosine and
serine residues, which changes its localization and its ability to bind to different proteins.
GSK-3 activity decreases in relationship to initiation of motility in epididymis [46].
GSK-3 phosphorylation is dynamic during epididymal maturation and its inhibitory
serine phosphorylation is higher in caudal epididymal sperm than caput spermatozoa.
These results suggest that GSK-3 might have regulatory role in sperm motility. GSK-3 is
one of the potential kinases responsible for regulation of glycolysis by enzyme
phosphorylation and therefore can have an indirect role in ATP production in sperm. This

43

study was undertaken to find out the localization of GSK-3 isoforms in mouse and bull
spermatozoa and its role in ATP production which might shed some light on its function
in sperm. First we looked at the subcellular distribution of GSK-3 isoforms by
immunoblotting after extracting sperm GSK-3 by different buffers. We used
homogenization buffer, HB+ which mainly extracts cytoplasmic proteins and RIPA+
buffer which has both sodium deoxycholate and NP-40, which are detergents allowing
extraction of the membrane proteins too. Immunoblotting by GSK-3 antibody showed
that most of GSK-3 is localized to membrane fraction of spermatozoa and can be
extracted by RIPA buffer. GSK-3 is a membrane bound enzyme in the somatic cells.
Sperm have highly compartmentalized areas with specific functions. The post acrosomal
region has been shown to be involved in sperm egg fusion. The sperm mid-piece is a site
of energy production in sperm with mitochondria wrapped around it. The principal piece
has fibrous sheath which has been speculated to provide support and as a site of signal
transduction mechanisms with glycolytic enzymes tightly bound to it [32, 33].
Immunocytochemistry analysis localized sperm GSK-3 to post acrosomal region. We
analyzed immature caput, caudal as well as ejaculated bovine spermatozoa to study the
change in localization if any. Sperm GSK-3 localization did not change during
epididymal maturation and during ejaculation. GSK-3 is regulated by its localization in
the somatic cells [67] but our preliminary results from immunocytochemistry suggest
localization might not play a role in regulation of GSK-3 in epididymal maturation. This
specific localization also suggests the GSK-3 may have a role in sperm-egg fusion based
on its localization in the post acrosomal region.

44

The antibody used to study GSK-3 by immuoblotting was raised against carboxy
terminus of GSK-3 which is conserved in several species. Surprisingly, mouse sperm
immunoblotting did not show antibody interaction. The mouse GSK-3 gene and protein
sequence were not annotated in NCBI. We annotated the mouse GSK-3 sequence and
on aligning it with other known gene sequences from human and rat, we found out that
GSK-3 carboxy terminus sequence is different than other species. This is the reason we
could detect mouse GSK-3 only when another antibody, raised against a different domain
in the GSK-3 sequence was used.
GSK-3 was discovered as an enzyme regulating the activity of glycogen synthase
and since then it has been found to be in involved in various cellular processes. Sperm
cells are specialized cells in the body which have varied metabolic requirements to
produce ATP to sustain motility needs. As GSK-3 is one of the potential kinases involved
in regulating the glycolytic enzymes, we wanted to analyze the role of GSK-3 in sperm
metabolism. To study the role of GSK-3 in sperm metabolism and motility we used GSK3 inhibitors to suppress GSK-3 activity and analyze its effect on sperm ATP levels and
motility. Various well characterized inhibitors of GSK-3 are available commercially. We
used lithium, bisindolylmaleimide-I, bisindolylmaleimide-V, bisindolylmaleimide-IX,
SB216763 and SB415286. Lithium was the only agent which reduced the percent
motility significantly. No significant effect of other GSK-3 inhibitors was observed on
sperm motility. ATP levels were measured after adding various GSK-3 inhibitors, but no
significant effect was observed on sperm ATP levels. GSK-3 activity is sensitive to
lithium with IC50 of lithium for GSK-3 being 10mM. IC50 represents the concentration

45

of an inhibitor that is required for 50% inhibition of the target. Lithium mediated motility
inhibition might be independent of its effect on GSK-3 as other GSK-3 inhibitors did not
produce the similar effect. Bis-I, Bis-IX were originally discovered as PKC inhibitors but
later studies also showed GSK-3 as their target.
These GSK-3 inhibitors did not show any apparent affects on motility and ATP
levels. Preliminary results from these studies suggest GSK-3 might not be involved in
sperm metabolism. More work is required to elucidate its role as metabolic enzyme.
GSK-3 might regulate motility by participating in other signaling pathways in sperm.

46

Fig. 16. Schematic presentation of how different modes of actions of DOG and
iodoacetamide affect mouse sperm ATP levels. TCA = Tricarboxylic acid
cycle; ETC = Electron transport Chain.

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