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

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ..v

INTRODUCTION .1

Background ........1 Aims .....13

METHODS ..14

RESULTS ....19

DISCUSSION ..38

REFERENCES.....47

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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

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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.

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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

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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].

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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

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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

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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].

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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-3-

phosphate 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

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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

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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

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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].

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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

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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

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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.

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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.

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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 GSK-

3 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

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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-lysine-coated 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.

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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

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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].

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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.

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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.

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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.

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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.

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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.

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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.

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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 a 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 GSK-

3 [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). Results shown are from a single

experiment.

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 mid-

piece 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 GSK-

3 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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Copy of VINAY THESIS- I.docCopy of 1.docBackground

Copy of 2.docSpermatozoon maturationSummary of Aims

Copy of 3.docMETHODSSperm Extract Preparation

Copy of 4.docThe 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

Copy of 5.docAim #1 Study the role of mitochondrial respiration and glycolysis in sperm ATP production and sperm motility.

Copy of 6.docThe glycolytic inhibitor, DOG decreases mouse sperm ATP levels and motility in a medium containing respiratory substrates.

Copy of 7.docCopy of 8.docCopy of 9.docCopy of 10.doc