Role of Glycolysis and Respiration in Sperm Metabolism and Motility

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

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 respiration……………………………8

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

lactate………………………………………………………………………22

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-3α antibody...... 28

Figure 10. Aligned GSK-3α peptide sequence of human, rat and mouse……...…...…30

Figure 11. Western blot of mouse sperm extracts probed with GSK-3α/β antibody.....31

Figure 12. Western blot of bovine sperm extracts probed with GSK-3α antibody…...32

Figure 13. Intracellular localization of GSK-3α/β in mouse sperm ………….…….…34

Figure 14. Intracellular localization of GSK-3α/β 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

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

1 2

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]. 3

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 4

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 5

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-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 7

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 9

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 α-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-3α (51 kDa) and GSK-3β (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.

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-3α and GSK-3β

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-3α/β 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 β-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-3α

was used to identify GSK-3α 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-3α (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-

3α and GSK-3β. 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 4ºC. 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 μl of 1 x 108 cells/ml) were attached to poly-L-lysine- coated coverslips and then fixed in 100% methanol for 5 min at -20ºC or cells in suspension were fixed with 4% formaldehyde in PBS for 30 min at 4ºC, 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 μl TTBS, then incubated

overnight with 200 μl blocking buffer (2.5% BSA and 5% normal goat serum in TTBS)

in a humidified chamber. GSK-3α/β (UBI) antibody was diluted in blocking buffer. The

cells were incubated in 200 μl of this diluted (1:2 to 1:200) primary antibody overnight at

4ºC in a humidified chamber. For the negative control, the primary antibody was omitted

and the cells were incubated in blocking buffer overnight at 4ºC. The cells were washed

three times with TTBS, then incubated, shielded from light, for 1 h at room temperature

in 200 μl 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 37°C 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 μl of the supernatants for each experiment was added to 100μl 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%) Whittingham’s

medium (99.3 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 0.36 mM NaH2PO4, 25 mM NaHCO3, 100 U/ml penicillin G-K salt, 50 µg/ml streptomycin sulfate, 25 mM sodium lactate, 0.50 mM sodium pyruvate, 5.55 mM glucose, pH 7.4) or 18

Whittingham’s 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 4μM 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

Kreb’s 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.

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.

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.

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 can’t 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.

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.

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.

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.

Aim #2 - Determine distribution and localization of GSK-3α and GSK-3β 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-3α in mouse caput and caudal RIPA sperm extracts. Bovine caput and caudal sperm extracts were used as positive controls. No bands corresponding to GSK-3α 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-3α antibody.

The apparent absence of GSK-3α in mouse sperm extracts was surprising since this antibody had been used to detect GSK-3α 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-3α which is conserved across several species.

There were no gene and peptide sequence of mouse GSK-3α in NCBI website

(http://www.ncbi.nlm.nih.gov/). Mouse GSK-3α gene and peptide sequences were computationally derived using the NCBI and www.ensembl.org websites. By using the known human GSK-3α sequence and the whole mouse genome in Blast search tool we were able to annotate the mouse GSK-3α sequence. This derived sequence was aligned with the known human and rat GSK-3α peptide sequence. We found that the conserved

(across different species such as human and rat) carboxy terminus domain

(WQSTDATPTLTNSS) of the GSK-3α against which the Zymed antibody was raised to be significantly different in mouse (Fig. 10).

Human GSK3α MSGGGPSGGGPGGSGRARTSSFAEPGGGGGGGGGGPGGSASGPGGTGGGKASVGAMGGGV Rat GSK3α MSGGGPSGGGPGGSGRARTSSFAEPGGGGGGGGGGPGGSASGPGGTGGGKASVGAMGGGV Mouse GSk3α MSGGGPSGGGPGGSGRARTSSFAVARRRRRRWWRRPGGSASGPGGTGGGKASVGAMGGGV *********************** . *************************

Human GSK3α GASSSGGGPGGSGGGGSGGPGAGTSFPPPGVKLGRDSGKVTTVVATLGQGPERSQEVAYT Rat GSK3α GASSSGGGPSGSGGGGSGGPGAGTSFPPPGVKLGRDSGKVTTVVATLGQGPERSQEVAYT Mouse GSk3α GASSSGGGPSGSGGGGSGGPGAGTSFPPPGVKLGRDSGKVTTVVATVGQGPERSQEVAYT *********.************************************:*************

Human GSK3α DIKVIGNGSFGVVYQARLAETRELVAIKKVLQDKRFKNRELQIMRKLDHCNIVRLRYFFY Rat GSK3α DIKVIGNGSFGVVYQARLAETRELVAIKKVLQDKRFKNRELQIMRKLDHCNIVRLRYFFY Mouse GSk3α DIKVIGNGSFGVVYQARLAETRELVAIKKVLQDKRFKNRELQIMRKLDHCNIVRLRYFFY ************************************************************

Human GSK3α SSGEKKDELYLNLVLEYVPETVYRVARHFTKAKLTIPILYVKVYMYQLFRSLAYIHSQGV Rat GSK3α SSGEKKDELYLNLVLEYVPETVYRVARHFTKAKLIIPIIYVKVYMYQLFRSLAYIHSQGV Mouse GSk3α SSGEKKDELYLNLVLEYVPETVYRVARHFTKAKLITPIIYIKVYMYQLFRSLAYIHSQGV ********************************** **:*:*******************

Human GSK3α CHRDIKPQNLLVDPDTAVLKLCDFGSAKQLVRGEPNVSYICSRYYRAPELIFGATDYTSS Rat GSK3α CHRDIKPQNLLVDPDTAVLKLCDFGSAKQLVRGEPNVSYICSRYYRAPELIFGATDYTSS Mouse GSk3α CHRDIKPQNLLVDPDTAVLKLCDFGSAKQLVRGEPNVSYICSRYYRAPELIFGATDYTSS ************************************************************

Human GSK3α IDVWSAGCVLAELLLGQPIFPGDSGVDQLVEIIKVLGTPTREQIREMNPNYTEFKFPQIK Rat GSK3α IDVWSAGCVLAELLLGQPIFPGDSGVDQLVEIIKVLGTPTREQIREMNPNYTEFKFPQIK Mouse GSk3α IDVWSAGCVLAELLLGQPIFPGDSGVDQLVEIIKVLGTPTREQIREMNPNYTEFKFPQIK ************************************************************

Human GSK3α AHPWTKVFKS-RTPPEAIALCSSLLEYTPSSRLSPLEACAHSFFDELRCLGTQLPNNRPL Rat GSK3α AHPWTKVFKS-RTPPEAIALCSSLLEYTPSSRLSPLEACAHSFFDELRSLGTQLPNNRPL Mouse GSk3α AHPWTKVFKSSKTPPEAIALCSSLLEYTPSSRLSPLEACAHSFFDELRRLGAQLPNDRPL ********** :************************************ **:****:***

Human GSK3α PPLFNFSAGELSIQPSLNAILIPPHLRSPAG-----TTTLTPSSQALTETPTSSDWQSTD Rat GSK3α PPLFNFSPGELSIQPSLNAILIPPHLRSPSG-----PATLTSSSQALTETQTGQDWQAPD Mouse GSk3α PPLFNFSPGELSIQPSLNAILIPPHLRSPAGPASPLTTSYNPSSQALTEAQTGQDWQPSD *******.*********************:* .:: ..*******: *..***..*

Human GSK3α AT-PTLTNSS Rat GSK3α AT-PTLTNSS Mouse GSk3α ATTATLASSS ** .**:.**

Fig. 10. Aligned GSK-3α 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-3α antibody (highlighted in green) and the UBI GSK-3α/β antibody (highlighted in pink) are shown.

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-3α/β 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-3α/β using another antibody. This antibody from Upstate Biotechnologies was against the polypeptide sequence KQLLHGEPNVSYICSRY, a region different from the conserved GSK-3α carboxy-terminus domain sequence (Fig. 10). With this antibody western blotting shows the presence of both GSK-3α and GSK-3β in the mouse caudal sperm extracts which can be recognized by their different molecular weights: GSK-3α, 47 KDa; GSK-3β, 51 KDa

(Fig. 11). The figure also shows that differences in extraction of GSK-3α/β in different 32

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

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-3α/β antibody shows almost all of the GSK-3α or GSK-3β 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.

Fig. 13. Intracellular localization of GSK-3α/β in mouse caput (A, B) and caudal (C, D) sperm. Left, indirect immunofluorescence using the GSK-3α/β

(UBI). Right, fluorescence image of the sperm head with the DNA binding dye, DAPI (4’-6 Diamidino-2-phenylindole). Bar in C represents 40 μm.

Fig. 14. Intracellular localization of GSK-3α/β in bovine caput (A, B), caudal (C,

D) and ejaculated (E, F) sperm. Left panels, bar is 100 μm. Right, additional image at higher magnification, bar is 20 μm.

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 37°C 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.

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 (5μM), Bis-V (1μM), Bis-IX (2μM), SB415286

(12.5μM), SB216763 (10μM). 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-6–deoxyglucose [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-3α 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-3α gene and protein

sequence were not annotated in NCBI. We annotated the mouse GSK-3α 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.

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.

REFERENCES

1. Jimmy D. Neill. Knobil and Neill’s Physiology of Reproduction, Volume 1,

Third Edition, 2005.

2. Yanagimachi R. Fertility of mammalian spermatozoa: its development and

relativity. Zygote 2(4): 371-2, 1994.

3. Smithwick EB, Young LG. Histological effects of androgen deprivation on the

adult chimpanzee epididymis. Tissue Cell 33(5): 450-61, 2001.

4. Jones RC, Clulow J. Interactions of sperm and the reproductive ducts of the male

tammar wallaby, Macropus eugenii (Macropodidae: Marsupialia). Reprod Fertil

Dev 6(4): 437-44, 1994.

5. Amann RP, Howards SS. Daily spermatozoal production and epididymal

spermatozoal reserves of the human male. J Urol 124(2): 211-5, 1980.

6. Kann ML, Serres C. Development and initiation of sperm motility in the hamster

epididymis. Reprod Nutr Dev 20(6): 1739-49, 1980.

7. Cornwall GA, Smyth TB, Vindivich D, Harter C, Robinson J, Chang TS.

Induction and enhancement of progressive motility in hamster caput epididymal

spermatozoa. Biol Reprod 35(4): 1065-74, 1986.

8. Soler C, Yeung CH, Cooper TG. Development of sperm motility patterns in the

murine epididymis. Int J Androl 17(5): 271-8, 1994.

9. Mann, T. and C. Lutwak-Mann. Male Reproductive Function and Semen. New

York: Springer-Verlag; 1981.

47 48

10. Hamamah S, Gatti JL. Role of the ionic environment and internal pH on sperm

activity. Hum Reprod 13 Suppl 4(20-30, 1998.

11. Geraci E, Giudice G. Sperm activation and sperm-egg interaction. J Submicrosc

Cytol Pathol 38(1): 11-20, 2006.

12. Fraser LR. Cellular biology of capacitation and the acrosome reaction. Hum

Reprod 10 Suppl 1(22-30, 1995.

13. Suarez SS. Hyperactivated motility in sperm. J Androl 17(4): 331-5, 1996.

14. Yamashita M, Yamagata K, Tsumura K, Nakanishi T, Baba T. Acrosome reaction

of mouse epididymal sperm on oocyte zona pellucida. J Reprod Dev 53(2): 255-

62, 2007.

15. Tash JS. Protein phosphorylation: the second messenger signal transducer of

flagellar motility. Cell Motil Cytoskeleton 14(3): 332-9, 1989.

16. Brokaw CJ. Direct measurements of sliding between outer doublet microtubules

in swimming sperm flagella. Science 243(4898): 1593-6, 1989.

17. Shingyoji C, Murakami A, Takahashi K. Local reactivation of Triton-extracted

flagella by iontophoretic application of ATP. Nature 265(5591): 269-70, 1977.

18. Smith EF. Regulation of flagellar dynein by calcium and a role for an axonemal

calmodulin and calmodulin-dependent kinase. Mol Biol Cell 13(9): 3303-13,

2002.

19. Wargo MJ, Smith EF. Asymmetry of the central apparatus defines the location of

active microtubule sliding in Chlamydomonas flagella. Proc Natl Acad Sci U S A

100(1): 137-42, 2003. 49

20. Nakano I, Kobayashi T, Yoshimura M, Shingyoji C. Central-pair-linked

regulation of microtubule sliding by calcium in flagellar axonemes. J Cell Sci

116(Pt 8): 1627-36, 2003.

21. Koefoed-Johnsen HH, Mann T. Studies on the metabolism of semen. 9. Effect of

surface active agents with special reference to the oxidation of succinate by

spermatozoa. Biochem J 57(3): 406-10, 1954.

22. Mann T, Lutwak-Mann C. Studies on the metabolism of semen. 4. Aerobic and

anaerobic utilization of fructose by spermatozoa and seminal vesicles. Biochem J

43(2): 266-70, 1948.

23. Mann T. Studies on the metabolism of semen: 2. Glycolysis in spermatozoa.

Biochem J 39(5): 458-65, 1945.

24. Harrison RAP. The metabolism of mammalian spermatozoa. Frontiers in

Reproduction and Fertility Control, ed. By RO Greep and MA Koblinsky, pp.

379-407, MIT press, Cambridge, 1977.

25. Van Dop C, Hutson SM, Lardy HA. Pyruvate metabolism in bovine epididymal

spermatozoa. J Biol Chem 252(4): 1303-8, 1977.

26. Peterson RN, Freund M. ATP synthesis and oxidative metabolism in human

spermatozoa. Biol Reprod 3(1): 47-54, 1970.

27. Frenkel G, Peterson RN, Freund M. Changes in the metabolism of guinea pig

sperm from different segments of the epididymis. Proc Soc Exp Biol Med 143(4):

1231-6, 1973. 50

28. Aalbers JG, Mann T, Polge C. Metabolism of boar semen in relation to sperm

motility and survival. J Reprod Fertil 2(42-53, 1961.

29. Hoskins DD, Patterson DL. Metabolism of rhesus monkey spermatozoa. J Reprod

Fertil 16(2): 183-95, 1968.

30. Turner RM. Tales from the tail: what do we really know about sperm motility? J

Androl 24(6): 790-803, 2003.

31. Storey BT, Kayne FJ. Energy metabolism of spermatozoa. V. The Embden-

Myerhof pathway of glycolysis: activities of pathway enzymes in hypotonically

treated rabbit epididymal spermatozoa. Fertil Steril 26(12): 1257-65, 1975.

32. Travis AJ, Foster JA, Rosenbaum NA, Visconti PE, Gerton GL, Kopf GS, Moss

SB. Targeting of a germ cell-specific type 1 hexokinase lacking a porin-binding

domain to the mitochondria as well as to the head and fibrous sheath of murine

spermatozoa. Mol Biol Cell 9(2): 263-76, 1998.

33. Mukai C, Okuno M. Glycolysis plays a major role for adenosine triphosphate

supplementation in mouse sperm flagellar movement. Biol Reprod 71(2): 540-7,

2004.

34. Miki K, Qu W, Goulding EH, Willis WD, Bunch DO, Strader LF, Perreault SD,

Eddy EM, O'Brien DA. Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-

specific glycolytic enzyme, is required for sperm motility and male fertility. Proc

Natl Acad Sci U S A 101(47): 16501-6, 2004.

35. Ford WC. Glycolysis and sperm motility: does a spoonful of sugar help the

flagellum go round? Hum Reprod Update 12(3): 269-74, 2006. 51

36. Tanaka H, Takahashi T, Iguchi N, Kitamura K, Miyagawa Y, Tsujimura A,

Matsumiya K, Okuyama A, Nishimune Y. Ketone bodies could support the

motility but not the acrosome reaction of mouse sperm. Int J Androl 27(3): 172-7,

2004.

37. Nass-Arden L, Breitbart H. Modulation of mammalian sperm motility by

quercetin. Mol Reprod Dev 25(4): 369-73, 1990.

38. Nimmo HG, Proud CG, Cohen P. The phosphorylation of rabbit skeletal muscle

glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-

monophosphate-dependent protein kinase. Eur J Biochem 68(1): 31-44, 1976.

39. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of

glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature

378(6559): 785-9, 1995.

40. Kapadia RM, Guntur AR, Reinhold MI, Naski MC. Glycogen synthase kinase 3

controls endochondral bone development: contribution of fibroblast growth factor

18. Dev Biol 285(2): 496-507, 2005.

41. Gong R, Rifai A, Dworkin LD. Activation of PI3K-Akt-GSK3beta pathway

mediates hepatocyte growth factor inhibition of RANTES expression in renal

tubular epithelial cells. Biochem Biophys Res Commun 330(1): 27-33, 2005.

42. Welsh GI, Wilson C, Proud CG. GSK3: a SHAGGY frog story. Trends Cell Biol

6(7): 274-9, 1996.

43. Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3.

Trends Biochem Sci 29(2): 95-102, 2004. 52

44. Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-

3/factor A. EMBO J 9(8): 2431-8, 1990.

45. Vijayaraghavan S, Stephens DT, Trautman K, Smith GD, Khatra B, da Cruz e

Silva EF, Greengard P. Sperm motility development in the epididymis is

associated with decreased glycogen synthase kinase-3 and protein phosphatase 1

activity. Biol Reprod 54(3): 709-18, 1996.

46. Vijayaraghavan S, Mohan J, Gray H, Khatra B, Carr DW. A role for

phosphorylation of glycogen synthase kinase-3alpha in bovine sperm motility

regulation. Biol Reprod 62(6): 1647-54, 2000.

47. Feng H, Sandlow JI, Sandra A. The c-kit receptor and its possible signaling

transduction pathway in mouse spermatozoa. Mol Reprod Dev 49(3): 317-26,

1998.

48. Somanath PR, Jack SL, Vijayaraghavan S. Changes in sperm glycogen synthase

kinase-3 serine phosphorylation and activity accompany motility initiation and

stimulation. J Androl 25(4): 605-17, 2004.

49. Hughes K, Nikolakaki E, Plyte SE, Totty NF, Woodgett JR. Modulation of the

glycogen synthase kinase-3 family by tyrosine phosphorylation. EMBO J 12(2):

803-8, 1993.

50. Frame S, Cohen P, Biondi RM. A common phosphate binding site explains the

unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol

Cell 7(6): 1321-7, 2001. 53

51. Smith GD, Wolf DP, Trautman KC, Vijayaraghavan S. Motility potential of

macaque epididymal sperm: the role of protein phosphatase and glycogen

synthase kinase-3 activities. J Androl 20(1): 47-53, 1999.

52. Vijayaraghavan S, Trautman KD, Goueli SA, Carr DW. A tyrosine-

phosphorylated 55-kilodalton motility-associated bovine sperm protein is

regulated by cyclic adenosine 3',5'-monophosphates and calcium. Biol Reprod

56(6): 1450-7, 1997.

53. Rieske JS, Zaugg WS. The inhibition by antimycin A of the cleavage of one of

the complexes of the respiratory chain. Biochem Biophys Res Commun 8(421-6,

1962.

54. Bachelard HS, Clark AG, Thompson MF. Cerebral-cortex hexokinase.

Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic

analysis. Biochem J 123(5): 707-15, 1971.

55. Hers I, Tavare JM, Denton RM. The protein kinase C inhibitors

bisindolylmaleimide I (GF 109203x) and IX (Ro 31-8220) are potent inhibitors of

glycogen synthase kinase-3 activity. FEBS Lett 460(3): 433-6, 1999.

56. Zhang HC, White KB, Ye H, McComsey DF, Derian CK, Addo MF, Andrade-

Gordon P, Eckardt AJ, Conway BR, Westover L, Xu JZ, Look R, Demarest KT,

Emanuel S, Maryanoff BE. Macrocyclic bisindolylmaleimides as inhibitors of

protein kinase C and glycogen synthase kinase-3. Bioorg Med Chem Lett 13(18):

3049-53, 2003. 54

57. Klein PS, Melton DA. A molecular mechanism for the effect of lithium on

development. Proc Natl Acad Sci U S A 93(16): 8455-9, 1996.

58. Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch

OL, Murphy GJ, Carter PS, Roxbee Cox L, Mills D, Brown MJ, Haigh D, Ward

RW, Smith DG, Murray KJ, Reith AD, Holder JC. Selective small molecule

inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene

transcription. Chem Biol 7(10): 793-803, 2000.

59. Narisawa S, Hecht NB, Goldberg E, Boatright KM, Reed JC, Millan JL. Testis-

specific cytochrome c-null mice produce functional sperm but undergo early

testicular atrophy. Mol Cell Biol 22(15): 5554-62, 2002.

60. Ford WC. Glycolysis and sperm motility: does a spoonful of sugar help the

flagellum go round? Hum Reprod Update 12(3): 269-74, 2006.

61. Ford WC, Waites GM. The effect of high doses of 6-chloro-6-deoxyglucose on

the rat. Contraception 24(5): 577-88, 1981.

62. Brown-Woodman PD, Mohri H, Mohri T, Suter D, White IG. Mode of action of

alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of

ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis.

Biochem J 170(1): 23-37, 1978.

63. Ford WC, Harrison A. The effect of 6-chloro-6-deoxysugars on adenine

nucleotide concentrations in and motility of rat spermatozoa. J Reprod Fertil

63(1): 75-9, 1981. 55

64. Sols A, Crane RK. Substrate specificity of brain hexokinase. J Biol Chem 210(2):

581-95, 1954.

65. Krzyzosiak J, Molan P, Vishwanath R. Measurements of bovine sperm velocities

under true anaerobic and aerobic conditions. Anim Reprod Sci 1999; 55: 163-173.

66. Bhat RV, Shanley J, Correll MP, Fieles WE, Keith RA, Scott CW, Lee CM.

Regulation and localization of tyrosine216 phosphorylation of glycogen synthase

kinase-3beta in cellular and animal models of neuronal degeneration. Proc Natl

Acad Sci U S A 2000; 97: 11074-11079.