msc thesis - matsushita-fournier_david_pharmacologytherapeutics (linkedin)

OXIDATIVE STRESS INDUCES REDOX-DEPENDENT MODIFICATIONS OF HUMAN SPERM AND SEMINAL PLASMA PROTEINS AND DAMAGES THE PATERNAL

GENOME

David Matsushita-Fournier

Supervisor: Dr. Cristian O’Flaherty

Department of Pharmacology and Therapeutics McGill University, Montreal

Quebec, Canada April 2015

A thesis submitted to McGill University in partial fulfillment of the requirement of the degree of Masters of Science

©David Matsushita-Fournier 2015

Matsushita-Fournier, 2

ABSTRACT

One in six couples are affected by infertility with 50% of cases traced back to the men.

High levels of reactive oxygen species (ROS) promote oxidative stress and are associated

with male idiopathic infertility. Physiologically high concentrations of ROS like

hydrogen peroxide (H2O2) and nitric oxide donors (i.e DaNONOate) have been shown to

impair sperm function such as motility and capacitation (CAP). ROS cause damage to

sperm DNA and is highly associated with male infertility. Peroxiredoxin (PRDX) family

of enzymes plays a significant role in the antioxidant protection of seminal plasma and

spermatozoa. Tubulin is a major component of sperm flagellum. CAP is necessary for

spermatozoon to become fertile and is dependent on actin polymerization. Retention of

histones in the nucleus may cause differential sensitization of neighboring DNA to

oxidative stress. We hypothesized that redox-dependent protein modifications of major

functional proteins of the semen and differential oxidation of sperm chromatin occurs in

spermatozoa under oxidative stress. We aimed to determine whether oxidative stress

alters sperm quality by determining redox-dependent modification of seminal plasma

PRDX1, spermatozoa tubulin and actin polymerization. We also aimed to determine

DNA oxidation and DNA nitration and their localization in human spermatozoa. Percoll-

washed spermatozoa were treated with increasing concentrations of either H2O2 or

DaNONOate. Afterwards, sperm were capacitated with albumin if needed. Actin

polymerization, DNA oxidation and nitration were determined by cytochemistry using

Phalloidin-Alexa Fluor 555 labeling, anti-8-hydroxydeoxyguanosine and anti-8-

nitroguanine antibody, respectively. S-glutathionylation, redox-dependent modification

of PRDX1 and tubulin was determined by SDS-PAGE under non-reducing conditions

and immunoblotting with specific antibodies. Seminal plasma PRDX1 and sperm tubulin

and actin underwent redox-dependent modifications upon H2O2 treatment. Actin

polymerization was inhibited by H2O2 treatment in capacitated spermatozoa. There was a

differential sensitivity of the nucleus to DNA damage causing unique distribution of both

8-hydroxydeoxyguanosine (8-OHdG) and 8-nitroguanine (NitroG). These results suggest

that seminal plasma antioxidant function may be irreversibly inhibited due to redox-

dependent modification. Spermatozoa impairment of motility and CAP by H2O2 may be


due to redox-dependent modification of tubulin and actin. Human sperm DNA has

differential sensitivity to oxidative stress possibly due to the nucleus’ heterogeneous

retention of histone during compaction with protamines.

In the preparation of this thesis, I participated in the experiment design, performed all

experiments and analysis of the resulting data.


RÉSUMÉ L’infertilité affecte un couple sur six et dans 50% des cas, l’homme en est la cause. Des

taux élevés d’espèces réactives de l’oxygène (ROS) favorisent le stress oxydatif et sont

associés avec l’infertilité masculine idiopathique. Il a été démontré que des

concentrations physiologiquement élevées de ROS tel que le peroxyde d’hydrogène

(H202) ou un donneur d’oxyde d’azote (DaNONOate), détériorent certaines capacités du

spermatozoïde, comme la motilité et la capacitation. Les ROS endommagent l’ADN du

spermatozoïde et sont fortement associées avec l’infertilité masculine. L’enzyme

peroxiredoxin-1 et la famille d’enzyme peroxiredoxin (PRDX) en général jouent un rôle

significatif dans la protection antioxydante du plasma séminal et des spermatozoïdes.

Sous de fortes conditions oxydatives, des modifications dans les spermatozoïdes

endommagent de façon irréversible la PRDX1. La Tubuline est une composante majeure

du flagelle du spermatozoïde, essentielle à la motilité. La capacitation est nécessaire pour

que le spermatozoïde devienne fertile. Une rétention d’histones et de protamines dans le

noyau peut causer une sensibilité inégale de l’ADN voisin au stress oxydatif. Notre

hypothèse est que la modification de protéines fonctionnelles majeures associées au

plasma séminal et aux capacités du sperme, ainsi que l’oxydation différentielle de la

chromatine du spermatozoïde surviennent dans des spermatozoïde pendant un stress

oxydatif. Notre but est de déterminer si le stress oxydatif altère la qualité du sperme en

déterminant les modifications redox-dépendant de la PRDX1 du plasma séminal, de la

tubuline du spermatozoïde et de la polymérisation de l’actine. Nous voulons aussi

déterminer l’oxydation et la nitration de l’ADN et leur localisation dans les

spermatozoïdes humains soumis à un stress oxydatif. Des spermatozoïdes sélectionnés

après gradient de Percoll ont été traités avec des concentrations croissantes de H2O2 ou de

DaNONOate. Ensuite, des populations isolées de spermatozoïdes traités et non-traités ont

été capacités avec de l’albumine. La polymérisation de l’actine, l’oxydation et la nitration

de l’ADN ont été déterminés par cytochimie en utilisant une étiquette Phalloidin-Alexa

Fluor 555 et des anticorps anti-8-hydroxydeoxyguanosine et anti-8-nitroguanine,

respectivement. La S-glutationylation, la modification redox-dépendante de PRDX1 et la

tubuline ont été déterminées par SDS-PAGE sous des conditions non-réductrices et par


immuno-buvardage avec certains anticorps spécifique. La PRDX1 du plasma séminal, la

tubuline et l’actine des spermatozoïdes ont subi des modifications redox-dépendantes

suivant un traitement H2O2. De plus, la polymérisation de l’actine a diminué dans des

spermatozoïdes capacités. Nous avons observé de fortes augmentations dose-dépendante

des niveaux de 8-hydroxydeoxyguanosine (8-OHdG) et de 8-nitroguanine (NitroG) dans

les spermatozoïdes traités au H202 et au DaNONOate, respectivement. Il y avait une

sensibilité inégale du noyau aux dommages de l’ADN, causant une distribution unique de

chacune des modifications d’ADN. Nos résultats suggèrent que la capacité antioxydante

du plasma séminal peut être irréversiblement inhibée par ces modifications. La

détérioration de la motilité et de la capacitation des spermatozoïdes par le H2O2 est

possiblement due aux modifications de la tubuline et de l’actine. L’ADN du spermatoïde

humain a une sensibilités inégale au stress oxydatif. Cela a possiblement pour cause la

rétention hétérogène d’histones dans le noyau durant le compactage avec les protamines.

Dans la préparation de cette thèse, j’ai participé à la conception expérimentale, réalisé

toutes les expériences et analysé les données résultantes.


TABLE OF CONTENTS 1 INTRODUCTION ............................................................................................................................ 12

1.1 OXIDATIVE STRESS IN MALE INFERTILITY ............................................................................................. 12 1.2 NECESSITY OF ADVANCED SELECTION OF SPERM DURING ASSISTED REPRODUCTIVE

TECHNOLOGY .......................................................................................................................................................... 15 1.3 CHARACTERIZING MALE FERTILITY ........................................................................................................ 16 1.4 SUBTYPES OF INFERTILITY ........................................................................................................................ 17 1.5 SPERM STRUCTURE AND MOTILITY ......................................................................................................... 17 1.6 SPERMATOGENESIS, MATURATION AND CAPACITATION ..................................................................... 20 1.7 SENSITIVITY OF SPERMATOZOA TO OXIDATIVE STRESS ...................................................................... 23 1.8 MAINTAINING REDOX BALANCE .............................................................................................................. 24

2 RESEARCH RATIONAL ................................................................................................................. 26 2.1 INFERTILITY AS A RESULT OF REDOX IMBALANCE ............................................................................... 26 2.2 ROS AND SPERMATOZOA IMPAIRMENT ................................................................................................. 27 2.2.1 ROS Impairment of Semen Antioxidant .................................................................................... 27 2.2.2 ROS Impairment Spermatozoa Motility ................................................................................... 27 2.2.3 ROS Impairment of Spermatozoa Capacitation .................................................................... 28 2.2.4 ROS Impairment of DNA Integrity .............................................................................................. 28 2.2.5 DNA Oxidation and Nitration ........................................................................................................ 28

3 HYPOTHESIS AND OBJECTIVES ................................................................................................ 30 4 MATERIALS AND METHODS ...................................................................................................... 31

4.1 REAGENTS AND MATERIALS ..................................................................................................................... 31 4.2 SUBJECTS ...................................................................................................................................................... 31 4.3 CASA ANALYSIS .......................................................................................................................................... 32 4.4 SPERM SAMPLE PREPARATIONS AND TREATMENTS ............................................................................ 32 4.5 INDUCTION OF IN VITRO OXIDATIVE STRESS IN SEMINAL PLASMA AND SPERMATOZOA ............. 32 4.6 INDUCTION OF SPERM CAPACITATION .................................................................................................... 33 4.7 WESTERN BLOTTING ................................................................................................................................. 33 4.8 DETERMINATION OF Β-‐ACTIN POLYMERIZATION ................................................................................. 34 4.9 DETERMINATION OF DNA OXIDATION AND NITRATION .................................................................... 34 4.10 STATISTICAL ANALYSIS ........................................................................................................................... 35

5 RESULTS .......................................................................................................................................... 36 5.1 GLUTATHIONYLATION OF SEMINAL PLASMA PROTEINS ..................................................................... 36


5.2 THIOL OXIDATION AND PROTEIN COMPLEX FORMATION OF SEMINAL PLASMA PRDX1 UNDER

OXIDATIVE STRESS ................................................................................................................................................. 38 5.3 THIOL OXIDATION AND PROTEIN COMPLEX FORMATION OF SPERMATOZOA TUBULIN UNDER

OXIDATIVE STRESS ................................................................................................................................................. 40 5.4 THIOL OXIDATION AND PROTEIN COMPLEX FORMATION OF β-‐ACTIN IN SPERMATOZOA UNDER

OXIDATIVE STRESS ................................................................................................................................................. 44 5.5 IMPAIRED Β-‐ACTIN POLYMERIZATION IN CAPACITATED SPERMATOZOA UNDER OXIDATIVE

STRESS 46 5.6 DIFFERENTIAL LOCALIZATION OF 8-‐OHDG AND NITROG IN SPERMATOZOA UNDER OXIDATIVE

STRESS 49

6 DISCUSSION .................................................................................................................................... 55 7 CONCLUSION .................................................................................................................................. 61 8 FUTURE DIRECTIONS .................................................................................................................. 62


LIST OF FIGURES Figure 1: Sources and clinical consequences of ROS in male infertility (Adapted from

Said et al., 2012) ....................................................................................................... 14

Figure 2: Structures of the mammalian sperm and components of the flagella (Adapted

from Eddy, 2006) ...................................................................................................... 19

Figure 3: Dose-dependent increase in high molecular weight GSS-R signal due to protein

complex formation in human seminal plasma following H2O2 treatment ................ 37

Figure 4: Thiol oxidation of PRDX1 results in high molecular weight protein complex

formation in human spermatozoa under H2O2-treatment .......................................... 39

Figure 5: Thiol oxidation of tubulin results in high molecular weight protein complex


Figure 6: Dose-dependent increase of insoluble tubulin found in the pellet under H2O2-

treatment ................................................................................................................... 43

Figure 7: Thiol oxidation of β-actin results in high molecular weight protein complex


Figure 8: β-Actin polymerization in capacitated spermatozoa determined by Phalloidin-

Alexa Fluor 555 labeling of F-actin ......................................................................... 47

Figure 9: β-Actin polymerization negatively impacted by H2O2-treatment during human

sperm capacitation .................................................................................................... 48

Figure 10: Dose-dependent increase of 8-OHdG intensity/area with H2O2-treatment ..... 51

Figure 11: Dose-dependent increase in NitroG intensity with DaNONOate-treatment ... 53

Figure 12: Comparison of differential localization of 8-OHdG and NitroG under strong

oxidative stress .......................................................................................................... 54


LIST OF ABBREVIATIONS Akt Protein Kinase B

AR Acrosome Reaction

ART Assisted Reproductive Technology

BSA Bovine Serum Albumin

BWW Biggers, Whitten and Whittingham medium

cAMP 3'-5'- Cyclic Adenosine Monophosphate

CAP Sperm Capacitation

CASA Computer Assisted Semen Analysis

Cys Cysteine

DaNONOate 1,1-diethyl-2-hydroxy-2-nitrosohydrazine

DNA Deoxyribonucleic Acid

DTT Diothiothreitol

ECL Chemiluminescence

ERK Extracellular Signal Regulator Kinase

eGPX Extracellular Glutathione Peroxidase

FCSu Fetal Cord Serum Ultrafiltrate

GSH Glutathione

GPX Glutathione Peroxidase

GRD Glutathione Reductase

GSS-R S-glutathionylation

H2O2 Hydrogen Peroxide

HBS HEPES Balanced Saline

LPC Lysophosphatidylcholine

NitroG 8-nitroguanosine

NitroY Tyrosine Nitration

NO� Nitric Oxide

O2•– Superoxide

ONOO– Peroxynitrite


PBS Phosphate Buffered Saline

PBS-T Phosphate Buffered Saline with 1% Triton-X100

PI3K Phosphatidylinositol-3-kinases

PKA Protein kinase A

PKC Protein Kinase C

PRDX Peroxiredoxin

PTK Protein Tyrosine Kinase

PUFA Polyunsaturated Fatty Acid

RDPM Redox-Dependent Protein Modifications

ROS Reactive Oxygen Species

SCSA Sperm Chromatin Structural Assay

SOD Superoxide Dismutase

TAC Total Antioxidant Capacity

TBARs Thiobarbituric Acid Reactive Substances

TTBS Tris-Buffered Saline with 0.1% Tween 20

Txndc Thioredoxin Domain-Containing Proteins

WHO World Health Organization

8-OHdG 8-Hydroxydeoxyguanosine


ACKNOWLEDGEMENT

Completing my Masters degree marks a great moment in my life. Using rational thought

and scientific experimentation to contribute to the breath of biological knowledge has

always been a goal of mine. This would not have been possible without the guidance and

support of some incredible people.

I would like to first thank my supervisor, Dr. Cristian O’Flaherty for allowing me to be a

part of his research, for his guidance and his patience. His passion for research is

inspirational.

I would like to thank my thesis committee members: Dr. Culty, Dr. Zini, and Dr.

DiBattista for their time and the endless help they provided me in building my research

and presenting my work.

For the infinite joy and motivation they provided on a day-to-day basis, I would like to

thank Krista, Burak and Connie. You made work a wonderful place. I felt at home

because of all the amazing people of H6 at the Royal Victoria Hospital. Thank you all.

My research would not have been possible without the donors who participated and

therefore I would like to extend my appreciation to them as well.

I would lastly like to thank my friends and my family who supported me throughout my

life. They showed me that there are many ways to contribute to this world and taught me

how important it is to follow your passion.


1 Introduction

1.1 Oxidative Stress in Male Infertility

Infertility is a global disease that impacts 15% of all couples of reproductive age. This

amounts to 60-80million couples worldwide (WHO, 2010). Although long-term analysis

of fertility is hard to assess due to other factor such as “Reduced Child-Seeking” behavior

of couples (Mascarenhas et al., 2012), studies analyzing semen from men around the

world show declining semen quality and their possible contribution to decreasing fertility

globally (Rolland et al., 2012, Aitken, 2013). Fertility issue can be traced back to the man

and women with equal incidence (Templeton et al., 1991, Jarow et al., 2002, Abid et al.,

2008). There are different causes of male infertility such as varicocele, cryptochordism,

cystic fibrosis, infections and tumors (Agarwal et al., 2008). There are also different risk

factors that appear to contribute to infertility indirectly such as smoking, inflammatory

disease and drug exposure amongst other (Afzelius et al., 1975, Anderson and

Williamson, 1988, Tournaye and Cohlen, 2012). Causes and risk factors for male

infertility commonly cause increased in oxidative stress in the semen (Agarwal et al.,

2008). For example, varicocele has been shown to increase nitric oxide (NO•) levels in

the spermatic veins of patients (Mitropoulos et al., 1996) while smoking is associated

with increased leukocyte concentration (leukocytes are a significant source of oxidative

stress in the semen) as well as increased concentrations of reactive oxygen species (ROS)

(as illustrated in Figure1) (Saleh et al., 2002c). For these and further reasons explored in

this literature review, oxidative stress research is becoming ever increasingly important in

the research of male fertility.

Oxidative stress is the result of a surplus of total ROS species due to either an increase in

their production and/or a decrease in the cell antioxidant scavenging capacity (Halliwell,

2006, Halliwell and Gutteridge, 2007b, Gong et al., 2012). Oxidative Stress results in

various redox-dependent modifications of its targets and has been shown to cause specific


damage to spermatozoa components: causing lipid peroxidation (Griveau et al., 1995,

Aitken, 1995), redox-dependent protein modifications (RDPM) (Morielli and O'Flaherty,

2015), DNA fragmentation (Zini et al., 2008b, Winkle et al., 2008, Talebi et al., 2008)

and DNA oxidation (Shen and Ong, 2000, Kao et al., 2008) (as illustrated in Figure 1).

Approximately 25% of infertile men have elevated levels of ROS in their semen (Iwasaki

and Gagnon, 1992, Zini et al., 1993). Oxidative stress is a common pathophysiological

mechanism in a wide range of disease (Lipinski, 2001, Aliev et al., 2002, Griendling and

FitzGerald, 2003, Chauhan and Chauhan, 2006). Oxidative stress has been identified as a

major contributing factor of infertility in men (Tremellen, 2008).

Diseases that are commonly associated with elevated concentrations of ROS in the semen

include inflammatory diseases such as varicocele (Ozbek et al., 2000, Zini et al., 2005,

Shiraishi and Naito, 2007). Cancer and chemotherapy are known to be associated with

DNA damage (O'Donovan, 2005) and source of ROS in semen (Said et al., 2012).

Leukocytes themselves are large sources of ROS during leukocytospermia (Saleh et al.,

2002a, Said et al., 2012). Immature spermatozoa are characterized by a residual

cytoplasmic droplet and an excess production of ROS. This excessive production of ROS,

the possibility of large numbers of these cells in close proximity to other spermatozoa

present in the semen results in immature sperm being a major source of oxidative stress

for healthy spermatozoa in some men (Ollero et al., 2001, Gil-Guzman et al., 2001, Said

et al., 2012).


Figure 1: Sources and clinical consequences of ROS in male infertility (Said et al.,

2012)

Sources of ROS and risk factors for oxidative stress in semen are diverse and numerous.

The above image highlights some of the major sources of oxidative stress as well as their

common mechanisms of damage and ultimate clinical consequences on the fertility that

result (Said et al., 2012).


1.2 Necessity of Advanced Selection of Sperm During Assisted

Reproductive Technology

Assisted reproductive technology (ART) is a common treatment option for couples

suffering from infertility. Assessment of ART success based on delivery rate of over

1300 infertile couples after multiple cycles of ART resulted in at least one live birth for

70% of couples within 5-years (Pinborg et al., 2009); however, ART is known to have a

one-time success rate of ~30% (Zini et al., 2008a). The practice of selecting the

spermatozoa for use during ART rely on primarily two methods; swim-up and density

gradient-centrifugation, both which rely on the motility of the sperm for selection

(Åkerlöf et al., 1987). The selection of sperm from infertile males and the tools utilized

during ART in clinical practice has been more or less unchanged since 1959 (Clark et al.,

2005, Lopez-Garcia et al., 2008).

The practice of selecting sperm based on motility exclusively does not directly take into

account other aspects of sperm dysfunction like morphology, apoptosis-like

manifestations, and maturation while positive selection for these characteristics results in

improved sperm-quality compared to motility alone (Said and Land, 2011). While

advanced selection methods of sperm may select higher quality sperm for ART, the

cost/benefit for health care and impact on reproductive outcome remain undetermined

(Yetunde and Vasiliki, 2013). Currently, assessment of DNA integrity in infertile males

is becoming increasingly important when patients and physicians decide on the best

treatment option as poor sperm DNA integrity has been associated with poor implantation

rates and increased negative health outcomes in offspring (Evenson et al., 1999, Spano et

al., 2000, Benchaib et al., 2007). This is even more significant as ART practice

circumvents the physiological selection process that occurs during natural pregnancy

(Zini and Libman, 2006). Though one cycle of ART allows for live births in ~30% of

infertile couples, it may propagate genetic defects to the offspring due to a damaged

paternal genome (Corabian and Hailey, 1999, Hansen et al., 2002, Agarwal et al., 2005,

Hansen et al., 2005). Following children conceived through ART, it is reported that they


are 20-30% more likely then children conceived naturally to have a spectrum of

developmental defects, behavioral issues or increased hospitalization in early childhood

(Hansen et al., 2005, Tournaye and Cohlen, 2012). Efforts to improve DNA integrity of

spermatozoa selected for ART using a microfluidic device is currently a focus in

infertility research (Nosrati et al., 2014).

1.3 Characterizing Male Fertility

Male fertility is clinically defined predominantly by the spermogram, an assessment of

total sperm number, sperm concentration, total and progressive motility and sperm

morphology (WHO, 2010). The assessments of these criteria are performed using a

microscope and a computer assisted semen analyzer (CASA) software (Tournaye and

Cohlen, 2012). Assays have been developed to determine functional capacity of the

sperm; however, used rather exclusively for basic research and not used clinically due to

time restrictions and their poorly characterized clinical value (Vasan, 2011).

Sperm morphology has been used as marker for fertility independent of sperm count and

motility (Kruger et al., 1986, Kruger et al., 1987). By utilizing a strict criteria for

morphology, Kruger demonstrated that infertile patient with normal morphology

(between 4-14% normal forms) had a significantly higher fertilization rate than those

patients with less than 4% normal forms (Kruger et al., 1988). More recently, the clinical

threshold of normal form for in vivo fertilization was estimated to be around 5% (Gunalp

et al., 2001). A strict criteria for intracytoplasmic morphology (under high magnification

of x6,000) was developed and showed correlation with DNA integrity, and may have

clinical value due to stricter sperm selection during ART (Maettner et al., 2014). This

inclusion of morphology during the selection process is known as intracytoplasmic

morphologically selected sperm injection (IMSI) (Lo Monte et al., 2013).


1.4 Subtypes of Infertility

The World Heath Organization (WHO) gives guidelines in regards to healthy semen

parameters. Lower reference limits are used as guidance; however, semen parameters

above these reference values do not guarantee fertility (Ayaz et al., 2012). Different

abnormal semen parameters will result in a different diagnosis. Asthenozoospermia is a

common cause of infertility in men. It is characterized by critically low sperm motility

and is seen in an average of 19% of infertile men while total asthenozoospermia is seen in

1 of 5000 men (Ortega et al., 2011). Asthenozoospermia is also associated with other

semen abnormalities such as low sperm concentration (oligo-asthenozoospermia),

abnormal sperm morphology (astheno-teratozoospermia) (Curi et al., 2003) and

leukocytospermia (Kortebani et al., 1992). Normozoospermic infertile males are men

who cannot conceive with a fertile female despite having a normal semen analysis and no

other detectable explanation for infertility. This type of patients represents ~15% of

idiopathic infertile men (Hamada et al., 2012), however, ranges between 6-37%

depending on the population and study (Templeton and Penney, 1982, Moghissi and

Wallach, 1983, Collins and Crosignani, 1992). Although normozoospermic infertile men

have no morphological or other semen abnormalities, they may still have significant

levels of sperm DNA fragmentation than their fertile counterpart as DNA fragmentation

is an abnormal sperm characteristic that is undetectable during routine semen analysis

(Saleh et al., 2002b).

1.5 Sperm Structure and Motility

The spermatozoon has the primary function to deliver the paternal genome to the female

oocyte. To accomplish this unique task, the spermatozoon has developed into a highly

specialized, highly compartmentalized, terminally differentiated cell (as illustrated in

Figure 2). The flagellum has the primary function of sperm motility while the head is the

site of the paternal genome (Yanagimachi, 1994, Yanagimachi, 2005). Proteomic analysis

of the head and tail reveal 721 and 521 unique proteins in the tail and in the head,

respectively (Aitken, 1995). The sperm head contains not only a highly condensed DNA


but also contains the acrosome (a sperm specific exocytotic vesicle), some remaining

cytoplasm and a cytoskeleton composed mainly by actin (Eddy, 2006).

Motility is a critical function of the sperm in that it is required to complete its function of

reaching the oocyte and ultimately for fertilization to occur (WHO, 2010). Poor motility

is commonly seen in infertile men and is associated with many other abnormal

parameters such as lipid peroxidation (Rao et al., 1989), increased mitochondrial and

structural abnormalities (such as abnormal flagella) (Baccetti et al., 1993).

Energy production is required for sperm motility and is produced largely by the sperm

mitochondria present in the midpiece of the flagella (illustrated in Figure 2) by a process

of oxidative phosphorylation (Olson and Winfrey, 1986, Olson and Winfrey, 1990).

Sperm contain specific isoforms of mitochondrial protein such as lactate dehydrogenase

C4 (Goldberg, 1963), allowing it to use more various substrates for the synthesis of ATP

compared to mitochondria of somatic cells. This biochemical flexibility is central in

allowing the sperm to maintain motility under the various conditions of the female

reproductive system (Piomboni et al., 2012).

Knock out models of various sperm proteins associated with motility; structural proteins

such as dynein and tubulin-associated proteins or metabolic proteins such as voltage-

dependent ion channels, results in various motility abnormalities such as truncated or

bent flagella and disorganized axoneme (Afzelius et al., 1975, Escalier, 2006).

One of the most prominent structures of the flagella is the axoneme core. It is composed

of a “9+2” complex of microtubules, which are composed of spermatid-specific α-tubulin

and β-tubulin (Eddy, 2006). Tubulin structure and related axonemal abnormalities

(assessed by electron microscopy) is frequently associated with male infertility such as

men with idiopathic oligo-astheno-teratozoospermia (iOAT) (El-Taieb et al., 2009).


Figure 2: Structures of the mammalian sperm and components of the flagella

(Adapted from Eddy, 2006)

The head (containing the paternal genome, the remaining cytoplasm and the acrosome) is

attached to the flagellum by the connecting piece. The Flagella contains different regions

such as the middle, principal and end piece. The middle piece houses the mitochondrial

sheath, containing the mitochondria. The image on the right represents the cytoskeletal

components of the flagellum. The Axoneme core consists of nine outer doublets of

microtubules, which surround a central pair of microtubules. These microtubules are

composed of primarily tubulin (modified image) (Eddy, 2006).


1.6 Spermatogenesis, Maturation and Capacitation

Spermatogenesis is a stepwise process of the male germ cell that ultimately results in the

terminally differentiated spermatozoa. The intermediate cell stages in this process are the

spermatogonia, spermatocytes and spermatids (Eddy, 2006). During spermatogenesis, the

Sertoli cell supports the germ cell development (Griswold and McLean, 2006) and the

Leydig cells maintains critical testosterone-levels within the testis (Stocco and McPhaul,

2006). A subsequent process of epididymal sperm maturation must occur before the

sperm acquires the ability to be motile, undergo capacitation, bind and ultimately fuse

with the oocyte (Robaire et al., 2006, Dacheux and Dacheux, 2014). As the spermatozoa

traverse the epididymis, it is exposed to varying protein compositions and concentrations

due to protein secretion, degradation, re-absorption and utilization by the spermatozoa

(Robaire et al., 2006). The spermatozoa undergo various changes during epididymal

sperm maturation including remodeling of its plasma membrane, active reabsorption of

its residual cytoplasm, changes in intracellular pH and ion concentrations and chromatin

condensation (Aitken and Vernet, 1998, Robaire et al., 2006, Cornwall and von Horsten,

2007).

Sperm chromatin is unique in its compaction with primarily protamine, a cysteine and

arginine-rich, basic proteins (Caron et al., 2005, Balhorn, 2007). During testicular

maturation, chromatin remodeling will occur resulting in replacing the majority of the

histones in the chromatin with protamines and with only about 10-15% histones

remaining in humans (Gatewood et al., 1987, Noblanc et al., 2013). Cross-linking

occurs during epidydimal transit between cysteine groups of the protamines, resulting in

a highly compact structure critical for normal fertilization (Kosower et al., 1992).

Excess nucleohistone presence in the chromatin and aberrant protamination are

characteristics of immature cells and renders these cells more susceptible to oxidative

stress and DNA damage (Sakkas et al., 1998, Aitken and De Iuliis, 2010). Both

hypocondensation and hypercondensation of sperm chromatin have been associated with

male infertility (Rodriguez et al., 1985, Rufas et al., 1991, Engh et al., 1992, Engh

et al., 1993) thus highlighting the need for a critical level of protamination required for


normal fertility. Epidydmal spermatozoa show spontaneous capacity to produce

superoxide (O2•–) (which dismutates to H2O2) through its surface NADPH oxidase. This

mechanism of peroxide generation is critical in downstream signaling that ultimately

results in chromatin condensation (Aitken and Vernet, 1998). Glutathione peroxidase 4

(GPX4) have the dual role of mediating the sulfoxidation events that result in protamine

cross-linking and chromatin compaction as well as scavenging of excess H2O2 during

epididymal maturation (Noblanc et al., 2011). Along with GPX4, peroxiredoxin 6

(PRDX6) participates in sperm chromatin condensation (Ozkosem et al., 2015). This

demonstrates that epididymal maturation is a redox-dependent process as well as how

ROS balance achieved by nuclear antioxidant enzymes is critical in maintaining DNA

integrity.

During ejaculation, epididymal spermatozoa are mixed with secretion from the male

accessary reproductive glands, the prostate gland, the seminal vesicles and the

bulbourethral glands (Risbridger and Taylor, 2006). Though ejaculated sperm are motile,

they must undergo the process of capacitation (CAP) before they are fertile

(Yanagimachi, 1994, de Lamirande et al., 2012). The CAP process is both temperature

and time-dependent and physiologically occurs in the oviduct of the female genital tract.

During CAP, the sperm will experience extensive changes in its intracellular ion

concentration, membrane fluidity and protein-phosphorylation status (de Lamirande et

al., 2012). CAP prepares the sperm for binding to the zona pellucida, for subsequent

Acrosomal Reaction (AR) and for oocyte fusion (Yanagimachi, 1994, Visconti and Kopf,

1998). CAP-associated protein tyrosine phosphorylation and membrane fluidity has been

shown to be compromised in asthenozoospermic patients (Buffone et al., 2005). CAP can

be induced with various combinations of substances including calcium ionophore, bovine

serum albumin (BSA), fetal cord serum ultrafiltrate (FCSu), progesterone and sodium

carbonate (Baldi et al., 1991, de Lamirande and Gagnon, 1995b, de Lamirande et al.,

1998a).

During the early events of CAP, the spermatozoa will experience an influx of calcium, a

rise in pH and will generate a low level of both O2•– and nitric oxide (NO•)


(Yanagimachi, 1994, de Lamirande and O’Flaherty, 2012). These three events will

activate adenylyl cyclase which leads to an elevation of intracellular cyclic adenosine

monophosphate (cAMP) and protein kinase A (PKA) activity (Parinaud and Milhet,

1996). ROS are also involved in the activation of protein kinase (PKC) and RAS proteins

as well as the inhibition of various phosphatases thus supporting the sperm progression

into late stages of CAP (O'Flaherty et al., 2006).

Late stages of CAP consist of phosphorylation of tyrosine residues predominantly in the

region of the fibrous sheath (Carrera et al., 1996) and actin polymerization in the post-

acrosomal region of the head (Brener et al., 2002). Actin polymerization is a critical step

during CAP of human and other mammalian spermatozoa while its rapid breakdown is

required for AR to occur (Brener et al., 2002). Actin polymerization is regulated by

protein phosphorylation events as inhibitors of protein kinases prevented it while

stimulators of tyrosine phosphorylation in sperm (sodium vanadate, H2O2, cAMP,

epidermal growth factor (EGF), etc.) triggered it (Spungin et al., 1995, Brener et al.,

2002).

The acrosome is an exocytotic vesicle derived from the Golgi apparatus. It is located in

the apical position of the head and contains a variety of hydrolytic enzymes such as

acrosine and hyaluronidase (Yanagimachi, 2005, Eddy, 2006). These enzymes will be

released during the AR, facilitating the penetration of the zona pellucida by the

spermatozoon. The AR can be induced in vitro with a variety of compounds such as

progesterone (Sagare-Patil et al., 2012). During CAP, phospholipase C will translocate to

the plasma membrane where it can activate calcium channels in both the outer acrosomal

membrane as well as the plasma membrane. With sustained high cytosolic calcium

concentrations, actin-severing proteins will be activated, breaking the intervening barrier

between the outer acrosomal membrane and the plasma membrane. Their fusion

ultimately results in the exocytosis of the acrosomal contents (Spungin et al., 1995). This

therefore illustrates the critical role of CAP and F-actin in AR.


Extracellular calcium controls O2•– synthesis during CAP. This differs from nitric oxide

synthesis, which is controlled by both intracellular and extracellular calcium

concentration (de Lamirande et al., 2009). The activation of these two ROS synthesis

have proven to be complex; PKC, protein tyrosine kinase (PTK), extracellular-signal-

regulated kinases (ERK), phosphatidylinositol 3-kinase (P13K) and protein kinase B

(Akt) activation increases NO• levels, while O2•– production appears to be upstream of

NO• production. Reciprocal activation of the two ROS demonstrates flexibility in the

system, allowing for compensatory action between the two when production of one is

impaired (de Lamirande et al., 2009, de Lamirande and Lamothe, 2009). The inhibition of

AR by superoxide dismutase (SOD) and catalase and stimulation of AR by H2O2

(generated by xanthine-xanthine oxidase system) indicate that AR is a ROS-dependent

process as CAP (de Lamirande et al., 1998b).

Observed extracellular generation of O2•– and its inhibition by SOD indicate that an

oxidase exists at the surface of the plasma membrane, however, has remained

undiscovered (de Lamirande and Gagnon, 1995a, O'Flaherty et al., 1999). While

extracellular O2•– can activate surface targets, O2

•– spontaneously dismutates to the

diffusible H2O2 that enters into the cell and activate intracellular targets like PKA and

PKC during CAP (Aitken et al., 1995, de Lamirande and Gagnon, 1995a, Rivlin et al.,

2004, O'Flaherty et al., 2006).

A sperm nitric oxide synthase (NOS) localized at the plasma membrane produces NO•

which activates surface and intracellular targets involved in CAP and other sperm

function such as motility (Lewis et al., 1996). Sperm CAP is inhibited by L-NAME, an

inhibitor of NOS (de Lamirande and O’Flaherty, 2012).

1.7 Sensitivity of Spermatozoa to Oxidative Stress

Not only are many aspects of sperm function dependent on proper redox signaling,

different physiological aspects of sperm make it uniquely sensitive to oxidative stress. As

many antioxidants are intracellular, the little volume of cytoplasm in spermatozoa gives


the spermatozoon little endogenous antioxidant protection (Zini et al., 1993).

Spermatozoa contain very little glutathione compared to somatic cells and compared to

the seminal plasma (Li, 1975, Evenson et al., 1993). The primary source of antioxidant

protection for the spermatozoa is from its environment of seminal plasma (Gong et al.,

2012). The plasma membrane of the spermatozoa contains high concentration of

polyunsaturated fatty acids (PUFAs). Due to their unsaturation, PUFAs are particularly

sensitive to oxidative stress rendering the plasma membrane vulnerable to lipid

peroxidation (Wathes et al., 2007). Lipid peroxidation has been utilized as a marker for

fertility using the Thiobarbituric acid reactive substances (TBARs) assay which measures

mainly malondialdehyde, a byproduct of lipid peroxidation (Kodama et al., 1996). The

spermatozoa have virtually no ability to produce de novo proteins by protein synthesis

and therefore cannot replace damaged proteins during oxidative stress (Zini et al., 1993).

1.8 Maintaining Redox Balance

Seminal plasma is the primary source of antioxidant protection due to its relative

abundance of antioxidants compared to that of the spermatozoa and therefore is key in

protecting the spermatozoa against deleterious oxidative stress. The antioxidant

protection of the seminal plasma is derived from both enzymatic and non-enzymatic

antioxidants and originates predominantly from the secretions of the male accessory

glands (Holmes et al., 1992, Zini et al., 2002).

The dismutation of O2•– to H2O2 can be both spontaneous and enzymatically catalyzed by

SOD. The seminal plasma exhibits strong SOD activity (as measured by the nitroblue

tetrazolium assay) and is heavily armed with both Cu/Zn-SOD (SOD1) and extracellular

SOD3 isoforms (Peeker et al., 1997). The spermatozoon has not been shown to possess

any significant amount of Cu/Zn-SOD due to the scarce cytosol; however, it exhibits

SOD-like activity (Zini et al., 2002)

H2O2 is considered a strong oxidizer and is actively removed by various antioxidants such

as catalase and other peroxidases (O'Flaherty, 2014). Catalase has been shown to be


absent or found in insignificant amounts in human spermatozoa and is therefore

considered not a major player in the elimination of H2O2 (O'Flaherty, 2014). Catalase-

like activity has been observed in spermatozoa (as measured by the H2O2-scavenging

ability) and therefore other peroxidases are considered responsible for the spermatozoa

H2O2 scavenging ability (Zini et al., 2002, Zini et al., 1993).

Peroxyredoxins (PRDXs) are a ubiquitously expressed, sulfhydryl-dependent, non-

selenium, non-heme peroxidases (Rhee et al., 2005). Although other peroxidases exist in

the semen such as glutathione peroxidases, PRDXs are regarded as highly protective due

to its rapid reduction of numerous peroxides (Flohé et al., 2011). The PRDX enzymes

contain one or two cysteine residues in there active sites and are used in their

classification: 2-Cys PRDXs (isoforms 1-4), atypical 2-Cys PRDX (isoform 5) and 1-Cys

PRDX (isoform 6) (O'Flaherty, 2014). PRDX isomers 1, 4, 5 and 6 are expressed in both

spermatozoa and seminal plasma; however, there is a specific localization of the PRDX

isoforms within the sperm sub-compartments (O'Flaherty and de Souza, 2011, O'Flaherty,

2014). PRDX6 has been shown to react with extremely low, physiological concentrations

of H2O2 (as low as 50µM) indicating it’s participation in physiological redox signaling as

well as pathological H2O2-scavenging (O'Flaherty and de Souza, 2011, O'Flaherty, 2014).


2 Research Rational

2.1 Infertility as a Result of Redox Imbalance

The impact of the semen’s failure to maintain physiological levels of ROS while avoiding

conditions of oxidative stress for the sperm is catastrophic. Thirty to 80% of infertile men

show elevated levels of ROS in their semen and ROS represent a major contributing

factor in their infertility. Elevated levels of ROS species that are commonly observed in

the semen of infertile men include O2•–, H2O2, NO• and peroxynitrite (ONOO–) (Iwasaki

and Gagnon, 1992, Saleh et al., 2003, Tremellen, 2008). Many antioxidants are

considered critical for normal fertility as knockout models of certain antioxidants like

PRDX6 or thioredoxin domain-containing proteins (Txndc1 and Txndc2) show

abnormal semen consistent with infertility such as abnormal sperm chromatin

compaction and DNA oxidation (Smith et al., 2013, Ozkosem et al., 2015). Due to the

clear impact of oxidative stress on male fertility, efforts to correct excessive ROS levels

in the semen using antioxidant supplementation have been developed (Lanzafame et al.,

2009, Choudhary et al., 2010, Showell et al., 2011, Gharagozloo and Aitken, 2011).

Treatment by specific antioxidant and antioxidant cocktails have demonstrated some

efficacy of semen parameter improvement, however, lack evidence from randomized

controlled trials (Showell et al., 2011) and there are consistently studies that fail to show

significant therapeutic effect on fertility (Agarwal et al., 2004). In light of sperm

physiology being highly dependent on redox signaling, it is becoming increasingly likely

that unspecific antioxidant supplementation may result in suppression of physiological

oxidative events (Agarwal et al., 2004). Antioxidant treatment has been shown to cause a

reduction in sperm DNA compaction by interfering with physiological protamine

disulphide bridges. This led to interference in paternal gene activity during

preimplantation development and possible cytoplasmic fragments in the embryo

(Evenson et al., 1980). It is therefore critical to better understand the targets of ROS in


the semen to develop more specific antioxidant and protection without interfering with

normal physiology.

2.2 ROS and Spermatozoa Impairment

2.2.1 ROS Impairment of Semen Antioxidant

During the elimination of H2O2 by PRDXs, the cysteine residues in the active site of the

enzyme become oxidized, rendering it inactive and requiring either the

thioredoxin/thioredoxin reductase system (for PRDX 1-5) (Rhee et al., 2005) or

glutathione/glutathione reductase system mediated by glutathione S-transferase (for

PRDX 6) to reactivate the enzyme (Manevich et al., 2004, Ralat et al., 2006). There is

also further evidence within spermatozoa that PRDX isoforms 1 and 6 undergo H2O2-

dependent high molecular mass complexes formation under strong oxidizing conditions

(O'Flaherty and de Souza, 2011). Complex formation due to PRDX hyperoxidation is an

irreversible process without sulfiredoxin and sestrin1 enzymes. Up to now, the presences

of these enzymes have not been reported in semen. This would therefore indicate that

spermatozoa PRDXs are permanently inactivated under strong oxidative stress, thus

incapable of scavenging future ROS. Reduced PRDX concentration in both the seminal

plasma and the spermatozoa and higher levels of PRDX thiol oxidation are associated

with impaired sperm quality in infertile men (Gong et al., 2012). It is not known whether

PRDX of the seminal plasma is being permanently impacted by oxidative stress in a

similar fashion due to thiol oxidation of its active site cysteine.

2.2.2 ROS Impairment Spermatozoa Motility

Oxidative stress is well known to cause impaired sperm motility (Plante et al., 1994,

Rosselli et al., 1995, Nobunaga et al., 1996, Balercia et al., 2004), however, the exact

component of the motility machinery that is targeted is yet to be determined. Tubulin

oxidation has been observed under oxidative stress in different cell types. The redox-

dependent modification of tubulin resulted in dimerization and higher-fold protein

complexation of tubulin. This in turn resulted in impaired polymerization of tubulin and

microtubule formation (Landino et al., 2011, Clark et al., 2014, Landino et al., 2014).


Therefore it is possible that tubulin is being modified similarly in spermatozoa under

oxidative stress, resulting in the observed impairment of sperm motility by ROS.

2.2.3 ROS Impairment of Spermatozoa Capacitation

Capacitation of spermatozoa requires a controlled level of ROS generation in order to

activate critical downstream signal transduction (de Lamirande and O’Flaherty, 2012);

however, excessive exposure to these same ROS species during oxidative stress results in

impaired capacitation in spermatozoa (Morielli and O'Flaherty, 2015). β-Actin is known

to undergo redox-dependent modified under oxidative stress conditions (Hung et al.,

2013), including glutathionylation of two of its cysteine residues (Terman and Kashina,

2013); however, modification in spermatozoa has not been well explored. Due to the

critical nature β-actin polymerization during CAP and AR, redox-dependent modification

of β-actin may very well be the mechanism being ROS-dependent impairment of sperm

capacitation.

2.2.4 ROS Impairment of DNA Integrity

DNA fragmentation has long been proposed as a marker for male infertility since, in

some cases, infertile males showed higher degree of DNA fragmentation index (DFI)

compared with men from fertile couples as measured by the sperm chromatin structural

assay (SCSA) (Evenson et al., 1980). Alternative measures of DNA damage has also

been show to correlate with poor fertility such as analysis of DNA fragmentation using a

single-cell gel electrophoresis (comet) assay (Irvine et al., 2000). Impaired DNA integrity

is also thought to be the product of abnormal protamine expression and compaction

resulting in excess ROS generation and abortive apoptosis during spermatogenesis

(Sakkas et al., 2003).

2.2.5 DNA Oxidation and Nitration

Poor DNA integrity has been well associated with elevated ROS concentrations (O'Brien

and Zini, 2005) and decreased antioxidant protection (Shamsi et al., 2009) in the semen

of infertile men. 8-hydroxydeoxyguanosine (8-OHdG) is the principal biomarker for

DNA oxidation as it is both precise and sensitive to oxidative stress (Kodama et al., 1997,

Shen et al., 1999). It has also been shown to negatively correlate with sperm motility,

sperm number and normal morphology (Shen et al., 1999). DNA oxidation can also


follow after accumulation of lipid peroxidases at the surface of the sperm (Twigg et al.,

1998). Recently, DNA modifications by nitrogen containing ROS has been gaining

interest as a separate measure of DNA damage in neurodegenerative diseases and cancer

(Thanan et al., 2014). 8-nitroguanine (NitroG) is a redox-dependent modified guanine

residue that is produced by NO• formation to ONOO– under oxidative stress (Kawanishi

et al., 2001). NitroG is believed to be highly mutagenic as DNA polymerase sensitivity to

NitroG sites resulted in high levels of point mutation during DNA synthesis (Wu et al.,

2006). This would be significant during embryonic development as there is extensive

DNA synthesis occurring. NitroG appears to co-localize with 8-OHdG in somatic cells

during oxidative stress (Thanan et al., 2014). Due to the protective nature of proper

protamination and condensation of the chromatin, differences in level of compaction may

cause differential sensitivity of the nucleus to oxidative stress. This would therefore

imply that the chromatin in the peripheral region of the nucleus to be more susceptible to

oxidative stress as it is known to retain more histones and is less compacted then other

parts of the nucleus (Ward, 2010). This differential sensitivity of the chromatin to

oxidative stress in the peripheral region of the nucleus was observed previously in mice

(Noblanc et al., 2013), however, yet to be shown in human spermatozoa. This also brings

into question the importance of what part of the nucleus and more specifically what genes

are being affected by oxidative stress and can this information be used to predict at

complications at different parts of embryonic development.


3 Hypothesis and Objectives

In this thesis, we hypothesized that oxidative stress results in redox-dependent

modification of functionally important proteins and sperm chromatin. To test our

hypothesis, our study had two aims 1) to determine the impact of oxidative stress on

principal sperm proteins critical to seminal plasma and sperm function by stepwise

analysis of redox-dependent protein modification and 2) to determine the impact of

oxidative stress on sperm chromatin by measuring the production and specific

localization of 8-OHdG and NitroG.


4 Materials and Methods

4.1 Reagents and Materials

Percoll was purchased from GE Healthcare (Baie d’Urfe, Qc, Canada). Mouse

monoclonal anti-GSS-R and anti-β-actin IgG antibodies were provided by Virogen (clone

G8, Watertown, MA, USA) and Sigma-Aldrich (Winston Park Dr. Oakville, Ontario,

Canada). Rabbit polyclonal anti-PRDX1 (ab41906) was purchased from AbCam

(Toronto, ON M5W 0E9, Ontario, Canada). Horseradish peroxidase-conjugated goat

anti-mouse IgG antibody was purchased from Cederlane Laboratories Ltd (Hornby, ON,

Canada). Nitrocellulose membranes (pore size, 0.22 mm) were purchased from

Osmonics, Inc (Westborough, MA, USA) and the chemoluminescence (ECL) Kit Lumi-

Light from Roche Molecular Biochemicals. Radiographic films (obtained from Fuji;

Minami-Ashigara, Japan) were used for immunodetection of blotted proteins. The anti-8-

OHdG antibody and the anti-NitroG antibody were purchased from StressMarq

Biosciences Inc (Victoria, BC, Canada) and from Dojindo Molecular Technologies Inc

(Rockville, Maryland, USA), respectively, Biotinylated horse anti-mouse IgG was

purchased from Vector Laboratories, Inc (Burlingame, CA, USA). Alexa Fluor 555

conjugate of streptavidin, Prolong Antifade and Alexa Fluor® 555 Phalloidin were

purchased from Life Technologies (Burlington ON L7L 5Z1, Canada). Diethylamine

NONOate (DaNONOate) was obtained from Calbiochem (San Diego, CA, USA). Other

chemicals used were of at least reagent grade.

4.2 Subjects

Healthy male donors (20-35 years old) were recruited in the Montreal, Quebec area. Prior

to their donation, males were asked to abstain from sex for 3 days. Samples were

collected in sterile containers and left at 37°C for 30 min to induce liquefaction. This

study has gained approval from the Ethics Board of the Royal Victoria Hospital-McGill


University health Centre and all participants have given informed consent for use of their

semen prior to participation.

4.3 CASA Analysis

Raw semen was analyzed by CASA (Sperm Vision HR software v1.01, Penetrating

Innovation, Ingersoll, ON, Canada) to assure that the sperm samples met the criteria of

normality established by the WHO 2010 Guidelines (WHO, 2010). Only semen reaching

the WHO standard was used for experiments.

4.4 Sperm Sample Preparations and Treatments

Four layer Percoll gradients (bottom to top, 95%-65%-40%-20%) were constructed with

100% Percoll and isotonic HEPES balanced saline (HBS) and were brought to room

temperature (RT) prior to use. Liquefied semen was loaded into the Percoll gradient and

centrifuged at 2,300xg at RT for 30min. Percoll gradient centrifugation is used to

separate out the seminal plasma and a highly motile population of spermatozoa (collected

from the 95% and 65-95% interface) from poorly motile, abnormal sperm and other cells

(e.g. leukocytes). Seminal plasma was collected from the top of the Percoll gradient and

centrifuged again at 13,000xg to pellet any remaining cells. The supernatant of the

seminal plasma was separated from any formed pellet and diluted 25x using HBS. The

concentration of 95%, highly motile sperm was reassessed using CASA and were diluted

to 100x106 using Biggers, Whitten and Whittingham medium (BWW, pH 8.0) (Biggers

et al., 1971).

4.5 Induction of In Vitro Oxidative Stress in Seminal Plasma and

Spermatozoa

Oxidative stress was induced in the seminal plasma and spermatozoa by exposing

aliquots of each samples to increasing concentrations of H2O2 for a period of 30 min at

37°C in BWW. DaNONOate (a NO• donor) was used to induce formation of NitroG in


sperm DNA. The H2O2 was washed out in the spermatozoa samples by centrifuging at

600xg for 5min at 20°C, discarding supernatant and suspending the sperm pellet in fresh

BWW.

4.6 Induction of Sperm Capacitation

Following H2O2 and DaNONOate treatment, the sperm were resuspended in fresh BWW

containing 3mg/ml bovine serum albumin (BSA) and 25mM sodium bicarbonate to

induce CAP. Spermatozoa were incubated in capacitating medium for 3.5 hours at 37°C.

Sperm capacitation was verified by levels of tyrosine phosphorylation (by

immunoblotting) and the increase on the levels of β-actin polymerization was assessed by

Phalloidin labeling of polymerized sperm β-actin (Brener et al., 2002).

4.7 Western Blotting

Seminal plasma and sperm suspensions were first mixed with sample buffer with or

without 100mM dithiothreitol (DTT) (reducing or non-reducing conditions, respectively),

boiled for 5 min and centrifuged at 13,000xg. Aliquots of 10µl of 1x106

spermatozoa/well or 1:25 diluted seminal plasma (10 µg/well) were loaded into 12%

polyacrylamide gels (Gong et al., 2012). They were subsequently electrophoresed and

electro-transferred onto nitrocellulose membranes in a 20% methanol transfer buffer. 5%

skim milk in 2mM Tris (pH 7.8)-buffered saline and 0.1% tween 20 (TTBS) was used to

block the membranes. Membranes were blocked for 30min at RT and subsequently

washed in fresh TTBS 3-times for 5min prior to immunoblotting. Membranes were

immunoblotted with primary antibodies anti-GSS-R, anti-PRDX1, anti-β-Actin or anti-

tubulin overnight.

The following day, membranes were washed with fresh TTBS 3-times for 5min.

membranes were then incubated for 1hour at RT with horseradish peroxidase-conjugated

secondary antibody. Membranes were then washed with fresh TTBS 3-times for 5min

before incubation with ECL. Positive immunoreactive bands were detected using Fuji


radiography films (Minami-Ashigara, Japan). Loading control was established using

colloidal silver and/or reblotting with anti-tubulin antibody. The relative intensity of each

band was determined using Un-Scan-It gel software version 5.1 (Silk Scientific

Corporation, Orem, Utah) and normalized to silver stain intensity.

4.8 Determination of β-‐Actin Polymerization

Following the in vitro oxidation and capacitation protocol (described above), 10µl of

sperm suspension were smeared onto Superfrost plus slides (Fisher Scientific, Montreal,

QC, Canada), allowed to air dry and fixed in a solution of 2% glutaraldehyde and

0.2%triton in phosphate buffered saline (PBS) for 10min. Sperm were then rehydrated in

fresh PBS for 5min. The slides were incubated overnight in the staining buffer of

50µg/mL lysophosphatidylcholine (LPC) and 5µl methanol-Phalloidin-Alexa Fluor 555

stock solution (resulting in final concentration of 2.5% per slide) in PBS (Brener et al.,

2002). The following day, slides were washed 3-times in TTBS, mounted with prolong

antifade with DAPI and sealed with a cover slip. Phallodin-Alexa Fluor 555 intensity was

assessed using ImageJ (NIH). Total fluorescence within the area of the head was

measured and normalized to the background fluorescence (Burgess et al., 2010, Burnett

et al., 2011). Minimum of two hundred cells were counted per sample.

4.9 Determination of DNA Oxidation and Nitration

Following the in vitro oxidation protocol (described above), 10µl of sperm suspension

were smeared onto superfrost plus slides (Thermo Fisher Scientific, Montreal, QC,

Canada), allowed to air dry and fixed in methanol at -20°C for 5min (O'Flaherty and de

Souza, 2011). Smears were then rehydrated with PBS for 10min. Sperm were

decondensed in a solution containing 1M DTT and 0.03µg/ml Heparin in PBS. Sperm

were decondensed to the point where about 80% of the heads swelled to about 5x their

original size (for DNA oxidation analysis) and about 2x (for DNA nitration analysis).

Sperm were then fixed in methanol at -20°C for 5min. Smears were rehydrated by

submerging them in PBS for 10min, blocked with 5% horse serum in PBS supplemented


with 1% Triton-X100 (PBS-T) for 30min and washed with fresh PBS. Slides were

incubated overnight with either anti-8-OHdG or anti-NitroG antibody. The following day,

slides were then washed with PBS-T and incubated for 1hour with a biotinylated horse

anti-mouse antibody. Slides were quickly washed of their antibodies 3-times using PBS-

T. Prolong antifade with DAPI was added and mounted with coverslip. Negative controls

were prepared in the same way except samples were not incubated with either anti-8-

OHdG or anti-NitroG antibody.

4.10 Statistical Analysis

Differences between treatments for relative tubulin, Phalloidin-Alexa Fluor 555, 8-OHdG

and NitroG intensities were analyzed by non-parametric Friedman Test and post hoc

Dunn’s multiple comparison test. Differences between capacitation and non-capacitation

using Phalloidin-Alexa Fluor 555 intensity were analyzed by T-test. A difference was

considered significant when the p value was equal or less than 0.05. Statistical analysis

was provided by prism version 6.0 by GraphPad Software, Inc. (7825 Fay Avenue, Suite

230 La Jolla, CA 92037 USA).


5 Results

5.1 Glutathionylation of Seminal Plasma Proteins

Glutathionylation levels of all seminal plasma proteins were first assessed to get a global

view of redox-dependent protein modifications under both mild (0.1mM H2O2) and

strong (0.5-2.0mM H2O2) oxidative stress. Untreated seminal plasma showed basal levels

of glutathionylation in both high and low molecular weight proteins (Figure 3). Upon

treatment with H2O2, there was immediate dose-dependent increase in GSS-R signal in

higher molecular weight (>130 kDa) proteins. There was also a concurrent decrease in

GSS-R signal in lower molecular weight proteins (15-25 kDa). Based on previous

evidence demonstrating that oxidative stress results in increased levels GSS-R and higher

molecular weight complex formation of proteins once oxidized (O'Flaherty and de Souza,

2011, Morielli and O'Flaherty, 2015) it was determined that this decrease in GSS-R

signal of lower molecular weight proteins was likely due to a upwards shift of molecular

weight due to protein complex formation. This evidence of high molecular mass complex

formation was specifically seen in the PRDX family of antioxidant enzymes (e.g. PRDX1

and PRDX6) in the spermatozoa, a family of enzymes also found abundantly in the

seminal plasma (O'Flaherty and de Souza, 2011). We therefore chose to assess the impact

of oxidative stress on seminal plasma PRDX oxidation to better explain the pattern of

GSS-R signal observed.


Figure 3: Dose-dependent increase in high molecular weight GSS-R signal due to

protein complex formation in human seminal plasma following H2O2 treatment

Human seminal plasma was diluted 1:25 with HBS1x and treated with increasing

concentrations of H2O2 for 30min at 37°C. 0.5 x106 sp/well were loaded, electrophoresed

in SDS polyacrylamide gel (under non-reducing conditions to preserve GSS-R protein

modifications) and immunoblotted with anti-GSS-R antibody (Western Blot, upper

panel). Silver stained sperm proteins were used as loading control (lower panel) and

absence of secondary antibody nonspecific binding was confirmed (not shown). The

experiment was repeated 3 other times with different healthy donors and a representative

blot is shown (n=4).


5.2 Thiol Oxidation and Protein Complex Formation of Seminal Plasma

PRDX1 Under Oxidative Stress

PRDX1 was seen to form protein complexes in human spermatozoa (O'Flaherty and de

Souza, 2011), thus we determined whether PRDX1 present in the seminal plasma is able

to form similar complexes. Untreated and H2O2-treated seminal plasma were tested for

reactivity to anti-PRDX1 antibody under both reducing and non-reducing conditions to

determine total amount and thiol oxidation of PRDX1, respectively. Under reducing

conditions, there were no changes in PRDX1 signal between treatments (see left image of

Figure 4). Under non-reducing conditions, however, we saw changes promoted by both

mild and strong oxidative stress (see right image of Figure 4). Under basal conditions,

two principal bands of 30 and ~46 kDa were observed. Under strong oxidative stress

(0.5-10mM H2O2), we saw a shift towards higher molecular mass proteins with stronger

signal at 46, 55 and 130-250 kDa bands compared to non-treated samples indicating

formation of thiol oxidized protein complexes.


Figure 4: Thiol oxidation of PRDX1 results in high molecular weight protein

complex formation in human spermatozoa under H2O2-treatment

Human seminal plasma was diluted 1:25 with HBS and treated with increasing

concentrations of H2O2 for 30min at 37°C. 0.5 x106 sp/well were loaded, electrophoresed

in SDS polyacrylamide gel (under both reducing (left) and non-reducing (right)) and

immunoblotted with anti-PRDX1 antibody (Western Blot). Silver stained sperm proteins

were used was used as loading control and absence of secondary antibody nonspecific

binding was confirmed (not shown). The experiment was repeated 3 other times with

different healthy donors and representative blots are shown (n=4).


5.3 Thiol Oxidation and Protein Complex Formation of Spermatozoa

Tubulin Under Oxidative Stress

H2O2-treatment has been known to impair sperm motility without affecting sperm

viability (Morielli and O'Flaherty, 2015). This indicates that H2O2 is targeting internal

motility machinery during its impairment of sperm motility. Therefore, we tested whether

tubulin is oxidized due to oxidative stress. We determined total amount and thiol

oxidation of tubulin by comparing sperm samples under reducing and non-reducing

conditions, respectively. Differences of running behavior of specific proteins during

electrophoresis of non-reduced samples (versus reduced samples) were concluded to be

caused by thiol oxidation as was previously demonstrated (O'Flaherty and de Souza,

2011). We observed no changes in tubulin intensity in the 55 kDa band and no changes in

its molecular weight under reducing conditions (see top left image of Figure 5). However,

the intensity of the tubulin band decreases and even disappears under strong oxidative

stress with 10mM H2O2 (see top right image of figure 5). Moreover, we see that the

strong oxidative treatment (2-10mM H2O2) promoted the formation of a ~200 kDa band

of tubulin indicating formation of thiol oxidized protein complexes. There was little

evidence of thiol oxidation occurring in tubulin at mild oxidative stress (0.1mM H2O2).

The pellet of the non-reducing sample was processed to test for the presence of tubulin.

The supernatant of the non-reducing sample was removed and the pellet was suspended

in an equal volume of reducing sample buffer (i.e. containing DTT), electrophoresed,

electrotransfered and immunoblotted with anti-tubulin antibody. Tubulin was found in

increasing concentration in the pellet with increasing exposure to H2O2 (see Figure 6).

This finding indicates that thiol oxidation decrease the solubility of tubulin likely due to

protein complex formation.


Figure 5: Thiol oxidation of tubulin results in high molecular weight protein


Percoll washed spermatozoa was diluted to 1x108/ml with BWW1x and was treated with

increasing concentrations of H2O2 for 30min at 37°C. 0.5 x106 sp/well were loaded in each


well, electrophoresed in SDS polyacrylamide gel (under both reducing (Top left) and non-

reducing (Top right)) and immunoblotted with anti-tubulin antibody (Western Blot).

Silver stain was used as loading control and used in normalizing the relative intensity of

bands (expressed as mean ± S.E.M, bottom right and bottom left graphs). Statistical

significance between treatments was found using non-parametric Friedman’s Test and

post hoc Dunn’s multiple comparison test. The experiment was repeated 3 other times

with different healthy donors and representative blots are shown (n=4).


Figure 6: Dose-dependent increase of insoluble tubulin found in the pellet under

H2O2-treatment

Pellet of non-reducing tubulin sample was resuspended in reducing sample buffer in

order to determine the presence of tubulin made insoluble by the H2O2 treatment (i.e.

thiol oxidation). Equal 10µl of pellet sample were loaded in each well and were

electrophoresed under reducing conditions. Immunoblotting with anti-tubulin revealed a

dose-dependent increase of insoluble tubulin in the pellet with increasing H2O2 treatment

(Western Blot). The experiment was repeated 2 other times with different healthy donors

and a representative blot is shown (n=3).


5.4 Thiol Oxidation and Protein Complex Formation of β-‐Actin in

Spermatozoa Under Oxidative Stress

β-Actin polymerization occurs during sperm capacitation (Breitbart et al., 2005). β-Actin

undergo post-translational modification including redox-dependent protein modifications

in somatic cells (Terman and Kashina, 2013, Su et al., 2013), thus we aimed to test the

impact of oxidative stress on β-actin in spermatozoa. Untreated and H2O2-treated

spermatozoa were tested for reactivity to anti-β-actin antibody under both reducing and

non-reducing conditions. β-actin showed no changes in molecular weight when

immunoblotted under reducing conditions (See left image of Figure 7). Under non-

reducing conditions, we detected two bands of 46 and 60 kDa were recognized by the

anti-β-actin antibody (See right image of Figure 7). Mild oxidative stress (0.1mM H2O2)

promoted an increase in the intensity of these bands, indicating an increase of thiol

oxidation of β-actin. The intensity of these bands decreased and a band of high molecular

mass (>205kDa) appeared when spermatozoa were challenged with a strong oxidative

stress (0.5-10mM H2O2) indicating formation of thiol oxidized protein complexes.


Figure 7: Thiol oxidation of β-actin results in high molecular weight protein


Percoll washed spermatozoa was diluted to 1x108/ml with BWW1x and was treated with

increasing concentrations of H2O2 for 30min at 37°C. 0.5 x106 sp/well were loaded in each

well, electrophoresed in SDS polyacrylamide gel (under both reducing (image left) and

non-reducing (image right)) and immunoblotted with anti-β-actin antibody. Silver stained

sperm proteins were used was used as loading control (image right, bottom panel) and

absence of secondary antibody nonspecific binding was confirmed (not shown).

Experiment was repeated 3 other times with different healthy donors and representative

blots are shown (n=4).


5.5 Impaired β-‐Actin Polymerization in Capacitated Spermatozoa Under

Oxidative Stress

To test the impact of thiol oxidation and redox-dependent protein modification on β-actin

polymerization during sperm CAP, a Phalloidin-Alexa Fluor 555-based cytochemistry

assay was employed. Phalloidin-Alexa Fluor 555 intensity was used to verify CAP in our

untreated, capacitated samples (Figure 8) (Liu et al., 1999, Brener et al., 2002) and used

as our positive control in our CAP experiment with prior H2O2 treatment (using the same

donor sample). Prior to CAP, spermatozoa were either untreated or treated with H2O2 to

induce oxidative stress and thiol oxidation of β-actin. There was a trend of inhibition of

CAP under mild oxidative stress (0.1mM H2O2, see right graph of figure 9). This CAP

inhibition becomes significant in spermatozoa previously exposed to a strong oxidative

stress (2.0-10mM H2O2, see Figure 9). Prior H2O2 treatment of capacitated spermatozoa

(0.1-10mM H2O2, Figure 9) resulted in no significant difference in Phalloidin intensity

compared to their uncapacitated controls (Figure 8).


Figure 8: β-Actin polymerization in capacitated spermatozoa determined by

Phalloidin-Alexa Fluor 555 labeling of F-actin

Percoll washed capacitated and uncapacitated spermatozoa were smeared, fixed with 2%

glutaraldehyde and stained overnight at 4°C with 5µl of stock phallotoxin-Alexa Fluor

555 solution per slide. Immunocytochemistry images were taken with fluorescence

microscopy (Top left and bottom left) at 400x magnification and 1sec exposure.

Phalloidin intensity was measured using ImageJ (expressed as mean ± S.E.M). Statistical

significance between samples was found using T-Test. Experiment was repeated 3 other

times with different healthy donors and representative images are shown (n=4).


Figure 9: β-Actin polymerization negatively impacted by H2O2-treatment during

human sperm capacitation

Percoll washed spermatozoa were first treated with H2O2 for 30min, washed then

capacitated for 3.5h in BWW (pH 8.0) supplemented with BSA and sodium bicarbonate

at 37°C. Spermatozoa were then smeared, fixed with 2% glutaraldehyde and stained

overnight at 4°C with 5µl of stock phallotoxin solution per slide. Immunocytochemistry

images were taken with phase contrast (top left images) and fluorescence microscopy

(bottom left images) at 400x magnification and 1sec exposure. Phalloidin-Alexa Fluor

555 intensity was measured using ImageJ (graph on the right, expressed as mean ±

S.E.M). Statistical significance between treatments was found using non-parametric

Friedman’s Test and post hoc Dunn’s multiple comparison test. The experiment was

repeated 3 other times with different healthy donors and representative images are shown

(n=4).


5.6 Differential Localization of 8-‐OHdG and NitroG in Spermatozoa

Under Oxidative Stress

In order to determine the impact of oxidative stress on sperm DNA, we measured the

levels of 8-OHdG and NitroG in samples treated with H2O2 or DaNONOate, respectively.

The immunocytochemistry-based technique developed here not only was useful to

quantify the total amount of DNA damage induced by these ROS but also to determine

their localization in the sperm nucleus.

Under strong oxidative stress (2.0-10mM H2O2), there was a significant and dose-

dependent increase in 8-OHdG levels present in the spermatozoa (Figure 10). When

looking at the individual sperm, the localization of DNA oxidation appears to be

primarily localized to the periphery of the nucleus and not uniformly distributed in the

nucleus (2.0-10mM H2O2, Figure 12).

When looking at DNA damage indicated by NitroG levels, we also see significant, dose-

dependent increase under strong DaNONOate treatment (2.0-10mM, Figure 11). Unlike

8-OHdG, the NitroG signal appeared initially localized in the post-acrosomal region of

the spermatozoa (Figure 12) and then signal appeared throughout the nucleus under even

stronger oxidative stress (2.0-10mM DaNONOate, figure 12).


A


Figure 10: Dose-dependent increase of 8-OHdG intensity/area with H2O2-treatment

A) Percoll washed spermatozoa were first treated with H2O2 for 30min, decondensed

using DTT (allowing sperm to swell to about 5times their original size in order to allow

antibody to penetrate the nucleus), fixed with 100% methanol and stained overnight at

4°C with anti-8-OHdG antibody. B) Images of at least 200 sperm were obtained using

fluorescence microscopy and total intensity was measured using ImageJ and normalized

to the area of the nucleus (expressed as mean ± S.E.M). Statistical significance between

treatments was found using non-parametric Friedman’s Test and post hoc Dunn’s

multiple comparison test. The experiment was repeated 3 other times with different

healthy donors and representative images are shown (n=4).

B


A


Figure 11: Dose-dependent increase in NitroG intensity with DaNONOate-treatment

A) Percoll washed spermatozoa were first treated with DaNONOate for 30min, minor

decondensed using DTT (allowing nucleus to decondensed without changing

significantly in size), fixed with 100% methanol and stained overnight at 4°C with anti-8-

OHdG antibody. B) Images of at least 200 sperm were obtained using fluorescence

microscopy and total intensity was measured using ImageJ and normalized to the area of

the nucleus (expressed as mean ± S.E.M). Statistical significance between treatments

was found using non-parametric Friedman’s Test and post hoc Dunn’s multiple

comparison test. The experiment was repeated 3 other times with different healthy donors

and representative images are shown (n=4).

B


Figure 12: Comparison of differential localization of 8-OHdG and NitroG under

strong oxidative stress

Percoll washed spermatozoa were first treated with either H2O2 (left image) or

DaNONOate (right image) for 30min, decondensed using DTT, fixed with 100%

methanol and stained overnight at 4°C with either anti-8-OHdG antibody (left image) or

anti-NitroG (right image). 8-OHdG staining labels predominantly the periphery of the

nucleus. NitroG staining labels primarily the post-acrosomal region (visible at 0.1 and

0.5mM DaNONOate treatment) appearing as a point of bright staining at one end of the

sperm, closest to the flagella.


6 Discussion

The aim of this study was to determine the effect of oxidative stress on functionally

important proteins and demonstrate that oxidative stress causes specific redox-dependent

modifications to proteins and DNA. Prior to this study, oxidative stress research in male

infertility focused on phenotypical changes (i.e. motility, morphology, fertilization, etc.).

Based on our knowledge, this study is the first in attempting to elucidate the molecular

mechanism of ROS mediated inhibition of both human seminal plasma and sperm

function.

Poor total antioxidant capacity (TAC) of the human semen is associated with oxidative

stress and poor fertility status in men (Pasqualotto et al., 2008, Mahfouz et al., 2009)

despite the total amount of antioxidants present in the seminal plasma of idiopathic

infertile males appear unchanged (Gong et al., 2012). This implies that the antioxidant

present in the seminal plasma may be inactivated, possibly due to redox-dependent

modification. Treatment with H2O2 resulted in dose-dependent increase in GSS-R in

high-molecular weight proteins and thiol oxidized protein complexes in seminal plasma

under non-reducing conditions (Figure 3). Thiol oxidized PRDX1 complexes form under

oxidative stress (evident from 0.5mM H2O2, Figure 4). These two redox-dependent

protein modifications (GSS-R and thiol oxidation) promote the inactivation of enzymatic

activity or function of the affected protein (i.e. receptors, structural proteins, ion

channels, etc) (Halliwell and Gutteridge, 2007a). Other seminal plasma antioxidant

enzymes, such as PRDX2, PRDX4, PRDX5 and PRDX6 (Pilch and Mann, 2006,

O'Flaherty and de Souza, 2011) and extracellular glutathione peroxidase (eGPX) and

glutathione reductase (GRD) (Pilch and Mann, 2006) could also be inactivated by

oxidative stress. Noteworthy, GRD is important to re-activate GPXs and PRDX6;

therefore its inactivation will prevent the reduction of eGPX and PRDX6 that will make

them unable to scavenge ROS produced in seminal plasma. The high molecular mass

complexes contain sulfonated form of PRDXs which are hyperoxidized and also inactive

as scavenger of ROS in human spermatozoa and other cell types (Lim et al., 2008,


O'Flaherty and de Souza, 2011). Hyperoxidized PRDXst can be only re-activated by

sulfiredoxin and sestrins (Lim et al., 2008). According to proteomic studies, these

enzymes are absent in human seminal plasma (Pilch and Mann, 2006), and thus,

hyperoxidized PRDXs present in seminal plasma will be irreversible inactive and unable

to protect spermatozoa as occur in idiopathic infertile men (Gong et al., 2012).

Altogether, these results indicate that PRDX1 and other seminal plasma proteins are

inactivated by thiol oxidation- and s-glutathionylation-dependent oxidative stress, unable

to protect spermatozoa and thus, considered as a plausible cause of infertility. The

inhibition of major antioxidant enzymes indicates that the thiol oxidation status of

seminal plasma proteins may be an important indicator of ability for seminal plasma to

protect spermatozoa from oxidative following oxidative stress. Thiol oxidation of PRDX

may also give important insight into the health of the semen by indicating the levels of

oxidative stress in seminal plasma of infertile men.

Sperm motility is one of the most important markers of male infertility, however, ROS

impact on motility is not well understood. Identification of modified proteins has become

increasingly relevant since motility has been recently shown to be inhibited with

significant levels of redox-dependent protein modification despite any decrease in sperm

vitality (Morielli and O'Flaherty, 2015). There are many potential target proteins of ROS

(Morielli and O'Flaherty, 2015); however, no protein of the motility machinery to be

modified under oxidative stress in spermatozoa has been reported yet. Tubulin is a major

structural protein of the sperm flagellum and is axonemal function during motility.

Abnormal axonemal morphology is associated with oxidative stress and male infertility

(de Lamirande and Gagnon, 1992, Escalier, 2006). In our study, H2O2-treatment caused

significant changes in thiol oxidation and solubility of tubulin (Figure 5 and 6). These

two changes indicate how redox-dependent modification of tubulin could be a major

contributing factor of the changes in axoneme and motility when spermatozoa are facing

oxidative stress conditions.

Although high-level of oxidative stress, produced at H2O2 concentrations of 2mM or

higher, causes tubulin thiol oxidation, sperm motility is impaired by lower ROS


concentrations (i.e. 0.5 mMH2O2) (Morielli and O'Flaherty, 2015). These findings

indicate that other proteins involved in the motility machinery are affected by oxidative

stress The sperm flagellum contains glycolytic enzymes such as glyceraldehyde 3-P

dehydrogenase and fructose 1,6 biphosphate aldolase that are associated with the fiber

sheath and the midpiece with mitochondria where the Krebs cycle and oxidative

phosphorylation takes place. Thus, the flagellum contains all the enzymatic machinery to

produce energy for motility (Eddy, 2006). Previous assessment of redox-dependent

protein modification in spermatozoa under ROS treatment showed strong labeling of s-

glutathionylated proteins in the tail and suggested that glyceraldehyde 3-P dehydrogenase

and enolase c and the Kreb’s cycle enzymes α-ketoglutarate dehydrogenase and malate

dehydrogenase are also likely targets for this type of modification (Morielli and

O'Flaherty, 2015).

Future studies could test populations of poorly motile spermatozoa from healthy male

populations (such as the sperm population isolated from the 40-65% Percoll interface) or

from asthenozoospermic males for signs of redox-modifications in tubulin.

Immunoprecipitation experiments can also provide evidence regarding changes in

protein-protein interactions of oxidized tubulin as well as identifying the specific redox-

dependent modifications that are occurring (i.e GSS-R, cross-linking, etc).

Tubulin is clearly affected by oxidative stress (Figure 5) and can be modified by different

redox-dependent protein modifications such as tyrosine nitration and GSS-R (Landino et

al., 2014). Thus it is possible that the reduced sperm motility observed in spermatozoa

under oxidative stress is due to thiol oxidation of tubulin. Based on these results, the

determination of tubulin thiol oxidation could be useful as an oxidative stress marker in

spermatozoa.

Sperm CAP is impaired in some cases of men infertility (Kholkute et al., 1992,

Oehninger et al., 1994). Similarly, exposure of spermatozoa to oxidative conditions

results in impaired capacitation (Morielli and O'Flaherty, 2015). Actin polymerization is

a critical event during CAP (Brener et al., 2002). Mild (0.1mM H2O2) and strong


oxidative conditions (0.5-10mM H2O2) resulted in thiol oxidation and formation of thiol

oxidized protein complexes of β-actin (Figure 7). Thiol oxidation of β-actin appears to

occur at lower concentrations of H2O2 compared to that of tubulin, which displays thiol

oxidation at higher H2O2 concentrations (equal or greater than 2mM). Similarly to

tubulin, actin can be modified by many redox-dependent modifications such as GSS-R

and NitroY (Terman and Kashina, 2013). The differences in localization of β-actin (in the

head) and tubulin (in the tail) may explain the apparent differential sensitivity of the

proteins to the same level of external oxidative stress as different sperm compartments

contain different antioxidants enzymes and antioxidant protection (O'Flaherty and de

Souza, 2011, O'Flaherty, 2014).

Capacitated sperm have increased levels of polymerized β-actin (Figure 8) (Brener et al.,

2002). β-Actin polymerization is impaired by previous treatment with strong oxidative

stress (2.0mM H2O2) in capacitating spermatozoa (Figure 9). This result indicates that

thiol oxidation of β-actin results in long-term impairment β-actin polymerization in

sperm under capacitating conditions and is consistent with H2O2 mediated impairment of

CAP (Morielli and O'Flaherty, 2015). Future experiments are needed to determine

whether populations of infertile men with impaired capacitation have significant levels of

oxidized β-actin and impaired β-actin polymerization.

β-Actin is the first protein of the capacitation pathway with clear evidence of redox-

dependent modification associated with capacitation-inhibiting oxidative conditions.

Redox-dependent functional impairment of β-actin polymerization is the first evidence to

explain the disturbance of a molecular mechanism due to oxidative stress that lead to

inhibition of capacitation.

Elevated levels of DNA damage is well associated with oxidative stress (O'Brien and

Zini, 2005) and male infertility (Zini et al., 2008b, Winkle et al., 2008, Talebi et al.,

2008). Recent studies in the mouse showed that histone retention in the peripheral regions

of the nucleus results in a differential sensitivity of neighboring DNA to oxidative stress

(Noblanc et al., 2013). The localization of DNA damage in the human spermatozoa


nucleus has not previously been identified. Utilizing an immunocytochemistry approach,

we have shown for the first time the localization of DNA oxidation and nitration in the

human spermatozoa under oxidative stress (Figure12). The elevated levels of 8-OHdG

measured by immunocytochemistry are comparable to data measured by others using

different approaches in H2O2-treated spermatozoa (Figure 10) (De Iuliis et al., 2009,

Aitken et al., 2014). 8-OHdG signal is localized primarily in the peripheral region of the

human spermatozoa nucleus, which is consistent with the mouse data (Figure 12).

Decondensation of about 5x the original size of the nucleus was required in order for the

antibody to penetrate the nucleus, as lower decondensation resulted in very week signal.

Decondensation of 5x or more (about 7x decondensation is the limit of decondensation

before rupturing the nucleus) consistently resulted in the same signal pattern and

intensity. Human spermatozoa with an increase in histone content have been associated

with male infertility (Zhang et al., 2006). It is possible, that due to a less compacted

sperm DNA due to high content of histones will allow the establishment of oxidative

stress-dependent damage. Future studies should test the co-localization of 8-OHdG and

histones in the human sperm nucleus. This result highlights the crucial role protamines to

ensure proper compaction to protect paternal genome from oxidative stress.

Total levels of NitroG increased when human spermatozoa was treated with increasing

concentrations of DaNONOate, a NO• donor (Figure 11). NitroG was consistently

present in the region of the post-acrosomal region following DaNONOate treatment

(Figure 12). The distribution pattern of NitroG in the nucleus was consistent irrespective

of the level of decondensation prior to immunostaining. Strong signal was achieved with

the anti-NitroG antibody despite less decondensation required for 8-OHdG, possibly due

to a higher affinity antibody.

8-OHdG and NitroG showed very different distribution patterns despite mouse data

suggesting that the periphery of the nucleus would be more sensitive to all types DNA

damage (Noblanc et al., 2013), explaining what we see with 8-OHdG but not what we see

with NitroG. NitroY, a redox-dependent protein modification localizes primarily in the

Triton-insoluble fraction (head and tail) of DaNONOate-treated spermatozoa (Morielli


and O'Flaherty, 2015). NitroY modified proteins may play a role in NitroG modification

and therefore explain their apparent co-localization. It is also possible that DaNONOate-

dependent damaged in the sperm DNA could be due to a direct effect of NO� and/or

peroxynitrite (ONOO-) on DNA bases or by altering nuclear proteins by NitroY

modification of the nuclear matrix or associated with the DNA.

Different genes have specific localization in the compacted sperm nucleus (Wykes and

Krawetz, 2003). Furthermore, histone-associated genes are involved in early embryonic

development (Gardiner-Garden et al., 1998). Preferential 8-OHdG modification of

histone-associated genes would promote mutations that will likely impact early

embryonic development. In the same line of thoughts, if NitroG modification occurs in

the post-acrosomal region and the periphery of the apical region of the sperm head, it

would likely impact later stages of embryo development first (Ward, 2010). The genes in

the post-acrosomal region have not been associated with a particular developmental step.

This information could be useful for prognostic purposes. If the nature of the DNA

damage or ROS is known, then we would be able to better predict when a problem (if

any) would arise during the reproductive process.


7 Conclusion Our results confirm that PRDX1 yielded a dose dependent increase in thiol oxidation and

high molecular weight complex levels following oxidative stress with H2O2. GSS-R and

other DTT-sensitive redox-dependent modifications (i.e. s-nitrosylation of cysteine

residues) may contribute to the impairment of PRDX1 antioxidant activity in seminal

plasma during oxidative stress due to the critical role of cysteine in the active site. Thiol

oxidation of tubulin resulted in increased levels of thiol oxidation and changes in

solubility possible due to protein-protein interactions, thus altering the normal

functioning of the protein in the motility machinery. β-Actin underwent thiol oxidation

under mild (0.1mM H2O2) and strong (equal or higher than 2mM H2O2) oxidative stress.

Changes in the level of F-actin in the head specifically may help elucidate the mechanism

behind the loss of ability to undergo capacitation. Higher 8-OHdG signal on the periphery

of the nucleus and higher 8-NitroG signal in the region of the post-acrosomal region

indicates a differential sensitivity of the DNA to oxidative stress.


8 Future Directions

Our future direction includes determining the types of the redox-dependent modifications

(i.e. GSS-R) occurring in PRDX1, tubulin and β-actin by immunoprecipitation. The

presence of thiol oxidized PRDX1, tubulin and β-actin will be assessed in infertile men

with impaired semen antioxidant protection, motility and capacitation, respectively. Other

spermatozoa protein undergoing redox-dependent protein modifications will also be

determined using mass spectroscopy (MALDI-TOF MS).

In the future, infertile male population should be tested for significant thiol oxidation of

PRDX1 and other antioxidant in the seminal plasma. Immunoprecipitation experiments

can also be used to determine the exact nature of the redox-dependent modifications

impacting β-actin, similar to what was suggested for tubulin. Furthermore, the flagella

itself should be considered a target itself for antioxidant treatment aiming to protect the

tubulin and other proteins from oxidative stress and restore sperm motility and male

fertility. Based on β-actin’s localization in the head, antioxidant therapy should target the

head of the sperm if the aim is to prevent β-actin oxidation and support it’s involvement

in CAP. Future research regarding DNA damage, 8-OHdG modifications should be tested

in DaNONOate treated spermatozoa and NitroG modification should be tested in H2O2

treated spermatozoa. We can then determine any overlap between the modifications and

ROS treatment as well as see if the pattern of localization is specific to the modification

and/or ROS.

Overwhelmingly, we see that different ROS produce specific redox-dependent

modifications in specific proteins and parts of the paternal genome. The study presented

in this thesis helps to better understand the molecular mechanisms that are affected in

human spermatozoa when they face oxidative stress conditions such as those occurring in

infertile men. This information will help to develop new diagnostic tools as well as

specific pharmacological and antioxidant treatments based on the nature of the ROS and

the ROS-dependent damage.


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