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
NOŸ 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).

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

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 NOŸ 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.

References:
ABID, S., MAITRA, A., MEHERJI, P., PATEL, Z., KADAM, S., SHAH, J., SHAH, R.,
KULKARNI, V., BABURAO, V. & GOKRAL, J. 2008. Clinical and laboratory
evaluation of idiopathic male infertility in a secondary referral center in India. J
Clin Lab Anal, 22, 29-38.
AFZELIUS, B. A., ELIASSON, R., JOHNSEN, O. & LINDHOLMER, C. 1975. Lack of
dynein arms in immotile human spermatozoa. J Cell Biol, 66, 225-32.
AGARWAL, A., MAKKER, K. & SHARMA, R. 2008. Clinical relevance of oxidative
stress in male factor infertility: an update. Am J Reprod Immunol, 59, 2-11.
AGARWAL, A., NALLELLA, K. P., ALLAMANENI, S. S. R. & SAID, T. M. 2004.
Role of antioxidants in treatment of male infertility: an overview of the literature.
Reproductive BioMedicine Online, 8, 616-627.
AGARWAL, P., LOH, S. K. E., LIM, S. B., SRIRAM, B., DANIEL, M. L., YEO, S. H.
& HENG, D. 2005. Two ‐ year neurodevelopmental outcome in children
conceived by intracytoplasmic sperm injection: prospective cohort study. BJOG:
An International Journal of Obstetrics & Gynaecology, 112, 1376-1383.
AITKEN, R., SMITH, T., JOBLING, M., BAKER, M. & DE IULIIS, G. 2014. Oxidative
stress and male reproductive health.
AITKEN, R. J. 1995. Free radicals, lipid peroxidation and sperm function. Reprod Fertil
Dev, 7, 659-68.
AITKEN, R. J. 2013. Falling sperm counts twenty years on: where are we now? Asian
journal of andrology, 15, 204.

AITKEN, R. J. & DE IULIIS, G. N. 2010. On the possible origins of DNA damage in
human spermatozoa. Molecular Human Reproduction, 16, 3-13.
AITKEN, R. J., PATERSON, M., FISHER, H., BUCKINGHAM, D. W. & VAN, D. M.
1995. Redox regulation of tyrosine phosphorylation in human spermatozoa and its
role in the control of human sperm function. Journal of Cell Science, 108 ( Pt 5),
2017-2025.
AITKEN, R. J. & VERNET, P. 1998. Maturation of redox regulatory mechanisms in the
epididymis. Journal of reproduction and fertility. Supplement, 53, 109-118.
ÅKERLÖF, E. V. A., FREDRICSON, B., GUSTAFSSON, O., LUNDIN, A., LUNELL,
N. O., NYLUND, L., ROSENBORG, L. & POUSETTE, Å. 1987. Comparison
between a swim-up and a Percoll gradient technique for the separation of human
spermatozoa. International Journal of Andrology, 10, 663-669.
ALIEV, G., SMITH, M. A., SEYIDOV, D., NEAL, M. L., LAMB, B. T., NUNOMURA,
A., GASIMOV, E. K., VINTERS, H. V., PERRY, G., LAMANNA, J. C. &
FRIEDLAND, R. P. 2002. The role of oxidative stress in the pathophysiology of
cerebrovascular lesions in Alzheimer's disease. Brain Pathol, 12, 21-35.
ANDERSON, J. B. & WILLIAMSON, R. C. 1988. Testicular torsion in Bristol: a 25-
year review. Br J Surg, 75, 988-92.
AYAZ, K., AHASAN, H. N., RAIHAN, M. R., MIAH, M. T., HAQUE, M. A. &
SIDDIQUE, A. A. 2012. Male Infertility – A Review.
BACCETTI, B., BURRINI, A., CAPITANI, S., COLLODEL, G., MORETTI, E.,
PIOMBONI, P. & RENIERI, T. 1993. Notulae seminologicae. 2. The ‘short
tail’and ‘stump’defect in human spermatozoa. Andrologia, 25, 331-335.

MSc Thesis - Matsushita-Fournier_David_PharmacologyTherapeutics (LinkedIn)

MSc Thesis - Matsushita-Fournier_David_PharmacologyTherapeutics (LinkedIn)

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to MSc Thesis - Matsushita-Fournier_David_PharmacologyTherapeutics (LinkedIn)

Similar to MSc Thesis - Matsushita-Fournier_David_PharmacologyTherapeutics (LinkedIn) (20)

MSc Thesis - Matsushita-Fournier_David_PharmacologyTherapeutics (LinkedIn)