Document 22440

Indian Journalof ExperimentalBiology
Vol. 43. November2005.pp 963-974
andpreventionof oxidative stress
in male reproductivephysiology
Ashok Agarwal* & Sushil A Prabakaran
Centerfor AdvancedResearchin HumanReproduction,Infertility, and SexualFunction,GlickmanUrological Institute,and
Departmentof ObstetricsandGynecology,The ClevelandClinic Foundation,9500EuelidAvenue,Desk A19.I, Cleveland,
Ohio, 44195,USA
Numerousfactors influence male fertility. Among thesefactors is oxidative stress(OS), wmcb bas elicited an enormous interest in researchersin recent period. Reactiveoxygen species(ROS) are continuouslyproducedby various metabolic and physiologicprocesses.OS occurswhen the delicatebalancebetweenthe productionof ROSand the inherentantioxidant capacity of the organism is distorted. Spermatozoaare particularly sensitiveto ROS as their plasma membrane
containspolyunsaturatedfatty acids (PUFA), which oxidizes easily.They also lack cytoplasmto generatea robustpreventive and repair mechanismagainstROS. The transition metal ions that are found in the body have a catalytic effect in the
generationof ROS. Lifestyle behaviourssuchas smokingand alcohol useand environmentalpollution further enhancethe
generationof ROS and thus,causedestructiveeffectson variouscellular organelleslike mitochondria.spermDNA etc. This
article analyzesthe detrimentaleffectsof OS on male ferdlity. measurement
of OS and effcctive ways to decreaseor eliminatethem completely. We have also provided information on oxidative stressin other systemsof the body, which may be
appliedto future researchin the field of reproductivebiology.
Keywords: Male fertility. Oxidative stress,Spermatozoa
As many as 15% of all couples living in the United
States have difficulty conceiving a child. Male factors
are responsible in at least 30% of the cases, and in
another 20%, the pathology is found both in men and
women. Approximately 6% of men between the age
of 15 and 50 suffer from male infertilityl. A metaanalysis of 61 studies worldwide found a downward
trend in sperm count and volume of seminal fluid over
the past 50 years2. In 1940, the average sperm count
was 113 million per mI. By 1990, the count had
dropped to 66 million per mI3. This decreasing trend
in sperm count has led to speculation that recent environmental, dietary and/or lifestyle changes are interfering with a man's ability to produce spermatozoa. It
is believed that these factors exert dleir detrimental
effects through oxidative stress (OS).
Oxidative stress (OS) and its significance-OS occurs as a consequence of an imbalance between the
production of reactive oxygen species (ROS) and the
available antioxidant defense against them4,S.ROS are
free radicals and peroxides that are derived from the
metabolismof oxygen and are presentin all aerobic
organisms.Theyray a significant role in many biological can affect the individual molecules and thus the entire is believed to
be one of the major causesof many humandiseases.
Some of the OS-mediatedpathologies are listed in
Table 1. Virtually every discipline of medicine is interestedin as and its implications.It is believed that
the new field of 'Oxidative Stress,Diagnostics and
Therapeutics'may have a significant impact on the
economics,scienceand the practiceof medicinein the
Table I-Diseases anddegenerativeprocessesmediatedby
Iron overload
Ischemic-reperrusion Rheumatoidarthritis
Maculardegeneration Segmentalprogeria
Cardiovascular Multiple sclerosis
Phone: (216) 444-9485.Fax: (216) 445-6049;
Cataractogenesis Musculardystrophy
E-mail: [email protected];
Website:www.clevelandclinic.orgjReproductiveResearchCenter Diabetes
Mechanism of ROS formation
Oxygen in the atmosphere has two unpaired electrons, and these unpaired electrons have parallel spins.
Oxygen is usually non-reactive to organic molecules
that have paired electrons with opposite spins. This
oxygen is considered to be in a ground (triplet or inactive) state and is activated to a singlet (active) state
by two different mechanisms:
a) Absorption of sufficient energy to reverse the
spin on one of the unpaired electrons.
Triplet oxygen(rr)
Singlet oxygen<r~)
(Highly reactive)
b) Monovalentreduction(accepta singleelectron).
Superoxideis formed in the flfSt monovalentreductionreaction,which undergoesfurther reductionto
form hydrogen peroxide. Hydrogen peroxide is further reducedto hydroxyl radicals in the presenceof
ferrous salts (Fe2+).This reaction was first described
by Fentonand later developedby Haberand Weiss.
Triplet oxygen (tt)
(Ground state)
Superoxide(i ~)
Fenton reaction:
Fe2++ H2~'
Origin of ROS in male reproductive system
Human semen consists of different types of cells
such as mature and immature spermatozoa, round
cells from different stages of the spermatogenic process, leukocytes and epithelial cells. Of these, leukocytes and immature spermatozoa are the two main
sourcesof ROS1°. Leukocytes,particularlyneutrophils and macrophages, have been associated with
excessive ROS production, and they ultimately cause
sperm dysfunctionll-18. ROS produced by leukocytes
forms the first line of defense in any infectious process. DNA and structural damage can be found in
I k
om eu ocytospenmc patients
Leukocytes act either directly by ROS synthesis or
indirectly by other nei~hboring white cells via soluble
factors as cytokinesl .22. ROS is significantly and
positively correlated with interleukin-S, a potent
stimulator of pro-inflammatory cytokines23.24.The
scavenging effect of antioxidants are greatly diminished under such infectious conditionsl4.
Another important source of ROS is immature
spermatozoa. A study from our group has demonstrated that ROS production is significantly and negatively correlated with semen quality2s. Other studies
have suggested that the defect in spermiogenesis that
results in retention of cytoplasmic droplets is a major
source of ROS26. There are two major systems of
ROS production in sperm. One is the NADPH oxidase
system at the level of the sperm plasma membrane
Table 2- Typesof reactiveoxygen speciesthat exist as radicals
and free radicals
Types of ROS
ROS representa broad categoryof moleculesthat
indicate the collection of radicals and non-radical
oxygen derivatives.Theseintermediatesmay participate in reactionsthat give rise to free radicals that
damageorganic substrates.Table 2 lists the types of
ROS that exist as radicals and free radicals in living
In addition, there is another class of free radicals
that are nitrogen derived called reactivenitrogen species (RNS). They are nitrogen-derivedmoleculesand
areconsidereda subclassof ROSS.9
(Table 3).
Nitric Oxide
Singlet Oxygen
Lipid peroxyl
Lipid peroxide
Table 3- Typesof reactivenitrogenspeciesthat induces
oxidative stress
Nitrous oxide
Nitrosyl carlon
Nitrogen dioxide
Dinitrogen trioxide
Nitrous acid
Nitroxyl anion
Nitryl chl,oride
and the other is NADH-dependent oxido-reductase
(diphorase)systemat the mitochondriallevef7. There
is a strong positive correlation between immature
spermatozoaand ROS production, which in turn is
negatively correlated with sperm quality. Furthermore, it has beennoticed that as the concentrationof
immature spermatozoain the human ejaculate increases,the concentrationof maturespermatozoawith
damagedDNA rises25.
nations to oxygen, fonning superoxide or hydrogen
peroxide, which is further reduced to an extremely
reactive OH radical that induces oxidative stress.
TMI, mainly iron, are involved in Fenton's reaction,
which produces highly reactive hydroxyl radicals.
Macrophages reduce TMI and facilitate monovalent
lipid hydroperoxide (LOOH) decomposition, which in
turn induces lipid peroxidation37. Both a deficiency
and an excess of manganese (Mn) causesneurotoxicity via ROS generation38.TMIs such as lead (Pb) and
Physiologicalrole of ROS
cadmium (C<t) also affect the male reproductive sysSpermatozoaacquire the capacity to mOveduring tem. These metals either directly affect the sperm
their transit through the epididymis, but lack the abilplasma membrane or catalyze the ROS formation. Pb
ity to fertilize. After ejaculation,they undergoa series causes relative zinc (Zn) deficiency, while Cd preof physiological changes called capacitation28.Ca- dominantly affects the distribution of Zn in the body.
pacitationis followed by acrosomereaction.which is
The authors of a study consisting of industrial
triggered by the zona pellucida of the ovum and alworkers have identified a decrease in the workers'
lows the sperm to fertilize the egg29.30.
Capacitation semen quality, semen volume, sperm count, sperm
and the acrosomereaction are redox-regulatedproc- motility and forward progression. The authors have
esses.Exogenousadministrationof 0,,'- or H2~ proalso reported an increase in abnormal sperm morpholmotescapacitationand the acrosomereactionwhereas ogy and an impairment of prostatic secretory function.
the addition of antioxidantspreventsthem from unThey have also observed that both Pb and Cd damage
dergoing theseevents.This demonstratesthe importhe DNA bases as measured by the levels 8-hydroxytanceofROS in humanspermfunctions3).
2'-deoxyguanosine (8-0HdG). Seminal plasma l~
Nitric oxide (NO1, a free radical with a relatively levels and artificial insemination cycle fecundity have
long half-life (7 s), promotescapacitation.No detect- a strong negative correlation39. Automobile pollution
able activity of nitric oxide synthase(NOS) is found increases the blood lead level, which decreasessperm
in spermatozoa,which suggeststhat it originatesfrom
count and viability.
tissuesor fluids from the female genital trac~. Low
Damage to lipids-Lipids
are most susceptible
concentrationsof NO causea significant increasein
macromolecules and are present in the spenD plasma
capacitation32and zona pellucida binding33. NO
membrane in the form of polyunsaturated fatty acids
regulates cyclic adenosinemonophosphate(cAMP)
(PUPA). ROS attack PUPA in the cell membrane
concentrationand induces capacitationof spermato- leading to a chain of chemical reactions called lipid
zoa through the action of adenyl cyclase.Finally, NO
peroxidation40. The reaction occurs in three distinct
alsoplays a role in spermhyperactivation34.
steps-initiation, propagation and termination. During
initiation, the free radicals react with fatty acid chain
Pathological role of ROS
(LH) and releases lipid free radical. This lipid radical
Effect of excessiveNO-NO is detrimental to
further reacts with molecular oxygen to form lipid
sperm motility in high concentrations35.
It may react .peroxyl radical. Peroxyl radicals again react with fatty
with superoxideor hydrogenperoxide,resultingin the acid to prrouce lipid free radical and this reaction is
formation of peroxynitrite, hydroxyl radical, N~ or
propagated. During tennination, the two radicals react
singlet oxygen, which causes oxidation of sperm with each other, and the process comes to an en~l.
membranelipids and thiol proteins36.In addition, NO
This process of fatty acid breakdown produces hydromay also inhibit cellular respiration by nitrosylation carbon gases(ethane or pentane) and aldehydes.
of heme in mitochondrial enzymes, aconitase and
This causes
a decreasein the level of adenoFinetriphosphate,
which therebyaffectsthe kineticsof Spermatozoa35.
Catalytic effectof different transition metal ionsTransition metals ions (TMI) can make electron do-
Damage 10 proleins-Sulphur-containing
acids, having thiol groups specifically, are very susceptible to OS. Aetivated oxygen can abstract H atom
from cysteine residues to form a thiyl radical that will
cross-link to a second thiyl radical to form disulphide
bridges. Alternatively, oxygen can add to a methionine residue to form methionine sulphoxide derivatives. Many amino acids undergo specific irreversible
modifications when a protein is oxidized. For example, tryptophan is readily cross-linked to form bityrosine products42. Oxidative damage can also lead to
cleavage of the polypeptide chain and formation of
cross-linked protein aggregates. Histidine, lysine,
proline, arginine and serine form carbonyl groups on
oxidation43. The oxidative degradation of protein is
enhanced in the presence of metal cofactors that are
capable of redox cycling, such as Fe.
Damage 10 DNA-Activated
oxygen and agents
that generate oxygen free radicals, such as ionizing
radiation, induce numerous lesions in DNA that lead
to deletions, mutations and other lethal genetic effects44.Pyrimidine bases are most susceptible to OS
as are purines and deoxyribose sugar. Oxidation of the"
sugar by the hydroxyl radical is the main cause for
DNA strand breaks4s. Oxidative damage can cause
base degradation, DNA fragmentation and crosslinking to protein46-48.In addition, incorporation of
oxidized deoxyribonucleoside triphosphate causes
gene mutation or altered gene expression. The rate of
DNA fragmentation is increased in the ejaculate of
infertile men49-S2
as indicated by the high level of 8OHdG, which is a product of DNA oxidation. Sperm
DNA is normally protected from oxidative insult by
two factors-the
antioxidants present in seminal
plasma. and the characteristic tight packaging of the
is programmed cell death
that occurs in a genetically determined fashionS4.It is
initiated by ROS-induced oxidative damage, but too
much OS can terminate apoptosis by inactivating the
. .dants can el.ther
caspase enzyme cascadeS5-S7
. A ntIoXl
suppress or facilitate apoptosiss8.The mitochondrion
is the most important cellular organellethat mediates
apoptosis.High levels of ROS disrupt the integrity of
the mitochondrial membrane,which in turn releases
cytochromec. It activatesthe caspaseenzymecascade
and triggers apoptosis.Studiesin infertile men have
shownthat high levels of cytochromec in the seminal
plasma indirectly reflect significant mitochondrial
damagecausedby high levels of ROS.Levels of ROS
in infertile men are correlatedpositively with apoptosis, which in turn is negatively correlatedwith conventional semen parameters49.S9-62.
Effects of life-style behavior and environment
OS associated with smoking-Tobacco contains
nearly 4000 harmful substancessuch as alkaloids,
nitrosamines,nicotine and hydroxycotinine.Many of
thesesubstancesgenerateROS and RNS63.64.
Smoking has been associatedwith decreasedsperm quality6S-67.
Reduction in motility is due to the damage
causedby ROS to the flagellum and axonemalstructures of the tail of spermatozoa.Spermmotility correlates negatively with the amount of cotinine and
hydroxycotininein the seminalplasma6s.68.
The presence of cotinine and hydroxycotinine in seminal
plasmaindicatesthat harmful substancesfound in tobaccosmokecanpenetratethe blood-testesbarrier.
Oxidativestressand alcohol-Many processesand
factors are involved in causing alcohol-inducedOS.
Alcohol metabolism results in the formation of
NADH, which enhancesactivity of the respiratory
chain, including heightened~ use and ROS formation. One of the by-productsof alcohol metabolism,
acetaldehyde,interacts with proteins and lipids to
form ROS. It is also capableof damagingthe mitochondrialeadingto decreasedA TP production.Alcohol also induceshypoxia that resultsin tissuedamage.
Excessivealcohol consumptionis associatedwith a
decreasein the percentageof nonnal spenDin asthenozoospermicpatients69.
However,better spenDmorpholop has beenobservedin men who drink moderately7.
OS and environment-Many environmentalfactors
such as radiation, medicationsand pollutants induce
ROS production)s.In a study conductedon Danish
greenhouseworkers,a high count of spennatozoawas
found among organic farmers who grew their products without the useof pesticidesor chemicalfertilizers. The group of blue-collar workers in the above
study had a low spenDcount in comparisonto the
abovegroup of workers7).Thesestudiescategorically
suggestthe needfor a healthyenvironment.
Measurement of ROS
Chemiluminescence method- The chemiluminescence method is the most commonly used technique
for measuring ROS produced by spennatozoan.73.The
assay determines the amount of ROS, not the level of
the sperm-damaging ROS present at any given time.
This method quantifies both intracellular and extracellular ROS. Luminol is a probe that reacts with different types of ROS. It can measure both intra- and
extracellular ROS whereas lucigenin can only measure the superoxide radical released extracellularly.
Hence, by using both the probes on the same sample,
it is possible to accurately identif~ intracellular and
extracellular generation of ROS74-6. However, there
are some serious limitations in measuring the superoxide formation; lucigenin undergoes redox cycling,
and there are some unknown sources of superoxide
Cytochrome c reduction or nitro blue tetrazolium
(NBT) reduction method- The chemiluminescence
method can indicate only the total amount of ROS
present in the semen and does not provide information
about the source of ROS. Cytochrome c reduction and
NBT reduction both can accurately predict whether
ROS have been produced by leukocytes or abnormal
spermatozoal',7'. The two most commonly used reagents to detect superoxide anion radicals are NBT and
ferricytochrome-c (Cyt)79. Superoxide formed by
electron transfer from a donor to molecular oxygen
can be quenched by NBT and Cyt, and these reagents
get reduced to diformazan and ferricytochrome-c, respectively. Detection of superoxide is confirmed
when addition of the enzyme superoxide dismutase
(SOD) causes a decrease in production of diformazan
from NBT or no production of ferricytochrome c from
cytochrome c. Hence, it is concluded that cytochrome c
reduction only measures extracellularly released superoxide, whereas NBT may be reduced bl, extracellular superoxide or other molecules as well .
Electron spin resonance and spin trapping-Electron spin resonance (ESR) spectroscopy-also known
as electron paramagnetic resonance (EPR}-is used to
detect electromagnetic radiation being absorbed in the
microwave region by paramagnetic species that are
subjected to an external magnetic field. This method
is significant in that it can directly detect free radicals81.It is, at present, the only analytic approach that
permits the direct detection of free radicals. This
technique reports on the magnetic properties of unpaired electrons and their molecular environment
Paramagnetic (unpaired) electrons can exist in two
orientations, either parallel (+1/2, -1/2) or antiparallel
with respect to an applied magnetic field. ESR utilizes
the electron spin states energy to obtain absorption
spectta. ESR cannot detect paramagentic species such
as NO, superoxide and hydroxyl radical (OH) as they
are short lived and very low in concentration. This
problem can be overcome by adding exogenous spin
ttap molecules to the unstable free radicals therebI
converting them to more stable secondary radica1s8.
A unique spectrum is obtained when a spin-trapped
free radical is exposed to an applied magnetic field.
Nitroxide and nitrone derivatives are the frequently
used spin traps and are used to measure the oxidative
stress on proteins and lipids. These spin traps can also
be used to label biomolecules and probe basal and
oxidative-induced molecular events in protein and
lipid microenvironments.
Aromatic traps-This method is considered superior to the spin trap, as it can be used to measure
ROS that are produced in vivo. Salicylate and phenylalanine are suitable for human consumption. They
react with the free radicals to form more stable products83-87.
Salicylate and phenylalanine react with OH
to yield 2,3-dihydroxybenzoate and tyrosine, respectively, which is not produced in vivoss.88.Many human studies have successfully used salicylate in vivo
to determine ROS levels89.90.
Efficiency of both ESR and aromatic trap depends
on their concentration at the sites of ROS formation.
Because the ROS molecules are less stable, the trap
molecules could compete with many other compounds. Both ESR and aromatic trap methods are not
used traditionally for the measurement of OS in the
male reproductive system; however, they are commonly used in the measurement of ROS in other systems like cardiovascular and cerebrovascular.
Flow cytometry-in flow cytometry, the fluorescent intensity of the compounds oxidized by ROS is
measured. Dihydrorhodamine 123 and 2, 7 dichlorofluorescin diacetate (DCFH-DA) are the few compounds that can diffuse into cells. They then become
deacetylated and lose their fluorescence. When oxidized by ROS, which is generated within the cell, they
beCome highly fluorescent. The fluorescence can be
quantified, which reflects the rate and quantity of the
ROS produced91.
Measurementor lipid peroxidation
Thiobarbituric acid assay-Lipid peroxidationis a
added to a free radical-generating system, the inhibition of the free radical action is measured and this
inhibition is related to the antioxidant capacity of the
sample. The FRAP assay measuresthe ferric reducing
ability of a sample. However, the results of these 3
methods are weakly correlated. In addition, these tests
do not measure T AC but instead predominantly
measure the low molecular weight, chain breaking
antioxidants, excluding the contribution of antioxidant
enzymes and metal binding proteins.
Enhanced chemiluminescence assay-In the enhanced chemiluminescence assay, horseradish peroxidase (HRP)-linked immunoglobulin is mixed with a
signal reagent (luminol plus para-iodophenol) - a
chemiluminescent substance to generate ROS. This
solution is then mixed with a substrate H2O2.The capacity of the antioxidants in the seminal plasma to
decreasethe chemiluminescence of the signal reagent
as measured by luminometer is compared with that of
Trolox (a water-soluble vitamin E analogue) and is
measuredas molar Trolox equivalents96.97.
Measurement of NO
Colorimetric assay- The colorimetric assay measGriessreaction-The Griessreactionis an indirect
measurementof NO. It measuresthe stable deriva- ures the absorbance of a sample with the help of
tives of NO such as NO2-and NO3-using a spectro- spectrophotometer. The results are compared with
photometer.The Griessreactionis a two-stepdiazoti- trolox (a vitamin E analogue) and are given in trolox
zation reaction.Autoxidation of NO and acidification equivalents. This assay is a rapid and reliable method
of nitrite (N~") resultsin the formation of dinitrogen for measuring seminal TAC and is less expensive than
trioxide (N2O3)'which reacts with sulfanilamide to
the enhancedchemiluminescence assay96.
form diazonium derivative. This intermediatecomROS-TAC score-Because each method for anapoundinteractswith N-l-naphthylethylenediamineto
lyzing ROS and T AC has its own set of disadvanyield a coloreddiazoproduct9S.
tages, it is difficult to accurately predict the OS status
Fluorescencespectroscopymethod-The fluores- of the semen. To overcome this problem, Sharma et
This is
cence spectroscopymethod is more advantageous aI. have proposed the ROS-TAC score98.99.
levthan the Griessreactionin that it has a greatersensitivity and specificity. The N2O3generatedfrom the els by principal components analysis. To predict the
autoxidation of NO or acidification of N~ - reacts OS level, a cutoff value of 30 was established using a
group of normal healthy controls. Most of the men
with aromatic 2,3-diaminonaphthalene(DAN) to
who had a ROS- T AC score below the cutoff value
yield highly fluorescent 2,3-naphthotriazole(NAT),
which is measuredusing a fluorescent spectropho- were found to be infertile. Further, it was observed
that an infertile male with a ROS-T AC score higher
Measurementof total antioxidant capacity (TAC) than 30 could eventually initiate a pregnancyl7.
complex processleading to the formation of various
aldehydes including malonaldehyde (MDA). The
thiobarbituric acid (TBA) assayis the most common
method used to assesschangesin MDA. The assay
detects TBA-reactive substancesvia high performance liquid chromatography (HPLC), spectrophotometry or spectrofluorescence92.
The simple TBA
test is highly unreliableas most TBA-reactive materials in humanbody fluids are not related to lipid peroxidation.
IsoprostaneiIsoP} method-The best available
lipid peroxidation biomarker is isoprostane(IsoP),
which is a specific end product of the peroxidationof
polyunsaturatedfatty acids93.94.
The IsoP marker is
advantageousin that it is stable and is not produced
by enzymatic pathways like cyclooxygenase and
lipoxygenase pathways of arachindonic acid. This
marker can be quantified in extracellular fluids such
as seminalplasmaor urine.
in semen-Assessmentof T AC is a complex process
becauseseveral different antioxidantsare presentin
the semen.Therefore, many methodshave been developed to analyzeTAC of the semen.The oxygen
radical absorbancecapacity (ORAC), the ferric reducing ability of plasma (FRAP) and the troloxequivalent antioxidant capacity (TEAC) assaysare
widely usedfor measuringT AC. Both the ORAC and
TEAC assaysare inhibition methods. A sample is
Antioxidants can fit into two broad categoriesenzymatic and non-enzymatic (Table 4). They provide the necessary defense against the OS generated
by ROS. Antioxidants are present in seminal plasma
and in sperm cells.
Seminal plasma contains three important enzymatic
antioxidants-SOD, catalaselOO
and GPXlGRD system.
Table 4- Different classesof antioxidantsthat scavengeROS
Emymatic antioxidants
Vitamin C, Vitamin E, Vitamin C and Vitamin A (carotenoids)
Proteinslike Albumin, Transferrin,HaptOgJobulin,
GJutadli~ (GSH)
. uiool
. -
In addition, it has an array of non-enzymatic antioxidants-ascorbateIOI, uratelO2,vitamin E1OJ,pyruvate6,
glutathionelO4,albumin, vitamin A, ubiquinollos, taurine and hypotaurinel(Wj. Seminal plasma is essential
in protecting the spermatozoa because they contain a
low volume of cytoplasm, which makes them less efficient in mounting a defense against ROS.
Human spermatozoa contain mainly enzymatic antioxidantsl(Wj.Of them, SOD plays a prominent role in
protecting spermatozoa against lipid peroxidation.
Superoxide dismutase (SOD)-SOD can be divided
into three different classes according to the catalytic
metal present at the active site. SOOt (CuZnSOD) is
found in the cytosol and contains copper (Cu) and Zn
as metal cofactors. SOD2 (MnSOD) is present in mitochondria and contains Mn. SOD3 (ECSOD) is present extracellularly. Of these, CufZn-SOD and MnSOD are the main forms. SOD catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen.
. HA+Oz
donatingan elecb"on.It containsseleniumat its active
center,which is required for its optimal functioning.
This explainsthe importanceof seleniumin male infertility. Four isozymesof GPX presentin humans
contain seleniumin their active center.The fi~ isozyme GPX1, preventsapoptosisinducedby OS. The
secondand the third isozymesare found in the gasb"ointestinaltract and in plasma, respectively. The
fourth fonD acts directly on membranephospholipid
hydroperoxidesand detoxifies them. It is present in
high levelsin the testis.
;:cGSH +NADpto
Net reaction
GRD stimulates the reduction of GSSG to GSH.
This ensuresa steady supply of the reductive substrate
(NADPH) to GPX. G6PD is required for the conversion of NADY to NADPH.
Catalase-Catalase detoxifies both intracellular
and extracellular hydrogen peroxide to water and
oxygen 109.In addition. catalase activates NO~induced
SpenD capacitation, which is a complex mechanism
involving H2~ 30.Catalase activity has also been detected in human spermatozoaand seminal plasmaloo.
+ Ih~
Seminal catalaseactivity in samplesof vasectomized men has been found to be similar to that of
non-vasectomizedmen, suggestingthat the origin of
the activity is neithertesticularnor epididyma111O.
SOD scavengesbodl intracellular and extracellular
superoxideradical and preventsthe lipid peroxidation
of plasmamembrane.However. it should be conjugated with catalaseor GPXI to prevent the action of
Non-enzymatic antioxidants
HzOz. which promotes the formation of hydroxyl
A variety of non-enzymatic antioxidants are presradicals'oo,SOD also prevents hyperactivation and ent in the semen, including vitamin C, vitamin E,
capacitationinduced by superoxideradicals'07,11ris glutathione, urate, ubiquinone and bilirubin are preswould suppressthe occunence of these ~ons
ent extracellularly in seminal plasma and are consid~maturely beforeejaculation"',
ered chain-breaking antioxidants.
Other nonGlutathione peroxidase/reductasesystem (GPX/ enzymatic antioxidants such as vitamin E, vitamin A,
GRD)-Importance of the GPXlGRD systemis cen- haptoglobulin, transferrin and ceruloplasmin are presterOOon its antilipoperoxidative defense in human ent in the plasma membrane of the spermatozoa and
GPX reactswith peroxidesand requires act as preventive antioxidants. Some of the effects of
reducedglutathione(GSH) as the reductivesubstance these antioxidants are reviewed below'!!.
Other antioxidantsmoleculessuch as N-acetyl Lcysteine,carotenoids,coenzymeQ 10 and carnitines
provide excellent antioxidant support. N-acetyl Lcysteineis a precursorof glutathionethat assistsin its
formation.It helpsto improvethe motility of spenD.It
also reducesthe DNA damagecausedby ROS47.123.
Carotenoidsplay an important role in protecting the
cells and organisms by scavengingthe superoxide
radicals124.CoenzymeQ is associatedwith low density lipoproteins (LDL) and hence,it protects lipids
It directly reactswith
oxygen and reduces superoxidegeneration. It also
reactswith peroxide radicals126.It is an energy promoting agent and improves spenDmotilityl27.Carnitine promotesmembranestability. It plays an important role in spenDmaturationand development.Like
coenzymeQ 10,it is an energypromotingagenr28.
Vitamin E- This is a major chain-breaking antioxidant present both in seminal plasma and in the
membranel12,It reacts directly with free radicals such
as the peroxy radical (ROO,) yielding lipid hydroperoxides. which can be removed by phospholipase
GSH-Px systems113, It also interrupts the lipid peroxidation process, which is why it is called a chainbreaking antioxidant, This antioxidant also prevents a
reduction in spenD motility. Finally, it scavenges all
three important types of ROS, namely superoxide,
H2~, and hydroxyl radicals 114,
Ascorbate-.oLike vitamin E, ascorbate is also a
chain-breaking antioxid3nt and is found both intracellularly and extracellularlyl12, It prevents lipid peroxidation due to peroxyl radicals. It also recycles vitamin E. It protects against DNA damage induced by
H2~ radical. Vitamin C has a paradoxical effect I 15as
it can also produce ROS by its action on transition
metal ions.
Aff + Fe 3+lCu2+
Fe}+/Cu +
.. A -+ Fe 2+/CU
~. + Fe3+/Cu
OH + OH' + Fe3+/Cu2+
is the most abundant
non-thiol protein in mammalian cells] ]6. It plays a
vital role in annihilating oxygen toxicity by interrupting the reaction leading to ~- formationl]7, In its
reduced form, it metabolizes H2O2 and OH, It is a
peptide composed of glutamate, cysteine and glycine
that exist in thiol-reduced glutathione (GSH) and oxidized glutathione (GSSG). Glutathione and selenium
play essential roles in the fonnation of phospholipid
hydroperoxide glutathione peroxidase-an enzyme
that is present in spermatids and forms the structural
part in the mid-piece of mature spermatozoa. A glutathione deficiency can lead to instability of the midpiece, resulting in defective motilityl]8,]19. It can restore the physiological constitution of polyunsaturated fatty acid in the cell membranel20.121.
In a study consisting of infertile men with unilateral varicocele or genital tract inflammation. glutathione led to a statistically significant improvement in
the sperm quality l04.122. T reatment WIth gIutathlone
was found to have a statistically significantly positive
effect on spenD motility (in particular forward progression) and on sperm morphology, among other
variables. Glutathione therapy was suggested as a
possible therapeutic option in cases where OS is the
probable cause of male infertility.
OS andits role in male infertility havebeenan area
of active researchover the past two decades.Many
free radicalsare the result of naturally occurringprocessessuch as oxygen metabolismand inflammation.
Environmental stimuli such as ionizing radiation
(from industry, excessiveexposureto solar radiations,
cosmic rays, and medical X-rays), environmental
toxins, altered atmosphericconditions (e.g. hypoxia
and hyperoxia), ozone and nitrous oxide (primarily
from automobileexhaust)as well as many infectious
conditionsgreatly enhanceROS production.Lifestyle
stressorssuch as cigarettesmoking and excessivealcohol consumptionare also known to affect levels of
free radicals.They playa vital role in the etiology of
many diseasesand degenerativeprocesses.Hence,it
is imperativeto have a soundknowledgeabout them
and the OS causedby them.
OS affects male fertility in many ways. The peroxidation of lipids, the cross-linking and inactivation
of proteins and fragmentationof DNA are typical
of free radicals. Becausethe reactions
occur quickly and are a part of complex chain reactions, we usually can detect only their "footprints".
This requires the need for accuratemeasurementof
the OS statusof the semen.The full potentialof innovative techniques like spin trapping and aromatic
trappingshouldbe utilized in the field of reproductive
science.Although, a wide rangeof antioxidants-both
naturaland syntheticexist, their routine usein clinical
conditionsis still controversial.This explainsthe need
to standardizea universalprotocol to avoid unethical
useof antioxidants.Furtherresearchis requiredin this
field to fully understandthe complicatedeventsassociated with OS and to eventually develop effective
strategiesto assist patients with OS-associatedmale
infertility .
The authorsthank the ClevelandClinic Glickman
Urological Institute for support of their research
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