Indian Journal of Experimental BiologyVol. 43. November 2005. pp 963-974

Review Article

Mechanism, measurement, and prevention of oxidative stressin male reproductive physiology

Ashok Agarwal* & Sushil A PrabakaranCenter for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Glickman Urological Institute, andDepartment of Obstetrics and Gynecology, The Cleveland Clinic Foundation, 9500Euelid Avenue, Desk A19.I, Cleveland,

Ohio, 44195, USANumerous factors influence male fertility. Among these factors is oxidative stress (OS), wmcb bas elicited an enor-

mous interest in researchers in recent period. Reactive oxygen species (ROS) are continuously produced by various meta-bolic and physiologic processes. OS occurs when the delicate balance between the production of ROS and the inherent anti-oxidant capacity of the organism is distorted. Spermatozoa are particularly sensitive to ROS as their plasma membranecontains polyunsaturated fatty acids (PUFA), which oxidizes easily. They also lack cytoplasm to generate a robust preven-tive and repair mechanism against ROS. The transition metal ions that are found in the body have a catalytic effect in thegeneration of ROS. Lifestyle behaviours such as smoking and alcohol use and environmental pollution further enhance thegeneration of ROS and thus, cause destructive effects on various cellular organelles like mitochondria. sperm DNA etc. Thisarticle analyzes the detrimental effects of OS on male ferdlity. measurement of OS and effccti ve ways to decrease or elimi-nate them completely. We have also provided information on oxidative stress in other systems of the body, which may beapplied to future research in the field of reproductive biology.

Keywords: Male fertility. Oxidative stress, Spermatozoa

metabolism of oxygen and are present in all aerobicorganisms. They ray a significant role in many bio-logical processes. as can affect the individual mole-cules and thus the entire organism. as is believed tobe one of the major causes of many human diseases.Some of the OS-mediated pathologies are listed inTable 1. Virtually every discipline of medicine is in-terested in as and its implications. It is believed thatthe new field of 'Oxidative Stress, Diagnostics andTherapeutics' may have a significant impact on theeconomics, science and the practice of medicine in thepresent century7.

Table I-Diseases and degenerative processes mediated byoxidative stress

Alzheimer'sdisease

Iron overload Parkinson's disease

IntroductionAs many as 15% of all couples living in the United

States have difficulty conceiving a child. Male factorsare responsible in at least 30% of the cases, and inanother 20%, the pathology is found both in men andwomen. Approximately 6% of men between the ageof 15 and 50 suffer from male infertilityl. A meta-analysis of 61 studies worldwide found a downwardtrend in sperm count and volume of seminal fluid overthe past 50 years2. In 1940, the average sperm countwas 113 million per mI. By 1990, the count haddropped to 66 million per mI3. This decreasing trendin sperm count has led to speculation that recent envi-ronmental, dietary and/or lifestyle changes are inter-fering with a man's ability to produce spermatozoa. Itis believed that these factors exert dleir detrimentaleffects through oxidative stress (OS).

Oxidative stress (OS) and its significance-OS oc-curs as a consequence of an imbalance between theproduction of reactive oxygen species (ROS) and theavailable antioxidant defense against them4,S. ROS arefree radicals and peroxides that are derived from the

Autoimmunedisease

Cancer

Ischemic-reperrusion Rheumatoid arthritisinjuryMacular degeneration Segmental progeria

disorders

AgingCardiovascular Multiple sclerosisdisease

Cataractogenesis Muscular dystrophy

Diabetes Pancreatitis

.Correspondent authorPhone: (216) 444-9485. Fax: (216) 445-6049;E-mail: Agarwaa@ccf.org;Website: www .clevelandclinic.orgjReproductiveResearchCenter

INDIAN J EXP BIOL. NOVEMBER 2005964

Mechanism of ROS formationOxygen in the atmosphere has two unpaired elec-

trons, and these unpaired electrons have parallel spins.Oxygen is usually non-reactive to organic moleculesthat have paired electrons with opposite spins. Thisoxygen is considered to be in a ground (triplet or in-active) state and is activated to a singlet (active) stateby two different mechanisms:

a) Absorption of sufficient energy to reverse thespin on one of the unpaired electrons.

.0-0. .0.-0:

Triplet oxygen (rr) Singlet oxygen <r~)(Ground state) (Highly reactive)

b) Monovalent reduction (accept a single electron).Superoxide is formed in the flfSt monovalent re-

duction reaction, which undergoes further reduction toform hydrogen peroxide. Hydrogen peroxide is fur-ther reduced to hydroxyl radicals in the presence offerrous salts (Fe2+). This reaction was first describedby Fenton and later developed by Haber and Weiss.

Origin of ROS in male reproductive systemHuman semen consists of different types of cells

such as mature and immature spermatozoa, roundcells from different stages of the spermatogenic proc-ess, leukocytes and epithelial cells. Of these, leuko-cytes and immature spermatozoa are the two mainsources of ROS 1°. Leukocytes, particularly neutro-phils and macrophages, have been associated withexcessive ROS production, and they ultimately causesperm dysfunctionll-18. ROS produced by leukocytesforms the first line of defense in any infectious proc-ess. DNA and structural damage can be found in

fr I k .. 19-21spermatozoa om eu ocytospenmc patients .Leukocytes act either directly by ROS synthesis orindirectly by other nei~hboring white cells via solublefactors as cytokinesl .22. ROS is significantly andpositively correlated with interleukin-S, a potentstimulator of pro-inflammatory cytokines23.24. Thescavenging effect of antioxidants are greatly dimin-ished under such infectious conditionsl4.

Another important source of ROS is immaturespermatozoa. A study from our group has demon-strated that ROS production is significantly and nega-tively correlated with semen quality2s. Other studieshave suggested that the defect in spermiogenesis thatresults in retention of cytoplasmic droplets is a majorsource of ROS26. There are two major systems ofROS production in sperm. One is the NADPH oxidasesystem at the level of the sperm plasma membrane

Monovalentreduction

Monovalentreduction

.0.-0:

Superoxide (i ~)(Reactive state)

.0-0.

Triplet oxygen (tt)(Ground state)

H:O-O:HHydrogen peroxide. Table 2- Types of reactive oxygen species that exist as radicals

and free radicalsFenton reaction:Fe2+ + H2~' Fe3+ +OH-+ OR'

Radic~ Non-RadicalsPeroxynitrite ONOO-on'Hydroxyl

H2O+~+ H+

~+OIr+OH'~...

NO.

RS.

R~.

LOQ'

HOC!

HA

-I~

~

LOOH

Hypochloric acid

Hydrogen Peroxide

Singlet OxygenOzone

Lipid peroxide

SuperoxideNitric Oxide

Thyl

Peroxyl

Lipid peroxyl

Table 3- Types of reactive nitrogen species that inducesoxidative stress

NO' NO.Nitrous oxide Nitrosyl carlon

Peroxynitrite OONO- Nitrogen dioxide N~ .Peroxynitrous acid ONOOH Dinitrogen trioxide NZO3

Nitroxyl anion NO- Nitrous acid HN~

Nitryl chl,oride NOzCl

Types of ROSROS represent a broad category of molecules that

indicate the collection of radicals and non-radicaloxygen derivatives. These intermediates may partici-pate in reactions that give rise to free radicals thatdamage organic substrates. Table 2 lists the types ofROS that exist as radicals and free radicals in livingorganisms.

In addition, there is another class of free radicalsthat are nitrogen derived called reactive nitrogen spe-cies (RNS). They are nitrogen-derived molecules andare considered a subclass of ROSS.9 (Table 3).

AGARWAL & PRABAKARAN: PREVENTION OF STRESS IN MALE REPRODUcrIVE PHYSIOLOGY 965

and the other is NADH-dependent oxido-reductase(diphorase) system at the mitochondriallevef7. Thereis a strong positive correlation between immaturespermatozoa and ROS production, which in turn isnegatively correlated with sperm quality. Further-more, it has been noticed that as the concentration ofimmature spermatozoa in the human ejaculate in-creases, the concentration of mature spermatozoa withdamaged DNA rises25.

Physiological role of ROSSpermatozoa acquire the capacity to mOve during

their transit through the epididymis, but lack the abil-ity to fertilize. After ejaculation, they undergo a seriesof physiological changes called capacitation28. Ca-pacitation is followed by acrosome reaction. which istriggered by the zona pellucida of the ovum and al-lows the sperm to fertilize the egg29.30. Capacitationand the acrosome reaction are redox-regulated proc-esses. Exogenous administration of 0,,'- or H2~ pro-motes capacitation and the acrosome reaction whereasthe addition of antioxidants prevents them from un-dergoing these events. This demonstrates the impor-tance ofROS in human sperm functions3).

Nitric oxide (NO1, a free radical with a relativelylong half-life (7 s), promotes capacitation. No detect-able activity of nitric oxide synthase (NOS) is foundin spermatozoa, which suggests that it originates fromtissues or fluids from the female genital trac~. Lowconcentrations of NO cause a significant increase incapacitation32 and zona pellucida binding33. NOregulates cyclic adenosine monophosphate (cAMP)concentration and induces capacitation of spermato-zoa through the action of adenyl cyclase. Finally, NOalso plays a role in sperm hyperactivation34.

nations to oxygen, fonning superoxide or hydrogenperoxide, which is further reduced to an extremelyreactive 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 monovalentlipid hydroperoxide (LOOH) decomposition, which inturn induces lipid peroxidation37. Both a deficiencyand an excess of manganese (Mn) causes neurotoxic-ity via ROS generation38. TMIs such as lead (Pb) andcadmium (C<t) also affect the male reproductive sys-tem. These metals either directly affect the spermplasma membrane or catalyze the ROS formation. Pbcauses relative zinc (Zn) deficiency, while Cd pre-dominantly affects the distribution of Zn in the body.

The authors of a study consisting of industrialworkers have identified a decrease in the workers'semen quality, semen volume, sperm count, spermmotility and forward progression. The authors havealso reported an increase in abnormal sperm morphol-ogy and an impairment of prostatic secretory function.They have also observed that both Pb and Cd damagethe DNA bases as measured by the levels 8-hydroxy-2'-deoxyguanosine (8-0HdG). Seminal plasma l~levels and artificial insemination cycle fecundity havea strong negative correlation39. Automobile pollutionincreases the blood lead level, which decreases spermcount and viability.

Damage to lipids-Lipids are most susceptiblemacromolecules and are present in the spenD plasmamembrane in the form of polyunsaturated fatty acids(PUPA). ROS attack PUPA in the cell membraneleading to a chain of chemical reactions called lipidperoxidation40. The reaction occurs in three distinctsteps-initiation, propagation and termination. Duringinitiation, the free radicals react with fatty acid chain(LH) and releases lipid free radical. This lipid radicalfurther reacts with molecular oxygen to form lipid

. peroxyl radical. Peroxyl radicals again react with fattyacid to prrouce lipid free radical and this reaction ispropagated. During tennination, the two radicals reactwith each other, and the process comes to an en~l.This process of fatty acid breakdown produces hydro-carbon gases (ethane or pentane) and aldehydes.

Pathological role of ROSEffect of excessive NO-NO is detrimental to

sperm motility in high concentrations35. It may reactwith superoxide or hydrogen peroxide, resulting in theformation of peroxynitrite, hydroxyl radical, N~ orsinglet oxygen, which causes oxidation of spermmembrane lipids and thiol proteins36. In addition, NOmay also inhibit cellular respiration by nitrosylationof heme in mitochondrial enzymes, aconitase andglyceraldehyde phosphate dehydrogenase. This causesa decrease in the level of adenoFine triphosphate,which thereby affects the kinetics of Spermatozoa35.

Catalytic effect of different transition metal ions-Transition metals ions (TMI) can make electron do-

Initiation:LH+R" L"+RH

Propagation:L.+~ LOO'

966 INDIAN 1 EXP BIOL, NOVEMBER 2005

is the most important cellular organelle that mediatesapoptosis. High levels of ROS disrupt the integrity ofthe mitochondrial membrane, which in turn releasescytochrome c. It activates the caspase enzyme cascadeand triggers apoptosis. Studies in infertile men haveshown that high levels of cytochrome c in the seminalplasma indirectly reflect significant mitochondrialdamage caused by high levels of ROS. Levels of ROSin infertile men are correlated positively with apopto-sis, which in turn is negatively correlated with con-ventional semen parameters49.S9-62.

Effects of life-style behavior and environmentOS associated with smoking-Tobacco contains

nearly 4000 harmful substances such as alkaloids,nitrosamines, nicotine and hydroxycotinine. Many ofthese substances generate ROS and RNS63.64. Smok-ing has been associated with decreased sperm qual-ity6S-67. Reduction in motility is due to the damagecaused by ROS to the flagellum and axonemal struc-tures of the tail of spermatozoa. Sperm motility cor-relates negatively with the amount of cotinine andhydroxycotinine in the seminal plasma6s.68. The pres-ence of cotinine and hydroxycotinine in seminalplasma indicates that harmful substances found in to-bacco smoke can penetrate the blood-testes barrier.

Oxidative stress and alcohol-Many processes andfactors are involved in causing alcohol-induced OS.Alcohol metabolism results in the formation ofNADH, which enhances activity of the respiratorychain, including heightened ~ use and ROS forma-tion. One of the by-products of alcohol metabolism,acetaldehyde, interacts with proteins and lipids toform ROS. It is also capable of damaging the mito-chondria leading to decreased A TP production. Alco-hol also induces hypoxia that results in tissue damage.Excessive alcohol consumption is associated with adecrease in the percentage of nonnal spenD in asthe-nozoospermic patients69. However, better spenD mor-pholop has been observed in men who drink moder-ately7 .

OS and environment-Many environmental factorssuch as radiation, medications and pollutants induceROS production)s. In a study conducted on Danishgreenhouse workers, a high count of spennatozoa wasfound among organic farmers who grew their prod-ucts without the use of pesticides or chemical fertiliz-ers. The group of blue-collar workers in the abovestudy had a low spenD count in comparison to theabove group of workers7). These studies categoricallysuggest the need for a healthy environment.

Damage 10 proleins-Sulphur-containing aminoacids, having thiol groups specifically, are very sus-ceptible to OS. Aetivated oxygen can abstract H atomfrom cysteine residues to form a thiyl radical that willcross-link to a second thiyl radical to form disulphidebridges. Alternatively, oxygen can add to a methio-nine residue to form methionine sulphoxide deriva-tives. Many amino acids undergo specific irreversiblemodifications when a protein is oxidized. For exam-ple, tryptophan is readily cross-linked to form bityro-sine products42. Oxidative damage can also lead tocleavage of the polypeptide chain and formation ofcross-linked protein aggregates. Histidine, lysine,proline, arginine and serine form carbonyl groups onoxidation43. The oxidative degradation of protein isenhanced in the presence of metal cofactors that arecapable of redox cycling, such as Fe.

Damage 10 DNA-Activated oxygen and agentsthat generate oxygen free radicals, such as ionizingradiation, induce numerous lesions in DNA that leadto deletions, mutations and other lethal genetic ef-fects44. Pyrimidine bases are most susceptible to OSas are purines and deoxyribose sugar. Oxidation of the"sugar by the hydroxyl radical is the main cause forDNA strand breaks4s. Oxidative damage can causebase degradation, DNA fragmentation and cross-linking to protein46-48. In addition, incorporation ofoxidized deoxyribonucleoside triphosphate causesgene mutation or altered gene expression. The rate ofDNA fragmentation is increased in the ejaculate ofinfertile men49-S2 as indicated by the high level of 8-OHdG, which is a product of DNA oxidation. SpermDNA is normally protected from oxidative insult bytwo factors-the antioxidants present in seminalplasma. and the characteristic tight packaging of theDNAs3.

Apoplosis-Apoptosis is programmed cell deaththat occurs in a genetically determined fashionS4. It isinitiated by ROS-induced oxidative damage, but toomuch OS can terminate apoptosis by inactivating the

ad S5-S7A . .dan .thcaspase enzyme casc e . ntIoXl ts can el er

suppress or facilitate apoptosiss8. The mitochondrion

AGARWAL Ii; PRABAKARAN: PREVEN'nON OF STRESS IN MALE REPRODUCTIVE PHYSIOLOGY 967

Paramagnetic (unpaired) electrons can exist in twoorientations, either parallel (+1/2, -1/2) or antiparallelwith respect to an applied magnetic field. ESR utilizesthe electron spin states energy to obtain absorptionspectta. ESR cannot detect paramagentic species suchas NO, superoxide and hydroxyl radical (OH) as theyare short lived and very low in concentration. Thisproblem can be overcome by adding exogenous spinttap molecules to the unstable free radicals therebIconverting them to more stable secondary radica1s8 .A unique spectrum is obtained when a spin-trappedfree radical is exposed to an applied magnetic field.Nitroxide and nitrone derivatives are the frequentlyused spin traps and are used to measure the oxidativestress on proteins and lipids. These spin traps can alsobe used to label biomolecules and probe basal andoxidative-induced molecular events in protein andlipid microenvironments.

Aromatic traps-This method is considered supe-rior to the spin trap, as it can be used to measureROS that are produced in vivo. Salicylate and phenyl-alanine are suitable for human consumption. Theyreact with the free radicals to form more stable prod-ucts83-87. Salicylate and phenylalanine react with OHto yield 2,3-dihydroxybenzoate and tyrosine, respec-tively, which is not produced in vivoss.88. Many hu-man studies have successfully used salicylate in vivoto determine ROS levels89.90.

Efficiency of both ESR and aromatic trap dependson their concentration at the sites of ROS formation.Because the ROS molecules are less stable, the trapmolecules could compete with many other com-pounds. Both ESR and aromatic trap methods are notused traditionally for the measurement of OS in themale reproductive system; however, they are com-monly used in the measurement of ROS in other sys-tems like cardiovascular and cerebrovascular.

Flow cytometry-in flow cytometry, the fluores-cent intensity of the compounds oxidized by ROS ismeasured. Dihydrorhodamine 123 and 2, 7 dichloro-fluorescin diacetate (DCFH-DA) are the few com-pounds that can diffuse into cells. They then becomedeacetylated and lose their fluorescence. When oxi-dized by ROS, which is generated within the cell, theybeCome highly fluorescent. The fluorescence can bequantified, which reflects the rate and quantity of theROS produced91.

Measurement of ROSChemiluminescence method- The chemilumines-

cence method is the most commonly used techniquefor measuring ROS produced by spennatozoan.73. Theassay determines the amount of ROS, not the level ofthe sperm-damaging ROS present at any given time.This method quantifies both intracellular and extra-cellular ROS. Luminol is a probe that reacts with dif-ferent types of ROS. It can measure both intra- andextracellular ROS whereas lucigenin can only meas-ure the superoxide radical released extracellularly.Hence, by using both the probes on the same sample,it is possible to accurately identif~ intracellular andextracellular generation of ROS74- 6. However, thereare some serious limitations in measuring the super-oxide formation; lucigenin undergoes redox cycling,and there are some unknown sources of superoxide

generation77.Cytochrome c reduction or nitro blue tetrazolium

( NBT) reduction method- The chemiluminescencemethod can indicate only the total amount of ROSpresent in the semen and does not provide informationabout the source of ROS. Cytochrome c reduction andNBT reduction both can accurately predict whetherROS have been produced by leukocytes or abnormalspermatozoal',7'. The two most commonly used rea-gents to detect superoxide anion radicals are NBT andferricytochrome-c (Cyt)79. Superoxide formed byelectron transfer from a donor to molecular oxygencan be quenched by NBT and Cyt, and these reagentsget reduced to diformazan and ferricytochrome-c, re-spectively. Detection of superoxide is confirmedwhen addition of the enzyme superoxide dismutase(SOD) causes a decrease in production of diformazanfrom NBT or no production of ferricytochrome c fromcytochrome c. Hence, it is concluded that cytochrome creduction only measures extracellularly released su-peroxide, whereas NBT may be reduced bl, extracel-lular superoxide or other molecules as well .

Electron spin resonance and spin trapping-Elec-tron spin resonance (ESR) spectroscopy-also knownas electron paramagnetic resonance (EPR}-is used todetect electromagnetic radiation being absorbed in themicrowave region by paramagnetic species that aresubjected to an external magnetic field. This methodis significant in that it can directly detect free radi-cals81. It is, at present, the only analytic approach thatpermits the direct detection of free radicals. Thistechnique reports on the magnetic properties of un-paired electrons and their molecular environment

Measurement or lipid peroxidationThiobarbituric acid assay-Lipid peroxidation is a

INDIAN J EXP DiaL. NOVEMBER 2005968

complex process leading to the formation of variousaldehydes including malonaldehyde (MDA). Thethiobarbituric acid (TBA) assay is the most commonmethod used to assess changes in MDA. The assaydetects TBA-reactive substances via high perform-ance liquid chromatography (HPLC), spectropho-tometry or spectrofluorescence92. The simple TBAtest is highly unreliable as most TBA-reactive materi-als in human body fluids are not related to lipid per-oxidation.

Isoprostane iIsoP} method-The best availablelipid peroxidation biomarker is isoprostane (IsoP),which is a specific end product of the peroxidation ofpolyunsaturated fatty acids93.94. The IsoP marker isadvantageous in that it is stable and is not producedby enzymatic pathways like cyclooxygenase andlipoxygenase pathways of arachindonic acid. Thismarker can be quantified in extracellular fluids suchas seminal plasma or urine.

added to a free radical-generating system, the inhibi-tion of the free radical action is measured and thisinhibition is related to the antioxidant capacity of thesample. The FRAP assay measures the ferric reducingability of a sample. However, the results of these 3methods are weakly correlated. In addition, these testsdo not measure T AC but instead predominantlymeasure the low molecular weight, chain breakingantioxidants, excluding the contribution of antioxidantenzymes and metal binding proteins.

Enhanced chemiluminescence assay-In the en-hanced chemiluminescence assay, horseradish peroxi-dase (HRP)-linked immunoglobulin is mixed with asignal reagent (luminol plus para-iodophenol) - a

chemiluminescent substance to generate ROS. Thissolution is then mixed with a substrate H2O2. The ca-pacity of the antioxidants in the seminal plasma todecrease the chemiluminescence of the signal reagentas measured by luminometer is compared with that ofTrolox (a water-soluble vitamin E analogue) and ismeasured as molar Trolox equivalents96.97.

Colorimetric assay- The colorimetric assay meas-ures the absorbance of a sample with the help ofspectrophotometer. The results are compared withtrolox (a vitamin E analogue) and are given in troloxequivalents. This assay is a rapid and reliable methodfor measuring seminal TAC and is less expensive thanthe enhanced chemiluminescence assay96.

ROS-TAC score-Because each method for ana-lyzing ROS and T AC has its own set of disadvan-tages, it is difficult to accurately predict the OS statusof the semen. To overcome this problem, Sharma etaI. have proposed the ROS-TAC score98.99. This isthe statistical score obtained from ROS and T AC lev-els by principal components analysis. To predict theOS level, a cutoff value of 30 was established using agroup of normal healthy controls. Most of the menwho had a ROS- T AC score below the cutoff valuewere found to be infertile. Further, it was observedthat an infertile male with a ROS- T AC score higherthan 30 could eventually initiate a pregnancyl7.

Measurement of NOGriess reaction-The Griess reaction is an indirect

measurement of NO. It measures the stable deriva-tives of NO such as NO2- and NO3- using a spectro-photometer. The Griess reaction is a two-step diazoti-zation reaction. Autoxidation of NO and acidificationof nitrite (N~") results in the formation of dinitrogentrioxide (N2O3)' which reacts with sulfanilamide toform diazonium derivative. This intermediate com-pound interacts with N-l-naphthylethylene diamine toyield a colored diazo product9S. .

Fluorescence spectroscopy method-The fluores-cence spectroscopy method is more advantageousthan the Griess reaction in that it has a greater sensi-tivity and specificity. The N2O3 generated from theautoxidation of NO or acidification of N~ - reacts

with aromatic 2,3-diaminonaphthalene (DAN) toyield highly fluorescent 2,3-naphthotriazole (NA T),which is measured using a fluorescent spectropho-tometers.

Measurement of total antioxidant capacity (TAC)in semen-Assessment of T AC is a complex processbecause several different antioxidants are present inthe semen. Therefore, many methods have been de-veloped to analyze T AC of the semen. The oxygenradical absorbance capacity (ORAC), the ferric re-ducing ability of plasma (FRAP) and the trolox-equivalent antioxidant capacity (TEAC) assays arewidely used for measuring T AC. Both the ORAC andTEAC assays are inhibition methods. A sample is

AntioxidantsAntioxidants can fit into two broad categories-

enzymatic and non-enzymatic (Table 4). They pro-vide the necessary defense against the OS generatedby ROS. Antioxidants are present in seminal plasmaand in sperm cells.

Seminal plasma contains three important enzymaticantioxidants-SOD, catalaselOO and GPXlGRD system.

~

AGARWAL & PRABAKARAN: PREVENTION OF STRESS IN MALE REPRODUCTIVE PHYSIOLOGY969

- -Table 4- Different classes of antioxidants that scavenge ROS

Emymatic antioxidantsSuperoxide dismutaseCatalaseGlutathione peroxidase/glutathione reductase (GPXKiRD)

Non-eazymadc antioxidantsVitamin C, Vitamin E, Vitamin C and Vitamin A (carotenoids)Proteins like Albumin, Transferrin, HaptOgJobulin,CeroJopasmin.GJutadli~ (GSH)Pyruvate. uiool

.,. . -

donating an elecb"on. It contains selenium at its activecenter, which is required for its optimal functioning.This explains the importance of selenium in male in-fertility. Four isozymes of GPX present in humanscontain selenium in their active center. The fi~ iso-zyme GPX1, prevents apoptosis induced by OS. Thesecond and the third isozymes are found in the gas-b"ointestinal tract and in plasma, respectively. Thefourth fonD acts directly on membrane phospholipidhydroperoxides and detoxifies them. It is present inhigh levels in the testis.

GSSG + NADPH + W GSH +NADpto;:cG~

2GSH + R(OOH)COOH .GSSG +GPX

R(OH)COOH + H2O

In addition, it has an array of non-enzymatic antioxi-dants-ascorbateIOI, uratelO2, vitamin E1OJ, pyruvate6,glutathionelO4, albumin, vitamin A, ubiquinollos, tau-rine and hypotaurinel(Wj. Seminal plasma is essentialin protecting the spermatozoa because they contain alow volume of cytoplasm, which makes them less ef-ficient in mounting a defense against ROS.

Human spermatozoa contain mainly enzymatic an-tioxidantsl(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 catalyticmetal present at the active site. SOOt (CuZnSOD) isfound in the cytosol and contains copper (Cu) and Znas metal cofactors. SOD2 (MnSOD) is present in mi-tochondria and contains Mn. SOD3 (ECSOD) is pres-ent extracellularly. Of these, CufZn-SOD and Mn-SOD are the main forms. SOD catalyzes the dismuta-tion of superoxide into hydrogen peroxide and oxy-

gen.

Net reactionNADPH + R(OOH)COOH +W . NADY

+ R(OH)COOH + H2O

GRD stimulates the reduction of GSSG to GSH.This ensures a steady supply of the reductive substrate(NADPH) to GPX. G6PD is required for the conver-sion of NADY to NADPH.

Catalase-Catalase detoxifies both intracellularand extracellular hydrogen peroxide to water andoxygen 109. In addition. catalase activates NO~inducedSpenD capacitation, which is a complex mechanisminvolving H2~ 30. Catalase activity has also been de-tected in human spermatozoa and seminal plasmaloo.

C~~l~~

.2HA H~ + Ih~SOD

20z-+2W . HA+OzSOD scavenges bodl intracellular and extracellular

superoxide radical and prevents the lipid peroxidationof plasma membrane. However. it should be conju-gated with catalase or GPXI to prevent the action ofHzOz. which promotes the formation of hydroxylradicals'oo, SOD also prevents hyperactivation andcapacitation induced by superoxide radicals '07, 11riswould suppress the occunence of these ~ons~maturely before ejaculation"',

Glutathione peroxidase/reductase system (GPX/GRD )-Importance of the GPXlGRD system is cen-terOO on its antilipoperoxidative defense in humanspennatoZ08. GPX reacts with peroxides and requiresreduced glutathione (GSH) as the reductive substance

Seminal catalase activity in samples of vasecto-mized men has been found to be similar to that ofnon-vasectomized men, suggesting that the origin ofthe activity is neither testicular nor epididyma111O.

Non-enzymatic antioxidantsA variety of non-enzymatic antioxidants are pres-

ent in the semen, including vitamin C, vitamin E,glutathione, urate, ubiquinone and bilirubin are pres-ent extracellularly in seminal plasma and are consid-ered chain-breaking antioxidants. Other non-enzymatic antioxidants such as vitamin E, vitamin A,haptoglobulin, transferrin and ceruloplasmin are pres-ent in the plasma membrane of the spermatozoa andact as preventive antioxidants. Some of the effects ofthese antioxidants are reviewed below'!!.

INDIAN J EXP BIOL, NOVEMBER 2005970

Other antioxidants molecules such as N-acetyl L-cysteine, carotenoids, coenzyme Q 10 and carnitinesprovide excellent antioxidant support. N-acetyl L-cysteine is a precursor of glutathione that assists in itsformation. It helps to improve the motility of spenD. Italso reduces the DNA damage caused by ROS47.123.Carotenoids play an important role in protecting thecells and organisms by scavenging the superoxideradicals 124. Coenzyme Q is associated with low den-sity lipoproteins (LDL) and hence, it protects lipidsagainst peroxidative damagel25. It directly reacts withoxygen and reduces superoxide generation. It alsoreacts with peroxide radicals 126. It is an energy pro-moting agent and improves spenD motilityl27. Carni-tine promotes membrane stability. It plays an impor-tant role in spenD maturation and development. Likecoenzyme Q 10, it is an energy promoting agenr28.

Vitamin E- This is a major chain-breaking anti-oxidant present both in seminal plasma and in themembranel12, It reacts directly with free radicals suchas the peroxy radical (ROO,) yielding lipid hydroper-oxides. which can be removed by phospholipaseGSH-Px systems 1 13, It also interrupts the lipid per-

oxidation process, which is why it is called a chain-breaking antioxidant, This antioxidant also prevents areduction in spenD motility. Finally, it scavenges allthree important types of ROS, namely superoxide,H2~, and hydroxyl radicals 114,

Ascorbate-.oLike vitamin E, ascorbate is also achain-breaking antioxid3nt and is found both intra-cellularly and extracellularlyl12, It prevents lipid per-oxidation due to peroxyl radicals. It also recycles vi-tamin E. It protects against DNA damage induced byH2~ radical. Vitamin C has a paradoxical effect I 15 as

it can also produce ROS by its action on transitionmetal ions.

OH + OH' + Fe3+/Cu 2+

Glutathione-Glutathione is the most abundantnon-thiol protein in mammalian cells] ]6. It plays avital role in annihilating oxygen toxicity by inter-rupting the reaction leading to ~- formationl]7, In itsreduced form, it metabolizes H2O2 and OH, It is apeptide composed of glutamate, cysteine and glycinethat exist in thiol-reduced glutathione (GSH) and oxi- .dized glutathione (GSSG). Glutathione and seleniumplay essential roles in the fonnation of phospholipid

hydroperoxide glutathione peroxidase-an enzymethat is present in spermatids and forms the structuralpart in the mid-piece of mature spermatozoa. A glu-tathione deficiency can lead to instability of the mid-piece, resulting in defective motilityl]8,]19. It can re-store the physiological constitution of poly-unsaturated fatty acid in the cell membranel20.121.

In a study consisting of infertile men with unilat-eral varicocele or genital tract inflammation. glutathi-one led to a statistically significant improvement inth uality l04.122 T " th I th ' e sperm q . reatment WI g uta lone

was found to have a statistically significantly positiveeffect on spenD motility (in particular forward pro-gression) and on sperm morphology, among othervariables. Glutathione therapy was suggested as apossible therapeutic option in cases where OS is theprobable cause of male infertility.

Aff + Fe 3+lCu 2+ .. A - + Fe 2+/CU +

Fe3+JCu+ + ~ ~. + Fe3+/Cu 2+

+H~Fe}+/Cu +

ConclusionOS and its role in male infertility have been an area

of active research over the past two decades. Manyfree radicals are the result of naturally occurring proc-esses such as oxygen metabolism and inflammation.Environmental stimuli such as ionizing radiation(from industry, excessive exposure to solar radiations,cosmic rays, and medical X-rays), environmentaltoxins, altered atmospheric conditions (e.g. hypoxiaand hyperoxia), ozone and nitrous oxide (primarilyfrom automobile exhaust) as well as many infectiousconditions greatly enhance ROS production. Lifestylestressors such as cigarette smoking and excessive al-cohol consumption are also known to affect levels offree radicals. They playa vital role in the etiology ofmany diseases and degenerative processes. Hence, itis imperative to have a sound knowledge about themand the OS caused by them.

OS affects male fertility in many ways. The per-oxidation of lipids, the cross-linking and inactivationof proteins and fragmentation of DNA are typicalconsequences of free radicals. Because the reactionsoccur quickly and are a part of complex chain reac-tions, we usually can detect only their "footprints".This requires the need for accurate measurement ofthe OS status of the semen. The full potential of inno-vative techniques like spin trapping and aromatictrapping should be utilized in the field of reproductivescience. Although, a wide range of antioxidants-bothnatural and synthetic exist, their routine use in clinicalconditions is still controversial. This explains the needto standardize a universal protocol to avoid unethical

AGARWAL & PRABAKARAN: PREVENTION OF STRESS IN MALE REPRODUCTIVE PHYSIOLOGY971

use of antioxidants. Further research is required in thisfield to fully understand the complicated events asso-ciated with OS and to eventually develop effectivestrategies to assist patients with OS-associated maleinfertility .

AcknowledgementThe authors thank the Cleveland Clinic Glickman

Urological Institute for support of their researchstudies.

16 Pasqualotto F F, Sharma R K, Potts J M et aI., Seminal oxi-dative stress in patients with chronic prostatitis, Urology, 55(2(xx) 881.

17 Sharma R K, Pasqualotto A E, Nelson D R et aI., Relation-ship between seminal white bl()(xf ceU counts and oxidativestress in men tteated at an infertility clinic, J Androl, 22(2001) 575.

18 Saleh R A. Agarwal A, Kandirali E et aI., Leukocytospemliais associated with increased reactive oxygen species produc-tion by human spermatozoa, Fertil Steril, 78 (2002) 1215.

19 Alvarez J G, Sharma R K, aUem M et aI., Increased DNAdamage in spenn from leukocytospermic semen samples asdetennined by the spenn chromatin SUUcture assay, FerrilSteril, 78 (2002) 319.

20 Aziz N, Agarwal A. Lewis-Jones I et aI., Novel associationsbetween specific spenn morphological defects and leukocy-tospennia. Ferril Sreril, 82 (2004) 621.

21 Shekarriz M, Sharma R K, Thomas A J, Jr. et aI., Positivemyeloperoxidase staining (Endtz test) as an indicator of ex-cessive reactive oxygen species fonnation in semen, J AssistReprod Genet, 12 (1995) 70.

22 Potts J M, Sharma R. Pasqualotto F et aI., Association ofureaplasma urealyticum with abnormal reactive oxygen spe-cies levels and absence of leukocytospermia. J Urol, 163(2(XX) 1775.

23 Potts J M & Pasqualotto F F, Seminal oxidative stress in pa-tients with chronic prostatitis, Andrologia, 35 (2003) 304.

24 NaUeUa K P, Allamaneni S S, Pasqualotto F F et aI., Rela-tionship of interleukin-6 with semen characteristics and oxi-dative stress in patients with varicocele, Urology, 64 (2004)1010.

25. GiI-Guzman E, aUero M, Lopez M C et al., Differentialproduction of reactive oxygen species by subsets of humanspermatozoa at different Stages of maturation, Hum Reprod,16 (2001) 1922.

26 Gomez E, Buckingham D W, Brindle J et aI., Developmentof an image analysis system to monitor the retention of re-sidual cytoplasm by human spermatozoa: Correlation withbiochemical markers of the cytoplasmic space, oxidativestress, and spenn function, J Androl, 17 (1996) 276.

27 Gavella M & Lipovac V, NADH-dependent oxidoreductase(diaphorase) activity and isozyme pattern of spenn in infer-tile men. Arch Androl, 28 (1992) 135.

28 Pasqualotto F F, Shanna R K, Nelson D R et aI., Relation-ship between oxidative stress. semen characteristics, andclinical diagnosis in men undergoing infertility investigation,Fertil Steril, 73 (2(xx) 459.

29 Lambert H. Overstreet J W, Morales P et al., SpenD capaci-tation in the human female reproductive tract, Fertil Steril,43 (1985) 325.

30 de Lamirande E, Leclerc P & Gagnon C, Capacitation as aregulatory event that primes spermatozoa for the acrosomereaction and fertilization, Mol Hum Reprod, 3 (1997) 175.

31 de Lamirande E. Tsai C, Harakat A et al., Involvement of re-active oxygen species in human spenD arcosome reaction in-duced by A23187, Iysophosphatidylcholine, and biological"fluid ultrafilttates. J Androl, 19 (1998) 585.

32 Zini A, De Lamirande E & Gagnon C, Low levels of nitricoxide promote human spenD capacitation in vitro, J Androl.16 (1995) 424.

References1 Purvis K & Christiansen E, Male infertility: Current con-

cepts, Ann Med, 24 (1992) 259.2 Carlsen E, Giwercman A, Keiding N et aL, Evidence for de-

creasing quality of semen during past 50 years, Bmj, 305(1992) ~.

3 Giwercman A. Carlsen E, Keiding N et aL, Evidence for in-creasing incidence of abnormalities of the human testis: a re-view, Environ Health Perspect, 101 Supp12 (1993) 65.

4 Sikka S C, Rajasekaran M & Hellstrom W J, Role of oxida-tive s~ and antioxidants in male infertility, J Androl, 16(1995) 464.

5 Sharma R K & Agarwal A, Role of reactive oxygen speciesin male infertility, Urology, 48 (1996) 835.

6 de Lamirande E & Gagnon C, Reactive oxygen species andhuman spennatoZ08. II. Depletion of adenosine triphosphateplays an important role in the inhibition of sperm motility, JAndrol, 13 (1992) 379.

7 Critical reviews of oxidative stress and aging. Advances inbasic science, diagnositcs and interventions, edited by R GCutler and H Rodriguez (World Scientific/National Aca-demic Press/Imperial College Press, New Jersey) 2002,1700. Critical reviews of oxidative s~ and aging.

8 Sikka S C, Relative impact of oxidative stress on male re-productive function, C,," Med Chem, 8 (2001) 851.

9 Darley-Usmar V, Wiseman H & Halliwell B, Nitric oxideand oxygen radicals: A question of balance, FEBS Len, 369(1995) 131.

10 Garrido N, Meseguer M, Simon C et al., Pro-oxidative andanti-oxidative imbalance in human semen and its relationwith male fertility, Asian J Androl, 6 (2004) 59.

11 Aitken R J & Baker H W, Seminal leukocytes: Passengers,terrorists or good samaritans?, Hum-Reprod, 10 (1995) 1736.

12 Shalika S, Dugan K, Smith R D et aL, The effect of positivesemen bacterial and Ureaplasma cultures on in vitro fertili-zation success, Hum Reprod, 11 (1996) 2789.

13 Aitken R J, Fisher H M, Fulton N et al., Reactive oxygenspecies generation by human spermatozoa is induced by ex-ogenous NADPH and inhibited by the flavoprotein inhibitorsdiphenylene iodonium and quinacrine, Mol Reprod Dev, 47(1997) 468.

14 Hendin B N, Kolettis P N, Sharma R K et al., Varicocele isassociated with elevated spermatozoal reactive oxygen spe-cies production and diminished seminal plasma antioxidantcapacity, J Urol, 161 (1999) 1831.

15 Ochsendorf F R. Infections in the male genital tract and re-IM:tive oxygen species, Hum Reprod Update, 5 (1999) 399.

INDIAN J EXP RIOL. NOVEMBER 2005972

tive stn:ss in men with idiopathic and male factor infertility.Fertil SteriI. 79 Suppl3 (2003) 1597.

53 Twigg J, Fulton N, Gomez E et aI., Analysis of the impact ofintracellular reactive oxygen species generation on thestructural and functional integrity of human spermato~Lipid peroxidation, DNA fragmentation and effectiveness ofantioxidants, Hum Reprod. 13 (1998) 1429.

54 Paasch U, Agarwal A, Gupta A K et aI., Apoptosis signaltransduction and the maturity status of human spermatozoa.Ann N Y Acad Sci, 1010 (2003) 486.

55 Hampton M B, Fadeel B & Orrenius S, Redox regulation ofthe Calo-pases during apoptosis. Ann N Y Acad Sci, 854 (1998)328.

56 Agarwal A, Saleh R A & Bedaiwy M A. Role of reactiveoxygen species in the pathophysiology of human reprodtM:-tion, Fertil Steril, 79 (2003) 829.

57 Wang X. Sharma R K, Sikka S C et al., Oxidative stress isassociated with increased apoptosis leading to spermat07A)8DNA damage in patients with male factor infertility. FertilSteril, 80 (2003) 531.

58 Halliwell B, Antioxidant defence mechanisms: From the be--ginning to the end (of the beginning), Free Radic Res, 31(1999) 261.

59 Padron 0 F, Brackett N 1., Sharma R K et al., Seminal re.:-tive oxygen species and sperm motility and nxxphology inmen with spinal cord injury, Fertil SteriI, 67 (1997) 1115.

60 Shekarriz M. Thomas A J, Jr. & Agarwal A. Incidence amlevel of seminal reactive oxygen species in normal nx:n.Urology, 45 (1995) 103.

61 Esfandiari N, Saleh R A. Blaut A Pet aI.. Effects of tem-perature on speno motion characteristics and reactive oxygenspecies. Int J Fertil Womens Med, 47 (2002) 227.

62 Aziz N. Saleh R A, Sharma R Ketal., Novel associationbetween spenD reactive oxygen species production, spennmorphological defects, and the speno deformity index, FertilSteriI, 81 (2004) 349.

63 Traber M G, van der Vliet A, Reznick A Z et aI., Tobacco-related diseases. Is there a role for antioxidant micronunientsupplementation'!, Clm Chest Med, 21 (2<XX» 173.

64 Cross C E. Halliwell B. Borish E T et aI.. Oxygen ooicalsand human disease. Ann Intern Med, 107 (1987) 526.

65 Vine M F, Tse C K. Hu P et aI., Cigarette smoking and se-men quality. Fertil SteriI. 65 (1996) 835.

66 Kunzle R. Mueller M D, Hanggi W et al., Semen quality ofmale smokers and non-smokers in infertile couples, FertilSteriI. 79 (2003) 287.

67 Saleh R A. Agarwal A. Sharma R K et al., Effect of cigarettesmoking on levels of seminal oxidative stress in infertilemen: a prospective study. Fertil Steril. 78 (2002) 491.

68 Pacifici R. AJtieri I, Gandini L et al., Nicotine. cotinine. aIxItrans-3-bydroxycotinine levels in seminal plasma of SDK)t-ers: Efects on sperm parameters, Ther Dnlg Monit, 15 (1993)358.

69 Goverde H J, Dekker H S. Janssen H J et al., Semen qualityand frequency of smoking and alcohol consumptioo-anexplorative study. Int J Fertil Menopausal StJId. 40 (1995)135.

70 Marinelli D, Gaspari 1., Pedotti Petal., Mini-review ofstudies on the effect of smoking and drinking habits 00 se-men parameters, Int J Hyg Environ Health. 207 (2004) 185.

33 Sengoku K, Tamate K. Yoshida T et aL. Effects of low con-centrations of nitric oxide on the zona pellucida bindingability ofhurnan spennatozoa. Fertil Steril. 69 (1998) 522.

34 Yeoman R R, Iones W D & Rizk B M, Evidence for nitricoxide regulation of hamster spenD hyperactivation, J Androl.19 (1998) 58.

35 Balercia G, Moretti S, Vignini A et aI., Role of nitric oxideconcenttations on human spenD motility, J Androl, 25 (2004)245.

36 Donnelly E T, Lewis S E, Thompson W et aL, SpenD nitricoxide and motility: The effects of nitric oxide synthasestimulation and inhibition. Mol Hum Reprod, 3 (1997) 755.

37 Gamer B. van Reyk D, Dean R T et aL, Direct copper re-duction by _~hages. Its role in low density lipoproteinoxidation, J BioI Chem.. 272 (1997) 6927.

38 Ali S F, Duhart H M, Newport G D et aI., Manganese-induced reactive oxygen species: Comparison between Mn+2and Mn+], Neurodegeneration, 4 (1995) 329.

39 Benoff S, Hurley I R. Millan C et aI.. Seminal lead concen-trations negatively affect outcomes of artificial insemination.Fertil Steril, 80 (2003) 517.

40 Shekarriz M. DeWire D M. Thomas A I,Ir. et al., A methodof human semen centrifugation to minimize the iatrogenicspeno injuries caused by reactive oxygen species, Eur Urol.28 (1995) 31.

41 Halliwell B, Gutteridge 1 M & Blake D. Metal ions and oxy-gen radical reactions in human inflammatory joint disease,Philos Trans R Soc Lond B BioI Sci, 311 (1985) 659.

42 Davies K I, Delsignore M E & Lin S W, Protein damage anddegradation by oxygen radicals. II. Modification of aminoacids, J BioI Chern, 262 (1987) 9902.

43 Stadtman E R & Levine R L. Free radical-mediated oxida-tion of free amino acids and amino acid residues in proteins,Amino Acids, 25 (2003) 207.

44 Tominaga H, Kodama S, Matsuda N et aI., Involvement ofreactive oxygen species (ROS) in the induction of genetic in-stability by radiation, J Radial Res (Tokyo), 45 (2004) 181.

45 Agarwal A & Saleh R A. Role of oxidants in male infertility;Rationale, significance, and treatment, Urol Clin North Am,29 (2002) 817.

46 Aitken R 1 & Krausz C. Oxidative stress, DNA damage andthe Y chromosome, Reproduction, 122 (2001) 497.

47 Lopes S, Iurisicova A, Sun 1 G et aL, Reactive oxygen spe-cies: Potential cause for DNA fragmentation in human sper-matozoa, Hum Reprod, 13 (1998) 896.

48 Imlay 1 A & Linn S, Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hy-drogen peroxide. J Bacteriol, 166 (1986) 519.

49 Moustafa M H. Sharma R K. Thornton 1 et aL, Relationshipbetween ROS production, apoptosis and DNA denaturationin spennatozoa from patients examined for infertility, HumReprtNl, 19 (2004) 129.

50 Agarwal A & Allamaneni S S. The effect of sperm DNAdamage on a§isted reproduction outcomes. A review, Min-erva Ginecol, 56 (2004) 235.

51 Agarwal A & Said T M, Role of sperm chromatin abnor-malities and DNA damage in male infel1ility, Hum ReprodUpdate. 9 (2003) 331.

52 Saleh R A. Agarwal A. Nada E A et aI.. Negative effects ofincreased sperm DNA damage in relation to seminal oxida-

AGARW AL & PRABAKARAN: PREVENTION OF STR~ IN MALE REPRODUcnVE PHYSIOLOGY 973

89 Tubaro M. Cavallo G. Pcnsa V et aL. ~ ordie rormation or hydroxyl r:MIicals in -=ute my<x:aldia) in-ran:tioo in man using salicylate as probe. Cardiology. IKJ(1992) 246-

90 luli~ L. Pratico D. Gbi'ielli A el ai. Re;k:tj(HJ or dipyrida-mole with die hydroxyl r:MIical. Lipids. 27 (1992) 349-

91 Bass D A. Pan:e J W. [Xchatelet L R el aL. I-low cytOIIK:Iricst1Kiies of oxidative product fOnllatMm by ~Is: Agra«bl JCSIX)nse to ~ SIiIIlUIaIjoo. J ,--, 130(1983) 1910.

92 Chirico S. Srojth C. MarchaIII C el aL. Lipid peroxidatKm inhyperlipidaerojc patients- A stlxiy or plasma using aD HPLC-based thiobarbituric acid lest. Free RIMlic Res u-. 19

(1993) 51-93 Romu L J & M(XTOW J D. ~ of F(2)-

i~ as an inck;x of oxjdalive stress ill viWI. FlURadic BioI Med. 28 (2(XX) 505-

94 FaIn S S & M(XTOW J D. The ~mES: U~ .-oo-:uof aIXhj(k)oic acid oxidatKMl-a review. CIUT Med a-. 10

{2003)I723-95 Tarpey M M. Wink D A & Grist-D M B. MedIIXIs rm- ck-

fectjon of ~ve DM:taboIiaes of oxygcu ami aibozea: 1ft

vilro and in viw1 aMlsick:rations. Ala J PIrysiol Rep/lllegrCDrJfl' Plrys~ 286 (2004-) R431-

96 Said T M. KanaI N. Sb8rma R K el aL. EIIhaIM:aI dK:oBlD-mi~ assay vs cokJrinaric -y fm- ~ ordie M)(aJ aDIioxidanl CaJ.:ity of huma8 seminal plasma. JAndrol. 24 (2003) 676-

97 Agarwal A. AllamalEOi S S & Said T M. Cl.:mllumiIK:S-cence lCdUIique fm- measuring ~ve oxygcu sp:cK:s. RL-prod Bu-d 0IrliIIe. 9 (2004) ~

98 Sharma R K. ~ F F. Nelson D R et IIL,"I1E ~-bve oxygen specics-tocal amioxidanl ~i a:..e is a -~ of oxidative stress to pmicI male iofatiIity, H-Reproti. 14 (1999) 2SO1-

99 Koleuis P N, ShamIa R K. PasquakJuo F F et m., Effect ofseminal oxidalive stress on fertility aftf:I" VasecI(.ny ~Feltil Steril. 71 (1999) 249-

100 Jeulin c. Soufir J C. WdX2" PetaL. r_a- iv-, ia --man 5IM:fDJafaI.Oa and scminal plasma. GtI8tte ~ 24(1989) 185-

101 Fraga C G. MddIOit P A. Shigeuaga M K et iii.. ~acid protects against ~ ox.id8live DNA ~in human 5fx:nn. Proc NIIIl ACI.I Sri USA. 88 ( 1991 )11003-

102 Thiele J J, FriesIeben H J. Fudas Jet IlL, AsaJItJic xid a8IUI3fr. in human seminal plasma: (ktcnniD3IDI iI8:nda-~ with clM:milumil~ ia w3sIa ~ H-Rep,oo. 10 (1995) 110-

103 Aitken R J & ClaJtSJD J S. SigllificalK% of ~ oxygcaspa:ies aIMi anboxidaocs in ~ning the efficacy of spermJKqmlation k:choiques, J Androl. 9 (1988) 367-

104 ~ A. PicaRk> M. Gandini Let at. GIutadJjcx.:: b~of dyspermia: Effm on the liPJlJeroxNtMion ~ H-Rep,oo. 9 (1994) 2044-

105 TauIx:r P F. Zaneveld L J. ~ng D et at. con..-:.n ofhuman split ej~ 1- Sp:I~ fnM:tI&:. ~globulins. albumin. lal:lDfelTin. b3R§ferriD aIM! odIa- plasmalXOfeios. J Reprod Feltil. 43 (1975) 249-

71 A~1l A. Ernst E & Bonde J P. High spenn density anKJngmembers of organic fanners' association. LallCel. 343 (1994)

1498.72 Agarwal A. Ikemoto I & Loughlin K R. Relationship of

spenn parameters with levels of reactive oxygen species insemen specimens.} Ural. 152 (1994) 107.

73 Shekarriz M. Thomas A J. Jr. & Agarwal A. Effects of timeand spenn concentration on reactive oxygen species fonna-tion in human semen. Arch Androl. 34 ( 1995) 69.

74 McKinney K A. uwis S E & Thompson W. ReaM:tive oxy-gen ~ies generation in human spenD: Luminol and loci-genin chemiluminescence probes, Arch Androl. 36 (1996)

119.75 Aitken R J. Buckingham D W & West K M, Reactive oxy-

gen species and human spennatozoa: Analysis of the cellularmechanisms involved in luminol- and locigenin-depelxientchemiluminescence.} Cell Physiol, 151 (1992) 466.

76 Said T M. Paasch U, Glander H J el aI.. Role of caspases inmale infertility, Hum Reprod Update, 10 (2004) 39.

77 Ford W C. Regulation of spenn function by reactive oxygenspecies, Hum Reprod Update. 10 (2004) 387.

78 Esfandiari N, Shamla R K. Saleh R A el aL, Utility of thenitroblue tetrazolium reduction test for assessment of re.:-tive oxygen species production by seminal leukocytes andspermatozoa. } Androl, 24 (2003) 862.

79 Kirby T W & Fridovich I. A picomolar &peCtrophotomebicassay for superoxide dismutase, Anal Biochem, 127 (1982)

435.80 Elferink J G. Measurement of the ~tabolic burst in human

neutrophils: A comparison.betWeen cytochrome c and NOTreduction, Res Commun Chem Pathol Phannacol, 43 (1984)

339.81 Weber G F, The ~rement of oxygen-derived free radi-

cals and related substances in medicine. } Clin Chem ClinBiochem. 28 (1990) 569.

82 Buettner G R. Spin trapping: ESR ~ters of spin ~-ducts. Free Radii: Bioi Med. 3 (1987) 259.

83 Kaur H. Edmonds S Eo Blake D R el aL. Hydroxyl radicalge~rarion by ~matoid blood and knee joint synovialfluid. Ann Rheum Du, 55 (1996) 915.

84 Kaur H & Halliwell B. Aromatic hydroxylation of phenyl-alanine as an assay for hydroxyl radicals. Measure~t ofhydroxyl radical fonnation from ozone and in blood frompremature babies using improved HPLC methodology, AnalBioclrem. 220 (1994) 11.

85 Halliwell B & Kaur H. Hydroxylarion of salicylate andphenylalanine as assays for hydroxyl radicals: A cautionarynote vi~ited for the third time. Free Radk Res, 27 (1997)239.

86 Coudray C & Favier A. Determination of salicylate by-droxylation products as an in vivo ox.idative stress marker.Free Radii: Bioi Med. 29 (2{xx) I~.

87 Themann C, Teismann P, Kuschinsky K el aL, Comparisonof two independent aromatic hydroxylation assays in combi-nation with intracerebral microdialysis to determine hy-droxyl free radicals.} Neuro.fci Melhods, 108 (2001) 57.

88 Li M. Carlson S, Kinzer J A el aL, HPLC and LC-MS stOO-ies of hydroxylation of phenylalanine as an assay for hy-droxyl radicals generated from Udenfriend's reagent. BiD-chem Biophys Res Commun, 312 (2003) 316.

INDIAN J EXP BIOI.., NOVEMBER 2OOS974

106 Alvarez J G &. Storey B T, Taurioo, hypotaurioe. epinepbri~and albumin inhibit lipid peroxidation in rabbit spennaIozoaand protect against loss of motility, Bioi Reprod, 29 (1983)548.

107 de Lamirande E & Gagnon C, Capacitation-associated pr0-duction of superoxide anion by human spennatozoa. FreeRadic Bioi Med, 18 (1995) 487.

108 Peeker R, Abramsson L &. Marklund S L. Superoxide dis-mutase isoenzymes in human seminal plasma and spermato-zoa, Mol Hum Reprod, 3 (1997) 1061.

109 Baker H W, Brindle J, Irvine D S et aL, Protective effect ofantioxidants on the impairment of sperm motility by acti-vated polymorphonuclear leukocytes, Fertil Steril, 65 (1996)411. -

110 Zini A, Fischer M A, Mak V et aI., Catalase-like and super-oxide dismutase-like activities in human seminal plasma.Ural Res, 30 (2002) 321.

III Sikka S C, Role of oxidative stress and antioxidants in an-drology and assisted reproductive technology, J Androl, 25(2004) 5.

112 Wang x. Falcone T, Attaran M et aI., Vitamin C and vitaminE supplementation reduce oxidative stress-induced embryotoxicity and improve the blastocyst development mte, Fel1ilSteril, 78 (2002) 1272.

113 Burton G W, Joyce A & Ingold K U, First proof that vitaminE is major lipid-soluble, chain-breaking antioxidant in hu-man blood plasma, Lancet, 2 (1982) 327.

114 Agarwal A, Nallella K p, Allamaneni S SetaL, Role of an-tioxidants in treatment of male infertility: An overview ofthe literature, Reprod Biomed Online, 8 (2004) 616.

115 Lutsenko E A, Carcamo J M & Golde D W, Vitamin C pre-vents DNA mutation induced by oxidative stress. J BioiChem, 277 (2002) 16895.

116 Irvine D S, Glutathione as a treatment for male infertility,Rev Reprod, 1 (1996) 6.

117 Y<xIa Y, Nakazawa M, Abe T et aI., Prevention of doxorubi-cin myocardial toxicity in mice by reduced glutathione, Can-cer Res, 46 (1986) 2551.

118 Hansen J C &. Deguchi Y, Selenium and fertility in animalsand man--A review, Acta Vet Scand, 37 (1996) 19.

119 Ursini F, Heirn S, Kiess M et aI., Dual function of d1e scle-ROprotein PHGPx during spenn maturation, Science, 285(1999) 1393.

120 Lenzi A, Gandini 1., Picardo M et aL, Lipoperoxidationdamage of SpemIatowa polyunsaturated fany acids (pUPA):Scavenger ~hanisms and JX)SSible scavenger dlerapies,Front Biosci, 5 (2(XX) EI.

121 Lenzi A, Gandini 1., Lombardo F et aI., Polyunsaturatedfany acids of genn cen membranes, glutathione and bluta-thione-dependent enzyme-PHGPx: from basic to clinic,Contraception, 65 (2002) 301.

122 Lenzi A, Lombardo F, Gandini L et aL, Glutathione dlerapyfor male infertility, Arch Androl, 29 (1992) 65.

123 Oeda T, Henkel R. Ohmori H et aI., Scavenging effect of N-acetyl-L-cysteine against reactive oxygen species in human~: A JX)SSible therapeutic mOOality for male factor in-fertility?, Andrologia, 29 (1997) 125.

124 Sies H &. Stahl W, Vitamins E and C, beta-carotene, andother caroteooids as antioxidants, Am J Clin Nutr, 62 (1995)1315S.

125 Frei B, Kim M C &. Ames B N, Ubiquinol-IO is an effectivelipid-soluble antioxidant at physiological concentrations,Proc Nad Acad Sci USA, 87 (1990) 4879.

126 MeUors A &. Tappel A 1., The inhibition of mitochondrialperoxidation by ubiquinone and ubiquinol, J Bioi Chern, 241(1966) 4353.

127 Lewin A &. Lavon H, The effect of coenzyme QIO on spermmotility and function. Mol Arpects Med, 18 Suppl (1997)S213.

128 Agarwal A &. Said T M, Carnitines and male infertility, Re-prod Biomed Online, 8 (2004) 376.