Mechanism, measurement, and prevention of oxidative stress in … · 2005. 12. 12. · Indian Journal of Experimental Biology Vol. 43. November 2005. pp 963-974 Review Article Mechanism,

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: [email protected];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

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

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~SOD20z-+2W . HA+Oz

SOD scavenges bodl intracellular and extracellularsuperoxide 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.

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