Indian Journalof ExperimentalBiology Vol. 43. November2005.pp 963-974 ReviewArticle Mechanism,measurement, 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 Introduction 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 processes.as can affect the individual molecules and thus the entire organism.as 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 presentcentury7. Table I-Diseases anddegenerativeprocessesmediatedby oxidativestress Alzheimer's disease Iron overload Autoimmune disease Ischemic-reperrusion Rheumatoidarthritis injury Maculardegeneration Segmentalprogeria disorders Cancer Cardiovascular Multiple sclerosis .Correspondentauthor disease Phone: (216) 444-9485.Fax: (216) 445-6049; Cataractogenesis Musculardystrophy E-mail: Agarwaa@ccf.org; Pancreatitis Website:www.clevelandclinic.orgjReproductiveResearchCenter Diabetes Parkinson'sdisease Aging INDIAN J EXP BIOL. NOVEMBER 2005 964 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. .0-0. .0.-0: Triplet oxygen(rr) (Groundstate) 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. Monovalent reduction Monovalent reduction .0-0. Triplet oxygen (tt) (Ground state) .0.-0: Superoxide(i ~) (Reactivestate) H:O-O:H Hydrogenperoxide . Fenton reaction: Fe2++ H2~' Fe3++OH-+ H2O+~+ 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 fr I k .. 19-21 spermatozoa 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 OR' H+ ~+OIr+OH' 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 organisms. 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). Non-Radicals Peroxynitrite ONOO- Radic~ Hydroxyl on' Superoxide Nitric Oxide ~... NO. Hypochloricacid HOC! HydrogenPeroxide HA Thyl RS. -I~ Peroxyl R~. LOQ' Singlet Oxygen Ozone Lipid peroxyl Lipid peroxide ~ LOOH Table 3- Typesof reactivenitrogenspeciesthat induces oxidative stress Nitrous oxide NO' Nitrosyl carlon NO. Peroxynitrite OONO- Nitrogen dioxide N~ Peroxynitrousacid ONOOH Dinitrogen trioxide NZO3 Nitrous acid HN~ Nitroxyl anion NO- Nitryl chl,oride NOzCl . AGARWAL & PRABAKARAN: PREVENTION OF STRESSIN MALE REPRODUcrIVE PHYSIOLOGY 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. 965 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 glyceraldehydephosphatedehydrogenase. 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- Initiation: LH+R" L"+RH Propagation: L.+~ LOO' 966 INDIAN 1 EXP BIOL, NOVEMBER 2005 Damage 10 proleins-Sulphur-containing amino 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 DNAs3. Apoplosis-Apoptosis 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. AGARWAL Ii; PRABAKARAN: PREVEN'nON OF STRESSIN MALE REPRODUCTIVEPHYSIOLOGY 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 generation77. 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 967 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 968 INDIAN J EXP DiaL. NOVEMBER 2005 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. the statistical score obtained from ROS and T AC 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 tometers. 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 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. ~ AGARWAL & PRABAKARAN: - PREVENTION OF STRESS IN MALE REPRODUCTIVE PHYSIOLOGY - Table 4- Different classesof antioxidantsthat scavengeROS Emymatic antioxidants Superoxidedismutase Catalase Glutathioneperoxidase/glutathione reductase(GPXKiRD) Non-eazymadcantioxidants Vitamin C, Vitamin E, Vitamin C and Vitamin A (carotenoids) Proteinslike Albumin, Transferrin,HaptOgJobulin, CeroJopasmin. GJutadli~ (GSH) Pyruvate . 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. SOD 20z-+2W . HA+Oz 969 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 GSSG+ NADPH + W G~ 2GSH+ R(OOH)COOH .GSSG + GPX R(OH)COOH + H2O Net reaction NADPH+ R(OOH)COOH +W . NADY + R(OH)COOH + H2O 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. C~~l~~ 2HA . H~ + 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 spennatoZ08. GPX reactswith peroxidesand requires act as preventive antioxidants. Some of the effects of reducedglutathione(GSH) as the reductivesubstance these antioxidants are reviewed below'!!. INDIAN J EXP BIOL, NOVEMBER 2005 970 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 againstperoxidativedamagel25. 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+ Fe3+JCu+ +~ Fe}+/Cu + +H~ .. A -+ Fe 2+/CU + ~. + Fe3+/Cu 2+ OH + OH' + Fe3+/Cu2+ Glutathione-Glutathione 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. . Conclusion 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 consequences 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 AGARWAL & PRABAKARAN: PREVENTION OF STRESS IN MALE REPRODUCTIVE PHYSIOLOGY useof antioxidants.Furtherresearchis requiredin this field to fully understandthe complicatedeventsassociated with OS and to eventually develop effective strategiesto assist patients with OS-associatedmale infertility . 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