Indian Journal of Experimental Biology Vol. 43, November 2005, pp 963-974

Review Article

Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology

Ashok Agarwal* & Sushil A Prabakaran

Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Glickman Urological Institute, and Department of Obstetrics and Gynecology, The Cleveland Clinic Foundation, 9500Euelid A venue, Desk A 19 .1, Cleveland,

Ohio, 44195, USA

Numerous factors influence male fertility. Among these factors is oxidative stress (OS), which has elicited an enor­mous interest in researchers in recent period. Reactive oxygen species (ROS) are continuously produced by various meta­bolic and physiologic processes. OS occurs when the delicate balance between the production of ROS and the inherent anti­oxidant capacity of the organism is distorted. Spermatozoa are particularly sensi tive to ROS as their plasma membrane contains polyunsaturated fatty acids (PUFA), which oxidizes easily. They also lack cytoplasm to generate a robust preven­tive and repair mechanism against ROS. The transition metal ions that are found in the body have a catalytic effect in the generation of ROS. Lifestyle behaviours such as smoking and alcohol use and environmental pollution further enhance the generation of ROS and thus, cause destructive effects on various cellular organelles like mitochondria, sperm DNA etc. This article analyzes the detrimental effects of OS on male fertility, measurement of OS and effective ways to decrease or elimi­nate them completely . We have also provided information on oxidative stress in other systems of the body, which may be applied to future research in the field of reproductive biology.

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 infertility' . 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 ml3

. 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 their 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

·5

. ROS are free radicals and peroxides that are derived from the

*Correspondent author Phone: (216) 444-9485, Fax: (216) 445-6049; E-mail : Agarwaa@ccf.org;

Website: www.clevelandclinic.org/ReproductiveResearchCenter

metabolism of oxygen and are present in all aerobic organisms. They play a significant role in many bio­logical processes6

. OS can affect the individual mole­cules and thus the entire organism. OS is believed to be one of the major causes of many human diseases. Some of the OS-mediated pathologies are listed in Table I. Virtually every discipline of medicine is in­terested in OS and its implications. It is believed that the new field of 'Oxidative Stress, Diagnostics and Therapeutics' may have a significant impact on the economics, science and the practice of medicine in the present century7

.

Table !-Diseases and degenerative processes mediated by oxidative stress

Alzheimer's Iron overload Parkinson's disease disease

Autoimmune Ischemic-reperfusion Rheumatoid arthritis disease injury

Cancer Macular degeneration Segmental progeria disorders

Cardiovascular Multiple sclerosis Aging di sease

Cataractogenesis Muscular dystrophy

Diabetes Pancreatitis

964 IND IAN J EXP B!OL. NOVEMBER 2005

Mechanism of ROS formation

Oxygen in the atmosphere has two unpaired elec­trons, and these unpaired electrons have parallel spins. Oxygen is usually non-reacti ve 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.

·o-o· ----IIJlo~ o·-o:

Triplet oxygen (fj) Singlet oxygen (j 1) (Ground state) (Highly reactive)

b) Monovalent reduction (accept a si ngle electron).

Superoxide is formed in the first monovalent re­duction reaction, which undergoes further reducti on to form hydrogen peroxide. Hydrogen peroxide is fur­ther reduced to hydroxyl radicals in the presence of ferrous salts (Fe2+). This reaction was first described by Fenton and later developed by Haber and Weiss .

Monovalent reduction

Monovalent reduction

·o-o· ____ ..,...., ·o ·--o: ____ ..,....,

Triplet oxygen (fi) (Ground state)

Fenton reaction :

Superoxide (j!) (Reactive state)

H:O-O:H Hydrogen peroxide

Fe2+ + H20 2· ----~IIJlo~ Fe3+ + OH- + OH·

Haber-Weiss reaction:

H20 2 + OH ·

H20 2 + o 2·-

Types ofROS

IIJlo H20+02+ W ___. 0 2+ OH- + OH·

ROS represent a broad category of molecules that indicate the collection of radicals and non-radical oxygen derivatives. These intermediates may partici­pate in reactions that give rise to free radicals that damage organic substrates. Table 2 li sts the types of ROS that exist as radicals and free radicals in living organi sms.

In addition, there is another class of free radicals that are nitrogen derived called reactive nitrogen spe­cies (RNS). They are nitrogen-derived molecules and are considered a subclass of ROS8

•9 (Table 3).

Origin of ROS in male reproductive system Human semen consists of different types of cells

such as mature and immature spermatozoa, round cells from di ffe rent stages of the spermatogenic proc­ess, leukocytes and epithelial cells. Of these, leuko­cytes and immature spermatozoa are the two main sources of ROS 10. Leukocytes, particularly neutro­phils and macrophages, have been associated with excessive ROS production, and they ultimately cause sperm dysfuncti on11

·1R. ROS produced by leukocytes

forms the first line of defense in any infectious proc­ess. DNA and structural damage can be found in spermatozoa from leukocytospermic patients 19-:21 . Leukocytes act e ither directly by ROS synthesis or indirectly by other neighboring white cells via soluble factors as cytoki nes 10'22

. ROS is significantly and positive ly correlated with interleukin-8, a potent stimulator of pro-inflammatory cytokines23·24 . The scavenging effect of antioxidants are greatly dimin­ished under such infectious conditions 14.

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 qualit/5

. 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- Types of reactive oxygen species that exist as radicals and free radica ls

Radicals Non-Radicals

Hydroxyl OH" Peroxynitrite oNoo-

Superoxide o2·- Hypochloric acid HOC I

Nitric Oxide NO" Hydrogen Perox ide H20 2

Thy I RS" Singlet Oxygen -lo2

Peroxyl R02. Ozone OJ

Lipid peroxy l Loo· Lipid peroxide LOOH

Table 3- Types of reacti ve nitrogen species that induces oxidati ve stress

Nitrous ox ide NO" Nitrosyl cation NO+

Peroxynitrite OONO- Nitrogen dioxide N02.

Peroxynitrous acid ONOOH Dinitrogen trioxide N20 J

Nitroxyl anion No- Nitrous acid HN02

Nitry l chloride N02CI

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

and the other is NADH-dependent oxido-reductase (diphorase) system at the mitochondrial level27

. There is a strong positive correlation between immature spermatozoa and ROS production, which in turn is negatively correlated with sperm quality. Further­more, it has been noticed that as the concentration of immature spermatozoa in the human ejaculate in­creases, the concentration of mature spermatozoa with damaged DNA rises25

.

Physiological role of ROS Spermatozoa acquire the capacity to move during

their transit through the epididymis, but lack the abil­ity to fertilize. After ejaculation, they undergo a series of physiological changes called capacitation28

. Ca­pacitation is followed by acrosome reaction, which is triggered by the zona pellucida of the ovum and al-

~' .,. h 29 30 c . . lows the sperm to 1ertt tze t e egg · . apacttatwn and the acrosome reaction are redox-regulated proc­esses . Exogenous administration of 0 2.- or Hz0 2 pro­motes capacitation and the acrosome reaction whereas the addition of antioxidants prevents them from un­dergoing these events. This demonstrates the impor­tance of ROS in human sperm functions 31

•

Nitric oxide (NO"), a free radical with a relatively long half-life (7 s), promotes capacitation. No detect­able activity of nitric oxide synthase (NOS) is found in spermatozoa, which suggests that it originates from tissues or fluids from the female genital tract30

. Low concentrations of NO cause a significant increase in

· · 32 II .d b. d. 33 NO capacttatton· and zona pe uct a m mg . regulates cyclic adenosine monophosphate (cAMP) concentration and induces capacitation of spermato­zoa through the action of adenyl cyclase. Finally, NO also plays a role in sperm hyperactivation34

Pathological role of ROS Effect of excessive NO-NO is detrimental to

sperm motility in high concentrations35. It may react

with superoxide or hydrogen peroxide, resulting in the formation of peroxynitrite, hydroxyl radical, N02 or singlet oxygen, which causes oxidation of sperm membrane lipids and thiol proteins36

. In addition, NO may also inhibit cellular respiration by nitrosylation of heme in mitochondrial enzymes, aconitase and glyceraldehyde phosphate dehydrogenase. This causes a decrease in the level of adeno~ine triphosphate, which thereby affects the kinetics of spermatozoa35

.

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

nations to oxygen, forming 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) causes neurotoxic­ity via ROS generation38

. TMis such as lead (Pb) and cadmium (Cd) also affect the male reproductive sys­tem. These metals either directly affect the sperm plasma membrane or catalyze the ROS formation. Pb causes relative zinc (Zn) deficiency, while Cd pre­dominantly affects the distribution of Zn in the body.

The authors of a study consisting of industrial workers have identified a decrease in the workers' semen quality, semen volume, sperm count, sperm motility and forward progression. The authors have also reported an increase in abnormal sperm morphol­ogy and an impairment of prostatic secretory function. They have also observed that both Pb and Cd damage the DNA bases as measured by the levels 8-hydroxy-2'-deoxyguanosine (8-0HdG). Seminal plasma lead levels and artificial insemination cycle fecundity have a strong negative correlation39

. Automobile pollution increases the blood lead level, which decreases sperm count and viability.

Damage to lipids-Lipids are most susceptible macromolecules and are present in the sperm plasma membrane in the form of polyunsaturated fatty acids (PUPA). ROS attack PUPA in the cell membrane leading to a chain of chemical reactions called lipid peroxidation40

. The reaction occurs in three distinct steps-initiation, propagation and termination. During initiation, the free radicals react with fatty acid chain (LH) and releases lipid free radical. This lipid radical further reacts with molecular oxygen to form lipid

. peroxyl radical. Peroxyl radicals again react with fatty acid to produce lipid free radical and this reaction is propagated. During termination, the two radicals react with each other, and the process comes to an end41

•

This process of fatty acid breakdown produces hydro­carbon gases (ethane or pentane) and aldehydes.

Initiation: LH + R· ____ ___...,. L· + RH

Propagation: L· + Oz ____ __...,. Loo·

966 INDIAN J EXP BIOL. NOVEMBER 2005

LH + Loo· ----•IJilo LOOH + L·

L +02 IJilo LOO·

Termination: L· +L· ------11J1lo~ LL

LOO + Loo· IJilo LOOL + Oz LOO· + L· IJilo LOOL

Damage to proteins-Sulphur-containing amino acids , having thiol groups specifically, are very sus­ceptible to OS. Activated oxygen can abstract H atom from cysteine res idues 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 irreversibk 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 to 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 breaks45. 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 men49.52 as indicated by the high level of 8-0HdG, 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 DNA 53.

Apoptosis-Apoptosis is programmed cell death that occurs in a genetically determined fashion 54. It is initiated by ROS-induced oxidative damage, but too much OS can terminate apoptosis by inactivating the caspase enzyme cascade55.57. Antioxidants can either suppress or facilitate apoptosis58. The mitochondrion

is the most important cellular organelle that mediates apoptosis . High levels of ROS disrupt the integrity of the mitochondrial membrane, which in turn releases cytochrome c. It activates the caspase enzyme cascade and triggers apoptosis. Studies in infertile men have shown that high levels of cytochrome c in the seminal plasma indirectly reflect significant mitochondrial damage caused by high levels of ROS. Levels of ROS in infertile men are correlated positively with apopto­sis, which in turn is negatively correlated with con-

. 49 59-62 vent10nal semen parameters ·- .

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

nearly 4000 harmful substances such as alkaloids, nitrosamines, nicotine and hydroxycotinine. Many of these substances generate ROS and RNS63

•64

. Smok­ing has been associated with decreased sperm qual­it/5.67. Reduction in motility is due to the damage caused by ROS to the flagellum and axonemal struc­tures of the tail of spermatozoa. Sperm motility cor­relates negatively with the amount of cotinine and hydroxycotinine in the seminal plasma65·68 . The pres­ence of cotinine and hydroxycotinine in seminal plasma indicates that harmful substances found in to­bacco smoke can penetrate the blood-testes barrier.

Oxidative stress and alcohol-Many processes and factors are involved in causing alcohol-induced OS. Alcohol metabolism results in the formation of NADH, which enhances activity of the respiratory chain, including heightened 0 2 use and ROS forma­tion. One of the by-products of alcohol metabolism, acetaldehyde, interacts with proteins and lipids to form ROS. It is also capable of damaging the mito­chondria leading to decreased A TP production. Alco­hol also induces hypoxia that results in tissue damage. Excessive alcohol consumption is associated with a decrease in the percentage of normal sperm in asthe­nozoospermic patients69

. However, better sperm mor­phology has been observed in men who drink moder­ately70.

OS and environment-Many environmental factors such as radiation, medications and pollutants induce ROS production 15. In a study conducted on Danish greenhouse workers, a high count of spermatozoa was found among organic farmers who grew their prod­ucts without the use of pesticides or chemical fertiliz­ers. The group of blue-collar workers in the above study had a low sperm count in comparison to the above group of workers71 . These studies categorically suggest the need for a healthy environment.

AGARWAL & PRABAKARAN: PREVENTION OF STRESS IN MALE REPRODUCTIV E PHYSIOLOGY 967

Measurement of ROS Chemiluminescence method- The chemilumines­

cence method is the most commonly used technique for measuring ROS produced by spermatozoan.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 identify intracellular and extracellular generation of ROS 74-76. However, there are some serious limitations in measuring the super­oxide formation ; lucigenin undergoes redox cycling, and there are some unknown sources of superoxide generation 77

.

Cytochrome c reduction or nitro blue tetrazolium ( N BT) 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

18 78 spermatozoa · . The two most commonly used rea-gents 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 di smutase (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 by extracel­lular superoxide or other molecules as well 80

.

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 tield. 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. Thi s technique reports on the magnetic properties of un­paired electrons and their molecular environment.

Paramagnetic (unpaired) electrons can exist in two orientations, either parallel (+ I /2 , -I /2) or anti parallel with respect to an applied magnetic field. ESR utilizes the electron spin states energy to obtain absorption spectra. ESR cannot detect paramagentic species such as NO, superoxide and hydroxy l radical (OH) as they are short lived and very low in concentration . This problem can be overcome by adding exogenous spin trap molecules to the unstable free radicals thereby converting them to more stable secondary radicals82

.

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 vivo85

·88

. Many hu­man studies have successfully used salicylate in vivo to determine ROS Ievels89·90.

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-ln flow cytometry, the fluores­cent intensity of the compounds oxidized by ROS is measured. Dihydrorhodami ne 123 and 2, 7 dichloro­tluorescin 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.

Measurement of lipid peroxidation Thiobarbituric acid assay- Lipid peroxidation is a

968 INDIAN J EXP BIOL, NOVEMBER 2005

complex process leading to the formation of various aldehydes including malonaldehyde (MDA). The thiobarbituric acid (TBA) assay is the most common method used to assess changes in MDA. The assay detects TBA-reactive substances via high perform­ance liquid chromatography (HPLC), spectropho­tometry or spectrofluorescence92

. The simple TBA test is highly unreliable as most TBA-reactive materi­als in human body fluids are not related to lipid per­oxidation.

lsoprostane (!soP) method-The best available lipid peroxidation biomarker is isoprostane (!soP), which is a specific end product of the peroxidation of polyunsaturated fatty acids93

•94

. The I soP marker is advantageous in 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 seminal plasma or urine.

Measurement of NO Griess reaction-The Griess reaction is an indirect

measurement of NO. It measures the stable deriva­tives of NO such as N02- and N03- using a spectro­photometer. The Griess reaction is a two-step diazoti­zation reaction. Autoxidation of NO and acidification of nitrite (N02") results in the formation of dinitrogen trioxide (N20 3), which reacts with sulfanilamide to form diazonium derivative. This intermediate com­pound interacts with N-1-naphthylethylene diamine to yield a colored diazo product95.

Fluorescence spectroscopy method-The fluores­cence spectroscopy method is more advantageous than the Griess reaction in that it has a greater sensi­tivity and specificity. The N20 3 generated from the autoxidation of NO or acidification of N02- reacts with aromatic 2,3-diaminonaphthalene (DAN) to yield highly fluorescent 2,3-naphthotriazole (NAT), which is measured using a fluorescent spectropho­tometer95.

Measurement of total antioxidant capacity (TAC) in semen-Assessment of T AC is a complex process because several different antioxidants are present in the semen. Therefore, many methods have been de­veloped to analyze T AC of the semen. The oxygen radical absorbance capacity (ORAC), the ferric re­ducing ability of plasma (FRAP) and the trolox­equivalent antioxidant capacity (TEAC) assays are widely used for measuring T A C. Both the ORAC and TEAC assays are inhibition methods. A sample is

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 measures the fen·ic 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 H20 2. The ca­pacity of the antioxidants in the seminal plasma to decrease the chemiluminescence of the signal reagent as measured by luminometer is compared with that of Trolox (a water-soluble vitamin E analogue) and is measured as molar Trolox equivalents%·97

•

Colorimetric assay-The colorimetric assay meas­ures the absorbance of a sample with the help of spectrophotometer. The results are compared with trolox (a vitamin E analogue) and are given in trolox equivalents. This assay is a rapid and reliable method for measuring seminal TAC and is less expensive than the enhanced chemiluminescence assay96

.

ROS-TAC score-Because each method for ana­lyzing ROS and T AC has its own set of disadvan­tages, it is difficult to accurately predict the OS status of the semen. To overcome this problem, Sharma et al. have proposed the ROS-TAC score98

'99

. This is the statistical score obtained from ROS and T AC lev­els by principal components analysis. To predict the OS level, a cutoff value of 30 was established using a group of normal healthy controls. Most of the men who had a ROS-T AC score below the cutoff value were found to be infertile. Further, it was observed that an infertile male with a ROS-TAC score higher than 30 could eventually initiate a pregnancy 17

•

Antioxidants Antioxidants can fit into two broad categories­

enzymatic 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, catalase 100 and GPX/GRD system.

AGARWAL & PRABAKARAN: PREVENTION OF STRESS IN MALE REPRODUCTIVE PHYSIOLOGY 969

Table 4-- Different classes of antioxidants that scavenge ROS

Enzymatic antioxidants Superoxide dismutase Catalase Glutathione peroxidase/glutathione reductase (GPX/GRD)

Non-enzymatic antioxidants Vitamin C, Vitamin E, Vitamin C and Vitamin A (carotenoids) Proteins like Albumin, Transferrin, Haptoglobulin, Cerulopasmin. Glutathione (GSH) Pyruvate Ubiquinol

In addition, it has an array of non-enzymatic antioxi­dants-ascorbate101, urate 102, vitamin E )(l3, pyruvate6

,

glutathione 104, albumin, vitamin A, ubiquinol 105, tau­rine and hypotaurine)(l6. 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 an­tioxidants106. 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. SODl (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, Cu/Zn-SOD and Mn­SOD are the main forms. SOD catalyzes the dismuta­tion of superoxide into hydrogen peroxide and oxy­gen.

SOD

SOD scavenges both intracellular and extracellular superoxide radical and prevents the lipid peroxidation of plasma membrane. However, it should be conju­gated with catalase or GPX/ to prevent the action of H20 2, which promotes the formation of hydroxyl radica!s 100. SOD also prevents hyperactivation and capacitation induced by superoxide radicals 107. This would suppress the occurrence of these reactions

lb c · 1 . 108 premature y e1ore eJacu at10n . Glutathione peroxidase/reductase system (GPXI

GRD )- Importance of the GPX/GRD system is cen­tered on its antilipoperoxidative defense in human spermatozoa. GPX reacts with peroxides and requires reduced glutathione (GSH) as the reductive substance

donating an electron. It contains selenium at its active center, which is required for its optimal functioning. This explains the importance of selenium in male in­fertility. Four isozymes of GPX present in humans contain selenium in their active center. The first, iso­zyme GPX 1, prevents apoptosis induced by OS. The second and the third isozymes are found in the gas­trointestinal tract and in plasma, respectively. The fourth form acts directly on membrane phospholipid hydroperoxides and detoxifies them. It is present in high levels in the testis.

GSSG + NADPH + W ... GSH + NADP+ GRD

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

R(OH)COOH + H20

Net reaction NADPH + R(OOH)COOH + W ... NADP+

+ R(OH)COOH + H20

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 conver­sion of NADP+ to NADPH.

Catalase-Catalase detoxifies both intracellular and extracellular hydrogen peroxide to water and oxygen 109. In addition, catalase activates No·-induced sperm capacitation, which is a complex mechanism involving H20/0. Catalase activity has also been de­tected in human spermatozoa and seminal plasma 100.

Catalase 2H202 ... H20 + Y2 02

Seminal catalase activity in samples of vasecto­mized men has been found to be similar to that of non-vasectomized men, suggesting that the origin of the activity is neither testicular nor epididymal 110.

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

ent in the semen, including vitamin C, vitamin E, glutathione, urate, ubiquinone and bilirubin are pres­ent extracellularly in seminal plasma and are consid­ered chain-breaking antioxidants. Other non­enzymatic antioxidants such as vitamin E, vitamin A, haptoglobulin, transferrin and ceruloplasmin are pres­ent in the plasma membrane of the spermatozoa and act as preventive antioxidants. Some of the effects of these antioxidants are reviewed below 111 .

970 INDIAN 1 EXP BIOL, NOVEMBER 2005

Vitamin E-This is a major chain-breaking anti­oxidant present both in seminal plasma and in the membrane 112

. It reacts directly with free radicals such as the peroxy radical (ROO.) yielding lipid hydroper­oxides, which can be removed by phospholipase GSH-Px systems 113

. It also inten·upts the lipid per­oxidation process, which is why it is called a chain­breaking antioxidant. This antioxidant also prevents a reduction in sperm motility. Finally, it scavenges all three important types of ROS, namely superoxide, H202, and hydroxyl radicals 114.

Ascorbate-Like vitamin E, ascorbate is also a chain-breaking antioxidant and is found both intra­cellularly and extracellularly 112

. It prevents lipid per­oxidation due to peroxyl radicals. It also recycles vi­tamin E. It protects against DNA damage induced by H202 radical. Vitamin C has a paradoxical effed 15 as it can also produce ROS by its action on transition metal ions.

AH" +Fe 3+1Cu 2+ --~IIJJo• A-+ Fe 2+/Cu +

Fe3+/cu+ + 0 2 IIJJo 0 2. + Fe3+/Cu 2+

Fe3+/Cu + + H202 IIJJo OH +OR+ Fe3+/Cu 2+

Glutathione-Glutathione is the most abundant non-thiol protein in mammalian cells 116. It plays a vital role in annihilating oxygen toxicity by inter­rupting the reaction leading to 0 2- formation 117

. In its reduced form, it metabolizes H20 2 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 formation 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 motiliti 18'119. It can re­store the physiological constitution of poly­unsaturated fatty acid in the cell membrane 120·121 .

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 104

'122

. Treatment with glutathione was found to have a statistically significantly positive effect on sperm 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.

Other antioxidants molecules such as N-acetyl L­cysteine, carotenoids, coenzyme Q 10 and carnitines provide excellent antioxidant support. N-acetyl L­cysteine is a precursor of glutathione that assists in its formation. It helps to improve the motility of sperm. It also reduces the DNA damage caused by ROS47·123

.

Carotenoids play an important role in protecting the cells and organisms by scavenging the superoxide radicals 124. Coenzyme Q is associated with low den­sity lipoproteins (LDL) and hence, it protects lipids against peroxidative damage 125

. It directly reacts with oxygen and reduces superoxide generation . It also reacts with peroxide radicals 126. It is an energy pro­moting agent and improves sperm motility 127

. Carni­tine promotes membrane stability. It plays an impor­tant role in sperm maturation and development. Like coenzyme Q 10, it is an energy promoting agent 128.

Conclusion OS and its role in male infertility have been an area

of active research over the past two decades. Many free radicals are the result of naturally occurring proc­esses 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), environmental toxins, altered atmospheric conditions (e.g. hypoxia and hyperoxia), ozone and nitrous oxide (primarily from automobile exhaust) as well as many infectious conditions greatly enhance ROS production. Lifestyle stressors such as cigarette smoking and excessive al­cohol consumption are also known to affect levels of free radicals. They play a vital role in the etiology of many diseases and degenerative processes. Hence, it is imperative to have a sound knowledge about them and the OS caused by them.

OS affects male fertility in many ways. The per­oxidation of lipids, the cross-linking and inactivation of proteins and fragmentation of DNA are typical consequences of free radicals. Because the reactions occur quickly and are a part of complex chain reac­tions, we usually can detect only their "footprints". This requires the need for accurate measurement of the OS status of the semen. The full potential of inno­vative techniques like spin trapping and aromatic trapping should be utilized in the field of reproductive science. Although, a wide range of antioxidants-both natural and synthetic exist, their routine use in clinical conditions is still controversial. This explains the need to standardize a universal protocol to avoid unethical

AGARWAL & PRABAKARAN: PREVENTION OF STRESS IN MALE REPRODUCTIVE PHYSIOLOGY 971

use of antioxidants. Further research is required in this field to fully understand the complicated evenis asso­ciated with OS and to eventually develop effective strategies to assist patients with OS-associated male infertility.

Acknowledgement The authors thank the Cleveland Clinic Glickman

Urological Institute for support of their research studies.

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