Investigation of antioxidative capacity in bovine seminal plasma. Effects of Omega-3 fatty acids

Contents

1 INTRODUCTION

2 REVIEW OF LITERATURE
2.1 Reactive Oxygen Species
2.1.1 Definition and existence of reactive oxygen species
2.1.2 Oxidative stress
2.1.3 Significance of oxidative stress in male reproduction
2.1.3.1 Sources of ROS in semen
2.1.3.2 Targets and pathological role of ROS in semen
Lipids of sperm plasma membrane and lipid peroxidation
Damage of DNA
Damage of proteins
Apoptosis
2.1.3.3 Physiological role of ROS in semen
2.2 Antioxidants
2.2.1 Enzymatic antioxidants
2.2.1.1 Superoxide Dismutase
2.2.1.2 Glutathione Peroxidase
2.2.1.3 Catalase
2.2.2 Non-enzymatic antioxidants
2.3 Seminal plasma and its antioxidative significance for male reproduction
2.3.1 Accessory glands and seminal plasma
2.3.2 Antioxidative properties of seminal plasma
2.4 Measurement of oxidative stress and antioxidants using chemiluminescence

3 OWN EXPERIMENTAL STUDIES
3.1 Establishment of a new assay for the determination of total antioxidative capacity of bovine seminal plasma
3.1.1 Abstract
3.1.2 Introduction
3.1.3 Materials and Methods
3.1.3.1 Chemicals
3.1.3.2 Semen collection, dilution and freezing
3.1.3.3 Handling of seminal plasma
3.1.3.4 Antioxidant assays
Total Antioxidant Capacity
Instrumentation and automated measurement of TAC SOD and GPx
Superoxide Dismutase (SOD) Assay
Glutathione Peroxidase (GPx) Assay
Determination of protein concentrations
Measurements of intra- and inter-assay Variations
3.1.3.5 Flow cytometric analyses Plasma Membrane Integrity and Acrosomal Integrity Lipid Peroxidation (LPO) Sperm Chromatin Structure Assay
3.1.3.6 Statistical analysis
3.1.4 Results
3.1.4.1 Reproducibility of TAC-, SOD- and GPx assays
3.1.4.2 TAC, SOD and GPx levels in seminal plasma
3.1.4.3 Volume and sperm concentration of ejaculates
3.1.4.4 Sperm quality
3.1.4.5 Relationships between antioxidant levels and between antioxidant levels and sperm quality
3.1.5 Discussion
3.2 Effects of feeding omega-3-fatty acids on sperm quality of Holstein Friesian bulls before and after cryopreservation: Effects on seminal plasma
3.2.1 Abstract
3.2.2 Introduction
3.2.3 Materials and Methods
3.2.3.1 Bulls
3.2.3.2 Dietary supplementation of bulls
3.2.3.3 Semen collection, dilution and freezing
3.2.3.4 Handling of seminal plasma and measurement of TAC, GPx and SOD
3.2.3.5 Flow cytometric analyses Plasma Membrane Integrity and Acrosomal Integrity Sperm Chromatin Structure Assay
3.2.3.6 Fatty acid extraction and analysis
3.2.3.7 Statistical analysis
3.2.4 Results
3.2.4.1 Fatty acid analysis
3.2.4.2 Dietary effects on sperm quality and fatty acid composition
3.2.4.3 Effect of feeding ALA on antioxidant levels of seminal plasma
3.2.4.4 Correlation between fatty acids and antioxidants
3.2.5 Discussion

4 SUMMARIZING DISCUSSION AND CONLUSIONS

5 SUMMARY

6 ZUSAMMENFASSUNG

7 REFERENCES

8 APPENDIX

List of abbreviations

illustration not visible in this excerpt

1 INTRODUCTION

Bovine semen has been cryopreserved since more than a half century for artificial insemination and nowadays it is being widely used all over the world. However, it is well known that the cryopreservation procedure is detrimental to sperm particularly because of chemical and physical stress factors which are occurring during this process (HAMMERSTEDT et al. 1990; WATSON 1995; CHATTERJEE and GAGNON 2001). One important factor is oxidative stress which, in turn, affects biological membranes and DNA of sperm (AITKEN and KRAUSZ 2001; BALL 2008). Bovine sperm themselves have only few amounts of endogenous antioxidants for the protection against reactive oxygen species (ROS) and the main antioxidant source is the seminal plasma (DAWRA and SHARMA 1985; BILODEAU et al. 2000). Therefore, the development of sensitive techniques for monitoring the activity of antioxidants in seminal plasma is of clinical importance. Sensitive chemiluminescence techniques have been employed to monitor total antioxidant capacity of human seminal fluid (SHARMA et al. 1999).

Polyunsaturated fatty acids (PUFA) play an important role in regulating sperm membrane fluidity and spermatogenesis (HAIDL and OPPER 1997; OLLERO et al. 2000). After freezing and thawing, the portion of PUFA in sperm plasma membrane decreases significantly due to lipid peroxidation (CEROLINI et al. 2001). Low portions of C20 and C22 PUFAs in sperm in old bulls were related to reductions in sperm quality and -fertilizing ability (KELSO et al. 1997b).

In various feeding experiments polyunsaturated fatty acids (PUFA) have been supplied to change the fatty acid composition of sperm membrane in order to improve sperm quality and fertility. Indeed, the fatty acid profile of sperm membranes can be modified with diet and an improvement of sperm quality was observed in a variety of livestock species including chicken, turkey, boar and stallion (KELSO et al. 1997a; ROOKE et al. 2001; BLESBOIS et al. 2004; BRINSKO et al. 2005). However, it is possible that feeding of PUFAs reduces also the antioxidative capacity of semen which, in turn, can (SURAI et al. 2000b; CASTELLINI et al. 2003) disturbs sperm quality in the case of excessive ROS production.

The aims of this study were: (i) to determine total antioxidant capacity of bovine seminal plasma and its relationship with other antioxidants and sperm quality; (ii) to ascertain whether feeding omega-3-fatty acid reduces the antioxidative status of seminal plasma.

2 REVIEW OF LITERATURE

2.1 Reactive Oxygen Species

2.1.1 Definition and existence of reactive oxygen species

A free radical is any species capable of independent existence that contains one or more unpaired electrons (HALLIWELL 1989). A radical can be neutral or positively or negatively charged. HALLIWELL and GUTTERIDGE (1989) defined reactive oxygen species (ROS) as a collective term which includes oxygen radicals (e.g. Superoxide radical and Hydroxyl radical) as well as some highly reactive derivates of O2 that do not contain unpaired electrons (non-radicals) such as hydrogene peroxide (H2O2), singlet oxygen ([1] O2) and hypochlorous acid (HOCl). ROS have different abilities to react with many structural and functional important molecules in living organisms.

There are some nitrogen-derived free radicals called reactive nitrogen species (RNS, e.g., nitrite oxide (NO.) and peroxinitrite anion (ONOO-) which are considered as a subclass of ROS (SIKKA 2001). Some of ROS and RNS are summarized in Table 1-1. The superoxide radical (O2.-) is the main free radical and an example of free radicals with intermediate reactivity (FRIDOVICH 1983; SIKKA 2001). One-electron reduction of O2 produces the superoxide radical, O2-. This is frequently written as O2.- where the dot denotes a radical species, that is an unpaired electron. It is negatively charged and produced in biological systems during electron transport (during respiration) in mitochondria. It can not rapidly cross the lipid membrane bilayer (KRUIDENIER and VERSPAGET 2002). However, superoxide is a precursor of other, more powerful ROS. It can participate in the production of more powerful radicals as an oxidizing agent or by donating an electron and thereby reducing Fe[3] + and Cu[2] + to Fe[2] + and Cu+, as follows:

O2.- + Fe[3] +/Cu[2] +  Fe[2] +/Cu+ + O2 (step 1, reduction of ferric ion to ferrous).

Further reactions between transition metals (Fe[2] + or Cu+) and H2O2 are the source of the hydroxyl radical (OH.) and called as Fenton reaction:

H2O2 + Fe[2] +/Cu+  OH. + OH- + Fe[3] +/Cu[2] + (step 2, fenton reaction).

The concerted action of O2.- and H2O2 to generate OH. (steps 1 and 2) is described as the “Haber-Weiss” reaction:

O2.- + H2O2  OH. + OH- (net reaction).

The Haber-Weiss reaction is catalysed by transition metal ions, especially iron ions. The hydroxyl radical is one of the biologically most important ROS intermediates and is a strong oxidizing species with an estimated half life of about 10-[9] seconds (HALLIWELL 1989; HALLIWELL and GUTTERIDGE 1990). O2.- and H2O2 are not considered to be as reactive as the hydroxyl radical. H2O2 is not a radical and relatively stable. H2O2 has high biological diffusion properties (e.g. mitochondrial) (SIKKA 2001; ORRENIUS 2007) whereas the O2.- can not cross biological membranes except in the protonated form (TURRENS 2003).

Table 1-1: Reactive oxygen and reactive nitrogen species modified from DARLEY-USMAR et al. (1995).

Reactive oxygen species (ROS)

illustration not visible in this excerpt

Reactive nitrogen species (RNS)

illustration not visible in this excerpt

2.1.2 Oxidative stress

All cells need O2 to produce efficient energy in mitochondria. The mitochondrial electron transport system consumes more than 85 % of all oxygen consumed by the cell. On the other hand, oxygen consumption generates by-products called ROS, because 1-3% of electrons escape from the chain of that transport system and univalent reduction of molecular oxygen results in superoxide formation (HALLIWELL 1990; HALLIWELL and CROSS 1994; HALLIWELL 2006).

Aerobic cells are normally exposed to ROS but they can survive under toxic conditions of oxygen because they have protector molecules against this oxygen toxicity, the antioxidants (HALLIWELL 2006). Normally, continuous production of ROS and activated oxygen species in the body is controlled tightly by antioxdants. If this sensitive equilibrium between oxidants and antioxidants is disordered, oxidative stress occurs which, in turn, increases the rates of cellular damage. SIES (1991; 1993) defined the oxidative stress as a disturbance in prooxidant-antioxidant balance in favor for prooxidant.

2.1.3 Significance of oxidative stress in male reproduction

2.1.3.1 Sources of ROS in semen

The production of ROS in sperm was noticed by MACLEOD (cited by AITKEN 1994) in 1943. However, a relationship between oxidative stress and male infertility was not observed before the 1980s. The major reasons for the occurence of oxidative stress are depletion of seminal antioxidants and an excess generation of free radicals by the spermatozoa themselves (WATHES et al. 2007).

MACLEOD (1943) reported a toxic effect of O2 towards sperm. He noticed that the increase of O2 concentration resulted in a more rapidly loss of sperm motility and suggested that H2O2, generated by the cells themselves from O2, was an actual toxic agent. Three years later, TOSIC and WALTON (1946) described deleterious effects of H2O2 on bovine sperm motility and viability (cited by VERNET et al. (2004)). Preliminary studies showed that processes used in assisted reproductive techniques like the removal of seminal plasma and centrifugation, respectively, induce a sudden burst of ROS production in sperm (IWASAKI and GAGNON 1992). All cells actively respiring generate ROS as a consequence of electron leakage from intracellular redox systems, such as the mitochondrial electron transport system. In addition, it is known that cells generate ROS as a by-product of enzymatic activities of some oxidases (amino acid oxidase, xanthine oxidase), peroxidases (horseradish peroxidase, thyroid peroxidase) and oxygenases (indolamine dioxygenase, cytochrome P450 reductase) (AITKEN and BAKER 2004). In sperm two major systems are responsible for the ROS production. The main system that produces ROS in sperm is the NADH dependent oxido- reductase (diphorase) at the level of mitochondria (GAVELLA and LIPOVAC 1992; KOPPERS et al. 2008). The other ROS producing system is the NADPH oxidase system that has been shown to be present at the level of sperm plasma membrane (AITKEN et al. 1992). TOSIC and WALTON (1950) have shown that in bovine semen, ROS are also generated via the oxidative deamination of aromatic acids (TOSIC and WALTON 1950). In agreement with the results of TOSIC and WALTON (1950), SHANNON and CURSON (1982) have detected an activity of L-amino acid oxidase (belongs to the family of oxidoreductases) located in the tail of bovine sperm.

There is evidence that immature spermatozoa are able to produce ROS that are negatively correlated with sperm quality (OLLERO et al. 2001; AGARWAL and SALEH 2002). In addition, peroxidase-positive leukocytes (mainly polymorphonuclear leukocytes and macrophages) are other sources of ROS in semen (Figure 1-1) (OCHSENDORF and FUCHS 1997).

illustration not visible in this excerpt

Figure 1-1: Role of oxidative stress in male infertility modified from SIKKA (2004) and TREMELLEN (2008).

2.1.3.2 Targets and pathological role of ROS in semen

The harmful effects of ROS on sperm function can occur in many ways. AITKEN et al. (1991) showed that there is an inverse relationship between ROS generation in semen and fertility in vitro and in vivo. IWASAKI and GAGNON (1992) reported that the ROS level correlates inversely with the percentage of motile spermatozoa.

Reactive oxygen species can attact all major classes of biomolecules, membrane lipids, proteins, nucleic acids and carbohydrates (AITKEN et al. 1989; SIKKA et al. 1995; AITKEN and BAKER 2004).

Lipids of sperm plasma membrane and lipid peroxidation

The plasma membrane of mammalian sperm contains high concentrations of polyunsaturated fatty acids (PUFAs) (LENZI et al. 1996). Almost 60% of fatty acids in bovine sperm is docosahexaenoic acid (SALEM et al. 1986). It contains 22 carbons and six unsaturated double bonds per molecule [22:6 (n-3)]. Counted from the end of the carbon chain its first double bond is at the third carbon position (n-3). Thus, it is referred to as ω−3 fatty acid. The α- linolenic acid (ALA), eicosapentaenoic acid [EPA; 20:5 (n-3)] and DHA are the most important, nutritionally-essential ω-3 fatty acids. The main dietary source of ω -3 fatty acids are fish, plants and nut oils. DHA and eicosapentaenoic acid (LEPAGE and ROY 1986) are found in cold-water fish. ALA is found in flaxseed, flaxseed oil, canola oil and soybean (FRIEDMAN and MOE 2006).

The short chain polyunsaturated fatty acid ALA is a precursor of important long chain polyunsaturated fatty acids such as DHA. It can not be synthesized by animal tissues, as they lack desaturase enzymes for their synthesis, and so must be supplied in the diet (WATHES et al. 2007). Furthermore, the mammalian body can convert ALA to another ω-3 using specific desaturase enzymes (Figure 1-2) (FRIEDMAN and MOE 2006).

DHA plays an important role in regulating sperm membrane fluidity and spermatogenesis (HAIDL and OPPER 1997; OLLERO et al. 2000). The amount of DHA in plasma membrane of sperm is decreased during the process of sperm maturation and there is a cell-to-cell variability in the concentration of DHA in sperm (OLLERO et al. 2000).

illustration not visible in this excerpt

Figure 1-2: The metabolic elongation pathway of omega-3 fatty acids modified from FRIEDMAN and MOE (2006).

The fluidity of plasma membrane is important for fusion events associated with fertilization (AITKEN and BAKER 2004). However, unsaturated fatty acids with two or more double bonds are particularly susceptible to free radical attack. The reason for susceptibility of unsaturated fatty acids to free radical attack is the presence of a double bond adjacent to a bis allylic methylene (CH2) group (BROUWERS and GADELLA 2003). This makes the methylene C-H bonds weaker. Therefore, hydrogen is more susceptible to abstraction on this site. The initiation of lipid peroxidation cascade is induced by the abstraction of this weakly bound hydrogen atom from the methylene group. Three distinct reaction steps have been elucidated for lipid peroxidation: initiation, propagation and termination (AGARWAL and PRABAKARAN 2005).

After the initiation step, a lipid alkyl radical (alkylradical) is formed. The lipid alkyl radical stabilizes by forming a conjugated diene radical and reacts easily with oxygen forming a lipid peroxyl radical (ROO.) by the propagation step (Table 1-2). This can abstract other hydrogen atoms of an unsaturated lipid, resulting in a free radical chain reaction and finally lipid hydroperoxides (ROOH or named as LOOH) are formed. The transition metals such as iron can accelerate the process of LPO. Propagation of LPO depends on the antioxidant defense systems of spermatozoa and seminal plasma (AGARWAL and SALEH 2002). The last process of LPO is the termination step. In general, the chain reaction stops at this step since two radicals react and produce a non-radical species. Chain breaking antioxidants like Vitamin E can speed up the termination by catching free radicals (BROUWERS and GADELLA 2003; AGARWAL and PRABAKARAN 2005).

One of the naturally occurring products of LPO is malondialdehyde. Many authors have determined its concentration to monitor the degree of peroxidative damage in spermatozoa (AITKEN and FISHER 1994). AITKEN et al. (1989; 1994) showed that the results of a malondialdehyde assay correlate well with defective sperm function in terms of motility and the capacity of sperm oocyte fusion. It has been shown that bulls with higher sperm lipid peroxidation have lower chance to sire calves (KASIMANICKAM et al. 2007). Furthermore, sperm lipid peroxidation was negatively correlated with sperm plasma membrane integrity (PMI) and DNA fragmentation index (KASIMANICKAM et al. 2007).

Table 1-2: Steps of lipid peroxidation adapted from AGARWAL and PRABAKARAN (2005).

illustration not visible in this excerpt

Damage of DNA

The integrity of DNA is important for the development of embryos (AITKEN and KRAUSZ 2001). Normally, the characteristic tight packaging of the sperm DNA and the antioxidants in seminal plasma constitute two important protecting factors of sperm DNA integrity (TWIGG et al. 1998a; TWIGG et al. 1998b).

If oxidative stress occurs as a result of an imbalance between excessive generation of ROS and antioxidants, it is one of the most important causes of abnormal DNA structure (ERENPREISS et al. 2006). DNA bases and phosphodiester backbones are very susceptible to peroxidation. It has been suggested that high levels of ROS are responsible for high frequencies of DNA strand breaks observed in the sperm of infertile men (FRAGA et al. 1996; AITKEN and KRAUSZ 2001). In bull, DNA fragmentation index of sperm evaluated by SCSA was negatively correlated with fertility (BALLACHEY et al. 1987; JANUSKAUSKA et al. 2001).

Damage of proteins

Amino acids containing thiol groups are very susceptible to oxidative stress. Damage of ROS to proteins has been implicated in the oxidative inactivation of several metabolic enzymes associated with aging (OLIVER et al. 1987). There is evidence that the protein oxidation of sperm is associated with decreased sperm motility and fertilizing ability (CHEN et al. 2001; DUTEAUX et al. 2004).

Modification of amino acid side chains to carbonyl derivates (i.e., aldehyde and ketones) and fragmentation of polypeptid chains are some possible outcomes of protein oxidation reactions. Determination of carbonyl content in proteins is a commonly used method to measure oxidative protein damage (FAGAN et al. 1999).

Apoptosis

Apoptosis is the process of programmed cell death that is a physiologically occurring event during an organism`s life cycle (HENGARTNER 1997). For example, apoptosis of testicular germ cells occurs throughout life. In this way, the overproliferation of germ cells can be controlled. Spontaneous apoptosis occurs in seminoferous epithelium and affects spermato- gonia, spematocytes, and spermatids (KERR et al. 1972; BILLIG et al. 1995; RODRIGUEZ et al. 1997).

ROS plays an important role in apoptosis induction under both physio- and pathological conditions. The release of cytochrome c from mitochondria, which in turn triggers caspase activation, is mediated by direct or indirect ROS action. It has been recently shown that apoptosis can be induced by cryopreservation. MARTIN et al. (2004) showed that cryopreservation causes apoptotic like mechanism in bovine sperm.

2.1.3.3 Physiological role of ROS in semen

After ejaculation, spermatozoa undergo a series of physiological changes in female genital tract. These changes called capacitation and acrosome reaction are triggered by the zona pellucida of the ovum (AUSTIN and BISHOP 1958; HUNTER and RODRIGUEZ- MARTINEZ 2004). In vitro studies demonstrated that administration of O2.- and H2O2 promotes capacitation and acrosome reaction and the addition of appropriate antioxidants prevents them from undergoing these events (DE LAMIRANDE et al. 1997). ZINI et al. (1995) have shown that low levels of NO. promote human sperm capacitation. Furthermore, it has been shown that NO. plays a role in sperm hyperactivation and in zona pellucida binding (DE LAMIRANDE et al. 1997; SENGOKU et al. 1998). Therefore, it has been mentioned that the acrosome reaction and capacitation are redox-regulated (free radical regulated) processes which allow the sperm to fertilize the egg.

2.2 Antioxidants

During the evolution living organisms developed specific mechanisms to protect themselves from the deleterious effects of ROS and RNS (HALLIWELL 1990). Antioxidants are substances that delay or prevent oxidation of that substrates (HALLIWELL 1990). They can counteract the damaging effects of free radicals in three major manners. Three different antioxidant defense systems are summarized in Table 1-3.

Table 1-3: Different levels of antioxidant defence in animal cells adapted from SURAI (1999).

illustration not visible in this excerpt

The antioxidative compounds are located in organelles, subcellular compartments or the extracellular space. They can be classified as enzymatic and non-enzymatic antioxidants:

Enzymatic antioxidants: superoxide dismutase (SOD), glutathione peroxidase

(GPx), catalase (CAT)

Non-enzymatic antioxidants: vitamin E, vitamin A, Selenium, vitamin C etc.

2.2.1 Enzymatic Antioxidants

2.2.1.1 Superoxide Dismutase (SOD)

Superoxide dismutase was discovered by MCCORD and FRIDOVICH in 1969 (MCCORD and FRIDOVICH 1969). This enzyme removes O2.- by catalyzing its dismutation, O2- being re-duced to H2O2 and O2 (2O2.- + 2H+  H2O2 + O2).

SOD scavenges both intracellular and extracellular O2.-, but it should be considered that SOD in cells work in conjunction with H2O2-removing enzymes such as GPx or CAT to prevent the

action of H2O2, which in turn, promotes the formation of hydroxyl radicals (HALLIWELL 1989; AGARWAL et al. 2005).

The SOD reacts with O2.- molecules at an extremely rapid rate and speed up the dismutation reaction remarkably, thus lowering the tissue concentration of O2.-. SOD in animal tisssues exists in multiple forms, e.g., a copper-zinc SOD (CuZn-SOD; available in cytosol), a mitochondrial manganese SOD (Mn-SOD), and an extracellular CuZn-SOD (FRIDOVICH 1995). The transition metals (Cupper, iron or mangan) are located at the active center of these enzymes.

The SOD has been detected in spermatozoa and seminal plasma of different species and the CuZn-SOD was the major form SOD. The presence of other forms is a matter of controversy (ABU-ERREISH et al. 1978; MENNELLA and JONES 1980; ALVAREZ and STOREY 1984). In mouse, about 9 % of sperm SOD activity was attributed to the Mn-SOD probably residing in the mitochondria (ALVAREZ and STOREY 1984). In contrast, KOBAYASHI et al. (1991) did not detect Mn-SOD activity in human sperm and seminal plasma.

2.2.1.2 Glutathione Peroxidase (GPx)

The major enzymatic system for the control of cellular peroxide levels consists of glutathione peroxidases and several ancillary enzymes required for the synthesis and reduction of glutathione (GSH). The GPx enzyme is made up of four protein subunits, each of them containing one atom of the selenium bound to a cysteine protein (selenocystein) at its active site (FORSTROM et al. 1978). However, there are some seleno-independent isoenzymes of GPx like GPx5 and GPx6 proteins. These isoenzymes contain a cystein residue instead of a selenocystein residue (HALL et al. 1998; HERBETTE et al. 2007).

GPx reduces H2O2 and lipidic or nonlipidic-hydroperoxides while oxidizing two molecules of its subtrate, glutathione (H2O2 + 2GSH  GSSG (ozidized GSH) + 2H2O). GSH is a low- molecular-weight thiol compound. As a simple tripeptide it is composed of glutamic acid- cysteine-glycine. A high concentration of GSH (~5 mM in animal cells) is present in almost every cell (HALLIWELL and GUTTERIDGE 1989). This takes place by reduction of GSSG to GSH by glutathione reductase (GRx). GRx contains two protein subunits, each with the flavin FAD at its active sites. FAD needs NADPH (Nicotinamide adenine dinucleotide phosphate) to reduce GSSG (MEISTER 1988) (GSSG + NADPH + H+ 2GSH + NADP+).

The NADPH is mainly provided in animal tissues by a complex metabolic pathway known as the oxidative pentose phosphate pathway (or called as Hexose Monophosphate Shunt). In this pathway, NADPH stems from the reaction (Figure 1-3) catalysed by glucose-6-phosphate dehydrogenase (HALLIWELL and GUTTERIDGE 1989).

illustration not visible in this excerpt

Figure 1-3: Schematic glutathione redox-cycle adapted from OCHSENDORF and FUCHS (1997): H2O2 is reduced by GPx, glutathione (GSH) is oxidized and thereafter reduced by glutathione reductase (GRx) utilizing NADPH. NADPH is available by the Hexosemonophosphate-shunt system.

In somatic cells, 60-75 % of GPx activity is found in the cytoplasm and 25-40 % in the mitochondria (ZAKOWSKI et al. 1978). At least four types of selenium-dependent GPx exist in mammalian body, called GPx1, GPx2, GPx3, and GPx4. Selenium is essential for catalysis in all four types of GPx (BRIGELIUS-FLOHE and TRABER 1999). The GPx4 is called phospholipid hydroperoxide glutathione peroxidase (PHGPx) because of its ability to reduce not only H2O2 but also fatty acid hydroperoxides to alcohols (FORESTA et al. 2002). On the other hand, in the male genital tract, an selenium-independent isoenzyme of GPx (GPx5) has been identified at both protein and mRNA level (HALL et al. 1998; VERNET et al. 2004; CHABORY et al. 2009).

In bovine seminal plasma a non-selenium-dependent GPx and a selenium-dependent GPx have been detected (KANTOLA et al. 1988; BILODEAU et al. 2000). It has been reported that the selenium-dependent GPx activity accounted for one third of total GPx activity and is negatively correlated with the malondialdehyde level and acrosomal damage (SLAWETA and LASKOWSKA-KLITA 1985; KANTOLA et al. 1988; SLAWETA et al. 1988).

2.2.1.3 Catalase

Catalase is present in all major body organs of animals, being especially localized in liver and erythrocytes. It removes H2O2 within cells according to the following equation: 2H2O2 2H2O + O2

It consists of four protein subunits, each of which contains a heme (Fe (III)-protoporphyrin) group bound to its active site (AEBI 1984). Each subunit also usually contains one molecule of NADPH bound to it. This helps to stabilize the enzyme (KIRKMAN et al. 1987). Disso- ciation of catalase into its subunits causes loss of its activity. Dissociations can occur easily on storage, freeze-drying, or exposure of the enzyme to acid or alkali (HALLIWELL and GUTTERIDGE 1989). The catalase activity of animal tissues is largely located in subcellular organelles known as peroxisomes. Mitochondria and the endoplasmic reticulum contain little catalase actvity. (MARKLUND et al. 1982; HALLIWELL and GUTTERIDGE 1989).

An activity of catalase has been found in seminal plasma of different species (BALL et al. 2000; BILODEAU et al. 2000; ZINI et al. 2002). However, its presence in sperm is a matter of controversy (TRAMER et al. 1998). Catalase activity in human sperm and seminal plasma was demonstrated using different methods (JEULIN et al. 1989; ZINI et al. 2002). BILODEAU et al. (2000) noticed a low level of catalase activity in bovine seminal plasma but they did not find any catalase activity in sperm.

2.2.2 Non-enzymatic antioxidants

In addition to enzymatic defence systems, there are extensive non-enzymatic antioxidant compounds consisting of several different types of lipid and water-soluble small molecules (SIKKA et al. 1995; AGARWAL et al. 2005). These molecules are able to scavenge free radicals. Lipid-soluble scavengers are especially vitamin E and β-Carotene. Water-soluble scavengers are vitamin C (ascorbic acid) and GSH. Furthermore, there are a lot of other antioxidant compounds like selenium, zinc, uric acid, ceruloplasmin, taurine etc. (SIES 1993). EVANS and BISHOP (1922) have discovered vitamin E as a “fertility vitamin (or fertility factor)”. They showed that some plant oils reduced the occurrence of foetal mortality in diet- restricted rats. This “fertility factor” was later isolated and characterized as tocopherol (BELL 1987). Vitamin E is a collective name of 8 fat-soluble vitamins (tocopherols and tocotrienols) with antioxidant properties (HERRERA and BARBAS 2001). Of these, -Tocopherol has the highest biological activity and is the most abundant form in nature (BRIGELIUS-FLOHE and TRABER 1999). The ability of vitamin E to quench ROS and its hydrophobicity have led to its common definition as the single most important essential lipid-soluble antioxidant (BURTON 1994). When tocopherol quenches a lipid peroxyl radical, it is oxidated to the tocopheroxy radical. Tocoperoxy radical accepts hydrogen to regenerate the tocopherol, which makes this first oxidation step fully reversible. Vitamin E is present both in sperm membranes and in seminal plasma (AGARWAL and PRABAKARAN 2005). Vitamin E and Selenium act synergistically and protect the biomembranes from oxidative attack. Vitamin E reduces alkyl peroxyl radicals of unsaturated lipids of cell membranes, thereby generating hydroperoxides that can be removed by the Se-dependent peroxidases (MAIORINO et al. 1989; BRIGELIUS-FLOHE and TRABER 1999).

Carotene is the precursor of vitamin A, an important lipophilic antioxidant in animal tissues. Carotenoid pigments such as β-Carotene are able to function as effective quenchers of ROS (AGARWAL et al. 2005). β-Carotene was found to act synergistically with tocopherol and it is capable of regenerating tocopherol from the tocopheroxyl radical (PALOZZA and KRINSKY 1992). Large amounts of carotenoid pigments occur in male gonads and accessory genital glands of mammals. The significance of vitamin A in developing and maintaining the normal germinal epithelium in animals has been emphasized by several studies (PALLUDAN 1966). It is an essential vitamin for spermatogenesis in rats (HUANG and HEMBREE 1979). Selenium, as a cofactor for glutathione peroxidase, is an important trace element and plays a crucial role in enzymatic antioxidantive defence system. It is an essential trace element for animals. Selenoenzymes (e.g. glutathione peroxidases, thioredoxin reductases) contain Se in the form of selenocysteine, an amino acid that is identical to cysteine, except that selenium replaces sulphur (PAPPAS et al. 2008). In spermatozoa, Selenium is abundantly localized in midpiece region (CALVIN 1981). In rat sperm, Selenium deficiency causes reduction of spermatogenesis and abnormal sperm morphology characterized by morphological midpiece alterations (WU et al. 1979).

In spermatids, selenium-containing PHGPx occurs as an active peroxidase. In mammalian tissues, the highest PHGPx acitivity was found in the testis (URSINI et al. 1995). It plays an important role during spermiogenesis, maturation of spermatozoa and embryonic development. On the other hand, this enzyme is transformed to an oxidatively inactivated protein in mature sperm and is contributed as a main constituent of the mitochondrial capsule in the midpiece (FORESTA et al. 2002). The activity of PHGPx is lower in sperm of infertile men sperm compared to that of fertile men (FORESTA et al. 2002).

Although the GPx activity in bovine seminal plasma is higher than in spermatozoa (BROWN et al. 1977; BILODEAU et al. 2000), selenium is found also in the spermatozoa in considerable concentrations. In bulls, the Selenium concentration in seminal plasma is about 10 times higher than in serum (SAARANEN et al. 1986). Furthermore, it is known that the[75] Se is faster incorporated into seminal plasma than into the spermatozoa of bulls (SMITH et al. 1979). This may explain the fact that seminal plasma GPx levels increase rapidly after an injection of Selenium to bulls (BARTLE et al. 1980).

2.3 Seminal plasma and its antioxidative role in male reproduction

2.3.1 Accessory glands and seminal plasma

Seminal plasma is the name for secretes originating from accessory glands (PETZOLT 2001). Immature sperm complete their passage through the epididymis and get matured. Thereafter, they will be transported via vas deferens and mixed with the seminal plasma before ejaculation. There are developmental differences of accessory glands between species. According to these differences, the amounts of secretions from the particular accessory glands vary between species (DÖCKE 1963). Seminal plasma is produced by epididymidis, ampulla ductus deferentis, prostate, vesicula seminalis, bulbo-urethral glands and urethral glands. The size and secretory output of accessory glands are regulated by testosterone (PETZOLT 2001). The amounts of seminal plasma vary considerably between species.

[...]