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Mechanisms involved in Reactive oxygen species induced sperm damages and their amelioration
- 1. Mechanisms involved in Reactive oxygen species induced sperm damages and their amelioration Rahul Katiyar PhD Scholar Germplasm Centre Div. of Animal Reproduction, IVRI
- 2. Introduction Role and types of ROS Common sources of ROS Pathogenesis of ROS mediated sperm damage Amelioration Conclusion Future prospects Outline
- 3. Introduction • Cryopreservation is known to degrade the potential fertility of the sperm cells by causing death of about 50 per cent of the cells and altered characteristics of many of the remaining cells. (Watson et al., 1995, Prasad et al., 1999) • The damages generally are the consequences of mechanical and osmotic phenomenon, cold shock and oxidative stress. • Reactive oxygen species (ROS) are produced in more quantities during cooling. • (Wang et al., 1991) • The levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. (Belodeau et al., 2000)
- 4. Role of ROS • Spermatozoa are susceptible to (ROS) attack. When manipulated in vitro, these cells run the risk of generating and being exposed to supra-physiological level of ROS. (Aitken et al., 2010) • The imbalance between the production of reactive oxygen species (ROS) and detoxification is known as oxidative stress (Agarwal et al., 2003) • An imbalance between ROS generation and scavenging activity is detrimental to the sperm and associated with male infertility. (Sharma & Agarwal, 1996)
- 5. (Aitken et al., 2015)
- 6. Type of ROS There are many types of radicals, but the most prominent in biological systems are derived from oxygen collectively known as Reactive Oxygen Species (ROS) Oxygen in its ground state has 2 unpaired electrons........Remember? O8: 1s2 2s2 2p4 So it is easy for Oxygen to accept electrons to form free radicals (Reactive Oxygen Species in this case!)
- 7. Common Sources of ROS ROS produced either intracellularly, originating from spermatozoa, or extracellularly, from environmental factors. Most common sources of ROS : Sperm cells themselves (Immature/defective/damaged/dead sperm). Leucocytes & other inflammatory cells. Semen processing techniques & egg yolk in diluted semen. Dissolved oxygen in extender.
- 8. ROS Cryostorage (Kim et al, 2010) Deficiency in antioxidant protection (Aitken and Curry, 2011) Presence of free radical– generating leukocytes in the male tract (Shi et al, 2012) Exposure to Electomagnetic radiation (Torres et al, 2010))
- 9. Pathogenesis of ROS mediated sperm damage Lipid peroxidation Apoptosis and DNA damage Motility impairment Protein damage
- 10. Pathogenesis in general (Agarwal et al., 2014)
- 11. Lipid peroxidation • Spermatozoa are known to be susceptible to loss of motility in the exogenous oxidant, as a consequence of LPO. • Spermatozoa are particularly vulnerable to lipid peroxidation because they contain high concentrations of unsaturated fatty acids, particularly docosahexaenoic acid with six double bonds per molecule. ( Jones et al., 1979)
- 12. Mechanism of lipid Peroxidation (Aitken et. al., 2016)
- 13. (Wathes et al., 2007) Lipid peroxidation cascade
- 14. LPO level in fresh and frozen- thawed buffalo spermatozoa Fresh stage Frozen- thawed LPO (nM MDA/109) 278.78 ± 18.2 364.67 ± 22.40 (Kadirvel et al., 2014) 238.90 ±3.09 478.83 ±3.35 (Balamurugan, 2015)
- 15. Apoptosis and DNA damage • DNA damage in spermatozoa has been linked with reduced rates of fertilization, impaired preimplantation development. ( Avendano and Oehninger, 2011) • A very early stage in this process, and possibly an initiating event, appears to be the induction of superoxide anion generation by the sperm mitochondria (Koppers et al, 2008, 2011)
- 16. Oxidation of vulnerable bases, particularly guanines Destabilizes the glycosyl bond, which attaches the base to the adjacent ribose unit Loss of the affected base and the generation of an abasic site ROS Mechanism of DNA damage (Agarwal et al., 2005)
- 17. The unique architecture of spermatozoa influences the impact of apoptosis on DNA integrity (Aitken et al., 2014)
- 18. Impact of oxidative stress in the male germ line upon the health and well-being of future generations Effect on male germ line Effect on future generation (Aitken et al., 2014)
- 19. Motility Impairment Reduction of sperm motility due to low ATP levels caused by DNA damage mechanisms. (Saraswat et al. 2012) ROS cause alteration in G-6-PDH thereby reducing the sperm motility due to low level of ATP. (Slater, 1984) ROS impaired sperm movement is mainly produced by inhibition / alteration of one or more enzymes involved in sperm cell metabolism.
- 20. Mechanism of Motility Impairment Lipid peroxidation chain reaction Formation of MDA, 4HNE, Acrolein Formation of adducts with the flagellar axonemal protein, dynein heavy chain Motility impairment (Baker et al., 2015)
- 21. Protein Damage • Superoxide anion -Sulphahydral oxidation • Per-hydroxyl radical- Fragmentation and cross-linking of soluble proteins • Hydroxyl radical - Protein fragmentation, amino acid modification and cross linking Adverse effect on freezability and fertilizing capacity of spermatozoa (Saraswat et al., 2013)
- 22. Assessment of Oxidative stress • The methods commonly used for measuring ROS can be categorized into: • 1) Reactions involving Nitroblue tetrazolium (NBT) or cyto- chrome c-Fe3+ complexes that measure ROS on the cell membrane surface • 2) Reactions that measure ROS (generated inside or outside the cell) utilizing chemiluminescence • 3) The electron spin resonance methods
- 23. • Assessment of lipid peroxidation : MDA can be assayed by the thiobarbituric acid reaction or BODIPY assay (Shannon and Curson, 1972; Priyadharshni et al. 2012) • Assessment of Superoxide anion radical : Spectrophotometric method based on the dismutase-inhibitable reduction of cytochrome C (Nash, 1953; Blake et al. 1987). • Assessment of hydroxyl radical: based on the determination of formaldehyde produced by the oxidation of dimethyl sulphoxide (DMSO) (Pontiki et al. 2006; Schraufstatter et al. 1986)
- 24. Amelioration of oxidative stress 1. Antioxidants Enzymatic Nonenzymatic 2. Biological ROS inhibitors Oviductal protein Seminal plasma HBP 3. Partial deoxygenation of extender
- 25. Antioxidants • Antioxidants are the agents, which break the oxidative chain reaction, thereby, reduce the oxidative stress. (Miller et al., 1993) Antioxidants Enzymatic Antioxidants • Superoxide dismutase (SOD) • Catalase • Glutathione peroxidase (GPx) • Glutathione reductase (GR) Non-Enzymatic Antioxidants • Vitamin C • Vitamin E • Glutathione • Glutamine • Cysteine
- 26. Catalase • Catalase @ 5 U/ml and Pyruvate @ 5 mM (Bilodeau et al., 2002) Added to EYT-G Prevented loss of sperm ATP Prevent production of MDA Increased activity of superoxide dismutase
- 27. Maintains the intracellular redox status Prevents leakage of intracellular enzymes and damage of chromatin. Added @ 5mM to the extender Preserve the sulfhydryl groups of protein which play an important role in sperm motility and metabolism Glutathione (Linderman et al., 1988)
- 28. Ascorbic acid • Biologically active reducing agent • Reduces and neutralizes free radicals • Improves carbohydrate metabolism and electron transport chain, thus the sperm motility • Added @ 10 Mm in extender prevents lipid peroxidation and helps maintain structural integrity of plasma membrane • Addition of ascorbic acid resulted in about 18% increase in the post-thaw motility, 11% increase in intact acrosome and prevented leakage of intracellular enzymes ( AST, ALT and AKP) ( Srivastava and Kumar, 2014)
- 29. α-tocopherol A chain breaking antioxidant It improves metabolic activity and cellular integrity of frozen- thawed semen Breaks the lipidperoxidation chain reaction through its interaction with lipid peroxyl and alkoxyl radicals Improves post-thaw motility, viability along with reduced peroxidative damage. (Beconi et al., 1993)
- 30. Manganese (Cheema et al., 2009)
- 31. Phosphodiesterase inhibitors • Butylated hydroxy toluene (BHT) Pentoxyfylline • Minimizes damage to the sperm motility and sperm cell membrane • Sustains sperm viability during freezing and thawing • Involved in the prevention of auto-oxidation reaction (Fusijava et al., 2004) • Reduces superoxide anions responsible for DNA apoptosis • Increases intracellular cAMP • Boosts sperm motility • Decreases lipid peroxidation (Zhang et al., 2005)
- 32. Addition of Oviductal protein (Kumaresan et al., 2006)
- 33. Addition of Seminal Plasma Heparin Binding Proteins • HBPs protect sperm from lipid peroxidation during cryopreservation. (Kumar et al., 2008) (Patel et al., 2015)
- 34. Deoxygenation of Extender • Levels of enzymatic antioxidants (SOD, GPx, CAT) and TAC in seminal plasma and LPO and ROS in spermatozoa at post-thaw stage of buffalo semen (Mean ± SE, N=30) Means bearing different superscripts (A, B & C) differ significantly (p<0.001) in column (Balamurugan, 2015) Groups Dissolved O2 (ppm) CAT (U/mg of protein) SOD (U/mg of protein) GPx (mmol/mi n/ml) TAC (mM) LPO (nmol/109 spermatozo a) ROS (Units of H2O2) Group I (Control) 8.50±0.07 A 0.00051±0.030C .193±0.005C 59.22±3.60C 1.421±0.021C 478.83±3.35A 197.16±2.77A Group II (LN2 Flushing) 3.71±0.02 B 0.0056±0.000A .257±0.002A 69.27±3.38A 1.667±0.015A 314.50±6.93C 123.50±1.461C Group III (Mechanic al method) 5.34±0.02C 0.0032±0.000B .215±0.006B 64.23±3.32B 1.532±0.014B 364.10±5.77B 146.66± 1.923B
- 35. Conclusion A balance between the benefits and risks from ROS and antioxidants appears to be necessary for the survival and normal functioning of spermatozoa. Increased oxidative damage to sperm membranes, proteins, and DNA is associated with alterations in signal transduction mechanisms that affect fertility. Excessive ROS formation can be controlled by use of antioxidants, biological ROS inhibitors or by using reduced level of dissolved oxygen in extender.
- 36. Future prospects To establish reference values for ROS in semen, above which antioxidants could be used for male infertility treatment. To simplify and validate the evaluation of ROS so that it can be performed routinely without the use of sophisticated equipment. To standardize the threshold value of dissolved oxygen in extender for optimum semen freezability and fertility.
Editor's Notes
- Semen cryopreservation and artificial insemination (AI) has long been practicing as these are beneficial tools for breed improvement. It is evident that the semen cryopreservation made substantial development to the livestock industry. On the other hand the process faces some hazards which decrease the fertility efficacy of cryopreserved semen.Various steps of cryopreservation such as processing, freezing and thawing exert physiological as well as chemical stress on the sperm membrane (Chatterjee et al., 2001). The major disadvantage of cryopreservation is death of the half of the sperm population during freezing and thawing procedure even if most adoptive preservation technique is followed . the seminal plasma contains a number of antioxidant components such as superoxide dismutase, catalase, glutathione peroxidase and free radical scavengers like vitamins C and E, hypotaurine, taurine, and albumin [8], [9] and [10]. With the progress in AI, semen is diluted to produce maximum number of doses from a single ejaculate but this practice has made the sperm more vulnerable by decreasing concentration of natural antioxidant components of semen. This aggravates the condition when spermatozoa are subjected to damage during cryopreservation. Therefore, it becomes imperative to restore the normal levels of antioxidants in semen.
- Although a significant physiological role of ROS during normal sperm function has been reported,5,6 such as they facilitate fusogenicity of the spermatozoa, which enables them to bind to the zona pellucida, undergo the acrosome reaction through membrane lipid peroxidation and phospholipase-A activity,7 traverse through the zona pellucida and fuse with oocyte membrane,8 but when the balance between ROS production and detoxification by antioxidants is disrupted, an excess of ROS create oxidative stress.
- Free radicals are a group of atoms or molecules that are highly reactive due to having one or more unpaired electrons [ 7 ] . As a result of having an incomplete outer valance shell, these molecules attempt to react with other molecules in their vicinity in order to gain one or more electrons. However, once a molecule loses an electron to a free radical, a chain reaction is created, as now the former molecule becomes a free radical itself (Fig. 13.1 ). The chain reaction created from free radicals can have catastrophic effects for living cells. ROS are a collection of radicals and nonradical derivatives of oxygen. Free radicals derived from nitrogen are called reactive nitrogen species (Table 13.1 ). The most common ROS that is produced by spermatozoa is the superoxide anion radical; this in turn forms hydrogen peroxide (strong oxidizer) on its own or by the action of superoxide dismutase [ 8 ] .
- High level of ROS disrupts the integrity of mitochondrial membrane which in turn releases cytochrome c. it activates the caspase enzyme cascade and triggers apoptosis
- The protective antioxidant systems in sperm are primarily of cytoplasmic origin and sperm discard most of their cytoplasm during terminal stages of differentiation [16]. Thus mammalian sperm lack a significant cytoplasmic component, which contains sufficient antioxidants to counteract the damaging effects of ROS and LPO. For this reason, sperm are susceptible to LPO during cryopreservation and thawing process (Bucak et al., 2010)
- A proposed mechanism by which unsaturated fatty acids might generate oxidative stress in human spermatozoa. An abundance of unsaturated fatty acids in the spermatozoa of infertile patients triggers free radical generation from a nonmitochondrial source, possibly the NADPH oxidase, NOX5, under the influence of calcium [167]. Excessive reactive oxygen species generation (ROS) then induces lipid peroxidation that, in turn, triggers phospholipase A2, precipitating the release of further unsaturated fatty acids and the induction of yet more ROS generation to perpetuate the oxidative stress.
- Abasic sites are well known to have a strong destabilizing effect on the DNA backbone, resulting in the induction of localized strand breaks.
- Conventional somatic cells feature a centrally placed nucleus surrounded by mitochondria embedded in the cytoplasm. Under these circumstances, endonucleases activated in the cytoplasm or released from the mitochondria during apoptosis are able to move into the nucleus (arrows) and cleave the DNA. (b) Spermatozoa are completely different from such somatic cells because their mitochondria (stained black) and most of their cytoplasm are located in the midpiece of the cell, physically separated from the nucleus. (c) As a consequence of this compartmentalization key effectors of apoptosis such as apoptosis inducing factor (AIF) or Endonuclease G (Endo G) remain resolutely locked in the sperm midpiece even when apoptosis is induced by the powerful PI3 kinase inhibitor, wortmannin and cannot move into the sperm nucleus. Because of this physical limitation, most DNA damage in mature spermatozoa is induced by membrane permeant reactive oxygen species emanating from the mitochondria, rather than nucleases.
- (1) A variety of primary factors can initiate oxidative stress in the male germ line including infection, age, obesity and exposure to a variety of adverse environmental influences. (2) This initial oxidative stress induces lipid peroxidation culminating in the production of lipid aldehydes such as 4HNE, which bind to proteins in the mitochondrial electron transport chain, stimulating the generation of reactive oxygen species (ROS). The latter stimulate yet more lipid peroxidation in a self‑propagating cycle that culminates in apoptosis. (3) One of the most sensitive targets of oxidative stress is the DNA in the sperm nucleus, generating 8‑hydroxy, 2’deoxyguanosine (8OHdG) base adducts. (4) The first enzyme in the base excision repair pathway, 8‑oxoguanine glycosylase 1 (OGG1), is present in spermatozoa and its activity creates abasic sites. The remainder of the DNA repair pathway is present in the oocyte. The oocyte has to repair the DNA damage brought into the zygote by the fertilizing spermatozoon before the initiation of S‑phase for the first mitotic division. (5) If the oocyte makes a mistake at this stage of DNA repair, it has the potential to create a mutation that will be represented in every cell in the body and could account for the range of pathologies seen in the offspring of fathers exhibiting high levels of oxidative DNA damage in their spermatozoa.
- The lipid peroxidation chain reactions initiated in spermatozoa may also result in the formation of a cascade of aldehyde by-products that include alkanals, such as malondialdehyde, and alkenals, such as 4HNE and acrolein. These compounds, particularly 4HNE and acrolein, are powerful electrophiles that form adducts with several proteins within the spermatozoa that, in turn, affect sperm function. For example, the formation of adducts with the flagellar axonemal protein, dynein heavy chain, may explain the effect of these aldehydes on sperm movement (Baker et al. 2015; Moazamian et al. 2015).
- Protein is the major component of seminal plasma and sperm cell organelles. The most of the enzymes responsible for various biochemical reactions of sperm are also proteins
- The luminol probe is more advantageous than the lucigenin probe for several reasons. Luminol measures both intracellular and extracellular ROS such as hydrogen peroxide, superoxide anion, and hydroxyl radical; on the other hand, lucigenin measures only the extracellular ROS, and in particular, superoxide anion. 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 spermatozoa
- Spermatozoa are protected by various antioxidants and antioxidant enzymes in the seminal plasma or in spermatozoa itself to prevent oxidative damage
- supplementation of Mn++ as an antioxidant to EYC-G during cryopreservation may maintain the enzyme system by its scavenging activity for free radicals or by reducing the oxidative stress and could reduce the effect of cryoinjury to cattle bull spermatozoa.
- BHT is a synthetic analog of Vitamin E, and is involved in the auto-oxidation reaction, thereby converting the peroxy radicals to hydroxyperoxides [11]. Pentoxifylline (PTX) works as a methylxanthine phosphor-diesterase inhibitor. It reduces superoxide anions and inhibits tumor-necrosis factor-alpha (TNF-alpha) responsible for DNA fragmentation and apoptosis or programmed cell death [23–25]. Additionally, it increases the intracellular cAMP [26], stimulates sperm motility, and improves the fertilization [10, 27]. Furthermore, Zhang et al. [28] showed that the application of PTX in clinical procedures leads to reduced lipid peroxidation associated sperm membrane damage and DNA apoptosis and scavenges the toxic reactive oxygen species.
- catalase activity was higher in nonluteal stage of estrous cycle in buffalo oviductal fluid (Kumaresan et al., 2004). This might be a reason for low MDA levels.
- declined level of MDA production in the spermatozoa treated with SP-HBP may be due to its interference with enzymatic reaction of inter and intracellular signaling responsible for ROS production.