Mechanisms involved in Reactive oxygen species induced sperm damages and their amelioration

Rahul KatiyarPhD ScholarGermplasm CentreDiv. of Animal Reproduction, IVRI

Introduction

Role and types of ROS

Common sources of ROS

Pathogenesis of ROS mediated sperm damage

Amelioration

Conclusion

Future prospects

Outline

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)

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)

(Aitken et al., 2015)

Type of ROSThere 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!)

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.

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))

Pathogenesis of ROS mediated sperm damage

Lipid peroxidation

Apoptosis and DNA damage

Motility impairment

Protein damage

Pathogenesis in general

(Agarwal et al., 2014)

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)

Mechanism of lipid Peroxidation

(Aitken et. al., 2016)

(Wathes et al., 2007)

Lipid peroxidation cascade

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)

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)

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)

The unique architecture of spermatozoa influences the impact ofapoptosis on DNA integrity

(Aitken et al., 2014)

Impact of oxidative stressin 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)

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.

Mechanism of Motility ImpairmentLipid 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)

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)

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

• 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)

Amelioration of oxidative stress 1. Antioxidants

Enzymatic Nonenzymatic

2. Biological ROS inhibitors Oviductal protein Seminal plasma HBP

3. Partial deoxygenation of extender

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

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

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)

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)

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)

Manganese

(Cheema et al., 2009)

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)

Addition of Oviductal protein

(Kumaresan et al., 2006)

Addition of Seminal Plasma Heparin Binding Proteins

• HBPs protect sperm from lipid peroxidation during cryopreservation. (Kumar et al., 2008)

(Patel et al., 2015)

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/min

/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 (Mechanical 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

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.

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.