Oxidants, Antioxidants, and Impact of the Oxidative Status in Male Reproduction

Oxidants, Antioxidants, and Impact of the Oxidative Status in Male Reproduction

Editors

Ralf Henkel

Luna Samanta

Ashok Agarwal

Table of Contents

Cover image

Title page

Copyright

Contributors

Foreword

Preface

Introduction

Part I. Basic Aspects

Chapter 1.1. Life Under Aerobic Conditions

Introduction

The Great Oxygenation Event

Biological Life and Environmental Evolution

Aerobic Respiration and the Mitochondria

Redox Biology and the Paradoxical Nature of Aerobic Life

Endogenous Antioxidants and Oxidative Stress

Mitochondrial Dysfunction

Conclusion

Glossary

List of Acronyms and Abbreviations

Chapter 1.2. The Oxidant Paradox

Introduction

Reactive Oxygen Species and Fertility

Oxidative Stress

Diabetes

Chapter 1.3. From Past to Present: An Historical Overview of the Concept of Spermatozoa, Reactive Oxygen Species, and Male-Factor Infertility

Definition of Reactive Oxygen Species

The Foundation of the Theory of Reactive Oxygen Species in Human Sperm Ejaculates

What Kind of Oxidase Are We Dealing With?

Other Measurements of Reactive Oxygen Species: The Challenge in Moving the Field Forward and Why We Cannot Continue to Do as We Have Done

Conclusion

Chapter 1.4. Basic Aspects of Oxidative Stress in Male Reproductive Health

Introduction

Pathological Effects of Oxidative Stress on Spermatozoa and Their Functional Capacity

Difference in Oxidative Stress Between Fertile and Infertile Men

Role of Oxidative Stress Assays in Unexplained Male Infertility

Selection of Non-DNA Damaged Sperm

Reversal of Oxidative Stress

Conclusion

Chapter 1.5. Leukocytes as a Cause of Oxidative Stress

Origin and Function of Seminal Leukocytes

Secretions of Leukocytes

Nature and Features of Reactive Oxygen Species

Oxidative Stress

Effect of Oxidative Stress on Sperm Functions and Male Fertility

Conclusion

Chapter 1.6. Human Spermatozoa and Interactions With Oxidative Stress

Introduction

What Are Reactive Oxygen Species?

Physiological Roles of Reactive Oxygen Species in Seminal Plasma

Reactive Oxygen Species Generation in Spermatozoa

Mechanism of Reactive Oxygen Species Production by Spermatozoa

NADPH Oxidases Generated Reactive Oxygen Species by Spermatozoa: Role in Capacitation

Nitric Oxide Synthase Generated Reactive Nitrogen Species by Spermatozoa During Capacitation

Reactive Oxygen Species and Immature Spermatozoa

Apoptosis and the Role of Reactive Oxygen Species Generation

Antioxidant Expression in Spermatozoa

Conclusion

Chapter 1.7. Causes of Reductive Stress in Male Reproduction

Introduction

Reactive Oxygen Species and the Male Reproductive System

The Antioxidant Paradox

Reducing Agents in Male Reproduction

Reductive Stress and the Male Reproductive System

Conclusion

List of Acronyms and Abbreviations

Chapter 1.8. Physiological Role of Reactive Oxygen Species in Male Reproduction

Introduction

Reactive Oxygen Species: Endogenous Generation and Sources

Reactive Nitrogen Species: Endogenous Generation and Sources

Physiological Levels of Reactive Oxygen Species and Sperm Function

Conclusion

List of Acronyms and Abbreviations

Part II. Clinical Aspects

Chapter 2.1. Epidemiology of Oxidative Stress in Male Fertility

Introduction

Idiopathic

Iatrogenic

Lifestyle

Environmental

Infection

Autoimmune

Testicular

Chronic Disease

Conclusion

List of Acronyms and Abbreviations

Chapter 2.2. Role of Oxidative Stress in the Etiology of Male Infertility and the Potential Therapeutic Value of Antioxidants

Introduction

Summary of Current Understanding in Relation to Human Spermatozoa

Oxidative Stress and Its Impact on Sperm Cell Structures and Functions

Sperm Nuclear Oxidative Stress and Its Developmental Impact

Therapeutic Value of Antioxidant Treatments

Chapter 2.3. Male Genital Tract Infections and Leukocytospermia

Definition

Etiology and Physiopathology

Clinical and Laboratory Findings

Treatment

Conclusions

Chapter 2.4. Varicocele

Introduction

Anatomy

Diagnosis

Varicoceles and Male Infertility

Mechanisms of Reactive Oxygen Species Production

Molecular Mechanisms

Treatment of Varicocele-Related Oxidative Stress

Conclusions

Chapter 2.5. Malnutrition and Obesity

Introduction

Key Nutrients Associated With Male Reproduction and Infertility

Human Adipose Tissue

Undernutrition and Underweight Males

Obesity: Clinical Definition, Risk Factors, and Epidemiology

Clinical Assessment of Adiposity and Fat Distribution

Pathophysiological Derangements of Obesity: The Metabolic Syndrome

Obesity, Metabolic Syndrome, and Male Reproduction Outcomes

Obesity and Micronutrient Deficiencies

Obesity, Oxidative Stress, and the Male Reproductive System

Nutrition, Weight Management Programs, and Male Reproductive Outcomes

Nutritional Supplementation and Antioxidants

Conclusion

Glossary

List of Acronyms and Abbreviations

Chapter 2.6. Role of Reactive Oxygen Species in Diabetes-Induced Male Reproductive Dysfunction

Introduction

Current Knowledge on the Effects of Diabetes Mellitus in Male Fertility

Diabetes Mellitus, Oxidative Stress, and Testicular Metabolism

Impact of Reactive Oxygen Species on Male Fertility: From Spermatogenesis to Sperm Maturation

Possible Role of Antioxidants in the Treatment of Diabetes-Induced Male Infertility

Conclusion

List of Acronyms and Abbreviations

Chapter 2.7. Thyroid Dysfunction and Testicular Redox Status: An Intriguing Association

Introduction

Thyroid Hormone and Its Mode of Action

Thyroid Hormone and Testes

Thyroid Dysfunction and Oxidative Stress in Testes

Thyroid Dysfunction and Antioxidant Defenses in Testes

Thyroid Dysfunction, Male Infertility, Antioxidant Treatment, and Testicular Function

Thyroid Dysfunction and Assisted Reproductive Technology Outcome

Conclusion

List of Abbreviations

Chapter 2.8. Aging

Introduction

Mitochondrial Free Radical Theory of Aging

Sperm DNA Damage/Fragmentation, Reactive Oxygen Species, and Aging

Antioxidants

Apoptosis

Reactive Oxygen Species, Aging, and Telomere Length

Conclusion

Glossary

List of Acronyms and Abbreviations

Part III. Clinical Methods to Determine and Treat Oxidative Stress

Chapter 3.1. Reactive Oxygen Species Methodology Using Chemiluminescence Assay

Introduction

Methods for Measuring Reactive Oxygen Species

Measurement of Reactive Oxygen Species by Chemiluminescence

Types of Luminometers

Instrument and Consumables

Assay Quality Control

Advantages and Disadvantages of Measuring Reactive Oxygen Species by Chemiluminescence

Limitations of Assay

Factors Affecting Chemiluminescent Signals

Newer Technique to Measure Oxidative Stress

Conclusion

Chapter 3.2. NBT Test

Introduction: Free Radicals: Two-Edged Molecules in Male Reproduction

Current Methods for Superoxide Detection in Semen

The NitroBlue Tetrazolium Test: A Colorimetric Marker of Superoxide in Semen

Clinical and Experimental Data: Principles and Interpretations

Existing Issues

Conclusion: Clinical Impact and Future Status of the NitroBlue Tetrazolium Test

Chapter 3.3. Total Antioxidant Capacity Measurement by Colorimetric Assay

Introduction

Methods for Measuring Antioxidants

Types of Assays for Total Antioxidant Measurement

Measurement of Antioxidant Capacity by Colorimetric Assay

Conclusions

Chapter 3.4. Oxidation-Reduction Potential Methodology Using the MiOXSYS System

Introduction

Clinical Significance of Oxidation-Reduction Potential

Methods for Measuring Oxidative Stress

Oxidation-Reduction Potential Measurement: Review of the Current Technology

Measurement of Oxidation-Reduction Potential by the MiOXSYS System

Instruments and Consumables

Assay Quality Control

Advantages and Disadvantages of Oxidation-Reduction Potential Measurement by MiOXSYS

Factors Affecting Measurement

Management of Abnormal Oxidation-Reduction Potential

Conclusion

Chapter 3.5. Treatment of Sperm Oxidative Stress: A Collaborative Approach Between Clinician and Embryologist

Introduction

Clinical Causes of Oxidative Stress and Their Treatment

Antioxidant Supplements to Augment Sperm Quality

Laboratory Procedures to Minimize Oxidative Damage to Sperm During Assisted Reproductive Technique Treatment

Concluding Remarks

List of Acronyms and Abbreviations

Part IV. Current Approaches: The OMICS

Chapter 4.1. Genomics and Epigenetics

Introduction

Epigenetics

Epigenetics in Male Reproduction

Oxidative Stress

Reactive Oxidative Species and Epigenetics

Final Thoughts

Chapter 4.2. Transcriptomics and Oxidative Stress in Male Infertility

Introduction

Methods Used in Transcriptomic Analyses

Transcriptomics and Male Infertility

Transcriptomic Studies on Male Infertility and Oxidative Stress

Conclusions

Glossary

List of Acronyms and Abbreviations

Chapter 4.3. Oxidative Stress and Sperm Dysfunction: An Insight Into Dynamics of Semen Proteome

Introduction

Proteomics: The Tools

Reactive Oxygen Species and Semen Proteome

Sperm Proteome in Pathophysiological Condition with Oxidative Predominance

Reactive Oxygen Species–Mediated Posttranslational Protein Modifications and Male Reproduction

Ubiquitination and Proteolysis

Concluding Remarks

Chapter 4.4. Metabolomics

The Current Situation of Male Fertility

Assisted Reproduction Techniques, Their Outcomes and Limitations

The Relevance of Male Factor in Reproductive Results

How to Estimate Male Fertility Potential, Its Limitations, and Potentially Useful Techniques to Be Applied

Exploring Male Fertility Biomarkers Through the Omics

Metabolomics in Male Fertility Studies

Conclusions

Index

Copyright

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Contributors

Ashok Agarwal,     American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

R. John Aitken,     The University of Newcastle (UoN), University drive, Callaghan, NSW, Australia

Marco G. Alves,     Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal

Mark A. Baker,     The University of Newcastle (UoN), University drive, Callaghan, NSW, Australia

Albert D. Bui,     American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

Meaghanne Caraballo,     American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

Gagan B.N. Chainy,     Utkal University, Bhubaneswar, Odisha, India

Shu-Jian Chen,     Rheinische Friedrich-Wilhelms University, Bonn, Germany

Julie W. Cheng,     Loma Linda University Medical Center, Loma Linda, CA, United States

Chak-Lam Cho,     Union Hospital, Hong Kong, China

Geoffrey N. De Iuliis,     The University of Newcastle (UoN), University drive, Callaghan, NSW, Australia

Joel R. Drevet,     Université Clermont Auvergne (UCA), Clermont-Ferrand, France

Damayanthi Durairajanayagam,     Universiti Teknologi MARA, Sungai Buloh, Selangor, Malaysia

Nicolás Garrido,     IVI Foundation, Valencia, Spain

Sezgin Gunes,     Ondokuz Mayis University, Samsun, Turkey

Sajal Gupta,     American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

Gerhard Haidl,     Rheinische Friedrich-Wilhelms University, Bonn, Germany

Ralf R. Henkel,     University of the Western Cape, Bellville, South Africa

James M. Hotaling,     University of Utah, Salt Lake City, UT, United States

Soumya Ranjan Jena,     Ravenshaw University, Cuttack, Odisha, India

Srikanta Jena,     Ravenshaw University, Cuttack, Odisha, India

Edmund Y. Ko,     Loma Linda University Medical Center, Loma Linda, CA, United States

Kristian Leisegang,     School of Natural Medicine, University of the Western Cape, Bellville, South Africa

Asli M. Mahmutoglu,     Ondokuz Mayis University, Samsun, Turkey

Jasmine Nayak,     Ravenshaw University, Cuttack, Odisha, India

Jacob Netherton,     The University of Newcastle (UoN), University drive, Callaghan, NSW, Australia

Pedro F. Oliveira,     Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal

Michael Owyong,     University of Miami Miller School of Medicine, Miami, FL, United States

Manesh Kumar Panner Selvam,     American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

Biren V. Patel,     University of Utah, Salt Lake City, UT, United States

Ranjith Ramasamy,     University of Miami Miller School of Medicine, Miami, FL, United States

Luís Rato,     (CICS-UBI) Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal

Rocío Rivera,     Instituto Universitario IVI Valencia, València, Spain

Edmund Sabanegh Jr. ,     Cleveland Clinic Foundation, Cleveland, OH, United States

Dipak Kumar Sahoo,     Iowa State University, Ames, IA, United States

Luna Samanta,     Ravenshaw University, Cuttack, Odisha, India

Rakesh Sharma,     American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

Suresh C. Sikka,     Tulane University, New Orleans, LA, United States

Branca M. Silva,     University of Beira Interior, Covilhã, Portugal

Michael C. Solomon Jr. ,     University of the Western Cape, Bellville, South Africa

Mário Sousa,     Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal

Nicholas N. Tadros,     Southern Illinois University, Springfield, IL, United States

Gülgez Neslihan Hekim Taşkurt,     Ondokuz Mayis University, Samsun, Turkey

Jaideep S. Toor,     Tulane University, New Orleans, LA, United States

Kelton Tremellen,     Flinders University, Bedford Park, SA, Australia

Eva Tvrda,     Slovak University of Agriculture in Nitra, Nitra, Slovak Republic

Sarah C. Vij,     Cleveland Clinic Foundation, Cleveland, OH, United States

Foreword

It is estimated that a male factor is identified in up to half of infertile couples. The etiology of male factor infertility is multifactorial yet studies have found that oxidative stress (OS) is a common mediator in the pathophysiology of this disease. It has been shown that 25% of unselected infertile men possess high levels of semen reactive oxygen species (ROS), whereas fertile men do not have high levels of ROS in semen. A controlled production of these ROS is required for normal sperm physiology (e.g., sperm hyperactivation, capacitation, and acrosome reaction) and for natural fertilization but the excessive production of ROS by immature germ cells and leukocytes causes sperm dysfunction (lipid peroxidation, loss of motility, and sperm DNA damage). Seminal OS is believed to be caused by an imbalance between ROS production and ROS scavenging by seminal antioxidants. However, whether it is an excess ROS production or a deficiency in antioxidant capacity that is the primary cause of semen OS remains a subject of controversy.

Spermatozoa are particularly susceptible to oxidative injury due to the abundance of plasma membrane polyunsaturated fatty acids. These unsaturated fatty acids provide fluidity that is necessary for membrane fusion events (e.g., the acrosome reaction and sperm–egg interaction) and for sperm motility. However, the unsaturated nature of these molecules predisposes them to free radical attack and lipid peroxidation of the sperm plasma membrane. Once this process has been initiated, accumulation of lipid peroxides occurs on the sperm surface (this results in loss of sperm motility) and oxidative damage to DNA can ensue. Natural protection from semen OS is afforded by sperm antioxidants and, to a greater extent, by seminal plasma, a fluid rich in both enzymatic and nonenzymatic antioxidants.

In this textbook, Oxidants, Antioxidants, and Impact of the Oxidative Status in Male Reproduction, co-editors Henkel, Samanta, and Agarwal have gathered an impressive group of experts (biologists and clinicians) to share their knowledge on the topic of OS. The drive to study OS in the male reproductive tract can be traced back to the research of Claude Gagnon and John Aitken, both pioneers in this field. The book is divided into three sections that cover this important area of reproductive biology: basic aspects, clinical aspects, and current approaches to OS. In the first section of the textbook, the biology of aerobic systems and the oxidant paradox is discussed. This is followed by several chapters that describe the etiology, physiology, and pathophysiology of OS in the male reproductive system, an active area of research.

A better understanding of the pathophysiology of male infertility and, in particular, the role of OS in male reproduction may help us more accurately define the causes of this condition. An ongoing area of research in the field of clinical andrology is the question of how specific health conditions (e.g., diabetes) contribute to male infertility. In the second section of the textbook, experts in the field discuss the epidemiology of OS and the clinical factors associated with OS in male reproduction. The techniques used to determine OS are also presented in this section. A detailed presentation on the role of in vivo and in vitro antioxidants is also explored. Antioxidant therapy remains a widely debated topic in male infertility. To date, studies suggest that antioxidants may be of benefit to infertile men although additional studies are needed to define the ideal antioxidant regimen and confirm the true advantage of this form of therapy. In the last section of the textbook, novel approaches to study ROS-mediated sperm function are presented. The interplay between OS in male reproductive health and genomics, epigenetics, and metabolomics are discussed. This ongoing area of research will help define the influence of OS on the metabolic profile of semen and sperm, the assembly and organization of the sperm chromatin, and the genomic integrity of sperm.

In order to better diagnose and treat male factor infertility we must further our understanding of the pathophysiology of this condition and, in particular, the role of OS in male reproductive health. The editors and authors of this book have done a superb job in assembling the most complete resource to date on the subject of oxidative stress in male reproduction and its clinical relevance. This textbook will undoubtedly be an important reference work that will guide biologists and clinicians on how to interpret and further investigate OS in male reproductive health.

Armand Zini, MD, FRCSC

Professor of Surgery, Department of Surgery, McGill University Associate Professor of Obstetrics and Gynecology University of Montreal Montreal, Canada (Email: ziniarmand@yahoo.com)

Preface

Male infertility is a global health concern because it affects not only the physical well-being of individual patients, but also the psychological health of couples who want to have children. It also has significant negative societal implications. In certain societies, for a man to not be able to father children means that he will suffer from ridicule, low self-esteem, and marginalization. On the other hand, in many male-dominated societies, women carry the burden of reproduction, including the blame if a marriage is barren. Generally, male infertility is the sole cause of a couple's infertility in 30% of cases, and in more than 50% of the cases if a female factor is also taken into consideration. Despite this large prevalence, male infertility, as a major public health concern, is not properly addressed, and the problem is not only underestimated by the patients themselves, but also by many health-care professionals. Even to this day, too many clinicians limit their evaluation of male infertility to the standard semen analysis, which has clearly been shown to be insufficient in predicting the fertilizing capacity of male germ cells. Consequently, many clinicians too often offer expensive assisted reproduction techniques instead of properly examining and treating problems of male infertility. Up to 40%–50% of male infertility cases in general and up to 80% in men with idiopathic infertility are due to an imbalance between oxidants and antioxidants in their male reproductive system in favor of the oxidants, thus causing oxidative stress. Although in recent years, scientists and clinicians have discovered that oxidative stress is a major cause and contributing factor in many cases of male infertility, the knowledge and understanding of its pathology and possible treatment options are lagging behind. This has the potential for unsatisfactory diagnoses and treatment of male infertility.

The book is organized in three main sections: basic aspects, clinical aspects, and current approaches. Renowned experts from 12 countries wrote 24 state-of-the-art chapters selected for this book. Thus our book provides detailed information on the role of reactive oxygen species in sperm physiology and pathology with essential, up-to-date, basic, and clinical information on these highly reactive compounds and oxidative stress. While the first part of the book provides basic, up-to-date background on the topic, including the chemical nature of reactive oxygen species, antioxidants, the antioxidant paradox, oxidative stress, and reductive stress, the second part deals with the clinical aspects, providing insightful information in the pathophysiological role of reactive oxygen species in various diseases such as aging, malnutrition, diabetes, and thyroid dysfunction. Furthermore, the most common diagnostic tests to determine oxidative stress are described in a simple and practical manner.

In light of the fact that many patients routinely take various dietary antioxidants in an uncontrolled manner as these are very much extolled for their beneficial effects, we included chapters on treatment options of oxidative stress in vitro and in vivo as well as on so-called reductive stress, which is due to an overdosage of antioxidants and is as dangerous as oxidative stress. While the latter is a new concept in andrology and an additional way of thinking and explaining certain aspects of male infertility, we also included practical diagnostic aspects and rounded these topics off with very recent developments using the omics platform. We feel that this book provides current and comprehensive information that addresses the physiology and pathology of oxidative stress as a cause of male infertility. This combination of basic and clinical information makes our book well suited for fertility practitioners, andrologists, urologists, medical doctors, reproductive professionals, and research students.

We are grateful to our contributors, who are distinguished leaders in the field with extensive experience from around the world.

This book would not have been possible without the excellent support of Elsevier. We are thankful to Tari Broderick, Senior Acquisition Editor, for her constant support, and Samuel Young, Editorial Project Manager, for managing this project. The editors are grateful to their families for their love and support.

We genuinely hope that this volume will support and enrich your practice in clinical andrology.

Ralf Henkel, BEd, PhD, Habil,     Bellville, South Africa

Luna Samanta, PhD,     Cuttack, India

Ashok Agarwal, PhD, HCLD (ABB), EMB (ACE),     Cleveland, OH, USA

Introduction

R. John Aitken,     University of Newcastle, Callaghan, NSW, Australia

Oxidants, Antioxidants, and Impact of the Oxidative Status in Male Reproduction provides readers with a definitive up-to-date account of our current understanding of the role played by oxidative stress in the etiology of male infertility. Within the pages of this book, Ralf Henkel, Luna Samanta, and Ashok Agarwal have orchestrated a thoughtful collection of authoritative reviews that demonstrate just how important the redox status of the ejaculate is in defining the quality of the human ejaculate. While the individual chapters reveal a fair level of consensus in terms of the overall clinical importance of reactive oxygen species (ROS) generation in the germ line, this accord is balanced by some enduring uncertainties over the source, nature, and detection of reactive oxygen metabolites as well as the significance of antioxidant supplementation in countering the oxidative stress they induce. Some differences of opinion in such a rapidly developing field are as inevitable as they are insightful. In order to understand the challenges being faced by this field the reader is initially presented with clear, well-articulated accounts of the fundamental chemistry of oxidative stress and then led through some of the hot spots of debate in the application of this knowledge to one of nature's most intractable problems—what factors are responsible for the very high levels of male infertility seen in our species?

It has been quite a journey. The initial steps on this long and winding road were taken by Tosic and Walton who, as long ago as 1946, published a paper in Nature, highlighting the sensitivity of bovine spermatozoa to hydrogen peroxide toxicity [1]. This was then succeeded by a much more extensive paper [2] in which they showed that bovine spermatozoa possess an aromatic L-amino acid oxidase (LAAO) system that in the presence of oxygen will affect the processes of deamination and dehydrogenation to generate H2O2 and ammonia [2]. Similar LAAO activity has been described in ram and stallion spermatozoa [3] where it may also play a role in creating oxidative stress, particularly in the presence of egg-yolk extenders containing high concentrations of aromatic amino acids (particularly phenylalanine). While a similar system is present in human spermatozoa it does not appear to play a significant role in the spontaneous generation of excess ROS by these cells [4]. Indeed, there is still great uncertainty in the field as to the sources of ROS that might contribute to disruption of human sperm function.

The answer to this question is unlikely to be simple. Several chapters in this volume allude to the significance of leukocyte contamination in defining the redox status human semen samples. There can be no doubt that in unfractionated ejaculates, leukocytes comprise the most significant source of ROS; however, the potential of these molecules to cause damage is countered by the antioxidant properties of seminal plasma. Indeed seminal plasma has probably evolved to perform just this role. At the moment of ejaculation spermatozoa will suddenly be exposed to an increase in oxygen tension and the ROS generated by leukocytes originating from the urethra, seminal vesicles, or prostate. Normally this oxidative attack is short-lived and the spermatozoa are protected by the powerful antioxidant properties of seminal plasma during the hiatus between ejaculation and colonization of the female reproductive tract. However, the seminal antioxidant pool may become exhausted if levels of leukocytic infiltration are high, leaving the spermatozoa vulnerable to a leukocyte-mediated oxidative attack. The degree of stress experienced by the spermatozoa under these circumstances will ultimately depend on the number of infiltrating leukocytes (particularly neutrophils and macrophages), their state of activation, and the details of how, when, and where they became activated. Measuring the balance of ROS generation within the ejaculate and the level of residual antioxidant protection offered by seminal plasma is an excellent approach to capturing this redox balance [5]. In making such measurements, we might reflect on whether the transient sojourn of spermatozoa in seminal plasma depleted of its antioxidant reserve is actually sufficient to compromise sperm function directly. Is it also possible that, in vivo, the presence of leukocytes in the ejaculate provides an indirect indication of long-term proinflammatory conditions prevailing in the male reproductive tract (or even systemically) that are actually responsible for the loss of sperm function seen in oxidative stress patients? There are many conditions where the level of oxidative stress in the ejaculate is reflective of a more generalized stress as in patients suffering from obesity, exposure to cigarette smoke, high alcohol intake, exposure to organic pesticides and herbicides, radiation, and other conditions. Moreover, several of the chapters in this volume highlight a growing range of clinical conditions including diabetes, chronic kidney disease, β-thalassemia, thyroid dysfunction, and hyperhomocysteinemia, which appear to be associated with a systemic oxidative stress of such intensity that fertility is compromised. In this context, oxidatively damaged spermatozoa may be the canaries in the coal mine, reflecting underlying conditions that influence an individual's entire health trajectory, including the ultimate risk of malignancy [6].

Once the spermatozoa have been separated from seminal plasma and purified, attention switches (or should switch) to gametes. Too many studies in this field have been compromised by a failure to account for the impact of contaminating leucocytes on the generation of ROS in washed sperm suspensions. Most commonly used sperm isolation techniques (including swim-up and discontinuous gradient density gradient centrifugation) do not succeed in removing all the leukocytes. The leukocyte-derived redox signals generated under such circumstances tend to overwhelm the limited ROS-generating capacity of spermatozoa, leading to potential errors in the literature in defining the source of ROS in spermatozoa. This has, for example, potentially impacted our view of the significance of NADPH oxidases in spermatozoa. At present, most evidence points to the sperm mitochondria as a major source of ROS [7] possibly supplemented with the activity of cytoplasmic enzymes such as the lipoxygenase, ALOX15 [8].

Another challenge that has plagued the field is the development of detection methods to determine the ROS-generating capacity of the ejaculate. Techniques such as chemiluminescence or nitroblue tetrazolium reduction are certainly sensitive but they do not report on a defined chemical entity and cannot readily discriminate the relative contributions from spermatozoa and leukocytes. Flow cytometry has the power to focus exclusively on the sperm population but specificity is again a problem. Most commonly used flow cytometry probes, such as dihydroethidium or dihydrodichlorofluorescein, are highly redox sensitive, but do not report on specific ROS. While we await the development of more specific probes we might focus on measuring the consequences of oxidative stress by assessing the products of lipid peroxidation. This could be achieved by flow cytometry mediated measurements of lipid oxidation products such as 4-hydroxynonenal, 4-hydroxyhexanal, acrolein, or malondiadhyde. A systematic analysis of the lipid peroxidation breakdown products generated as a consequence of an oxidative attack on human spermatozoa would be valuable in selecting an appropriate marker (see Chapter 1).

The role of oxidative stress in damaging sperm DNA and thereby influencing the mutational load subsequently carried by any offspring is another area of investigation highlighted in this book that has important implications for assisted reproductive technologies. It is particularly relevant to the increasing use of intracytoplasmic sperm injection (ICSI) as the default insemination technique, since the inadvertent fertilization of oocytes with oxidatively damaged spermatozoa is inevitable with this approach. The consequences of such action, in terms of the normality and health trajectory of the offspring, will be an important focus for future studies in this area. A recent study demonstrating an increase in autism spectrum disorder in offspring conceived by ICSI [9] may be a harbinger for bad news yet to come.

Ultimately ROS are a two-edged sword as far as the cell biology of human spermatozoa is concerned. On the positive side, they are important modulators of sperm capacitation, but when the antioxidant factors that protect these vulnerable cells are overwhelmed, they are mediators of cell senescence and death. For these and other aspects of oxidative stress in the male germ line this book provides expert comprehensive coverage and should be read by anybody interested in this rapidly emerging field.

References

[1] Tosic J, Walton A. Formation of hydrogen peroxide by spermatozoa and its inhibitory effect on respiration. Nature. 1946;158:485.

[2] Tosic J, Walton A. Metabolism of spermatozoa. The formation and elimination of hydrogen peroxide by spermatozoa and effects on motility and survival. Biochem J. 1950;47:199–212.

[3] Aitken J.B, Naumovski N, Curry B, Grupen C.G, Gibb Z, Aitken R.J. Characterization of an L-amino acid oxidase in equine spermatozoa. Biol Reprod. 2015;92:125.

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Part I

Basic Aspects

Outline

Chapter 1.1. Life Under Aerobic Conditions

Chapter 1.2. The Oxidant Paradox

Chapter 1.3. From Past to Present: An Historical Overview of the Concept of Spermatozoa, Reactive Oxygen Species, and Male-Factor Infertility

Chapter 1.4. Basic Aspects of Oxidative Stress in Male Reproductive Health

Chapter 1.5. Leukocytes as a Cause of Oxidative Stress

Chapter 1.6. Human Spermatozoa and Interactions With Oxidative Stress

Chapter 1.7. Causes of Reductive Stress in Male Reproduction

Chapter 1.8. Physiological Role of Reactive Oxygen Species in Male Reproduction

Chapter 1.1

Life Under Aerobic Conditions

Kristian Leisegang     School of Natural Medicine, University of the Western Cape, Bellville, South Africa

Abstract

Following the rise of prokaryote life dependent on anaerobic respiration, oxygen was produced as a toxic by-product. This steadily and significantly contributed to the formation of an oxygen-rich environment that resulted in a shift in atmospheric and oceanic redox status. This necessitated the evolution of aerobic respiration, and mitochondria seemingly arose independently of bacterial life before becoming incorporated into eukaryote cells. Aerobic respiration allowed for a significant increase in adenosine triphosphate production, which is critical to meet the energy demands required for complex multicellular life to evolve. This molecular process also results in a leakage of potentially toxic reactive oxygen species (ROS) from the mitochondria. Although ROS have evolved as critical molecular and cellular regulators, excessive ROS damages cellular molecules and components. Therefore, endogenous antioxidants evolved as an important regulator of the redox state within the cellular environment in order to optimize the beneficial roles of redox biology.

Keywords

Aerobic respiration; Antioxidants; Evolution; Great oxygenation event; Mitochondria; Reactive oxygen species

Introduction

Biology on earth has had a significant impact on planetary evolution. This is prominently evident through planetary redox evolution due to changing atmospheric and oceanic gaseous environments [1]. In the modern era, climate change is based on the evolutionary recent rapid rise of CO2 in the atmosphere, significantly driven by Homo sapiens' influence through industrialization and unrestricted utilization of fossil fuel (carbonaceous) resources [2]. However, in the ancient organic rich environment of the late Archean (4–2.5  Ga: giga anuum = 1 billion years ago) and Proterozoic (2.5–0.5  Ga) periods, significant climate change occurred in which the atmosphere was being polluted by rising oxygen (O2) concentrations [2]. Although a rapid rise of O2 is evident in the records, it remains a challenge for geobiologists to fully understand the role of biological life in the oxygenation of the planet. This is due to scarce access to the well-preserved rocks from these times as well as a poor biological record of early biota [1]. However, it is evident that biological life significantly contributed to the changing environment, in which aerobic respiration evolved as a prominent event for the development of multicellular organisms due to the ability to produce energy more efficiently [1,2].

The Great Oxygenation Event

The early biosphere of earth, around 3.8  Ga in which life arose, is generally accepted to have been a low O2 environment [2] dominated by carbon dioxide (CO2) and nitrogen (N2). This chemical composition would have created an environment with mild reducing conditions, providing the main energy source for exclusively unicellular organisms consisting mainly of hyperthermophiles [1,3]. These organisms utilized chemical sources of energy through metabolic CO2 assimilation mechanisms, and from this photosynthesis appears to have arisen early in the evolution of life. O2 is a toxic molecular by-product of this anaerobic respiration [3]. Following this, geographical evidence suggests a significant global rise in atmospheric O2 approximately 2.3  Ga, known as the Great Oxygenation Event (GOE) [4]. In addition to biological life, environmental and climatic events such as glaciations and intense continental weathering that resulted in oxidation of the ocean surface water also contributed to the rise in O2 levels [5].

However, as early as 3  Ga, there was an atmospheric change in which oxygenic photosynthesis (oxygenic phototrophy) evolved through ancient cyanobacteria as a significant microbial biochemical innovation, providing the most significant source of modern atmospheric O2 concentrations [6]. Following the GOE, a final rise in atmospheric O2 was observed approximately 0.5  Ga [1].

It is clear, therefore, that the biological production of O2 dramatically modified the environment into the Proterozoic period, resulting in more oxidizing conditions compared to that of the Archean period [1,7]. Importantly, the associated change in the environmental redox state modified the bioavailability of metals, including iron, copper and molybdenum [8]. The bioavailability of O2 provided early biota with an oxidant environment, which arguably necessitated the evolution of aerobic respiration [8].

Biological Life and Environmental Evolution

The emergence of O2 in the evolution of Earth required at least one biological pathway to have been active and present, as abiotic generation of O2 (e.g., photodissociation of water vapor with hydrogen escaping to space) would have been too ineffective [9]. With abiotic production of O2 from water via UV light considered as a minor contributor, it is proposed that biological photosynthesis in cyanobacteria and photosynthetic eukaryotes greatly contributed to modern atmospheric O2 levels [10].

In addition to oxygenic photosynthesis, biological sources of O2 generation include reactive oxygen species (ROS) detoxification, chlorate generation, and nitrogen-driven anaerobic methane oxidation [9]. O2 production from detoxification of ROS is generally regarded to have evolved after oxygenic photosynthesis, and initial mechanisms of defense to ROS were physical barriers as opposed to enzymatic antioxidant systems [11]. Although there is some debate, aerobic respiration would have likely evolved after that of oxygen photosynthesis [1]. However, prior to this evolution, O2 may have been produced by chemosynthetic sources, such as possible nitrite anaerobic methane oxidation by oxygenic bacteria, primordial chlorate metabolism by microbial chlorate dismutation, catalase-like enzymes converting radioactivity produced peroxides, or via nitric oxide dismutation on volcanic nitric oxide in the presence of abundant oceanic iron to release environmental oxygen [12]. Increased subaerial volcanic activity further provided oxidized gases such as CO2 and SO2, whereas submarine volcanoes provided more reducing gases such as H2, CO, CH4, and H2S [1]. Although surface oceanic water may have become oxygen-rich earlier, the deep oceans remained anoxic until atmospheric O2 reached near present-day levels around 0.5  Ga [1]. This period ended the dominance of prokaryotes on earth and allowed complex and multicellular life to evolve [1].

With the evolution of oxygenic phototrophs, molecular O2 became the major electron acceptor on Earth [12]. Other electron acceptors exist; for example nitrate, which has an oxidative potential that is similar to that of O2 through the process of denitrification [12]. Both aerobic respiration and denitrification reactions share some common machinery and enzyme mediators, resulting in the production of potentially damaging highly reactive molecules [12].

Although there is more research required for geochemists to refine the understanding of earth's redox evolution in atmospheric and oceanic gasses [1,6], particularly the timing of oxygenation and the GOE currently dated at 2.3  Ga [1,4,6], the increasing generation of O2 in the atmosphere during this period was in fact a major atmospheric pollutant that affected biological life on earth [13]. Increasing atmospheric O2 led to the development of aerobic niches for advanced eukaryote organisms to develop into more complex multicellular life observed in the present-day biosphere [3].

Aerobic Respiration and the Mitochondria

Fundamental to cellular activity, respiration critically involves a molecular series of redox reactions. This consists of the transfer of electrons through membrane-associated cofactors and protein complexes, from an electron donor to an electron acceptor [10]. Aerobic respiration appears to have evolved after O2 increased in the atmosphere following climate transformation via anaerobic life forms [10]. The evolution of aerobic respiration was therefore driven by the increasing environmental source of O2 [12].

All known eukaryotes either have mitochondria or evolved from ancestors with mitochondria. With the energy demands of multicellular life being very high, aerobic respiration was a critical step in the development of biological evolution [10]. Aerobic respiration is in fact the most effective exergonic biological process known. Furthermore, this process plays central roles in molecular cycles of carbon, nitrogen, and sulfur in addition to oxygen [14]. In mitochondrial-driven aerobic respiration, the electron transfer is to the dioxygen molecule, driven via the oxygen reductase members of the heme-copper oxidoreductase family, which conserves some of the free energy released from O2 reduction [15]. The energy released in this biological process leads to the generation of an electrochemical gradient, known as the mitochondrial membrane potential (MMP), required for adenosine triphosphate (ATP) production [10].

Mitochondria are cytoplasmic organelles found in all eukaryote cells, and through cellular aerobic respiration (oxidative phosphorylation) provide the principle molecular energy supply in ATP. This happens primarily via the metabolism of simple sugars and long chain fatty acids [16]. In addition to energy production and recycling of adenosine diphosphate (ADP) into ATP, steroid hormones (pregnenolone) and lipids are synthesized. Ancient and distinct mitochondrial DNA (mtDNA) is replicated and hundreds of biochemical processes essential for human life occur [17]. Furthermore, mitochondria have significant roles in molecular signaling, cellular differentiation, cellular growth and division, immune signaling, and apoptosis [17].

Energy producing metabolic pathways originated very early during the evolution of life, and proteins associated with aerobic respiration chains have been found in early bacteria and Archaea, and may have arisen from a common ancestor [18]. Mitochondria themselves are considered to be of bacterial origin, arising from α-proteobacteria [19]. It has been argued that mitochondria, as well as chloroplasts, evolved from free-living bacteria within the eukaryotic cell as a symbiotic relationship (endosymbiont hypothesis) [20]. However, a significant amount of the mitochondrial proteome appears to have evolved outside α-proteobacteria, and has followed diverse evolutionary pathways through different eukaryote lineages creating a genomic and functional mosaic [19]. Hence, a debate remains whether mitochondria evolved after eukaryote cells arose or originated at the same time as the cells containing these organelles [19], with two fundamentally different themes: (1) the archezoan scenario, where primitive mitochondria were hosted in a hypothetical primitive eukaryote termed archezoan; and (2) the symbiogenesis scenario, where a single endosymbiotic event occurred, leading to the development of the mitochondria [19].

Including the double membrane, mitochondrial structure is divided into four compartments: the outer membrane, intermembrane space, inner membrane (folded into cristae), and matrix. The inner membrane is folded into cristae, where ATP synthase and protein complexes that regulate cellular respiration occur [17]. Rather than a static producer of energy, the mitochondria are dynamic, and the mitochondrial membrane is continuously remodeled in response to cellular signaling [17]. However, it is important to note that mitochondria across different species show significant variations in morphological appearance, coding capacity, and modes of expression [19]. In mammals, mtDNA has been sequenced from numerous species. In addition to ATP synthase, mtDNA encodes approximately 13 protein subunits of the mitochondrial electron transport chain as well as rRNA and tRNA units for the mitochondrial translation system [19]. Only the most ancestral mtDNAs encode for almost all of this gene set, for example Reclinomonas americana [21], with other organisms using as little as only three protein genes (e.g., mtDNA of Plasmodium falciparum).

The mitochondrial biomachinery and related redox activity is operated by five mitochondrial protein complexes [22]. These consist of nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I), the quinine pool, bc1 complex (complex III), and cytochrome c as the back bone for aerobic respiratory chain, with complex IV as a terminal oxidase [12]. Furthermore, based on the membrane associated bioelectrical gradient, the MMP is an important and reliable marker for mitochondrial function. A range of −136 to −140  mV is considered optimal for ATP production in all living organisms [23]. However, a detailed understanding of MMP generation has still not been completely elicited. Under normal cellular conditions, this potential appears to be exclusively generated by electron flow through electron transport chain complexes I–IV, terminating as a direct electrical current that terminates in subunit II of complex IV that gradually charges the MMP [23].

Redox Biology and the Paradoxical Nature of Aerobic Life

Following the GOE and the environment becoming O2-rich, the molecule has become critical for aerobic life on Earth, specifically through ATP-generation via oxidative phosphorylation ) and peroxide molecules [28]. Hydrogen peroxide (H2O2), however, is not an unstable radical although it is termed a ROS [28]. In this context, we also must consider that O2 itself is a diradical, a characteristic that explains its chemical reactivity.

in the mitochondria, producing H2O2 (a major cellular regulator of redox sensing and signaling)  [30].

ROS are known to regulate various cellular functions, such as apoptosis, ion transport systems, immune regulation and inflammatory responses, cellular responses to growth factor signaling, and regulation of genetic expression [28]. These small reactive molecules can further react with and damage cellular macromolecules, particularly proteins and lipids [28]. Molecular redox (phosphorylation/dephosphorylation) switches are the adaption mechanisms for physiological concentrations of ROS, which then function as mechanisms of cellular signaling [31]. Oxidative stress (OS) is therefore defined as a state of excess ROS production and/or the reduction in savaging antioxidants, which results in pathophysiological changes similar to the general adaption syndrome of cellular stressors [22,31].

Endogenous Antioxidants and Oxidative Stress

As excessive ROS production is toxic to cellular molecules and structures, a balance between ROS generation and metabolism by endogenous and exogenous antioxidants is crucial. Internal and external disturbances to this balance can lead to OS within cells and broader biological systems [27]. Over time, OS induces cellular damage that may accumulate, which is increasingly associated with complex pathophysiology known to mediate chronic disease. This includes a prominent role in ageing and age-related chronic diseases, including but not limited to obesity, metabolic syndrome, and associated type-2 diabetes mellitus, cardiovascular diseases, various malignancies, and neurodegenerative diseases including Alzheimer's disease [28]. OS has also been implicated as major cause or contributing factor of male infertility [32,33].

Due to the potentially detrimental effects of OS, cells have evolved defense mechanisms through the scavenging and neutralizing properties of endogenous antioxidants that reduced ROS with various cofactors. These regulating enzymes include catalase, glutathione peroxidase, and the SOD family [13]. Members of the SOD family include SOD1 (copper and zinc), SOD2 (Manganese), and the extracellular SOD3. Additional examples of well-studied endogenous antioxidant families include peroxiredoxins, glutaredoxins, and thioredoxins [13,34].

Contrary to the concept of OS, reductive stress (RS) involves a cellular shift of the redox status into the reduced state, which is also detrimental for cells as they are not able to induce regulatory redox switches [35]. RS is considered as dangerous for cells as OS [36], associated with cardiac damage, neurological diseases [37], and dysregulations of embryogenesis [38]. RS is also increasingly associated with male infertility [39,40].

Mitochondrial Dysfunction

Through the production of ATP via aerobic respiration, mitochondria are a significant source for the generation of excessive ROS, which further results in mitochondrial damage and dysfunction [41]. Underlying this dysfunction are the minor variations in MMP, which results in a dramatic reduction in ATP synthesis and a further increase in ROS production, thus reflecting the concept of mitochondrial dysfunction [23].

Mitochondrial dysfunction is receiving increasing attention and is associated with a wide range of human pathology, particularly involving non-communicable chronic and degenerative diseases. This includes obesity, metabolic syndrome, type-2 diabetes mellitus, cardiovascular disease, numerous cancers, and neurodegeneration [42,43]. Furthermore, mitochondrial dysfunction and associated OS is considered to be a major underlying mechanism of the ageing process, including negative impact on spermatogenesis and steroidogenesis in males [41,43]. However, there is evidence to suggest that ROS are not always deleterious, and may even promote longevity pathways, and therefore play a complex role in health and disease [41].

Conclusion

The evolution of aerobic respiration was necessitated by the toxic evolution of the planet due to oxygen generated as a by-product of prokaryote-based anaerobic respiration. This allowed for the evolution of aerobic respiration and a significant increase in energy generation for cellular activities, and multicellular eukaryote life evolved. In this process, mitochondria have emerged as a significant organelle associated with numerous cellular activities beyond energy production. Due to leakage of electrons in aerobic respiration, physiologically critical yet potentially toxic molecules termed ROS arise. It is important for a cellular redox steady state to be maintained for appropriate cellular functioning, with numerous endogenous antioxidants having evolved for this regulation. Oxidative and reductive stress arise out of an imbalance between ROS and total antioxidant capacity (TAC), and the understanding of this relationship in the molecular pathology across a wide range of diseases is of critical importance. Additionally, dysfunctional mitochondria and changes in MMP are associated with cellular or tissue ROS:TAC mismatch, and is emerging as an important mediator in numerous disease processes.

Glossary

Aerobic respiration   Process of developing cellular energy involving oxygen.

Anaerobic respiration   Cellular respiration in the absence of oxygen.

Antioxidant   Any molecule that inhibits oxidation and scavenges free radicals and reactive oxygen species.

Archaea   Microorganisms that are similar in morphology to bacteria with different molecular organization that constitute an ancient group between bacteria and eukaryotes.

Archean period   Denoting the eon that constitutes the early to middle Precambrian period in which there was no life on Earth.

Cyanobacteria   A division of prokaryote microorganisms related to bacteria that are capable of photosynthesis, representing the earliest known form of life on Earth.

Exergonic   A chemical or metabolic process accompanied by the release of energy.

Eukaryote   Cells that have genetic material in the form of DNA, including all living organisms except eubacteria and archaea.

Free radical   An uncharged highly reactive and short-lived molecule having an unpaired valence electron.

Geobiology   The study of interactions that occur between the living organisms and their biological products (biosphere) and the geosphere.

Great Oxygenation Event   The appearance of dioxygen in the Earth's atmosphere induced predominantly by biological activities of living organisms.

Oxidative stress   An imbalance between increased free radicals and/or reduced antioxidant scavengers that contributes to cellular cytotoxicity.

Oxygenic phototrophy   Metabolic respiration process using photons for energy generation (photosynthesis) that releases oxygen as a by-product, found in some plants, algae, and cyanobacteria.

Prokaryote   Microscopic single-cell organism with no distinct nucleus or other specialized organelles, including bacteria and cyanobacteria.

Proterozoic period   The eon that constitutes the later part of the Precambrian, between the Archean eon and the Cambrian period, in which the earliest forms of life evolved.

Proteobacteria   The largest and most diverse group (phylum) of bacteria that are gram-negative that show metabolic diversity including chemoautotrophic, chemoorganotrophic, and phototrophic. This phylum includes a variety of pathogens such as Escherichia, Salmonella, Vibrio, Helicobacter, and others.

Reactive oxygen species (ROS)   Chemically reactive molecules containing oxygen that are formed as a normal by-product of aerobic respiration.

Redox (reduction-oxidation)   The chemical processes of oxidation (process of losing or donating one or more electrons) and reduction (process of gaining or accepting one or more electrons) considered together as complementary processes.

Redox reaction   One atom or molecule receives electrons from another atom or compound.

Reductive stress   An imbalance between reduced free radicals and/or increased antioxidant scavengers that contributes to cellular cytotoxicity.

List of Acronyms and Abbreviations

ADP   adenosine diphosphate

AMP   adenosine monophosphate

ATP   adenosine triphosphate

CO2   carbon dioxide molecule

Ga   Giga-anuum (one billion years ago)

H2O2   hydrogen peroxidase

N2   nitrogen molecule

MMP   mitochondrial membrane potential

mtDNA   mitochondrial DNA

mV   millivolts

NADH   nicotinamide adenine dinucleotide

NO   nitrous oxide

O2   oxygen molecule

   superoxide radical

OH   hydroxyl radical

OS   oxidative stress

ROS   reactive oxygen species

SO2   sulphide

SOD   superoxide dismutase

References

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