**Meet the editor**

Bin Wu, Ph.D., HCLD (ABB) is currently a scientific laboratory director at Arizona Center for Reproductive Endocrinology and Infertility, USA. He received his training in genetics and reproductive biology at the Northwest Agricultural University in China and Cornell University, New York and post-doctor training at University of Guelph, Canada. He was promoted as a professor

at the Northwest Agricultural University. As an embryologist, he later joined in the Center for Human Reproduction in Chicago. Dr. Wu is a member of many professional associations, such as American Society for Reproductive Medicine; International Embryo Transfer Society; Society for the Study of Reproduction; American Association of Bioanalysts and European Society of Human Reproduction and Embryology. Also, he has obtained some significant research awards from these professional associations.

## Contents

### **Preface XI**


### Chapter 7 **Protein Kinase A and Protein Kinase C Connections: What Could Angiogenesis Tell Us? 169**

Beatriz Veleirinho, Daniela Sousa Coelho, Viviane Polli, Simone Kobe Oliveira, Rosa Maria Ribeiro-Do-Valle, Marcelo Maraschin and Paulo Fernando Dias

### **Section 3 Emrbyo Technology 183**

Chapter 8 **A Novel Discipline in Embryology — Animal Embryo Breeding 185** Bin Wu, Linsen Zan, Fusheng Quan and Hai Wang

Chapter 9 **Assisted Reproductive Technologies in Safeguard of Feline Endangered Species 199** Natascia Cocchia, Simona Tafuri, Lucia Abbondante, Leonardo Meomartino, Luigi Esposito and Francesca Ciani

Chapter 10 **Antiluteolytic Strategy for Bovine Embryo Transfer Programmes 231** Néstor Isaías Tovío Luna, Arturo Duica Amaya and Henry Alberto Grajales Lombana

### Preface

Chapter 7 **Protein Kinase A and Protein Kinase C Connections: What**

Chapter 8 **A Novel Discipline in Embryology — Animal Embryo**

Bin Wu, Linsen Zan, Fusheng Quan and Hai Wang

Chapter 9 **Assisted Reproductive Technologies in Safeguard of Feline**

Meomartino, Luigi Esposito and Francesca Ciani

Chapter 10 **Antiluteolytic Strategy for Bovine Embryo Transfer**

Natascia Cocchia, Simona Tafuri, Lucia Abbondante, Leonardo

Néstor Isaías Tovío Luna, Arturo Duica Amaya and Henry Alberto

Beatriz Veleirinho, Daniela Sousa Coelho, Viviane Polli, Simone Kobe Oliveira, Rosa Maria Ribeiro-Do-Valle, Marcelo Maraschin and

**Could Angiogenesis Tell Us? 169**

Paulo Fernando Dias

**Section 3 Emrbyo Technology 183**

**VI** Contents

**Breeding 185**

**Endangered Species 199**

**Programmes 231**

Grajales Lombana

Animal individual life begins at the combination of sperm and oocyte. This combination re‐ sults in the embryogenesis, which is a complex process from ovum fertilization through to fetal stage. Long time ago, many studies focused on this field to reveal the fertilization, em‐ bryo formation and development, which formed a branch discipline of biology science, i.e., embryology. To date, embryology has been enriched and developed greatly in the terms of its contents and forms. It not only includes oogenesis, spermiogenesis, embryogenesis, im‐ plantation and fetal formation mechanism, but also involves in pharmacology, basic scientif‐ ic research, and regenerative medicine. Although this subject has been studied for more than a century, it is still a pioneering field with many alternative aspects such as embryonic stem cell, somatic cell cloning, and many novel discoveries appear continuously. Particularly, some novel embryo biotechnologies have initiated a new era in the fields of medical science and agriculture owing to their enormous biomedical and commercial potential. Thus, this book contains some novel discoveries and theories on the embryology field in last decade.

Section 1, recent investigation has showed that sperm DNA integrity is vital for successful fertilization, embryo development, pregnancy, and transmission of genetic material to the offspring. DNA fragmentation is the most frequent DNA anomaly present in the male gam‐ ete, which is associated with poor semen quality, low fertilization rates, impaired embryo quality and reduced clinical outcomes in assisted reproduction procedures. Also, numerous studies have shown that oxidative stress plays a role in the pathophysiology of infertility and assisted fertility. Reactive oxygen species (ROS) is important mediator of normal sperm function, such as signal transduction mechanisms that affect fertility. Spermatozoa are par‐ ticularly susceptible to ROS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids and their cytoplasm contains low concentrations of the scavenging enzymes. Here, the first two chapters are listed to describe the relationship of the ROS and sperm DNA fragmentation with male fertility. Chapter One summarizes the causes of fragmentation in spermatic DNA and its relation with seminal parameters, male aging and outcome in assisted reproduction technology (ART). Chapter Two provides new information on the relationship of ROS with spermatic DNA fragmentation and dysfunc‐ tion. Furthermore, the third chapter describes the influence of ROS on ovarian functions and provides some evidences of oxidative stress in ovarian physiopathology, which influences folliculogenesis and steroidogenesis in the fluid follicular environment. Thus, increasing the knowledge of the mechanisms whereby the effect of ROS and endogenous antioxidant sys‐ tem on the reproductive processes will be beneficial to the optimal application of exogenous antioxidants to fertility treatment. For instance, adding the treatment of antioxidant enzyme, such as catalase, glutathione peroxidase, and the superoxide dismutase isoforms for testis sperm or ovarian oocytes, will be able to maintain low levels of oxidative stress for improv‐ ing sperm and ovarian oocyte development.

Section 2, due to new technology application, especially three-dimensional imaging (3D), to human embryo analysis, some novel discoveries have been recorded in human embryologi‐ cal area. In this part, three chapters present new theory and concept in human embryology. Chapter Four puts forward a novel concept of Fundus-Ovary-Salpinx-Para-aorta Implanta‐ tion Promoting Unit (FOSPa-IP unit) during human embryo implantation. In humans, the corpus luteum, which is formed from the ovulated follicle, produces progesterone that indu‐ ces adequate endometrial differentiation for embryo implantation. During pregnancy, the embryo trophoblast cells secrete human chorionic gonadotropin (HCG) that stimulates the maternal corpus luteum to sustain progesterone production. In turn, it acts on the endome‐ trium to maintain embryo implantation in the uterus. Thus, human embryo implantation is mainly regulated by the endocrine system. Chapter Five used newly high resolution imag‐ ing 3D technique to measure many embryo and fetal morphologies and development proc‐ ess, and re-profile human embryology atlas, which proposes the future direction for human embryo analysis. Chapter Six describes another novel cellular and molecular interactions during limb development by the studies on the Split Hand—Foot congenital malformation. It illustrates the pathway centered on the master transcription factor p63, which impacts the regulation of signaling molecule controlling growth and shape of the normal limb.

Section 3, there is a very close relationship between embryology and new developed embryo biotechnologies. Thus, this section will concentrate on discussing the application of embry‐ ology and its techniques to animal breeding and production. Chapter Eight puts forward a novel concept of "Animal Embryo Breeding" to describe this discipline formation, develop‐ ment and application in animal genetic improvement and domestic breeding. The relation‐ ship of embryo breeding with other disciplines has been profiled. Thus, animal scientists and breeder can easily understand and apply embryo breeding theory and its biotechnolo‐ gies to accelerate animal genetic improvement, to modify genetic construction and animal population and to design and create new animal individual or breed. Chapter Nine de‐ scribes the application of the assisted reproductive technologies (ART) in Safeguard of Fe‐ line endangered species. This new method in ART may greatly improve these endangered animal reproductive efficiency. In Chapter Ten, a new antiluteolystic strategy for bovine em‐ bryo transfer programs has been put forward to increase the successful rate for bovine em‐ bryo transfer.

Thus, this book will greatly update some novel knowledge in embryology field and it also provides some basic theories and technologies for animal scientists and breeder as well as embryologist and anthropologists.

Great thanks go to all authors who gladly contributed their time and expertise to prepare these outstanding chapters included in this book.

> **Bin Wu, Ph.D., HCLD (ABB)** Arizona Center for Reproductive Endocrinology and Infertility Tucson, Arizona USA

**Section 1**

**Gamete Biology**

sperm or ovarian oocytes, will be able to maintain low levels of oxidative stress for improv‐

Section 2, due to new technology application, especially three-dimensional imaging (3D), to human embryo analysis, some novel discoveries have been recorded in human embryologi‐ cal area. In this part, three chapters present new theory and concept in human embryology. Chapter Four puts forward a novel concept of Fundus-Ovary-Salpinx-Para-aorta Implanta‐ tion Promoting Unit (FOSPa-IP unit) during human embryo implantation. In humans, the corpus luteum, which is formed from the ovulated follicle, produces progesterone that indu‐ ces adequate endometrial differentiation for embryo implantation. During pregnancy, the embryo trophoblast cells secrete human chorionic gonadotropin (HCG) that stimulates the maternal corpus luteum to sustain progesterone production. In turn, it acts on the endome‐ trium to maintain embryo implantation in the uterus. Thus, human embryo implantation is mainly regulated by the endocrine system. Chapter Five used newly high resolution imag‐ ing 3D technique to measure many embryo and fetal morphologies and development proc‐ ess, and re-profile human embryology atlas, which proposes the future direction for human embryo analysis. Chapter Six describes another novel cellular and molecular interactions during limb development by the studies on the Split Hand—Foot congenital malformation. It illustrates the pathway centered on the master transcription factor p63, which impacts the

regulation of signaling molecule controlling growth and shape of the normal limb.

Section 3, there is a very close relationship between embryology and new developed embryo biotechnologies. Thus, this section will concentrate on discussing the application of embry‐ ology and its techniques to animal breeding and production. Chapter Eight puts forward a novel concept of "Animal Embryo Breeding" to describe this discipline formation, develop‐ ment and application in animal genetic improvement and domestic breeding. The relation‐ ship of embryo breeding with other disciplines has been profiled. Thus, animal scientists and breeder can easily understand and apply embryo breeding theory and its biotechnolo‐ gies to accelerate animal genetic improvement, to modify genetic construction and animal population and to design and create new animal individual or breed. Chapter Nine de‐ scribes the application of the assisted reproductive technologies (ART) in Safeguard of Fe‐ line endangered species. This new method in ART may greatly improve these endangered animal reproductive efficiency. In Chapter Ten, a new antiluteolystic strategy for bovine em‐ bryo transfer programs has been put forward to increase the successful rate for bovine em‐

Thus, this book will greatly update some novel knowledge in embryology field and it also provides some basic theories and technologies for animal scientists and breeder as well as

Great thanks go to all authors who gladly contributed their time and expertise to prepare

**Bin Wu, Ph.D., HCLD (ABB)**

Tucson, Arizona

USA

Arizona Center for Reproductive Endocrinology and Infertility

ing sperm and ovarian oocyte development.

VIII Preface

bryo transfer.

embryologist and anthropologists.

these outstanding chapters included in this book.

### **Chapter 1**

### **Sperm DNA Fragmentation and Its Relation With Fertility**

Javier García-Ferreyra

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60825

### **Abstract**

Sperm DNA integrity is vital for successful fertilization, embryo development, pregnancy, and transmission of genetic material to the offspring. DNA fragmentation is the most frequent DNA anomaly present in the male gamete that has been associated to poor semen quality, low fertilization rates, impaired embryo quality, and preim‐ plantation development and reduced clinical outcomes in assisted reproduction procedures. This work summarizes the causes of fragmentation in the spermatic DNA, and its relation with seminal parameters, male aging, and results in assisted repro‐ duction procedures.

**Keywords:** Spermatozoa, DNA fragmentation, seminal parameters, ROS, IVF, ICSI

### **1. Introduction**

Semen quality is frequently used as an indirect measure of male infertility. Ejaculate volume, sperm concentration, motility, and morphology determined according to the World Health Organisation (WHO) are the most important parameters evaluated in infertility centers as part of routine semen analysis. The genetic composition in a newborn is the results of oocyte and sperm DNA information, and it should be intact for further embryo and fetal development that will result in a healthy offspring. Any type of damage present in the DNA of male or female gametes can lead to an interruption of the reproductive process. Sperm DNA frag‐ mentation might be the most frequent cause of paternal DNA anomaly transmission to progeny and is found in a high percentage of spermatozoa from subfertile and infertile men.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Several hypotheses have been proposed as to the molecular mechanism of sperm DNA fragmentation, the most important ones being: apoptosis, abnormal chromatin packaging, and reactive oxygen species [1]. Several studies show that spermatozoa with DNA fragmentation are able to fertilize an oocyte [2-4], but are related to abnormal quality embryo, block in the blastocyst development, and lower pregnancy rates either natural or using IUI, IVF, or ICSI procedures [5-10]. Various studies demonstrate that the oocytes and the embryo retain the ability to repair DNA damage that may be present in the paternal genome; however, it is not yet clear if all types of damage can be repaired. For instance, double-stranded DNA breaks appear to be less repairable than single-stranded breaks and, therefore, have a greater impact on embryo quality and/or embryo development. Additionally, the capacity of oocyte to repair DNA damage will depend on factors like maturity, maternal age, and external factors. This review summarizes the causes that produce sperm DNA fragmentation, its relation to seminal parameters, paternal age, and effect on assisted reproduction procedures.

### **2. Human sperm chromatin structure**

Germ cells mediate the transfer of genetic information from generation to generation and are thus pivotal for the maintenance of life. Spermatogenesis is a continuous and precisely controlled process that involves extremely marked cellular, genetic and chromatin changes resulting in a generation of highly specialized sperm cells (Figure 1). Spermatogonial stem cells replicate and differentiate into primary spermatocytes that undergo genetic recombination to give rise to round haploid spermatids [11]. Round spermatids then undergo a differentiation process called spermiogenesis where marked cellular, epigenetic, and chromatin remodeling takes place [12, 13]. The nucleosomes are disassembled and the histones are removed and replaced by the high positively charged protamines forming tight toroidal complexes, organizing 85—95% of the human sperm DNA [14]. Human spermatozoa have two types of protamine (P1 and P2). P2 has fewer thiol groups for disulfide bonding and this makes human sperm chromatin less stable [15]. Finally, during the transit in the epididymis the cysteines become progressively oxidized forming inter- and intraprotamine disulfide bonds that, along with zinc bridges, stabilize and compact completely the chromatin [16, 17]. All these interac‐ tions make mammalian DNA the most condensed eukaryotic DNA [18], adjusting to the extremely limited volume of the sperm nucleus [19].

Chromatin organization plays an important role during the fertilization process and early embryo development. The sperm chromatin is a crystalline, insoluble, compact, and wellorganized structure in DNA loop domains with an average length of 27 kilobytes. These loops, which can be visualized by using fluorescent in situ hybridization (FISH), are attached at their bases to the nuclear matrix. During sperm decondensation the DNA remains anchored to the base of the tail, suggesting the presence of a nuclear annulus-like structure in human sperm [20]. This DNA organization permits the transfer of the very tightly packaged genetic infor‐ mation to the egg and ensures that the DNA will be delivered in a physical and chemical form that allows the developing embryo to access the genetic information [1].

**Figure 1.** Espermatogenesis. **a:** Spermatogonia (2n); **b:** primary spermatocyte (2n); **c: s**econdary spermatocyte (n); **d:** spermatid (n); **e:** spermatozoa

### **3. Causes of DNA fragmentation**

Sperm DNA fragmentation can be caused by apoptosis, defects in chromatin remodeling during the process of spermiogenesis, and oxygen radical-induced DNA damage.

### **3.1. Apoptosis**

Several hypotheses have been proposed as to the molecular mechanism of sperm DNA fragmentation, the most important ones being: apoptosis, abnormal chromatin packaging, and reactive oxygen species [1]. Several studies show that spermatozoa with DNA fragmentation are able to fertilize an oocyte [2-4], but are related to abnormal quality embryo, block in the blastocyst development, and lower pregnancy rates either natural or using IUI, IVF, or ICSI procedures [5-10]. Various studies demonstrate that the oocytes and the embryo retain the ability to repair DNA damage that may be present in the paternal genome; however, it is not yet clear if all types of damage can be repaired. For instance, double-stranded DNA breaks appear to be less repairable than single-stranded breaks and, therefore, have a greater impact on embryo quality and/or embryo development. Additionally, the capacity of oocyte to repair DNA damage will depend on factors like maturity, maternal age, and external factors. This review summarizes the causes that produce sperm DNA fragmentation, its relation to seminal

Germ cells mediate the transfer of genetic information from generation to generation and are thus pivotal for the maintenance of life. Spermatogenesis is a continuous and precisely controlled process that involves extremely marked cellular, genetic and chromatin changes resulting in a generation of highly specialized sperm cells (Figure 1). Spermatogonial stem cells replicate and differentiate into primary spermatocytes that undergo genetic recombination to give rise to round haploid spermatids [11]. Round spermatids then undergo a differentiation process called spermiogenesis where marked cellular, epigenetic, and chromatin remodeling takes place [12, 13]. The nucleosomes are disassembled and the histones are removed and replaced by the high positively charged protamines forming tight toroidal complexes, organizing 85—95% of the human sperm DNA [14]. Human spermatozoa have two types of protamine (P1 and P2). P2 has fewer thiol groups for disulfide bonding and this makes human sperm chromatin less stable [15]. Finally, during the transit in the epididymis the cysteines become progressively oxidized forming inter- and intraprotamine disulfide bonds that, along with zinc bridges, stabilize and compact completely the chromatin [16, 17]. All these interac‐ tions make mammalian DNA the most condensed eukaryotic DNA [18], adjusting to the

Chromatin organization plays an important role during the fertilization process and early embryo development. The sperm chromatin is a crystalline, insoluble, compact, and wellorganized structure in DNA loop domains with an average length of 27 kilobytes. These loops, which can be visualized by using fluorescent in situ hybridization (FISH), are attached at their bases to the nuclear matrix. During sperm decondensation the DNA remains anchored to the base of the tail, suggesting the presence of a nuclear annulus-like structure in human sperm [20]. This DNA organization permits the transfer of the very tightly packaged genetic infor‐ mation to the egg and ensures that the DNA will be delivered in a physical and chemical form

that allows the developing embryo to access the genetic information [1].

parameters, paternal age, and effect on assisted reproduction procedures.

**2. Human sperm chromatin structure**

4 New Discoveries in Embryology

extremely limited volume of the sperm nucleus [19].

During spermiogenesis, apoptosis allows the monitoring of the germ cell population that will be sustained by Sertoli cells [21], to regulate the overproduction of sperm cell and the elimi‐ nation of abnormal cells [22]. Sperm apoptosis is mediated by type Fas proteins [23], and their concentration is above 50% in males with abnormal seminal parameters [24]. Generally, cells marked with Fas proteins are phagocytized and eliminated by Sertoli cells to which these are associated [25, 26]. However, a percentage of defective germ cells undergo sperm remodeling during spermiogenesis, appearing later on the ejaculate, showing normal morphology but are genetically altered [27]. Apoptosis entails cell membrane disruption, cytoskeletal rearrange‐ ment, nuclear condensation and intranucleosomal DNA fragmentation in numerous frag‐ ments ≥185 bp [28].

### **3.2. Damage during chromatin packing in the spermiogenesis**

Sperm chromatin structure has a complex arrangement of DNA and sperm nuclear protein with different levels of compaction to shrink the nuclear volume and head size [29]. Then, DNA fragmentation may be the result of unresolved strand breaks created during the normal process spermiogenesis in order to relieve the torsional stresses involved in packaging a very large amount of DNA into the very small sperm head. These physiological strand breaks are corrected through H2Ax phosphorylation and activation of nuclear poly (ADP-ribose) polymerase and topoisomerase [30].

### **3.3. Oxygen radical-induced DNA damage by reactive oxygen species**

ROS or free radicals are oxidizing agents that are generated as byproducts of the metabolism of oxygen. Due to the presence of at least one unpaired electron, they form highly reactive molecules (e.g. hydroxyl ion [OH], superoxide ion [O2 - ], nitric oxide [NO], peroxyl [RO2], lipid peroxyl [LOO], and Thyl [RS]) and non-radical molecules (singlet oxygen [O2], hydrogen peroxide [H2O2], hypochloric acid [HOCl], lipid peroxide [LOOH], and ozone [O3]) [31].

It has been reported that the chromatin in the sperm nucleus is vulnerable to oxidative damage, leading to base modifications and DNA fragmentation [32]. De luliis *et al*. [33] showed that electromagnetic radiation induces ROS production, resulting in DNA damage and decreased motility and vitality in human spermatozoa. Moreover, several toxins released from structural materials or industrial products (e.g., benzene, methylene chloride, hexane, toluene, trichloro‐ ethane, styrene, heptane, and phthalates) and toxins in the form of metals (e.g. cadmium, chromium, lead, manganese, and mercury) increase ROS production in the testes, impairing the spermatogenesis and inducing sperm DNA fragmentation [34-36]. Additionally, con‐ sumption of tobacco and alcohol leads to higher rates of ROS production and high levels of DNA strand breaks [37], decreasing in sperm motility [38] and apoptosis.

Furthermore, the activation of sperm caspases and endonucleases by ROS induce sperm DNA fragmentation. Studies by Cui *et al.* [39] and Banks *et al*. [40] showed that *in vivo* exposure of mouse testis at 40º—42ºC results in a significant increase in DNA fragmentation, occurring in the epididymis by activation of caspases and endonucleases. The potential damage that sperm may experience during passage through the epididymis could be limited by removing them before that passage. Patients with high levels of DNA fragmentation in semen and repeated IVF failure can increase their clinical outcomes using testicular sperm obtained by testicular sperm extraction (TESE or TESA) [41].

Human sperm chromatin becomes cross-linked under conditions of oxidative stress and exhibits increased DNA strand breakage [42]. When DNA is minimally damaged, spermatozoa can undergo self-repair and potentially regain the ability to fertilize the oocyte and proceed with development [43]. In fact, the oocyte is also capable of repairing damaged sperm DNA; but when the oocyte machinery is not sufficient to repair DNA damage the embryo may fail to develop or implant in the uterus.

### **4. Age and DNA fragmentation**

The increase in life expectancy, women's entry into the labor market and the popular use of contraception has contributed to the social phenomena of delaying family planning and parenthood to the mid or late thirties. This has also had a significant impact on males. In Germany, the median age of married fathers has increased from 31.3 years in 1991 to 33.1 years in 1999 [44]. The same trend has also been seen in England. In 1993, fathers aged 35—54 years accounted for 25% of live births. Ten years later, these percentages grew to 40% [45]. Among couples seeking pregnancy through assisted reproduction technologies (ART), fathers are significantly older compared with those not needing ART (36.6 vs. 33.5 years) [46].

In Western societies, advanced paternal age is a phenomenon that parallels advanced maternal age and is associated with various reproductive hazards including decrease of testicular volume, alterations in testicular histomorphology, and a decrease in the inhibin B/FSH ratio consistent with a reduced Sertoli cell mass [47]. Other observable patterns include risk of chromosomal disorders, decline in semen volume, progressive motility, and daily sperm production with advanced age [48].

On the other hand, García-Ferreyra *et al*. [49] evaluated the effect of age on fertility and showed that the sperm DNA fragmentation, progressive motility, and spermatozoa morphology are associated with advanced paternal age. They analyzed seminal samples of 217 infertile patients between 21 and 68 years, which were distributed into four groups: <30 years, 30—39 years, 40 —49 years and ≥50 years. The results showed an age-dependent increase in sperm DNA fragmentation, which was statistically significant starting at 40 years old (Table 1). Patients ≥ 50 years old had morphologically normal spermatozoa, significantly lower compared to those men <40 years (Figure 2).


**a** *P*<0.05 in relation to the group <30 years

large amount of DNA into the very small sperm head. These physiological strand breaks are corrected through H2Ax phosphorylation and activation of nuclear poly (ADP-ribose)

ROS or free radicals are oxidizing agents that are generated as byproducts of the metabolism of oxygen. Due to the presence of at least one unpaired electron, they form highly reactive

peroxyl [LOO], and Thyl [RS]) and non-radical molecules (singlet oxygen [O2], hydrogen peroxide [H2O2], hypochloric acid [HOCl], lipid peroxide [LOOH], and ozone [O3]) [31].

It has been reported that the chromatin in the sperm nucleus is vulnerable to oxidative damage, leading to base modifications and DNA fragmentation [32]. De luliis *et al*. [33] showed that electromagnetic radiation induces ROS production, resulting in DNA damage and decreased motility and vitality in human spermatozoa. Moreover, several toxins released from structural materials or industrial products (e.g., benzene, methylene chloride, hexane, toluene, trichloro‐ ethane, styrene, heptane, and phthalates) and toxins in the form of metals (e.g. cadmium, chromium, lead, manganese, and mercury) increase ROS production in the testes, impairing the spermatogenesis and inducing sperm DNA fragmentation [34-36]. Additionally, con‐ sumption of tobacco and alcohol leads to higher rates of ROS production and high levels of

Furthermore, the activation of sperm caspases and endonucleases by ROS induce sperm DNA fragmentation. Studies by Cui *et al.* [39] and Banks *et al*. [40] showed that *in vivo* exposure of mouse testis at 40º—42ºC results in a significant increase in DNA fragmentation, occurring in the epididymis by activation of caspases and endonucleases. The potential damage that sperm may experience during passage through the epididymis could be limited by removing them before that passage. Patients with high levels of DNA fragmentation in semen and repeated IVF failure can increase their clinical outcomes using testicular sperm obtained by testicular

Human sperm chromatin becomes cross-linked under conditions of oxidative stress and exhibits increased DNA strand breakage [42]. When DNA is minimally damaged, spermatozoa can undergo self-repair and potentially regain the ability to fertilize the oocyte and proceed with development [43]. In fact, the oocyte is also capable of repairing damaged sperm DNA; but when the oocyte machinery is not sufficient to repair DNA damage the embryo may fail

The increase in life expectancy, women's entry into the labor market and the popular use of contraception has contributed to the social phenomena of delaying family planning and parenthood to the mid or late thirties. This has also had a significant impact on males. In


], nitric oxide [NO], peroxyl [RO2], lipid

**3.3. Oxygen radical-induced DNA damage by reactive oxygen species**

DNA strand breaks [37], decreasing in sperm motility [38] and apoptosis.

molecules (e.g. hydroxyl ion [OH], superoxide ion [O2

polymerase and topoisomerase [30].

6 New Discoveries in Embryology

sperm extraction (TESE or TESA) [41].

to develop or implant in the uterus.

**4. Age and DNA fragmentation**

**<sup>b</sup>***P*<0.05 in relation to the group 30—39 years

**c** *P*<0.05 in relation to the group 40—49 years

García-Ferreyra et al. Sperm DNA fragmentation. JFIV Reprod Med Genet 2012

**Table 1.** Sperm DNA fragmentation according to male age

In males, germ cells divide continuously. It has been estimated that 30 spermatogonial stem cell divisions take place before puberty, when they begin to undergo meiotic divisions. From then on, 23 meiotic divisions per year occur, resulting in 150 replications by the age of 20 and 840 replications by the age of 50 [50]. Because of these numerous divisions of stem cells, older men may have an increased risk of errors in DNA transcription. Furthermore, germ cells are continuously under attack from endogenous and exogenous factors that can induce a wide range of DNA lesions, thereby affecting normal cellular processes such as transcription, recombination and replication [51]. One of the main theories of aging states that aging results

 *García-Ferreyra et al. Sperm DNA fragmentation. JFIV Reprod Med Genet 2012*  García-Ferreyra et al. Sperm DNA fragmentation. JFIV Reprod Med Genet 2012

 In males, germ cells divide continuously. It has been estimated that 30 spermatogonial stem cell divisions take **Figure 2.** Scatter graph illustrating the associations between age and DNA fragmentation (a; *r=*0.106; *p=*0.0001) and morphology (b; *r=*0.054; *p=*0.0017)

from an accumulation of unrepaired DNA lesions; such lesions have been routinely linked to aging in many tissues including the brain, the liver, and the testis [52, 53]. Paul *et al*. [53] showed that there is an age-related accumulation of DNA damage in the testis, particularly caused by oxidative stress in the form of 8-oxodG lesions. Furthermore, aging seems to lower the capacity of germ cells to repair such DNA damage, resulting in the production of spermatozoa with increased DNA damage. This is likely to lead to a decline in genome quality that may be passed on to future generations, specifically the offspring of older males. place before puberty, when they begin to undergo meiotic divisions. From then on, 23 meiotic divisions per year occur, resulting in 150 replications by the age of 20 and 840 replications by the age of 50 [50]. Because of these numerous divisions of stem cells, older men may have an increased risk of errors in DNA transcription. Furthermore, germ cells are continuously under attack from endogenous and exogenous factors that can induce a wide range of DNA lesions, thereby affecting normal cellular processes such as transcription, recombination and replication [51]. One of the main theories of aging states that aging results from an accumulation of unrepaired DNA lesions; such lesions have been routinely linked to aging in many tissues including the brain, the liver, and the testis [52, 53]. Paul *et al*. [53] showed that there is an age-related accumulation of DNA damage in the testis, particularly caused by oxidative stress in the form of

8-oxodG lesions. Furthermore, aging seems to lower the capacity of germ cells to repair such DNA damage, resulting in the production of spermatozoa with increased DNA damage. This is likely to lead to a decline in genome quality that may

progressive motility [54], and has been associated with infertility and low fertilization rates in conventional IVF procedures

#### **5. Spermatozoa morphology and DNA fragmentation** be passed on to future generations, specifically the offspring of older males.

Teratozoospermia is defined as ≤ 4% normal sperm morphology at semen analysis with normal sperm count and normal progressive motility [54], and has been associated with infertility and low fertilization rates in conventional IVF procedures [55, 56]. **Spermatozoa morphology and DNA fragmentation**  Teratozoospermia is defined as ≤ 4% normal sperm morphology at semen analysis with normal sperm count and normal

Several studies indicate that DNA damage is associated with abnormalities in conventional semen parameters [24, 57-59]. Irvine *et al*. [57] found a stronger inverse correlation between DNA damage with concentration (-0.54) and Saleh *et al*. [60] showed an inverse correlation with the motility (-0.47). Larson-Cook *et al*. [61] demonstrated that only three of the 10 men with high levels of DNA damage had asthenozoospermia and/or oligozoospermia. In the study of García-Ferreyra *et al*. [49] evaluating the effect of age on semen parameters in infertile men, it was shown that the advanced paternal age was related to high percentages of fragmented DNA and low values of spermatic concentration, motility and morphology. Recently, García-Ferreyra *et al*. [62] assessed the quality of spermatic DNA according to spermatozoa morphol‐ ogy in 196 men, concluding that high levels of DNA damage were related to abnormal sperm morphology (Figure 3). Besides, when splitting the patients into a group of normozoospermic men and a group of men with at least one impaired conventional semen parameter or infertile men, the two groups were significantly different from each other in DNA fragmentation, [55, 56]. Several studies indicate that DNA damage is associated with abnormalities in conventional semen parameters [24, 57-59]. Irvine *et al*. [57] found a stronger inverse correlation between DNA damage with concentration (-0.54) and Saleh *et al*. [60] showed an inverse correlation with the motility (-0.47). Larson-Cook *et al*. [61] demonstrated that only three of the 10 men with high levels of DNA damage had asthenozoospermia and/or oligozoospermia. In the study of García-Ferreyra *et al*. [49] evaluating the effect of age on semen parameters in infertile men, it was shown that the advanced paternal age was related to high percentages of fragmented DNA and low values of spermatic concentration, motility and morphology. Recently, García-Ferreyra *et al*. [62] assessed the quality of spermatic DNA according to spermatozoa morphology in 196 men, concluding that high levels of DNA damage were related to abnormal sperm morphology (Figure 3). Besides, when splitting the patients into a group of normozoospermic men and a group of men with at least one impaired conventional semen parameter or infertile men, the two groups were significantly different from

7

motility, and morphology percentages (Table 2). Similar results were reported by Levitas *et al*. [63], Cardona *et al*. [64], Molina *et al*. [65], and Brahem *et al*. [66] while Winkle *et al*. [67] only reported a decrease in sperm motility.


*\*P<0.05* in relation to the Normozoospermic group

from an accumulation of unrepaired DNA lesions; such lesions have been routinely linked to aging in many tissues including the brain, the liver, and the testis [52, 53]. Paul *et al*. [53] showed that there is an age-related accumulation of DNA damage in the testis, particularly caused by oxidative stress in the form of 8-oxodG lesions. Furthermore, aging seems to lower the capacity of germ cells to repair such DNA damage, resulting in the production of spermatozoa with increased DNA damage. This is likely to lead to a decline in genome quality that may be passed

**Figure 2.** Scatter graph illustrating the associations between age and DNA fragmentation (a; *r=*0.106; *p=*0.0001) and

 In males, germ cells divide continuously. It has been estimated that 30 spermatogonial stem cell divisions take place before puberty, when they begin to undergo meiotic divisions. From then on, 23 meiotic divisions per year occur, resulting in 150 replications by the age of 20 and 840 replications by the age of 50 [50]. Because of these numerous divisions of stem cells, older men may have an increased risk of errors in DNA transcription. Furthermore, germ cells are continuously under attack from endogenous and exogenous factors that can induce a wide range of DNA lesions, thereby affecting normal cellular processes such as transcription, recombination and replication [51]. One of the main theories of aging states that aging results from an accumulation of unrepaired DNA lesions; such lesions have been routinely linked to aging in many tissues including the brain, the liver, and the testis [52, 53]. Paul *et al*. [53] showed that there is an age-related accumulation of DNA damage in the testis, particularly caused by oxidative stress in the form of 8-oxodG lesions. Furthermore, aging seems to lower the capacity of germ cells to repair such DNA damage, resulting in the production of spermatozoa with increased DNA damage. This is likely to lead to a decline in genome quality that may

0

> 20 30 40 50 60 70 age (years)

2

4

6

morphology (%)

8

10

Teratozoospermia is defined as ≤ 4% normal sperm morphology at semen analysis with normal sperm count and normal progressive motility [54], and has been associated with infertility and

Teratozoospermia is defined as ≤ 4% normal sperm morphology at semen analysis with normal sperm count and normal progressive motility [54], and has been associated with infertility and low fertilization rates in conventional IVF procedures

 Several studies indicate that DNA damage is associated with abnormalities in conventional semen parameters [24, 57-59]. Irvine *et al*. [57] found a stronger inverse correlation between DNA damage with concentration (-0.54) and Saleh *et al*. [60] showed an inverse correlation with the motility (-0.47). Larson-Cook *et al*. [61] demonstrated that only three of the 10 men with high levels of DNA damage had asthenozoospermia and/or oligozoospermia. In the study of García-Ferreyra *et al*. [49] evaluating the effect of age on semen parameters in infertile men, it was shown that the advanced paternal age was related to high percentages of fragmented DNA and low values of spermatic concentration, motility and morphology. Recently, García-Ferreyra *et al*. [62] assessed the quality of spermatic DNA according to spermatozoa morphology in 196 men, concluding that high levels of DNA damage were related to abnormal sperm morphology (Figure 3). Besides, when splitting the patients into a group of normozoospermic men and a group of men with at least one impaired conventional semen parameter or infertile men, the two groups were significantly different from

Several studies indicate that DNA damage is associated with abnormalities in conventional semen parameters [24, 57-59]. Irvine *et al*. [57] found a stronger inverse correlation between DNA damage with concentration (-0.54) and Saleh *et al*. [60] showed an inverse correlation with the motility (-0.47). Larson-Cook *et al*. [61] demonstrated that only three of the 10 men with high levels of DNA damage had asthenozoospermia and/or oligozoospermia. In the study of García-Ferreyra *et al*. [49] evaluating the effect of age on semen parameters in infertile men, it was shown that the advanced paternal age was related to high percentages of fragmented DNA and low values of spermatic concentration, motility and morphology. Recently, García-Ferreyra *et al*. [62] assessed the quality of spermatic DNA according to spermatozoa morphol‐ ogy in 196 men, concluding that high levels of DNA damage were related to abnormal sperm morphology (Figure 3). Besides, when splitting the patients into a group of normozoospermic men and a group of men with at least one impaired conventional semen parameter or infertile men, the two groups were significantly different from each other in DNA fragmentation,

7

on to future generations, specifically the offspring of older males.

**a b**

20 30 40 50 60 70 age (years)

 *García-Ferreyra et al. Sperm DNA fragmentation. JFIV Reprod Med Genet 2012* 

García-Ferreyra et al. Sperm DNA fragmentation. JFIV Reprod Med Genet 2012

**5. Spermatozoa morphology and DNA fragmentation**

be passed on to future generations, specifically the offspring of older males.

low fertilization rates in conventional IVF procedures [55, 56].

**Spermatozoa morphology and DNA fragmentation** 

[55, 56].

20

morphology (b; *r=*0.054; *p=*0.0017)

30

40

DNA fragmentation (%)

50

60

8 New Discoveries in Embryology

García-Ferreyra et al. Sperm DNA fragmentation JFIV Reprod Med Genet 2014

**Table 2.** Relation between DNA fragmentation, motility and morphology.

García-Ferreyra et al. Sperm DNA fragmentation JFIV Reprod Med Genet 2014

**Figure 3.** Scatter graph illustrating associations between DNA fragmentation and morphology (r=2.464; p=0.000)

### **6. IVF/ICSI procedures and sperm DNA fragmentation**

Sperm DNA contributes half of the offspring's genomic material and abnormal DNA can lead to derangements in the reproductive process. Several studies provide good evidence that sperm DNA and chromatin damage are associated with male infertility and reduced natural conception rates [6, 68, 69]. In humans, high levels of sperm DNA damage have been related

2

to low fertility potential, failure to obtain blastocysts, blockage in embryo development after embryo implantation, increased risk of recurrent miscarriages, reduced chances of successful implantation, and negative effects on the health of the offspring [70-72].

Studies of Virro *et al*. [73], Huang *et al*. [59], and Borini *et al*. [76] showed a negative correlation between fertilization rates and high levels of sperm DNA fragmentation. However, if the type and extent of DNA damage can be balanced by the reparative ability of the oocyte, it is possible to achieve fertilization even in the presence of elevated sperm DNA fragmentation rates [74, 75]. Given that, excessive damage in sperm DNA may result in early reproductive failures and during the 4 to 8 cell stage, when the paternal genome is switched on, the development of the embryo will be affected by sperm DNA integrity causing apoptosis, fragmentation, and difficulty to reach the blastocyst stage [19, 76].

An inverse relationship has been reported between the likelihood of achieving pregnancy either by natural intercourse and intrauterine insemination (IUI), but there are conflicting results with IVF/ICSI procedures and the presence of high sperm DNA fragmentation levels [72, 74, 77, 78]. An extended study by Bungum *et al*. [79] performed on a total of 998 IUI cycles showed significantly lower odds ratios for clinical pregnancy and delivery when the male partner had a DNA fragmentation index >30% measured by SCSA. On the other hand, published studies suggest conflicting results of the influence of sperm DNA fragmentation on embryo quality and development capacity in the outcomes of IVF and ICSI [3, 5, 7, 60].

Two meta-analyses made by Evenson and Wixon [80] and Li *et al*. [81] evaluating the relation of sperm DNA fragmentation and assisted reproduction outcomes reported different results; the first one showed that the clinical outcomes in IIU, IVF, and ICSI were closely related to DNA fragmented; whereas the other one suggested only negative effect on IVF procedures. A possible explanation for these differences is the different methods used to detect DNA integrity and the lack of standardization of methods used to evaluate sperm DNA fragmentation. Recently, Zini *et al*. [82] performed a systematic review of 28 studies to examine the influence of sperm DNA fragmentation on embryo quality and/or embryo development at IVF and ICSI (8 IVF, 12 ICSI, and 8 mixed IVF-ICSI). In 11 of 28 studies there was a positive relation between DNA fragmented and poor embryo quality/development. Sperm DNA fragmentation was associated with poor embryo development in 7 of 11 positive studies, and with poor embryo quality in 5 of the 11 positive studies. Moreover, according to ART procedures the sperm DNA fragmentation was associated only with 1/8 IVF studies (poor embryo quality), and 5/12 ICSI studies (poor quality and/or delayed development). These data suggest that the effect of sperm DNA fragmentation on embryo quality/development is more dramatic in ICSI compared to IVF, probably because with ICSI the natural selection barriers are bypassed entirely and the fertilization with highly DNA fragmented sperm is possible, which does not occur in IVF where the integrity of sperm DNA is closely related to sperm motility and sperm membrane characteristics important during the natural selection process reducing the probability of fertilization with DNA-damage sperm at IVF [83, 84]. Finally, the majority of studies indicate that sperm DNA fragmentation has negative effects on pregnancy rate, embryo quality, live birth, and early pregnancy loss.

### **7. Conclusions**

to low fertility potential, failure to obtain blastocysts, blockage in embryo development after embryo implantation, increased risk of recurrent miscarriages, reduced chances of successful

Studies of Virro *et al*. [73], Huang *et al*. [59], and Borini *et al*. [76] showed a negative correlation between fertilization rates and high levels of sperm DNA fragmentation. However, if the type and extent of DNA damage can be balanced by the reparative ability of the oocyte, it is possible to achieve fertilization even in the presence of elevated sperm DNA fragmentation rates [74, 75]. Given that, excessive damage in sperm DNA may result in early reproductive failures and during the 4 to 8 cell stage, when the paternal genome is switched on, the development of the embryo will be affected by sperm DNA integrity causing apoptosis, fragmentation, and

An inverse relationship has been reported between the likelihood of achieving pregnancy either by natural intercourse and intrauterine insemination (IUI), but there are conflicting results with IVF/ICSI procedures and the presence of high sperm DNA fragmentation levels [72, 74, 77, 78]. An extended study by Bungum *et al*. [79] performed on a total of 998 IUI cycles showed significantly lower odds ratios for clinical pregnancy and delivery when the male partner had a DNA fragmentation index >30% measured by SCSA. On the other hand, published studies suggest conflicting results of the influence of sperm DNA fragmentation on embryo quality and development capacity in the outcomes of IVF and ICSI [3, 5, 7, 60].

Two meta-analyses made by Evenson and Wixon [80] and Li *et al*. [81] evaluating the relation of sperm DNA fragmentation and assisted reproduction outcomes reported different results; the first one showed that the clinical outcomes in IIU, IVF, and ICSI were closely related to DNA fragmented; whereas the other one suggested only negative effect on IVF procedures. A possible explanation for these differences is the different methods used to detect DNA integrity and the lack of standardization of methods used to evaluate sperm DNA fragmentation. Recently, Zini *et al*. [82] performed a systematic review of 28 studies to examine the influence of sperm DNA fragmentation on embryo quality and/or embryo development at IVF and ICSI (8 IVF, 12 ICSI, and 8 mixed IVF-ICSI). In 11 of 28 studies there was a positive relation between DNA fragmented and poor embryo quality/development. Sperm DNA fragmentation was associated with poor embryo development in 7 of 11 positive studies, and with poor embryo quality in 5 of the 11 positive studies. Moreover, according to ART procedures the sperm DNA fragmentation was associated only with 1/8 IVF studies (poor embryo quality), and 5/12 ICSI studies (poor quality and/or delayed development). These data suggest that the effect of sperm DNA fragmentation on embryo quality/development is more dramatic in ICSI compared to IVF, probably because with ICSI the natural selection barriers are bypassed entirely and the fertilization with highly DNA fragmented sperm is possible, which does not occur in IVF where the integrity of sperm DNA is closely related to sperm motility and sperm membrane characteristics important during the natural selection process reducing the probability of fertilization with DNA-damage sperm at IVF [83, 84]. Finally, the majority of studies indicate that sperm DNA fragmentation has negative effects on pregnancy rate, embryo quality, live

implantation, and negative effects on the health of the offspring [70-72].

difficulty to reach the blastocyst stage [19, 76].

10 New Discoveries in Embryology

birth, and early pregnancy loss.

Sperm DNA fragmentation is an important factor that should be evaluated in subfertile and infertile men because several studies have shown that it has an important impact, independent of the parameters of classic semen analysis, on the reproductive process in both natural and assisted reproduction. Particularly, it affects the embryo quality and/or embryo development that decrease the implantation rates and increase the rates of early miscarriage in ART. Finally, it is important to obtain a clear diagnosis and the application of adequate methods of sperm selection pre—ART when high levels of sperm DNA fragmentation are observed to increase the possibilities to achieve the pregnancy in couples with high sperm DNA fragmentation and repeated assisted reproduction failures.

### **Author details**

Javier García-Ferreyra

Address all correspondence to: jgarciaf@fertilab.pe

FERTILAB Laboratory of Assisted Reproduction, Lima, Perú

### **References**


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16 New Discoveries in Embryology


### **Reactive Oxygen Species (ROS) and Male Fertility**

Simona Tafuri, Francesca Ciani, Eugenio Luigi Iorio, Luigi Esposito and Natascia Cocchia

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60632

### **Abstract**

Oxidative energy production is inevitably associated with the generation of reactive oxygen species (ROS), excessive concentrations of which can lead to cellular pathol‐ ogy. A free radical may be defined as any molecule that has one or more unpaired electrons. The superoxide anion, the hydroxyl radical, and the hypochlorite radical are some of the highest reactive radicals of oxygen. Owing to their high reactivity and to their capability of initiating an uncontrolled cascade of chain reactions, ROS produce extensive protein damage and cytoskeletal modifications and inhibit cellular mechanisms. Aerobic organisms are equipped with a powerful battery of mechanisms that protect them from the adverse effects of lipid peroxidation (LPO) and other manifestations of oxygen toxicity. Defective sperm function frequently causes male infertility, due to abnormal flagella movement, failure to recognize the zona, and inhibition of sperm-oocyte fusion. ROS are fundamental mediators of physiological sperm function, such as signal transduction mechanisms that have an effect on fertility. ROS can have positive effects on sperm and the concentration functions depending on the nature and the concentration of the ROS involved. They are necessary in regulating the hyperactivation and the ability of the spermatozoa to undergo acrosome reaction. An increased amount of superoxide anion (O2 - ) is one of the first steps required by the spermatozoa for induction and development of hyperactivation and capacitation. Numerous studies have shown that oxidative stress plays an important role in the pathophysiology of infertility and assisted fertility. The paternal genome is of primary importance in the normal embryo and fetal develop‐ ment. ROS-induced sperm damage during sperm translation, such as signal trans‐ duction through the seminiferous tubules and epididymis, is one of the most important mechanisms leading to sperm DNA damage. Male germ cells are extremely

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

vulnerable to oxidative stress as the sperm membrane is rich in unsaturated fatty acids and lacks the capacity for DNA repair. Spermatozoa are particularly susceptible to ROS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFA) and their cytoplasm contains low concentrations of the scavenging enzymes. Many clinical and research institutes are investigating the usefulness of antioxidant supplementation and their role in prevention of the infertility problems. Incubation under oxygen in vitro was detrimental to human spermatozoa, decreasing motility and viability. Since then, many reports have associated ROS with impaired sperm function, including decreased motility, abnormal morphology, and decreased sperm-egg penetration. Increasing knowledge of the mechanisms whereby ROS and endogenous antioxidant systems influence reproductive processes can assist to optimize the application of exogenous antioxi‐ dants to fertility treatment.

**Keywords:** ROS, Fertility, Oxidative stress

### **1. Introduction**

### **1.1. Mammalian testis and reproduction**

The primary sex organs of the male reproductive system are the two testes in which sperm is produced [1, 2]; the testis contains seminiferous tubules that consist of germinal epithelium and peritubular tissue [2, 3]. The epithelium contains two basic cell types, the somatic and germinal cells [4]. At different developmental stages, germ cells, including spermatogonial stem cells and differentiated cells formed during and following meiosis, are primary and secondary spermatocytes and spermatids, respectively.

These cells are located within invaginations of somatic Sertoli cells, with which maintain an intimate and cooperative relationship [3, 4]. Sertoli cells form the blood-testis barrier and are implicated in phagocytosis, secretion of testicular fluid for sperm transport, production of endocrine and paracrine substances that regulate spermatogenesis, and secretion of androgenbinding protein [5].

The development of the testis is a paradigm for the development of other organs, incorporating mechanisms for determining organ shape, size, internal architecture, vascularization, and interaction with other tissues physically, hormonally, and neurally. In the testis's develop‐ ment, several cells are bipotential, since the genital ridges must be able to differentiate into testes or ovaries depending on signals received; the differentiation of these cell lineages does not proceed independently, but it follows from differentiation of Sertoli cells, which then orchestrate the behavior of all other cell types [6]. Finally, the testis is built from a combination of innate precursors and immigrant cells such as germ cells.

Testosterone-secreting Leydig cells are found in the intertubular tissue surrounding the capillaries and have an important role in the spermatogenesis and the differentiation of sexual organs and secondary male sex characteristics. The Leydig cell is a polyhedral epithelioid cell with a single ovoid nucleus that contains one to three nucleoli and abundant dark-staining peripheral heterochromatin. The acidophilic cytoplasm contains many membrane-bound lipid droplets and a large amount of smooth endoplasmic reticulum. Testicular Leydig cells are the principal source of androgens in the male.

Spermatogenesis occurs in the seminiferous tubules, and it is a dynamic and metabolically active biological process during which haploid spermatozoa are produced through a gradual transformation of germ cells. These cells migrate from the basal compartment toward the luminal regions of the tubules, passing the blood-testis barrier.

The secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary, under the influence of gonadotropin-releasing hormone (GnRH) released by the hypothalamus, affects the male reproductive function. LH stimulates Leydig cells to produce testosterone, which exerts a negative feedback on GnRH and gonadotropin secretion. FSH stimulates Sertoli cell proliferation, a necessary step for the maturation of germ cells, given that the number of Sertoli cells largely determines the number of germ cells that can be correctly nurtured in the testis. During spermatogenesis, FSH and testosterone act in synergy [7, 8].

The early development of gonads has a higher energy requirement than ovaries [9]. The presence of many mitochondria in male germ cells highlights their importance in testicular metabolism [10, 11]. The germ cells's survival in the adult testis is dependent from carbohy‐ drate metabolism, including glycolysis and mitochondrial oxidative phosphorylation. During spermatogenesis, many changes in the energy metabolism of germ cells are involved, mainly due to the blood-testis barrier and changes to the surrounding medium.

The spermatogonia, mature sperm, and the somatic Sertoli cells show high glycolytic activity, whereas spermatocytes and spermatids produce adenosine triphosphate (ATP) by mitochon‐ drial oxidative phosphorylation [12, 13]. During spermatogenesis, three types of mitochondria are identified: the mitochondria in Sertoli cells, spermatogonia, and preleptotene and leptotene spermatocytes; the intermediate form in zygotene spermatocytes; and the condensed form in pachytene spermatocytes, secondary spermatocytes, and early spermatids [7]. The physiolog‐ ical death of germ cells via apoptosis occurs in the spermatogenic process and can be increased by hormone deprivation, heat, and toxin exposure [3]. Therefore, mitochondria play a central and important role in Leydig cell steroidogenesis.

### **1.2. ROS and male fertility**

vulnerable to oxidative stress as the sperm membrane is rich in unsaturated fatty acids and lacks the capacity for DNA repair. Spermatozoa are particularly susceptible to ROS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFA) and their cytoplasm contains low concentrations of the scavenging enzymes. Many clinical and research institutes are investigating the usefulness of antioxidant supplementation and their role in prevention of the infertility problems. Incubation under oxygen in vitro was detrimental to human spermatozoa, decreasing motility and viability. Since then, many reports have associated ROS with impaired sperm function, including decreased motility, abnormal morphology, and decreased sperm-egg penetration. Increasing knowledge of the mechanisms whereby ROS and endogenous antioxidant systems influence reproductive processes can assist to optimize the application of exogenous antioxi‐

The primary sex organs of the male reproductive system are the two testes in which sperm is produced [1, 2]; the testis contains seminiferous tubules that consist of germinal epithelium and peritubular tissue [2, 3]. The epithelium contains two basic cell types, the somatic and germinal cells [4]. At different developmental stages, germ cells, including spermatogonial stem cells and differentiated cells formed during and following meiosis, are primary and

These cells are located within invaginations of somatic Sertoli cells, with which maintain an intimate and cooperative relationship [3, 4]. Sertoli cells form the blood-testis barrier and are implicated in phagocytosis, secretion of testicular fluid for sperm transport, production of endocrine and paracrine substances that regulate spermatogenesis, and secretion of androgen-

The development of the testis is a paradigm for the development of other organs, incorporating mechanisms for determining organ shape, size, internal architecture, vascularization, and interaction with other tissues physically, hormonally, and neurally. In the testis's develop‐ ment, several cells are bipotential, since the genital ridges must be able to differentiate into testes or ovaries depending on signals received; the differentiation of these cell lineages does not proceed independently, but it follows from differentiation of Sertoli cells, which then orchestrate the behavior of all other cell types [6]. Finally, the testis is built from a combination

Testosterone-secreting Leydig cells are found in the intertubular tissue surrounding the capillaries and have an important role in the spermatogenesis and the differentiation of sexual

dants to fertility treatment.

**1.1. Mammalian testis and reproduction**

**1. Introduction**

20 New Discoveries in Embryology

binding protein [5].

**Keywords:** ROS, Fertility, Oxidative stress

secondary spermatocytes and spermatids, respectively.

of innate precursors and immigrant cells such as germ cells.

Oxygen is essential for animal life. Most of the body's energy is produced by the enzymatically controlled reaction of oxygen with hydrogen in oxidative phosphorylation occurring in the mitochondria during oxidative respiration. In controlled reaction steps, hydrogen is provided in the form of reducing equivalent and the energy produced is conserved in the form of highenergy phosphates. A four-electron reduction of molecular oxygen to water involving cytochrome oxidase occurs in the mitochondria. During this stepwise, enzymatic reduction of oxygen, free radicals are formed [14].

Free radicals were first described more than a century ago [15]; more than 30 years later, it was showed that all oxidation reactions involving organic molecules would be mediated by free radicals [16]. Then, free radicals were found in biological systems and were involved in many pathological processes and aging [17-19]. Subsequently, their signaling function was evaluat‐ ed, and then it was found that they were regulated by hormones like insulin and were regulators of metabolic pathways [20-22].

They are short-lived reactive chemical intermediates, which contain one or more electrons with unpaired spin. Free radicals are highly reactive and oxide lipids in membranes, carbohydrates and amino acids in proteins, and damage nucleic acids. Free radicals are active participants in different processes, and they cannot be considered only damaging agents, but real players in many normal functions of living organisms. They are normal by-products in various metabolic and physiological processes, whereas excessive production of them results in the oxidative stress.

The dioxygen molecule (O2) is a biradical, because it contains two electrons with the same spin in an external antibonding molecular orbital. Molecular oxygen can be reduced via a fourelectron mechanism with acceptance of four protons yielding two water molecules. In this case, the free biradical is simply converted to a non-radical species due to acceptance of the four electrons and four protons. However, there is another way to reduce molecular oxygen; this is one-electron successive reduction. Receiving one electron, O2 is converted to the superoxide anion radical (O2 ⋅-), containing one unpaired electron in an external antibonding orbital. Accepting a second electron and two protons converts the superoxide anion radical into hydrogen peroxide (H2O2); H2O2 has a non-radical nature and is chemically more active than molecular oxygen but less active than O2 ⋅-.

The formation of the most reactive of oxygen species, the hydroxyl radical (HO<sup>⋅</sup> ), results from the further reduction of H2O2 leading to its dismutation. Finally, acceptance of a fourth (final) electron and one more proton HO<sup>⋅</sup> forms a water molecule. Since O2 ⋅-, H2O2, and HO<sup>⋅</sup> are chemically more reactive than molecular oxygen, they are collectively called ROS, but only O2 ⋅- and HO<sup>⋅</sup> are actually free radicals, whereas H2O2 is not. Therefore, in biological research, the term "free radicals" is frequently replaced by "reactive oxygen species" (ROS), which is a more general term and includes both free radical and non-radical species.

ROS formation and redox signaling play a role in physiology and in a variety of pathologies, including inflammatory, infectious, and degenerative disorders, either in humans or in animals [23-25]. ROS are involved in a variety of pathophysiological conditions of the testis,

and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The disruption of redox signaling and control and imbalance in favor of prooxidant species define oxidative stress [26, 27].

Free radicals were first described more than a century ago [15]; more than 30 years later, it was showed that all oxidation reactions involving organic molecules would be mediated by free radicals [16]. Then, free radicals were found in biological systems and were involved in many pathological processes and aging [17-19]. Subsequently, their signaling function was evaluat‐ ed, and then it was found that they were regulated by hormones like insulin and were

They are short-lived reactive chemical intermediates, which contain one or more electrons with unpaired spin. Free radicals are highly reactive and oxide lipids in membranes, carbohydrates and amino acids in proteins, and damage nucleic acids. Free radicals are active participants in different processes, and they cannot be considered only damaging agents, but real players in many normal functions of living organisms. They are normal by-products in various metabolic and physiological processes, whereas excessive production of them results in the oxidative

The dioxygen molecule (O2) is a biradical, because it contains two electrons with the same spin in an external antibonding molecular orbital. Molecular oxygen can be reduced via a fourelectron mechanism with acceptance of four protons yielding two water molecules. In this case, the free biradical is simply converted to a non-radical species due to acceptance of the four electrons and four protons. However, there is another way to reduce molecular oxygen; this is one-electron successive reduction. Receiving one electron, O2 is converted to the superoxide

Accepting a second electron and two protons converts the superoxide anion radical into hydrogen peroxide (H2O2); H2O2 has a non-radical nature and is chemically more active than

the further reduction of H2O2 leading to its dismutation. Finally, acceptance of a fourth (final)

chemically more reactive than molecular oxygen, they are collectively called ROS, but only

the term "free radicals" is frequently replaced by "reactive oxygen species" (ROS), which is a

ROS formation and redox signaling play a role in physiology and in a variety of pathologies, including inflammatory, infectious, and degenerative disorders, either in humans or in animals [23-25]. ROS are involved in a variety of pathophysiological conditions of the testis,

⋅-.

The formation of the most reactive of oxygen species, the hydroxyl radical (HO<sup>⋅</sup>

more general term and includes both free radical and non-radical species.

⋅-), containing one unpaired electron in an external antibonding orbital.

forms a water molecule. Since O2

are actually free radicals, whereas H2O2 is not. Therefore, in biological research,

), results from

are

⋅-, H2O2, and HO<sup>⋅</sup>

regulators of metabolic pathways [20-22].

22 New Discoveries in Embryology

molecular oxygen but less active than O2

electron and one more proton HO<sup>⋅</sup>

stress.

O2

⋅- and HO<sup>⋅</sup>

anion radical (O2

Oxidative stress is a state in which an oxidant-generating system overcomes an antioxidant defense system, a process that is involved in many diseases including male factor infertility and/or subfertility. ROS are products of normal cellular metabolism and are formed during the normal enzymatic reactions of intercellular and intracellular signaling [28]. ROS overpro‐ duction can be induced through physiological or pathological mechanisms, including ROS generation by leukocytes as a cytotoxic mechanism of host defense, during hypoxic states leading to high levels of ROS, as well as by drugs with oxidizing effects on cells. Then, when mitochondria become a target of elevated levels of ROS, the process of oxidative phosphory‐ lation might be affected because of a possible damage of proteins and membrane lipids. Lipids are present in the sperm plasma membrane in the form of polyunsaturated fatty acids (PUFAs) that contain more than two carbon-carbon double bonds. ROS attacks PUFA in the cell membrane, leading to a cascade of chemical reactions called lipid peroxidation.


At low concentrations, ROS are metabolic intermediates in the metabolism of prostanoids, in gene regulation and cellular growth and in signal transduction [29, 30]. At high concentrations, ROS exert bionegative effects and damage all major classes of biomolecules.

During reproduction, ROS are involved in many important mechanisms of sperm physiology. An increase in ROS generation at the beginning of capacitation is followed by an increase in tyrosine phosphorylation [31]. The motility was associated with the generation of superoxide anion and a phosphorylation of tyrosine residues.

Furthermore, the acrosome reaction was associated with an extracellular superoxide anion of spermatozoa [32]. In the male genital tract, ROS are generated by spermatozoa and leukocytes including neutrophils and macrophages. In the semen, sperm cells are one of the major cellular sources of ROS. The male germ cells produced a small amount of ROS from the earliest stages of the development [33]. They are involved in the sperm chromatin condensation, regulating the number of germ cells by induction of apoptosis or proliferation of spermatogonia [34]. In the mature sperm, ROS play an important role in the capacitation, acrosome reaction and sperm motility, and they can also function as signaling molecules. There are at least two mechanisms of their production: the membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an enzyme complex that is contained in the cell membrane, and the mitochondria.

Furthermore, many studies have demonstrated that low and physiological levels of ROS play an important role in processes such as capacitation, hyperactivation, acrosome reaction, and sperm-oocyte fusion in order to ensure appropriate fertilization, whereas high levels of ROS cause sperm pathologies such as ATP depletion and loss of sperm motility and viability [35]. When the ROS overcomes the antioxidant defense systems and disrupts the intricate balance between ROS and antioxidants, pathological defects occur that causes significant damage to biomolecules such as lipids, proteins, nucleic acids, and carbohydrates [36]. The ROS found in the seminal plasma originates from various endogenous and exogenous sources; there are many endogenous sources of ROS in the seminal plasma such as peroxidase-positive leuko‐ cytes including polymorphonuclear leukocytes and macrophages [37]. Most of these peroxi‐ dase-positive leukocytes derive from the prostate and seminal vesicles; if these sources of ROS are triggered by many intracellular or extracellular stimuli, as inflammation or infection, they can increase ROS and the NADPH production via the hexose monophosphate shunt [38, 39]. An increase in proinflammatory cytokines, such as interleukin (IL)-8, and a decrease in the antioxidant superoxide dismutase (SOD) can result in a respiratory burst, production of high levels of ROS, and oxidative stress. Between exogenous sources of ROS, there are toxins, phthalates, and others [40]. Infections lead to an excessive ROS production, resulting in an oxidative burst from neutrophils/macrophages as a first-line defense mechanism. When there is an infection, an imbalance of prooxidants and antioxidants favors the oxidative stress that damages the sperm functions such as motility and fertilization. In the testis and epididymis infections, the ROS produced are very detrimental to the spermatozoa because of the long contact time and the loss of antioxidant protection.

During the final phase of the ejaculation, only high numbers of ROS-producing leukocytes are harmful to sperm functions. An infection which involves ROS in the epididymis, prostate gland, and/or seminal vesicles could indirectly damage sperm functions [41].

In 1943, a paper was published showing the effect of high oxygen tensions on motility and prevention of this phenomenon by adding catalase, which suggested the involvement of oxygen overload in motility of spermatozoa [42]. Indeed, ROS generation was dependent on the oxygen tension; higher oxygen tensions increased ROS generation, mainly from leukocytes, whereas low oxygen tensions improved the survival rate and penetration capacity [43].

Oxidative stress has been considered a main cause to male infertility, but studies have showed that low and verified concentrations of ROS play a pivotal role in sperm physiological processes such as capacitation, hyperactivation, acrosome reactions, and signaling processes to provide a suitable fertilization, but an increase in oxidative stress leads to male infertility by the induction of peroxidative damage to the sperm plasma membrane, DNA damage, and apoptosis. ROS must be maintained at appropriate levels to ensure appropriate physiological function while preventing pathological damage to the spermatozoa. ROS is thought to influence fertility by affecting sperm membranes and sperm DNA. They reduce sperm motility and its ability to fuse with the oocyte and compromise paternal genomic contribution to the embryo; in fact, sperm are vulnerable to oxidative stress-induced damage due to the high portion of PUFA and also due to the low concentrations of scavenging enzymes in their cytoplasm, both contributing to the defective sperm function observed in a high percentage of infertility.

There are many agents that cause an increase in testicular oxidative stress, such as environmental toxins or conditions such as varicocele, orchitis, cryptorchidism, and aging, all of which leads to an increase in germ cell apoptosis and hypospermatogenesis. ROSinduced DNA damage may also potentiate germ cell apoptosis, leading to a decrease in sperm count and thus to the decline of semen quality, both of which are associated with male infertility [39]. Large amounts of pathogenic mutant mtDNA accumulate in the testis; the resulting mitochondrial respiratory dysfunction in spermatogenic cells leads to a decrease in energy production that ultimately induces meiotic arrest and abnormalities in sperm morphology, stressing the importance of mitochondrial respiratory function in mammalian spermatogenesis [44].

### **2. Apoptosis and oxidative stress**

(NADPH) oxidase, an enzyme complex that is contained in the cell membrane, and the

Furthermore, many studies have demonstrated that low and physiological levels of ROS play an important role in processes such as capacitation, hyperactivation, acrosome reaction, and sperm-oocyte fusion in order to ensure appropriate fertilization, whereas high levels of ROS cause sperm pathologies such as ATP depletion and loss of sperm motility and viability [35]. When the ROS overcomes the antioxidant defense systems and disrupts the intricate balance between ROS and antioxidants, pathological defects occur that causes significant damage to biomolecules such as lipids, proteins, nucleic acids, and carbohydrates [36]. The ROS found in the seminal plasma originates from various endogenous and exogenous sources; there are many endogenous sources of ROS in the seminal plasma such as peroxidase-positive leuko‐ cytes including polymorphonuclear leukocytes and macrophages [37]. Most of these peroxi‐ dase-positive leukocytes derive from the prostate and seminal vesicles; if these sources of ROS are triggered by many intracellular or extracellular stimuli, as inflammation or infection, they can increase ROS and the NADPH production via the hexose monophosphate shunt [38, 39]. An increase in proinflammatory cytokines, such as interleukin (IL)-8, and a decrease in the antioxidant superoxide dismutase (SOD) can result in a respiratory burst, production of high levels of ROS, and oxidative stress. Between exogenous sources of ROS, there are toxins, phthalates, and others [40]. Infections lead to an excessive ROS production, resulting in an oxidative burst from neutrophils/macrophages as a first-line defense mechanism. When there is an infection, an imbalance of prooxidants and antioxidants favors the oxidative stress that damages the sperm functions such as motility and fertilization. In the testis and epididymis infections, the ROS produced are very detrimental to the spermatozoa because of the long

During the final phase of the ejaculation, only high numbers of ROS-producing leukocytes are harmful to sperm functions. An infection which involves ROS in the epididymis, prostate

In 1943, a paper was published showing the effect of high oxygen tensions on motility and prevention of this phenomenon by adding catalase, which suggested the involvement of oxygen overload in motility of spermatozoa [42]. Indeed, ROS generation was dependent on the oxygen tension; higher oxygen tensions increased ROS generation, mainly from leukocytes, whereas low oxygen tensions improved the survival rate and penetration capacity [43].

Oxidative stress has been considered a main cause to male infertility, but studies have showed that low and verified concentrations of ROS play a pivotal role in sperm physiological processes such as capacitation, hyperactivation, acrosome reactions, and signaling processes to provide a suitable fertilization, but an increase in oxidative stress leads to male infertility by the induction of peroxidative damage to the sperm plasma membrane, DNA damage, and apoptosis. ROS must be maintained at appropriate levels to ensure appropriate physiological function while preventing pathological damage to the spermatozoa. ROS is thought to influence fertility by affecting sperm membranes and sperm DNA. They reduce sperm motility and its ability to fuse with the oocyte and compromise paternal genomic contribution to the embryo; in fact, sperm are vulnerable to oxidative stress-induced damage due to the high

gland, and/or seminal vesicles could indirectly damage sperm functions [41].

mitochondria.

24 New Discoveries in Embryology

contact time and the loss of antioxidant protection.

Oxidative stress is implicated between causes of male infertility. ROS production and its effects on semen quality have been widely clarified. Oxygen is essential to sustain life, and physio‐ logical levels of ROS are necessary to maintain normal cell functions. However, products of oxygen such as ROS can be detrimental to cell function and survival [45].

ROS are detrimental to sperm survival and function due to its adverse effects on sperm membrane and genetic material. High frequency of single- and double-stranded DNA breaks due to oxidative stress activates apoptosis by inducing cytochrome c and caspases 9 and 3 [46]. Disruption of inner and outer mitochondrial membranes results in release of cytochrome c, a protein which activates caspases and induces apoptosis. Mitochondrial exposure to ROS results in the release of apoptosis-inducing factor, which directly interacts with the DNA and leads to DNA fragmentation [46]. Seminal oxidative stress, sperm DNA damage, and apoptosis constitute a unified pathogenic molecular mechanism in infertility. Therefore, apoptosis in semen could be a useful indicator of semen quality.

### **3. Antioxidants in male fertility**

Antioxidants are substances, enzymatic and nonenzymatic, which serve to eliminate ROS. Enzymatic oxidants, or natural oxidants, include glutathione reductase (GSH), superoxide dismutase (SOD), and catalase, while some non-enzymatic oxidants include vitamins (C, E, and B), carotenoids, carnitines, cysteines, pentoxifylline, metals, taurine, and albumin [47]. Glutathione reductase and peroxidase are the principal reducing agents in the body and behave as antioxidant scavengers in the epididymis and testes [48]. Their action on sperm membranes confers protection on to the lipid components, preserving the sperm viability and motility [49]. Preceding in vitro studies have demonstrated that GSH reduces lipid peroxida‐ tion and improves the sperm membrane characteristics [50]. The main antioxidant enzyme system in the semen includes SOD, catalase, and glutathione peroxidase.

SODs are metalloenzymes that catalyze the dismutation reactions of the superoxide anion and are present in intracellular and extracellular forms; two of the intracellular forms are copperzinc SOD, which is localized in the cytoplasm and contains copper and zinc (Cu, ZnSOD, SOD1) in the active site, and manganese SOD, which is located primarily in the mitochondrial matrix and contains manganese in the active site (MnSOD, SOD2). The extracellular form of SOD (EC-SOD, SOD3) acts in the extracellular space and it is related to the surface polysac‐ charides though it may also be present in a free form [51]. SOD presents high activity in the seminal plasma with 75% of its activity connected to the activity of SOD1 and the remaining 25% to SOD3; these isoenzymes are maybe derived from the prostate [52]. SOD and catalase protect sperm from superoxide anions catalyzing the conversion of superoxide into oxygen and H2O2, thereby preventing lipid peroxidation and enhancing motility [53].

$$\cdot \text{O}\_{2^{-}} \text{\text{+} \cdot \text{O}\_{2^{-}} \text{\text{\textbullet2H}^{+}} \rightarrow \text{H}\_{2}\text{O}\_{2} \text{\textbullet O}\_{2}$$

SOD and catalase assist in removing ROS that has the potential to damage sperm. Catalase catalyzes the conversion of H2O2 to O2 and H2O and presents a heme group with a central iron atom. It acts mainly in the endoplasmic reticulum, peroxisomes, mitochondria, and cytosol in many cell types [54]. Catalase was found in the human and rat sperm cells and in the seminal plasma; the prostate seems to be its source [55]. The sperm cell capacitation induced by nitric oxide is activated by catalase [56].

due to oxidative stress activates apoptosis by inducing cytochrome c and caspases 9 and 3 [46]. Disruption of inner and outer mitochondrial membranes results in release of cytochrome c, a protein which activates caspases and induces apoptosis. Mitochondrial exposure to ROS results in the release of apoptosis-inducing factor, which directly interacts with the DNA and leads to DNA fragmentation [46]. Seminal oxidative stress, sperm DNA damage, and apoptosis constitute a unified pathogenic molecular mechanism in infertility. Therefore, apoptosis in

Antioxidants are substances, enzymatic and nonenzymatic, which serve to eliminate ROS. Enzymatic oxidants, or natural oxidants, include glutathione reductase (GSH), superoxide dismutase (SOD), and catalase, while some non-enzymatic oxidants include vitamins (C, E, and B), carotenoids, carnitines, cysteines, pentoxifylline, metals, taurine, and albumin [47]. Glutathione reductase and peroxidase are the principal reducing agents in the body and behave as antioxidant scavengers in the epididymis and testes [48]. Their action on sperm membranes confers protection on to the lipid components, preserving the sperm viability and motility [49]. Preceding in vitro studies have demonstrated that GSH reduces lipid peroxida‐ tion and improves the sperm membrane characteristics [50]. The main antioxidant enzyme

SODs are metalloenzymes that catalyze the dismutation reactions of the superoxide anion and are present in intracellular and extracellular forms; two of the intracellular forms are copperzinc SOD, which is localized in the cytoplasm and contains copper and zinc (Cu, ZnSOD, SOD1) in the active site, and manganese SOD, which is located primarily in the mitochondrial matrix and contains manganese in the active site (MnSOD, SOD2). The extracellular form of SOD (EC-SOD, SOD3) acts in the extracellular space and it is related to the surface polysac‐ charides though it may also be present in a free form [51]. SOD presents high activity in the seminal plasma with 75% of its activity connected to the activity of SOD1 and the remaining 25% to SOD3; these isoenzymes are maybe derived from the prostate [52]. SOD and catalase protect sperm from superoxide anions catalyzing the conversion of superoxide into oxygen

<sup>+</sup> O + O ×+2H H O +O 2 2 - - 22 2 ×× ®

SOD and catalase assist in removing ROS that has the potential to damage sperm. Catalase catalyzes the conversion of H2O2 to O2 and H2O and presents a heme group with a central iron atom. It acts mainly in the endoplasmic reticulum, peroxisomes, mitochondria, and cytosol in many cell types [54]. Catalase was found in the human and rat sperm cells and in the seminal plasma; the prostate seems to be its source [55]. The sperm cell capacitation induced by nitric

system in the semen includes SOD, catalase, and glutathione peroxidase.

and H2O2, thereby preventing lipid peroxidation and enhancing motility [53].

semen could be a useful indicator of semen quality.

**3. Antioxidants in male fertility**

26 New Discoveries in Embryology

oxide is activated by catalase [56].

2 2 2 2 2H O O + 2H O

Glutathione peroxidase (GPX), another antioxidant enzyme in the semen, catalyzes the reduction of H2O2 and organic peroxides [51]. GPX contains selenium in the form of seleno‐ cysteine in its active site. It is located in the sperm in the mitochondrial matrix [52] but has also been found to have a nuclear form that preserves sperm DNA from oxidative damage and enters in the process of chromatin condensation. It was found in the seminal plasma; therefore, it could originate from the prostate [57, 58].

Between nonenzymatic antioxidants, there are vitamin E which encompass a group of potent, lipid-soluble, chain-breaking antioxidants. Structural analyses have revealed that molecules having vitamin E antioxidant activity include four tocopherols (α, β, γ, δ) and four tocotrienols (α, β, γ, δ). Vitamin E (α-tocopherol), a chain-breaking antioxidant in the sperm's cell mem‐ brane, neutralizes H2O2 and quenches free radicals, therefore stopping chain reactions that develop lipid peroxides and protecting the membrane from the oxidative damage [48]. Vitamin E improves the activity of other scavenging oxidants and helps to keep motility and morphol‐ ogy of the sperm [54]. It preserves the spermatogenesis in male rats and fails to conserve zygotes in female rats. Selenium deficiency can induce male infertility and could thus support an antioxidant function of vitamin E in the reproductive system. Therefore, vitamin E and selenium can act in synergy in membrane protection from oxidative stress. Vitamin E is known to readily reduce alkyl peroxy radicals of unsaturated lipids, thereby generating hydroperox‐ ides that are reduced by the selenoperoxidases, in particular by phospholipid-hydroperoxide glutathione peroxidase.

Vitamin C or L-ascorbic acid, or ascorbate (the anion of ascorbic acid), is an essential nutrient for humans and many animals. Vitamin C is a major chain-breaking antioxidant and is present in the extracellular fluid. It neutralizes hydroxyl, superoxide, and hydrogen peroxide radicals

and prevents sperm agglutination [53]. It also helps to recycle vitamin E. It plays a significant role in removing oxidative stress in the seminal plasma. It reacts with OH- , O2 - , and H2O2 in the extracellular fluid, thus protecting sperm viability and motility [59].

Carnitine, a water-soluble antioxidant, participates in sperm motility and prevents lipid oxidation; it protects the sperm DNA and membranes from oxidative damage and maintains sperm viability and motility [60].

Carotenoids are a family of pigmented compounds that are synthesized by plants and microorganisms, but not animals. They are present as micro-components in fruits and vegetables and are responsible for their yellow, orange, and red colors. Carotenoids are thought to be responsible for the beneficial properties of fruits and vegetables in preventing diseases including cardiovascular diseases, cancer, and other chronic diseases. Carotenoids (βcarotene and lycopene) are very efficient singlet molecular oxygen quenchers; they prevent peroxidation in the seminal plasma [59].

Cysteines, precursors of intracellular GSH, increase the amount of GSH synthesized that prevents oxidative damage to the cell membrane and DNA. There are a few other minor antioxidants that contribute to relieving oxidative stress, such as albumin, taurine/hypotaur‐ ine, inositol, and some metals. Albumin, a plasma protein, interacts with peroxyl radicals and inhibits the chain reactions that generate ROS production and preserve motility and viability of sperm.

and prevents sperm agglutination [53]. It also helps to recycle vitamin E. It plays a significant

Carnitine, a water-soluble antioxidant, participates in sperm motility and prevents lipid oxidation; it protects the sperm DNA and membranes from oxidative damage and maintains

Carotenoids are a family of pigmented compounds that are synthesized by plants and microorganisms, but not animals. They are present as micro-components in fruits and vegetables and are responsible for their yellow, orange, and red colors. Carotenoids are thought to be responsible for the beneficial properties of fruits and vegetables in preventing diseases including cardiovascular diseases, cancer, and other chronic diseases. Carotenoids (βcarotene and lycopene) are very efficient singlet molecular oxygen quenchers; they prevent

Cysteines, precursors of intracellular GSH, increase the amount of GSH synthesized that prevents oxidative damage to the cell membrane and DNA. There are a few other minor

, O2 -

, and H2O2 in

role in removing oxidative stress in the seminal plasma. It reacts with OH-

the extracellular fluid, thus protecting sperm viability and motility [59].

sperm viability and motility [60].

28 New Discoveries in Embryology

peroxidation in the seminal plasma [59].

Taurine, a non-enzymatic antioxidant, scavenges ROS; inositol enhances GSH activity and preserves normal sperm morphology.

Selenium is an important component in the regular development and maturation of the testes and contributes to the protection of sperm DNA and cell membranes, particularly when used as an adjunct to vitamin E. The specific role of selenium in spermatogenesis appears to be related to phospholipid hydroperoxide glutathione peroxidase, which is expressed depending on the developmental state of spermatids.

Zinc acts as a chelator and binds ROS; manganese enhances sperm motility and viability [61, 62]. Chrome, another essential micronutrient, is a component of enzymes involved in carbo‐ hydrate metabolism. Its supplementation reduces fat deposition in rats, preventing obesity, the initial phase of inflammation, and oxidative stress [63]. Although seminal plasma contains a range of protective antioxidants such as SOD, catalase, and glutathione peroxidase, these defenses are less abundant in the sperm and seem to be impaired in cases of male infertility [64].

### **4. Measurement of ROS**

Oxidative stress results from an imbalance between ROS production and the intracellular and extracellular antioxidants that scavenge ROS. There are many direct assays that measure the oxidation of the sperm cell membrane. The most used assay measures malondialdehyde (MDA), one of the final products of sperm cell membrane lipid peroxidation [65, 66]. Increased levels of MDA correlate with decreased sperm parameters.

Quantification of sperm DNA damage has also been used as assay for intracellular ROSinduced oxidant injury by measuring a specific product of oxidant-induced DNA damage, 8 oxo-7, 8, -dihydro 2′ deoxyguanoside (8-OHdG), used as a specific marker of oxidative injury to sperm DNA [67].

The most used method for measurement of seminal ROS is the indirect chemiluminescence assay. Luminol (5-amino-2, 3, dihydro 1, 4, phthalazinedione), or lucigen, can be used for quantification of redox activities of spermatozoa [68]. Lucigen measures only extracellular superoxide radicals, while luminol is used to measure extracellular and intracellular levels of ROS.

The nitroblue tetrazolium assay requires a light microscope and allows differentiation of spermatic and leukocytic ROS without the steps required in chemiluminescence assays. Nitroblue tetrazolium interacts with superoxide radicals in the sperm and leukocytes by changing to diformazan, a blue pigment. The concentration of diformazan correlates with the concentration of intracellular ROS [68].

The antioxidant levels of the semen can also be determined by chemiluminescence assay or by a colorimetric assay. Antioxidant levels are measured through the addition of a known concentration of ROS to the semen, leading to the development of the chemiluminescence signal or a color change. This assay allows the antioxidants in the semen to scavenge the known ROS and then the measurement of residual ROS level. The intensity of the signal produced is inversely correlated with the total antioxidant capacity of the sample [69].

Another method for measuring oxidative stress can be carried through the measurement of lipid peroxidation in the whole sperm by a commercially assay kit (LP Sperm Test, Diacron International, Grosseto, Italy). The assay is based on the ability of peroxides to promote the oxidation of Fe2+ to Fe3+; the product of peroxidation (Fe3+) binds to the thiocyanate, developing a colored complex measured photometrically [70].

### **5. ROS In Vitro Fertilization (IVF) or artificial insemination**

**4. Measurement of ROS**

30 New Discoveries in Embryology

to sperm DNA [67].

ROS.

concentration of intracellular ROS [68].

levels of MDA correlate with decreased sperm parameters.

Oxidative stress results from an imbalance between ROS production and the intracellular and extracellular antioxidants that scavenge ROS. There are many direct assays that measure the oxidation of the sperm cell membrane. The most used assay measures malondialdehyde (MDA), one of the final products of sperm cell membrane lipid peroxidation [65, 66]. Increased

Quantification of sperm DNA damage has also been used as assay for intracellular ROSinduced oxidant injury by measuring a specific product of oxidant-induced DNA damage, 8 oxo-7, 8, -dihydro 2′ deoxyguanoside (8-OHdG), used as a specific marker of oxidative injury

The most used method for measurement of seminal ROS is the indirect chemiluminescence assay. Luminol (5-amino-2, 3, dihydro 1, 4, phthalazinedione), or lucigen, can be used for quantification of redox activities of spermatozoa [68]. Lucigen measures only extracellular superoxide radicals, while luminol is used to measure extracellular and intracellular levels of

The nitroblue tetrazolium assay requires a light microscope and allows differentiation of spermatic and leukocytic ROS without the steps required in chemiluminescence assays. Nitroblue tetrazolium interacts with superoxide radicals in the sperm and leukocytes by changing to diformazan, a blue pigment. The concentration of diformazan correlates with the

The antioxidant levels of the semen can also be determined by chemiluminescence assay or by a colorimetric assay. Antioxidant levels are measured through the addition of a known concentration of ROS to the semen, leading to the development of the chemiluminescence New studies are underway to find new methods for supporting longer storage of cooled animal semen. All aerobic organisms require oxygen for life; although it is an essential element, oxygen is responsible for ROS production. It is known that high concentrations of ROS cause sperm pathology. Low concentrations of ROS play an important role in sperm physiology, while higher concentrations are detrimental. A study showed the influence of ROS on capacitation and the acrosome reaction in frozen-thawed bull spermatozoa; they concluded that ROS is required in the capacitation process and that hydrogen peroxide may participate as an inducer of the acrosome reaction [71, 72].

ROS act as second messengers and are involved in the sperm capacitation, acrosome reaction, and oocyte fertilization. They regulate the increase of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) activation, and phosphorylation of PKA substrates (arginine-X-X-(serine/threonine) motif), phosphorylation of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase kinase (MEK) proteins and threonine-glutamatetyrosine motif, and tyrosine phosphorylation of fibrous sheath proteins [73]. When ROS increase, the endogenous antioxidant defenses of gametes decrease and oxidative stress is induced [74]. High concentrations of ROS induce changes in sperm cell functions, altering fluidity and integrity of sperm membranes due to lipid peroxidation. Furthermore, ROS can damage DNA in the sperm nucleus, deplete ATP in mitochondria, and cause loss of sperm motility, viability, and capacity for fertilization [75]. Oxidative stress may be a cause of male infertility and contribute to DNA fragmentation in spermatozoa. There are few studies on the effects of antioxidant addition to extenders during cooling and/or freezing mammalian spermatozoa. Spermatozoa are subjected to peroxidative damage due to an excess of ROS because of the high presence of polyunsaturated fatty acids in membrane phospholipids. The antioxidant systems control the balance between production and neutralization of ROS and protect spermatozoa against peroxidative damage [76]. Recent studies moreover show a physiological SOD activity in human seminal plasma [77, 78]. SOD is an important antioxidant defense in all cells exposed to oxygen. Their use as additives in semen extenders has had controversial effects [79, 80]. SOD is responsible for H2O2 and O2 production, by dismutation of superoxide radicals. The addition of SOD to semen improves the quality of semen and reduces ERK activation [81, 82]. The addition of SOD to the semen extender could prolong storage of stallion semen, allowing longer distance shipments and a more precise timing of insemination, increasing the high rates of fertility. Furthermore, the antioxidant addition might also bring benefits to spermatozoa in the female reproductive tract [83]. ROS are responsible for the deterioration in quality of semen stored at 5°C, and the addition of SOD to the semen extender improves the quality of cold-stored semen.

Another work evaluated the effect of SOD supplementation in ovary transport media during 4°C storage of cat ovaries at different time intervals on the occurrence of ovarian apoptosis and on the ability to undergo in vitro oocyte development. The authors by immunohisto‐ chemical analysis, reverse transcriptase polymerase chain reaction (RT-PCR) analysis, and viability test analysis have demonstrated that SOD supplementation in transport media of domestic cat ovaries reduces cellular apoptosis and enhances COC survival and in vitro embryo production (IVEP) [84].

### **6. Conclusion**

Oxidative stress has been extensively studied for about four decades. Substantial progress has been achieved to date from descriptive characterization of this process to delineation of molecular mechanisms underlining adaptive responses and targeted manipulations of expected responses. Oxygen toxicity is an inherent challenge to aerobic life, including sper‐ matozoa, the cells responsible for propagation of the species. The oxidative damage to sperm membranes, proteins, and DNA is connected with changes in signal transduction mechanisms that affect fertility.

Spermatozoa and oocytes possess an inherent but limited capacity to generate ROS to aid in the fertilization process. Although a variety of defense mechanisms including antioxidant enzymes, vitamins, and biomolecules are available, a balance of the benefits and risks from ROS and antioxidants appears to be necessary for the survival and function of spermatozoa.

The antioxidants α-tocopherol (Vitamin A), ascorbic acid (Vitamin C), and retinoids (Vitamin A) are all potent scavengers of reactive oxygen species. Many studies have investigated the role of these and other antioxidants in improving sperm parameters.

The origin and the etiologies of increased ROS in males with suboptimal sperm quality are increasingly clear, presenting many pathways for a potential therapy. However, well-designed randomized controlled trials will be required to evaluate the potential of antioxidant systems. Furthermore, prooxidative and antioxidative properties of therapeutics are currently receiving more attention as part of anti-infectious therapies too.

ROS production might be beneficial or harmful for living organisms; this also applies in spermatozoa, which require low levels of ROS to show their full capacity in fertilizing. Conversely, oxidative stress is damaging for spermatozoa and many other cellular types; an excess of ROS has been associated with many diseases including diabetes, cancer, atheroscle‐ rosis, and Parkinson disease.

Oxidative stress might also be a consequence of unhealthy lifestyles such as smoking, alcohol abuse, or exposure to chemical or electromagnetic pollution. ROS are important contributors to the regulation of sperm function in both a positive and a negative sense. Thus, these cells generate low levels of ROS in order to promote capacitation and the functional evolution of sperm behaviors needed for fertilization, including hyperactivation and the presentation of zona recognition molecules on their surface. If fertilization does not occur, the continued generation of ROS activates the intrinsic apoptotic cascade.

Future progress in the field needs identification of the most crucial cellular targets for ROS action as well as discovery of the underlying mechanisms and consequences of the interaction between ROS and cellular components.

The mechanisms responsible for removing ROS and their regulation would be the second hot topic for ongoing studies of ROS metabolism.

In recent years, it was discovered that ROS and ROS-regulated pathways are actively involved in modification of diverse cellular processes starting from core metabolism and hormonal signaling through to complicated processes such as fertilization and development. The latter along with some biotechnological avenues would also extend ROS-related studies in practical directions. Therefore, much remains to be learned about the effects of ROS on biological systems, the adaptive strategies that overcome ROS attack, and the natural use of ROS in the signaling and regulation of metabolism.

### **Author details**

storage of stallion semen, allowing longer distance shipments and a more precise timing of insemination, increasing the high rates of fertility. Furthermore, the antioxidant addition might also bring benefits to spermatozoa in the female reproductive tract [83]. ROS are responsible for the deterioration in quality of semen stored at 5°C, and the addition of SOD to the semen

Another work evaluated the effect of SOD supplementation in ovary transport media during 4°C storage of cat ovaries at different time intervals on the occurrence of ovarian apoptosis and on the ability to undergo in vitro oocyte development. The authors by immunohisto‐ chemical analysis, reverse transcriptase polymerase chain reaction (RT-PCR) analysis, and viability test analysis have demonstrated that SOD supplementation in transport media of domestic cat ovaries reduces cellular apoptosis and enhances COC survival and in vitro

Oxidative stress has been extensively studied for about four decades. Substantial progress has been achieved to date from descriptive characterization of this process to delineation of molecular mechanisms underlining adaptive responses and targeted manipulations of expected responses. Oxygen toxicity is an inherent challenge to aerobic life, including sper‐ matozoa, the cells responsible for propagation of the species. The oxidative damage to sperm membranes, proteins, and DNA is connected with changes in signal transduction mechanisms

Spermatozoa and oocytes possess an inherent but limited capacity to generate ROS to aid in the fertilization process. Although a variety of defense mechanisms including antioxidant enzymes, vitamins, and biomolecules are available, a balance of the benefits and risks from ROS and antioxidants appears to be necessary for the survival and function of spermatozoa.

The antioxidants α-tocopherol (Vitamin A), ascorbic acid (Vitamin C), and retinoids (Vitamin A) are all potent scavengers of reactive oxygen species. Many studies have investigated the

The origin and the etiologies of increased ROS in males with suboptimal sperm quality are increasingly clear, presenting many pathways for a potential therapy. However, well-designed randomized controlled trials will be required to evaluate the potential of antioxidant systems. Furthermore, prooxidative and antioxidative properties of therapeutics are currently receiving

ROS production might be beneficial or harmful for living organisms; this also applies in spermatozoa, which require low levels of ROS to show their full capacity in fertilizing. Conversely, oxidative stress is damaging for spermatozoa and many other cellular types; an excess of ROS has been associated with many diseases including diabetes, cancer, atheroscle‐

role of these and other antioxidants in improving sperm parameters.

more attention as part of anti-infectious therapies too.

rosis, and Parkinson disease.

extender improves the quality of cold-stored semen.

embryo production (IVEP) [84].

**6. Conclusion**

32 New Discoveries in Embryology

that affect fertility.

Simona Tafuri1 , Francesca Ciani1\*, Eugenio Luigi Iorio2 , Luigi Esposito1 and Natascia Cocchia1

\*Address all correspondence to: ciani@unina.it

1 Department of Veterinary Medicine and Animal Productions - University of Naples Feder‐ ico II, Naples, Italy

2 International Observatory of Oxidative Stress, Salerno, Italy

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[84] Cocchia N., Corteggio A., Altamura G., Tafuri S., Rea S., Rosapane I., Sica A., Landol‐ fi F., Ciani F. Superoxide dismutase (SOD) addition to transport media during stor‐ age of cat ovaries 4°C. Reproductive Biology, 2015; 15(1):56-64.

### **Chapter 3**

### **Influence of ROS on Ovarian Functions**

### Francesca Ciani, Natascia Cocchia, Danila d'Angelo and Simona Tafuri

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61003

#### **Abstract**

[84] Cocchia N., Corteggio A., Altamura G., Tafuri S., Rea S., Rosapane I., Sica A., Landol‐ fi F., Ciani F. Superoxide dismutase (SOD) addition to transport media during stor‐

age of cat ovaries 4°C. Reproductive Biology, 2015; 15(1):56-64.

40 New Discoveries in Embryology

High level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress (OS) an emerging health risk factor involved in the aging and in many diseases, either in humans or in animals. ROS are a double-edged sword – they serve as key signal molecules in physiological processes, but also have a role in pathological processes involving the female reproductive tract.

ROS affect multiple physiological processes in reproduction and fertility, from oocyte maturation to fertilization, embryo development and pregnancy. Several studies indicate that follicular atresia in mammalian species due to the accumulation of toxic metabolites often results from oxidative stress. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Conversely, increase of ROS levels is primarily responsible for the alteration of macromolecules, such as lipids, proteins and nucleic acids, that lead to significant damage of cell structures and thereby cause oxidative stress. To prevent damage due to ROS, cells possess a number of non-enzymatic and enzymatic antioxidants. Non-enzymatic antioxidants include vitamin C, glutathione and vitamin E. Enzymatic antioxidants consist of superoxide dismutases (MnSOD and Cu/ZnSOD) that convert superoxide into hydrogen peroxide; glutathione peroxidase (GPX) and catalase (CAT) which neutralize hydrogen peroxide. Intracellular homeostasis is ensured by the complex interaction between pro-oxidants and antioxidants.

This chapter describes gathering evidence that oxidative stress is involved in ovarian physio-pathology caused by diverse stimuli. There is strong evidence that ROS are involved in initiation of apoptosis in antral follicles caused by several chemical and

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

physical agents, in the fluid follicular environment, influencing the folliculogenesis and the steroidogenesis. Although less attention has been focused on the roles of ROS in primordial and primary follicle death, several studies have shown protective effects of antioxidants and/or evidence of oxidative damage, suggesting that ROS may play a role in these smaller follicles as well. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity. It has been investigated the role of antioxidant enzymes, such as catalase, glutathione peroxidase and the SOD isoforms in maintaining low levels of oxidative stress.

The literature provides some evidence of oxidative stress influencing the entire reproductive cycle. OS plays a role in multiple physiological processes from oocyte maturation to fertilization and embryo development. An increasing number of published studies have pointed towards increased importance of the role of OS in female reproduction. Of course, there is much to learn about this topic, whereby it cannot be underestimated.

**Keywords:** Assisted reproductive technologies (ART), reactive oxygen species, ova‐ ry functions

### **1. Introduction**

High level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress (OS) an emerging health risk factor involved in the aging and in many diseases, either in humans or in animals. ROS are a double-edged sword – they serve as key signal molecules in physiological processes, but also have a role in pathological processes involving the female reproductive tract.

ROS affect multiple physiological processes in reproduction and fertility, from oocyte matu‐ ration to fertilization, embryo development and pregnancy. Several studies indicate that follicular atresia in mammalian species due to the accumulation of toxic metabolites often results from oxidative stress. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Conversely, increase of ROS levels is primarily responsible for the alteration of macromole‐ cules, such as lipids, proteins and nucleic acids, that lead to significant damage of cell structures and thereby cause oxidative stress. To prevent damage due to ROS, cells possess a number of non-enzymatic and enzymatic antioxidants. Non-enzymatic antioxidant include vitamin C, glutathione and vitamin E. Enzymatic antioxidants consist of superoxide dismutases (MnSOD and Cu/ZnSOD) that convert superoxide into hydrogen peroxide; glutathione peroxidase (GPX) and catalase (CAT) which neutralize hydrogen peroxide. Intracellular homeostasis is ensured by the complex interaction between pro-oxidants and antioxidants.

This chapter describes gathering evidence that oxidative stress is involved in ovarian physiopathology caused by diverse stimuli. There is strong evidence that ROS are involved in initiation of apoptosis in antral follicles caused by several chemical and physical agents, in the fluid follicular environment, influencing the folliculogenesis and the steroidogenesis. Al‐ though less attention has been focused on the roles of ROS in primordial and primary follicle death, several studies have shown protective effects of antioxidants and/or evidence of oxidative damage, suggesting that ROS may play a role in these smaller follicles as well. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity. It has been investigated the role of antioxidant enzymes, such as catalase, glutathione peroxidase and the SOD isoforms in maintaining low levels of oxidative stress.

The literature provides some evidence of oxidative stress influencing the entire reproductive cycle. OS plays a role in multiple physiological processes from oocyte maturation to fertiliza‐ tion and embryo development. An increasing number of published studies have pointed towards increased importance of the role of OS in female reproduction. Of course, there is much to learn about this topic, whereby it cannot be underestimated.

### **2. Follicular development and ovary functions**

physical agents, in the fluid follicular environment, influencing the folliculogenesis and the steroidogenesis. Although less attention has been focused on the roles of ROS in primordial and primary follicle death, several studies have shown protective effects of antioxidants and/or evidence of oxidative damage, suggesting that ROS may play a role in these smaller follicles as well. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity. It has been investigated the role of antioxidant enzymes, such as catalase, glutathione peroxidase and the SOD

The literature provides some evidence of oxidative stress influencing the entire reproductive cycle. OS plays a role in multiple physiological processes from oocyte maturation to fertilization and embryo development. An increasing number of published studies have pointed towards increased importance of the role of OS in female reproduction. Of course, there is much to learn about this topic, whereby it

**Keywords:** Assisted reproductive technologies (ART), reactive oxygen species, ova‐

High level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress (OS) an emerging health risk factor involved in the aging and in many diseases, either in humans or in animals. ROS are a double-edged sword – they serve as key signal molecules in physiological processes,

ROS affect multiple physiological processes in reproduction and fertility, from oocyte matu‐ ration to fertilization, embryo development and pregnancy. Several studies indicate that follicular atresia in mammalian species due to the accumulation of toxic metabolites often results from oxidative stress. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Conversely, increase of ROS levels is primarily responsible for the alteration of macromole‐ cules, such as lipids, proteins and nucleic acids, that lead to significant damage of cell structures and thereby cause oxidative stress. To prevent damage due to ROS, cells possess a number of non-enzymatic and enzymatic antioxidants. Non-enzymatic antioxidant include vitamin C, glutathione and vitamin E. Enzymatic antioxidants consist of superoxide dismutases (MnSOD and Cu/ZnSOD) that convert superoxide into hydrogen peroxide; glutathione peroxidase (GPX) and catalase (CAT) which neutralize hydrogen peroxide. Intracellular homeostasis is

but also have a role in pathological processes involving the female reproductive tract.

ensured by the complex interaction between pro-oxidants and antioxidants.

isoforms in maintaining low levels of oxidative stress.

cannot be underestimated.

ry functions

42 New Discoveries in Embryology

**1. Introduction**

The study of folliculogenesis and factors involved in its function is important in order to develop techniques able to increase the effectiveness of therapies or biotechniques included in assisted reproductive technologies (ART).

The follicle and oocyte development in mammals starts in fetal life. Briefly the primordial germinal cells undergo to mitosis until the ovogonias formed become primary oocytes. The meiotic development starts and at the birth the progression stops to the diplotene phase of the first meiotic division [1]. It will continue at the puberty. During the period of meiosis inter‐ ruption the chromosomes become relaxed and nuclear structure so formed is named germinal vescicle (GV). At the puberty the GV disappears, the chromatin is recondensed, the pairs of homologous chromosomes are separated and half of them are expelled forming the first polar body. At this point the meiosis is interrupted again (metaphase II - MII). In this moment the oocyte is mature and fertile [2-4]. Luteinizing hormone (LH) is responsible of resumption of meiosis [5, 6]. The oocytes included in primordial follicles form a finite stock which leave this stage just when they are stimulated [7]. However, it was found that young adult rats have mitotic activity in germinative cells in order to maintain the follicular pool. The mechanisms involved in growing are not yet known [8].

During folliculogenesis the ovarian steroids, estradiol (E2) and progesterone (P), and the peptide hormone, inhibin, are synthesized in the granulosa cells and theca cells. These hormones feed back to regulate the synthesis and secretion of GnRH, LH, and FSH. The majority of ovarian follicles do not ovulate, but undergo an apoptotic process of degeneration called atresia at the small antral follicle stage [9].

Growth of the antral follicles, in most cases, can be divided into two phases. In the first phase, characterized by slow growth stage, early growth of follicles can be attributed to an increase in the number of granulose cells and therefore an increase in the surface of the granulose layer [10]; this stage is critical for the development of oocyte capacity, in which it reaches the final size and competence [11, 12]. In the second phase, characterized by fast growth, in follicles larger than 2-5 mm, follicular growth appears to result from antrum development rather than an increase of the number of granulosa cells. This exponential increase in the antrum surface extends up to a possible ovulation of this follicle [13]. Modest are the information about the endocrine dependence or influence on the growth of small antral follicles. Several were the experiments performed to determine which hormone(s) is involved in this process. In cows, the immunization of GnRH, hence inactivation of the hormone, demonstrated that the first stage of the antral follicular growth can occur in an environment characterized by basal levels of follicle stimulation hormone (FSH) and without luteinizing hormone (LH) pulses [14-16]. It has not been demonstrated how the growth of small antral follicles is possible under basal levels of FSH. In mice the follicular wall is not responsive to FSH up to follicles develop from pre-antral stage to small antral follicles [17]. In any case, the second phase is absolutely under FSH control and adequate pulse of LH [18]. Stimulation of preovulatory follicle development in rodents via injection of equine chorionic gonadotropin (eCG, also called pregnant mare's serum gonadotropin), which has FSH and LH receptor-binding activity, followed 46–48 h later by an ovulatory dose of human chorionic gonadotropin (hCG), which has only LH receptorbinding activity, is commonly used in experiments assessing the effects of gonadotropin hormones on ovarian gene expression and other endpoints and for generating preovulatory follicles or ovulated oocytes for other studies [19].

In mammalian species, the main function of the corpus luteum (CL) is the synthesis of progesterone which is required for the establishment of a uterine environment suitable for the development of peri-implantation conceptus (embryo and associated extra-embryonic membranes) and the successful progression and maintenance of pregnancy [20]. Progesterone acts on the endometrium to regulate the synthesis of growth factors, cytokines, transport and adhesion proteins, protease inhibitors, hormones and enzymes which are primary regulators of conceptus implantation, survival and development [21]. Thus, compromised CL proges‐ terone production Although the mechanisms of CL rescue from cell death and maintenance of progesterone production are very complex and vary among mammalian species [22], there is substantial evidence that reactive oxygen species (ROS) are key factors in determining the CL lifespan [23] and that antioxidants play significant roles in CL physiology during the oestrous/menstrual cycle [24-27]. Luteal ROS production and propagation depends upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to CL function. In addition, the sequence of events leading to the functional and structural luteal regression at the end of the oestrous/menstrual cycle is still not clear. The scarce in-vivo reports studying the CL of rats [29], women [28] and sheep [28, 29] have shown the importance of antioxidant enzymes in the control of CL function during the peri-implantation period. As a luteal phase defect can impact fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of ROSscavenging antioxidant enzymes in the control of mammalian CL function and integrity [30].

majority of ovarian follicles do not ovulate, but undergo an apoptotic process of degeneration

Growth of the antral follicles, in most cases, can be divided into two phases. In the first phase, characterized by slow growth stage, early growth of follicles can be attributed to an increase in the number of granulose cells and therefore an increase in the surface of the granulose layer [10]; this stage is critical for the development of oocyte capacity, in which it reaches the final size and competence [11, 12]. In the second phase, characterized by fast growth, in follicles larger than 2-5 mm, follicular growth appears to result from antrum development rather than an increase of the number of granulosa cells. This exponential increase in the antrum surface extends up to a possible ovulation of this follicle [13]. Modest are the information about the endocrine dependence or influence on the growth of small antral follicles. Several were the experiments performed to determine which hormone(s) is involved in this process. In cows, the immunization of GnRH, hence inactivation of the hormone, demonstrated that the first stage of the antral follicular growth can occur in an environment characterized by basal levels of follicle stimulation hormone (FSH) and without luteinizing hormone (LH) pulses [14-16]. It has not been demonstrated how the growth of small antral follicles is possible under basal levels of FSH. In mice the follicular wall is not responsive to FSH up to follicles develop from pre-antral stage to small antral follicles [17]. In any case, the second phase is absolutely under FSH control and adequate pulse of LH [18]. Stimulation of preovulatory follicle development in rodents via injection of equine chorionic gonadotropin (eCG, also called pregnant mare's serum gonadotropin), which has FSH and LH receptor-binding activity, followed 46–48 h later by an ovulatory dose of human chorionic gonadotropin (hCG), which has only LH receptorbinding activity, is commonly used in experiments assessing the effects of gonadotropin hormones on ovarian gene expression and other endpoints and for generating preovulatory

In mammalian species, the main function of the corpus luteum (CL) is the synthesis of progesterone which is required for the establishment of a uterine environment suitable for the development of peri-implantation conceptus (embryo and associated extra-embryonic membranes) and the successful progression and maintenance of pregnancy [20]. Progesterone acts on the endometrium to regulate the synthesis of growth factors, cytokines, transport and adhesion proteins, protease inhibitors, hormones and enzymes which are primary regulators of conceptus implantation, survival and development [21]. Thus, compromised CL proges‐ terone production Although the mechanisms of CL rescue from cell death and maintenance of progesterone production are very complex and vary among mammalian species [22], there is substantial evidence that reactive oxygen species (ROS) are key factors in determining the CL lifespan [23] and that antioxidants play significant roles in CL physiology during the oestrous/menstrual cycle [24-27]. Luteal ROS production and propagation depends upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to CL function. In addition, the sequence of events leading to the functional and structural luteal regression at the end of the oestrous/menstrual cycle is still not clear. The scarce in-vivo reports studying the CL of rats [29], women [28] and sheep [28, 29] have shown the importance

called atresia at the small antral follicle stage [9].

44 New Discoveries in Embryology

follicles or ovulated oocytes for other studies [19].

### **3. Reactive Oxygen Species (ROS): Chemical and Oxidative Stress (OS)**

Free radicals are believed to play an important role in regulating the metabolic activity and functioning of some organs. There is a complex interaction of the pro-oxidants (free radicals) and antioxidants, resulting in the maintenance of the intracellular homeostasis. Whenever there is an imbalance between the pro-oxidants and antioxidants, favorable to free radicals, a state of oxidative stress (OS) is initiated. It is an emerging health risk factor involved in the aging and in many diseases, either in humans or in animals. Under normal conditions, paired electrons create stable bonds in biomolecules. A free radical is defined as any species capable of independent existence that contains one or more unpaired electrons in the outer orbit, independently upon the expressed electric charge. Depending on the distribution of the charge (electron cloud) and/or of its redox potential, free radicals have a more or less marked reactivity, linked to the spontaneous tendency to exist as entities having all the electrons arranged in pairs. This state corresponds to the chemical stability. The radicals are not equally reactive, in general the increase of charge and volume ratio of free radicals is directly propor‐ tional to their reactivity, therefore, they will tend to reach their own stability stripping electrons to any chemical species with which they are in contact and oxidize them [31].

Free radicals are classified on the basis the nature of the atom to which it belongs the orbital with the unpaired electron. There are, therefore, free radicals centered on oxygen, carbon, nitrogen, or chlorine, and so on. The present chapter, however, will reference mainly to free radicals centered on the oxygen, known more simply as oxygen free radicals. The latter, in fact, besides being one of quantitatively the most important elements of living matter, as well as the primary source of life itself, through a variety of mechanisms – not last the same cellular respiration – induces continuously the formation of chemical species with reactivity charac‐ teristics.

The oxygen free radicals are included into more large family of reactive oxygen species (ROS). This term indicates a class of reactive chemical species derived from oxygen, not necessarily radical, all united by more or less marked tendency to oxidize various organic substrates (carbohydrates, lipids, amino acids, proteins, nucleotides, etc.). Classic examples of radical origin of ROS are singlet oxygen and hydroxyl radical. The ozone and hydrogen peroxide, however, are not radical reactive oxygen species.

In living organisms, ROS are generated during normal cellular metabolic activity; some exogenous agents, however, can increase production, even with direct mechanism. It is possible to identify at least five sources of primary metabolic free radicals, in relation to the cellular site mainly interested in the production of ROS: the plasma membrane, mitochondria, peroxisomes, the smooth endoplasmic reticulum (microsomes) and the cytosol. In each of these locations ROS are produced either spontaneously or as a result of reactions catalyzed by enzymes or by transition metals (eg. iron or copper) [31].

The free radicals can be generated by different mechanisms and, once formed, generally give rise to a series of chain reactions, in the course of which the radicalic site can be transferred or inactivated [31, 32].

Free radicals are mainly generated by homolytic cleavage or interaction with the transition metals. The term homolytic cleavage refers to the division of the covalent bond of a molecule as effect of the administration energy (thermal or radiant), with generation of two new chemical species, each one with an unpaired electron, distinctive element of free radicals. A classic example of homolytic cleavage is the radiolysis or photolysis of water that generates an atom of hydrogen and a hydroxyl radical. This chemical reaction is different from the ionization observed, for example, after dissolved in water molecules having at least one covalent bond polarized (eg. HCl). In this case, the water molecules, because of their polarity and without any administration of energy, are able to crack one of the polarized covalent bonds of the molecule solute generating two chemical species loaded of opposite sign, a cation and an anion (H+ and Cl- , respectively, in the example considered). The ionization, unlike the homolytic cleavage, the doublet electronic binding of the original molecule is not separated but remains in one of the new ionic species (anion) [33].

In the interaction with the transition metals, the electron generated by oxidation of a metal transition in ionic form (eg. from Fe2+ to Fe3+ or Cu+ to Cu2+) breaks a covalent bond to a target molecule generating a radical free and an anion. Alternatively, the electron required for reducing a transition metal in ionic form (eg. from Fe3+ to Fe2+ or Cu2+ to Cu+ ) is extracted from the covalent binding of a target molecule, which is decomposed into a free radical and a cation. Through this mechanism, for example, iron (Fe2+/Fe3+ ) or copper (Cu+ /Cu2+) act as catalysts in a sequence of redox reactions generating alkoxy radicals (RO\*) and peroxyl (R-O-O\*) from peroxides (R-O-O-R). In the simplest case - described for the first time by Fenton - one ferrous ion (Fe2+), oxidizes to ferric ion (Fe2+), transfers its electron to a molecule of hydrogen peroxide (H2O2) and it breaks one of covalent bonds, generating a free radical (the hydroxyl radical, HO\*) and an anion (hydroxyl ion). In turn, the ferric ion (Fe3+) is reduced - regenerating as any catalyst – to ferrous ion (Fe2+), ripping an electron from a second molecule of hydrogen peroxide, which is split into a free radical (radical perhydroxyl (HOO\*), and a cation (a hydrogen ion, H+ ). Similarly, the hydroperoxides are split, for catalytic action of the iron, in the radical alkoxyl (RO\*) and peroxyl (ROO\*). In the absence of catalysts, the split of peroxides - which gives rise to a single species radical, the alkoxy - can take place only with energy consumption. A method of great biological relevance that gives rise to the formation of free radicals, includes the decomposition of nitrocompounds. In fact, alkyl radicals originate following the removal of molecular nitrogen (N2) [31].

Once a radical reaction is triggered, it tends to propagate chain. There are four basic mecha‐ nisms of propagation of radical reactions: transfer, addition, fragmentation and rearrange‐ ment. The most common among these is the transfer. In this mode, the free radical - generated by one of previous reactions - attacks a molecule subtracting to it one of its atoms (generally a hydrogen atom). The result is the formation of a new reactive species and, in practice, radical site has been transferred. With this mechanism, for example, the hydroxyl radical (HO\*), attacking an organic molecule (R-H), rips to this one atom of hydrogen and generates, with a molecule of water (H2O), an alkyl radical (R\*). With this mechanism, the radical site is transferred from the hydroxyl radical to the alkyl one.

Finally, a radical reaction chain may stop (term) by two mechanisms: combination or dispro‐ portion. In particular, in the combination, which is the homolytic cleavage of the reverse reaction, two radicals react with each other giving rise to a molecule not more reactive. The first radical acts as the oxidant, while the second acts as a generic antioxidant. This mechanism is exploited to block a radical reaction, and in general, any radical process chain can be interrupted by the intervention of agents called, generically, antioxidants.

In living organisms ROS are generated during normal cellular metabolic activity; some exogenous agents, however, may increase production, even with direct mechanism (figure 1).

As mentioned above, it is possible to identify at least 5 of primary metabolic free radical sources, in relation to cellular site: the plasma membrane, the mitochondria, peroxisomes, smooth endoplasmic reticulum (microsomes) and the cytosol (figure 2).

**Figure 1.** General mechanism of ROS production.

locations ROS are produced either spontaneously or as a result of reactions catalyzed by

The free radicals can be generated by different mechanisms and, once formed, generally give rise to a series of chain reactions, in the course of which the radicalic site can be transferred or

Free radicals are mainly generated by homolytic cleavage or interaction with the transition metals. The term homolytic cleavage refers to the division of the covalent bond of a molecule as effect of the administration energy (thermal or radiant), with generation of two new chemical species, each one with an unpaired electron, distinctive element of free radicals. A classic example of homolytic cleavage is the radiolysis or photolysis of water that generates an atom of hydrogen and a hydroxyl radical. This chemical reaction is different from the ionization observed, for example, after dissolved in water molecules having at least one covalent bond polarized (eg. HCl). In this case, the water molecules, because of their polarity and without any administration of energy, are able to crack one of the polarized covalent bonds of the molecule solute generating two chemical species loaded of opposite sign, a cation and

homolytic cleavage, the doublet electronic binding of the original molecule is not separated

In the interaction with the transition metals, the electron generated by oxidation of a metal

molecule generating a radical free and an anion. Alternatively, the electron required for

the covalent binding of a target molecule, which is decomposed into a free radical and a cation.

a sequence of redox reactions generating alkoxy radicals (RO\*) and peroxyl (R-O-O\*) from peroxides (R-O-O-R). In the simplest case - described for the first time by Fenton - one ferrous ion (Fe2+), oxidizes to ferric ion (Fe2+), transfers its electron to a molecule of hydrogen peroxide (H2O2) and it breaks one of covalent bonds, generating a free radical (the hydroxyl radical, HO\*) and an anion (hydroxyl ion). In turn, the ferric ion (Fe3+) is reduced - regenerating as any catalyst – to ferrous ion (Fe2+), ripping an electron from a second molecule of hydrogen peroxide, which is split into a free radical (radical perhydroxyl (HOO\*), and a cation (a

the radical alkoxyl (RO\*) and peroxyl (ROO\*). In the absence of catalysts, the split of peroxides - which gives rise to a single species radical, the alkoxy - can take place only with energy consumption. A method of great biological relevance that gives rise to the formation of free radicals, includes the decomposition of nitrocompounds. In fact, alkyl radicals originate

Once a radical reaction is triggered, it tends to propagate chain. There are four basic mecha‐ nisms of propagation of radical reactions: transfer, addition, fragmentation and rearrange‐ ment. The most common among these is the transfer. In this mode, the free radical - generated by one of previous reactions - attacks a molecule subtracting to it one of its atoms (generally a hydrogen atom). The result is the formation of a new reactive species and, in practice, radical

reducing a transition metal in ionic form (eg. from Fe3+ to Fe2+ or Cu2+ to Cu+

, respectively, in the example considered). The ionization, unlike the

) or copper (Cu+

). Similarly, the hydroperoxides are split, for catalytic action of the iron, in

to Cu2+) breaks a covalent bond to a target

) is extracted from

/Cu2+) act as catalysts in

enzymes or by transition metals (eg. iron or copper) [31].

but remains in one of the new ionic species (anion) [33].

transition in ionic form (eg. from Fe2+ to Fe3+ or Cu+

Through this mechanism, for example, iron (Fe2+/Fe3+

following the removal of molecular nitrogen (N2) [31].

inactivated [31, 32].

46 New Discoveries in Embryology

an anion (H+

hydrogen ion, H+

and Cl-

**Figure 2.** Primary source of ROS cell production

The plasma membrane is one of the most important sources of ROS, particularly (but not exclusively) in polymorphonuclear leukocytes (PMNs). In fact, in the plasma membrane of PMNs are located several enzymes, such as the NADPH oxidase and lipoxygenase, whose activation is accompanied by the production, respectively, of superoxide anion and metabolic intermediates with chemical characteristics of peroxides. The NADPH oxidase is an enzyme that catalyzes the formation of superoxide anion by NADPH (H+ ) and molecular oxygen, after specific stimulation of PMNs, due, for example, to endotoxins, bacteria, or antibodies).

The reaction is made possible by the increased availability of NADPH (H+ ), for the increased oxidation of glucose through the shunt of hexoses, and of molecular oxygen, under the socalled "respiratory burst". The system of lipoxygenase, localized also at the level of the plasma membrane, includes three enzymes, the 5-, 12-, and 15- lipoxygenase, which catalyze the formation, from arachidonic acid, of 5-, 12-, and 15-HPETE (hydroperoxyeicosatetraenoic acid), respectively. These substances are chemically hydroperoxides acids, theybelong to a group of ROS named ROM (reactive oxygen metabolites, ie metabolites or derived reactive oxygen). The production of ROS at the level of PMNs plasma membrane for activation NADPH oxidase and/or lipoxygenase, takes place, typically, in the course of reactive processes (eg. infections, immunoreactions pathogenic, inflammation) [31].

The mitochondria are the primary metabolic source of ROS because the enzyme complexes of respiratory chain are localized on their crests and are involved in oxidative phosphorylation. Ideally, the transfer of electrons from reduced NAD to cytochrome C and from the latter to oxygen should end with the production of H2O, once synthesized ATP, (reduction tetravalent of molecular oxygen). However, already in normal conditions, this process is not perfect so, for not easily controllable reasons, a certain amount of electrons (1-2%) escapes the system transport of various coenzymes (eg. ubiquinone, flavoproteins, cytochromes, etc.) and reacts directly with molecular oxygen, generating, thus, superoxide anion and/or hydrogen peroxide (reduction uni- and bivalent molecular oxygen). In fact, this process, during a intense exercise in skeletal muscle, this electronic shunt can reach 15% of the oxygen used by mitochondria due to the intense stimulation of cellular metabolism. The phenomenon of the reduction in one or bivalent molecular oxygen takes place, in the mitochondria, without the intervention of enzymes, as opposed to what is observed in other cell locations. In other words, from a purely chemical point of view, the production of free radicals during oxidative phosphorylation is not just a mode of enzymatic production of reactive species. In fact, as it has just been men‐ tioned, the generation of free radicals in living organisms is closely related to vital phenomena and, therefore, constitutes a "physiological" phenomenon that takes place continuously in the course of redox reactions through both enzymatic and non-enzymatic mechanisms. It should be stressed that, in addition to mitochondria, there are other sources of non-enzymatic free radicals in cells. For example, peroxynitrite spontaneously generates hydroxyl and nitroxide radicals. However, the most important non-enzymatic reactions from a biological standpoint for the production of free radicals are those catalyzed by transition metals. In these reactions, which generally require iron or copper in the reduced state (respectively Fe2+ and Cu+ ), hydrogen peroxide is split into hydroxyl radical and hydroxyl ion for incorporation of the electron ripped to transition metal, which is released in the oxidized form (Fe3+ and Cu2+, respectively), according to the mechanism discussed above of the interaction with transition metals. Hydroperoxides undergo a similar reaction, which generate the alkoxy radical. The enzymes that regenerate the transition metals in the reduced state constitute a complex indicated with MCO (metal-catalyzed oxidation systems). They include xanthine oxidase, NADPH and NADH oxidase, nicotinic acid hydroxylase, the cytochrome P450 system, the NADH reductase (with coenzyme quinone), the succinic-reductase (with coenzyme quinone) and an amount of iron-sulfur proteins non-heme. The quinones and reduced flavin prosthetic groups generated by these enzymes in their turn reduce the transition metals, resulting in the direct reduction of molecular oxygen to hydroxyl radical and/or peroxide hydrogen (through the mediation or not of superoxide anion).

The plasma membrane is one of the most important sources of ROS, particularly (but not exclusively) in polymorphonuclear leukocytes (PMNs). In fact, in the plasma membrane of PMNs are located several enzymes, such as the NADPH oxidase and lipoxygenase, whose activation is accompanied by the production, respectively, of superoxide anion and metabolic intermediates with chemical characteristics of peroxides. The NADPH oxidase is an enzyme

specific stimulation of PMNs, due, for example, to endotoxins, bacteria, or antibodies).

oxidation of glucose through the shunt of hexoses, and of molecular oxygen, under the socalled "respiratory burst". The system of lipoxygenase, localized also at the level of the plasma membrane, includes three enzymes, the 5-, 12-, and 15- lipoxygenase, which catalyze the formation, from arachidonic acid, of 5-, 12-, and 15-HPETE (hydroperoxyeicosatetraenoic acid), respectively. These substances are chemically hydroperoxides acids, theybelong to a group of ROS named ROM (reactive oxygen metabolites, ie metabolites or derived reactive oxygen). The production of ROS at the level of PMNs plasma membrane for activation NADPH oxidase and/or lipoxygenase, takes place, typically, in the course of reactive processes (eg.

The mitochondria are the primary metabolic source of ROS because the enzyme complexes of respiratory chain are localized on their crests and are involved in oxidative phosphorylation. Ideally, the transfer of electrons from reduced NAD to cytochrome C and from the latter to oxygen should end with the production of H2O, once synthesized ATP, (reduction tetravalent of molecular oxygen). However, already in normal conditions, this process is not perfect so, for not easily controllable reasons, a certain amount of electrons (1-2%) escapes the system transport of various coenzymes (eg. ubiquinone, flavoproteins, cytochromes, etc.) and reacts directly with molecular oxygen, generating, thus, superoxide anion and/or hydrogen peroxide (reduction uni- and bivalent molecular oxygen). In fact, this process, during a intense exercise in skeletal muscle, this electronic shunt can reach 15% of the oxygen used by mitochondria due to the intense stimulation of cellular metabolism. The phenomenon of the reduction in one or bivalent molecular oxygen takes place, in the mitochondria, without the intervention of enzymes, as opposed to what is observed in other cell locations. In other words, from a purely chemical point of view, the production of free radicals during oxidative phosphorylation is not just a mode of enzymatic production of reactive species. In fact, as it has just been men‐ tioned, the generation of free radicals in living organisms is closely related to vital phenomena and, therefore, constitutes a "physiological" phenomenon that takes place continuously in the course of redox reactions through both enzymatic and non-enzymatic mechanisms. It should be stressed that, in addition to mitochondria, there are other sources of non-enzymatic free radicals in cells. For example, peroxynitrite spontaneously generates hydroxyl and nitroxide radicals. However, the most important non-enzymatic reactions from a biological standpoint for the production of free radicals are those catalyzed by transition metals. In these reactions, which generally require iron or copper in the reduced state (respectively Fe2+ and Cu+

hydrogen peroxide is split into hydroxyl radical and hydroxyl ion for incorporation of the electron ripped to transition metal, which is released in the oxidized form (Fe3+ and Cu2+,

) and molecular oxygen, after

), for the increased

),

that catalyzes the formation of superoxide anion by NADPH (H+

48 New Discoveries in Embryology

infections, immunoreactions pathogenic, inflammation) [31].

The reaction is made possible by the increased availability of NADPH (H+

In addition to the plasma membrane and mitochondria, peroxisomes also represent an important source of ROS. In these cell organelles, in fact, a particular process of fatty acid oxidation takes place, which is different from the conventional way (beta-oxidation). In the first stage of this sequence of reactions, a flavoprotein extracts a pair of hydrogen atoms from one molecule of activated fatty acid (acyl-CoA) by transferring it directly to molecular oxygen, with the formation of hydrogen peroxide (subsequently inactivated by catalase).

In the endoplasmic reticulum (microsomes) production of reactive species passes through the cytochrome P450. The latter plays a major role in detoxification processes. The cytochrome P450 acts as immediate donor of electrons in many reactions of hydroxylation, particularly those that take place within the hepatocytes and that are aimed to inactivation of hormones (eg. steroid) and not physiological compounds (xenobiotics, such as toxic and hydrophobic drugs which are thereby made more soluble and less toxic). The P450 is a heme iron protein localized not only in the endoplasmic reticulum of the liver but also in the mitochondria of the adrenal cortex that, in a process very complex and not yet fully clarified, acts as connection between NADPH (H+ ) (electron donor) and the substrate that should be hydroxyled. In this complex reaction, a substrate able to be hydroxylated (SH) reacts with NADPH (H+ ) and molecular oxygen (O2) to form the corresponding hydroxylated derivative (S-OH), plus NADP + and water. A production of free radicals in the cell also occurs in the course of many other biochemical reactions, such as during oxidation of hypoxanthine to xanthine and xanthine to uric acid, which mark the final phase of the catabolism of purine nucleotides. Both of these reactions are catalyzed by xanthine dehydrogenase, a molybdenum enzyme. Under special conditions, such as during the so-called ischemia-reperfusion, xanthine dehydrogenase is converted to xanthine oxidase (probably for proteolytic cleavage calcium-dependent). The latter, using as a final electron acceptor the oxygen, generates hydrogen peroxide and super‐ oxide anion, starting, respectively, from hypoxanthine and xanthine.

Other reactions that generate free radicals are described in the synthesis of catecholamines.

From the above, it is clear that ROS represent intermediate obligated cellular metabolism. And since their production is closely linked to the vital phenomena, they have been called "irre‐ placeable companions" of our existence.

It appears evident that in each cell site, the production of reactive species has its own specific function. In fact, it has been recognized that ROS play an important role "in the service of life" because they are not only involved in cell metabolism but also in the "reactive processes" such as infection and inflammation. Actually, the superoxide anion and other ROS are generated on the outer surface of the plasma membrane of activated leukocytes. These reactive species attack extraneous components such as bacteria, weakening the wall and making them more readily accessible to phagocytosis and, ultimately, to their destruction. These "immunological" activities are expressed not only in respect of extraneous components but also against "self" components, such as tissues or transplanted organs (rejection reaction). This strategy is also used in the course of healing of organs or tissues subject to trauma. In fact, the leukocytes migrate to the injured, are activated and begin bombing damaged cells with free radicals, that accelerate their destruction, remove lysis products, and promote the recovery (regeneration). The production of free radicals by the cells may sometimes undergo a considerable increase depending on external stimuli. In fact, physical, chemical and biological agents, alone or in combination, may also induce the generation of ROS or increase the "physiological" production through a specific metabolic stimulation. Ionizing and UV radiation are reported to be physical agents. Both these sources of energy can induce the phenomenon of homolytic cleavage of water, also called radiolysis or photolysis, depending on the type of radiation involved.

In this reaction, the water molecule absorbs energy and uses it to break one of its two covalent bonds with the hydrogen: the products will be two free radicals, the hydroxyl radical and the hydrogen atom. Considering that a living organism is made up primarily of water and he spends most of his life under the influence of radiation (UV or ionizing they are) it is clear how this phenomenon affects substantially the production of free radicals.

As chemical agent, capable of stimulating the production of free radicals, ozone (ROS) is to be quoted. It directly generates peroxyl radicals by interaction with phenolic compounds. The two cases considered so far (radiation and ozone) are examples of direct production of reactive species. Other chemical agents, however, such as polycyclic aromatic hydrocarbons, or certain drugs, induce increased production of free radicals through an indirect mechanism, activating the cytochrome P450 microsomal level. Biological agents that typically lead to increased production of ROS for metabolic activation are bacteria, as part of the physiological process of defense against infection, and certain antibodies, as part of some reactions immune-pathogen. In these cases, as mentioned with regard to the plasma membrane, the PMNs are directly implicate. They, in fact, possess NADPH oxidase and a series of enzymes directly involved in the production and, in part, inactivation of reactive chemical species, such as superoxide dismutase (SOD), myeloperoxidase (MPx), catalase (CAT) and glutathione peroxidase (GPx).

SOD catalyzes the conversion of superoxide anion into hydrogen peroxide which, in turn, can be inactivated to water by CAT or GPx. However, the availability of chlorides - even at physiological concentrations - makes the hydrogen peroxide a substrate for MPx. The end result is the production of a highly oxidising agent, the hypochlorous acid (HClO). The HClO can attack numerous organic substrates and, in particular, amino acids and proteins, to produce chloramines, a potential source of alkoxyl and peroxyl radicals. Finally, an increase in free radical production may be observed in "physiological " situations, such as after an intense muscular effort or in the course of many diseases. In the latter case, often, it is not clear how far the ROS are the cause or the effect of a certain pathology [31].

### **4. The antioxidant defense system**

because they are not only involved in cell metabolism but also in the "reactive processes" such as infection and inflammation. Actually, the superoxide anion and other ROS are generated on the outer surface of the plasma membrane of activated leukocytes. These reactive species attack extraneous components such as bacteria, weakening the wall and making them more readily accessible to phagocytosis and, ultimately, to their destruction. These "immunological" activities are expressed not only in respect of extraneous components but also against "self" components, such as tissues or transplanted organs (rejection reaction). This strategy is also used in the course of healing of organs or tissues subject to trauma. In fact, the leukocytes migrate to the injured, are activated and begin bombing damaged cells with free radicals, that accelerate their destruction, remove lysis products, and promote the recovery (regeneration). The production of free radicals by the cells may sometimes undergo a considerable increase depending on external stimuli. In fact, physical, chemical and biological agents, alone or in combination, may also induce the generation of ROS or increase the "physiological" production through a specific metabolic stimulation. Ionizing and UV radiation are reported to be physical agents. Both these sources of energy can induce the phenomenon of homolytic cleavage of water, also called radiolysis or photolysis, depending on the type of radiation involved.

50 New Discoveries in Embryology

In this reaction, the water molecule absorbs energy and uses it to break one of its two covalent bonds with the hydrogen: the products will be two free radicals, the hydroxyl radical and the hydrogen atom. Considering that a living organism is made up primarily of water and he spends most of his life under the influence of radiation (UV or ionizing they are) it is clear how

As chemical agent, capable of stimulating the production of free radicals, ozone (ROS) is to be quoted. It directly generates peroxyl radicals by interaction with phenolic compounds. The two cases considered so far (radiation and ozone) are examples of direct production of reactive species. Other chemical agents, however, such as polycyclic aromatic hydrocarbons, or certain drugs, induce increased production of free radicals through an indirect mechanism, activating the cytochrome P450 microsomal level. Biological agents that typically lead to increased production of ROS for metabolic activation are bacteria, as part of the physiological process of defense against infection, and certain antibodies, as part of some reactions immune-pathogen. In these cases, as mentioned with regard to the plasma membrane, the PMNs are directly implicate. They, in fact, possess NADPH oxidase and a series of enzymes directly involved in the production and, in part, inactivation of reactive chemical species, such as superoxide dismutase (SOD), myeloperoxidase (MPx), catalase (CAT) and glutathione peroxidase (GPx).

SOD catalyzes the conversion of superoxide anion into hydrogen peroxide which, in turn, can be inactivated to water by CAT or GPx. However, the availability of chlorides - even at physiological concentrations - makes the hydrogen peroxide a substrate for MPx. The end result is the production of a highly oxidising agent, the hypochlorous acid (HClO). The HClO can attack numerous organic substrates and, in particular, amino acids and proteins, to produce chloramines, a potential source of alkoxyl and peroxyl radicals. Finally, an increase in free radical production may be observed in "physiological " situations, such as after an intense muscular effort or in the course of many diseases. In the latter case, often, it is not clear

this phenomenon affects substantially the production of free radicals.

how far the ROS are the cause or the effect of a certain pathology [31].

ROS are chemical species potentially detrimental. For this reason, living organisms have developed over millennia of evolution a complex antioxidant defense system, consisting of a set of enzymes, vitamins, trace elements and other vitamin-like substances. These antioxidants may be classified according to different criteria: on the basis of the origin, in endogenous and exogenous, on the basis of the chemical nature, in the enzymatic and non-enzymatic, and on the basis of the solubility in fat-soluble and water-soluble. On the basis, however, of the mechanism of action prevalent, physiological antioxidants can be easily assembled into four main groups: preventive antioxidants, scavenger, shelter agents and adaptation agents [34].

Preventive antioxidants are agents that, through various mechanisms, such as the chelation of transition metals, prevent the formation of reactive species.

The scavengers act through different mechanisms. They may be of hydrophilic nature (albumin, urate, ascorbate, urate) or lipophilic (carotenoids, vitamin E, ubiquinol). According to some researchers, the scavenger should be distinguished from antioxidants proper. In fact, while the scavenger (eg. A-tocopherol) are agents that reduce the concentration of free radicals removing them from the medium in which they are located, antioxidants (eg. Diphenylamine) are agents that inhibit the auto-oxidation process, e.g. the fat rancidity. This phenomenon, well known in food science, is called auto-oxidation since it occurs through a sequence of autoca‐ talytic radical reactions in the presence of oxygen. Alternatively, you can use the term peroxidation, as the same process generates intermediates with characteristics of peroxides (R-O-OR).

Through this process some dietary fat rancid and cellular membranes of living organisms are oxidized.

Shelter agents include only enzymes involved after the damage from reactive species has been established. Their action - often sequential - provides first the identification of the molecular segment oxidized, then the separation of the fragment unusable and, finally, the synthesis and the insertion of a new segment in substitution of the damaged one. The category of shelter antioxidants includes hydrolases (glycosidases, lipases, proteases), and the transferase and polymerases, all essential for the repair of free radical damage of important molecules or cellular structures (eg. DNA, membranes, etc.).

Finally, the agents of adaptation include all substances or techniques or procedures through which it is possible to strengthen the physiological antioxidant system of an organism. For example, a proper physical exercise or the adoption of a proper and balanced diet are measures by itself able to check the oxidative metabolism by reducing the production of reactive species, and induction of enzymes with antioxidant activity.

The antioxidant defense system is regularly distributed in the body, both at the extracellular and intracellular levels.

In plasma, the set of substances potentially able to give equivalent reducing (hydrogen atoms or single electrons) so as to meet "the greed of electrons" that makes free radical constitutes unstable is the so-called barrier antioxidant. In the plasma, all protein and, in particular, albumin, bilirubin, uric acid, cholesterol, and various exogenous antioxidants introduced with food or in the form of dietary supplements (ascorbate, tocopherol, polyphenols etc.) are part of it. The thiol groups (-SH), commonly found in the cysteine side chain, play a role of particular importance in the context of this barrier. In addition, thiol groups, are the most chemically reactive sites on proteins, such as albumin, and have strong reducing properties [35, 36].

Inside the cells, the antioxidant system of cell defense has its precise compartmentalization (figure 3). The antioxidant system includes some enzymes (glutathione, superoxide dismutase, catalase) and a series of substances taken from outside (vitamins and substances similar to antioxidant activity, such as polyphenols, trace elements etc.). Some of these agents are fatsoluble (eg. tocopherols) and, entering the team of biomembranes, constitute the first line of defense against the attack of free radicals. Others, however, are water soluble (eg. ascorbate) and intervene especially in the context of soluble matrix of the cytoplasm and cellular organ‐ elles.

**Figure 3.** Compartmentalization of antioxidant system

Glutathione (GSH) is a tripeptide (L-g-glutamyl-L-cysteinyl-glycine, with multiple biological functions and that has been found in all mammalian cells [37-39]. Its biological activity is primarily related to the active thiol group of the cysteine residue [40]. The reduced and oxidized forms of glutathione (GSH and GSSG) act in concert with other redox-active com‐ pounds (e.g., NAD(P)H) to regulate and maintain cellular redox status. It is an abundant lowmolecular-mass thiol antioxidant, which either interacts directly with reactive oxygen and nitrogen species (ROS and RNS, respectively) or serves as a cofactor for many antioxidant and associated enzymes such as peroxidases and transferases [41]. The chemical structure of GSH determines its potential functions and its broad distribution among all living organisms reflects its important biological role Probably most importantly, GSH is responsible for protection against ROS and RNS, and detoxification of endogenous and exogenous toxins of an electro‐ philic nature. Depletion of GSH results in DNA damage and increased H2O2 concentrations; as such, GSH is an essential antioxidant. During the reduction of H2O2 to H2O and O2, GSH is oxidized to GSSG by glutathione peroxidase (GPx). Glutathione reductase participates in the reverse reaction, and utilizes the transfer of a donor proton from NADPH to GSSG, thus, recycling GSH [42]. Vitamin E (α-tocopherol) protects GPx4-deficient cells from cell death. In addition, glutathione is (1) a storage form of cysteine in the cells and for interorgan transfer; (2) a storage form and transporter of nitric oxide (as GSNO); (3) involved in the metabolism of estrogens, leukotrienes, and prostaglandins, reduction of ribonucleotides to deoxyribonu‐ cleotides, and maturation of iron–sulfur clusters of proteins; (4) involved in the regulation of certain transcription factors from the environment to cellular transcription machinery; (5) involved in the detoxification of many endogenous compounds and xenobiotics (the mercap‐ turate pathway); and (6) copper and iron transfer. Glutathione also can be used even for the detoxification of ions of transition metals such as chromium [43, 44].

unstable is the so-called barrier antioxidant. In the plasma, all protein and, in particular, albumin, bilirubin, uric acid, cholesterol, and various exogenous antioxidants introduced with food or in the form of dietary supplements (ascorbate, tocopherol, polyphenols etc.) are part of it. The thiol groups (-SH), commonly found in the cysteine side chain, play a role of particular importance in the context of this barrier. In addition, thiol groups, are the most chemically reactive sites on proteins, such as albumin, and have strong reducing properties [35, 36].

Inside the cells, the antioxidant system of cell defense has its precise compartmentalization (figure 3). The antioxidant system includes some enzymes (glutathione, superoxide dismutase, catalase) and a series of substances taken from outside (vitamins and substances similar to antioxidant activity, such as polyphenols, trace elements etc.). Some of these agents are fatsoluble (eg. tocopherols) and, entering the team of biomembranes, constitute the first line of defense against the attack of free radicals. Others, however, are water soluble (eg. ascorbate) and intervene especially in the context of soluble matrix of the cytoplasm and cellular organ‐

Glutathione (GSH) is a tripeptide (L-g-glutamyl-L-cysteinyl-glycine, with multiple biological functions and that has been found in all mammalian cells [37-39]. Its biological activity is primarily related to the active thiol group of the cysteine residue [40]. The reduced and oxidized forms of glutathione (GSH and GSSG) act in concert with other redox-active com‐ pounds (e.g., NAD(P)H) to regulate and maintain cellular redox status. It is an abundant lowmolecular-mass thiol antioxidant, which either interacts directly with reactive oxygen and nitrogen species (ROS and RNS, respectively) or serves as a cofactor for many antioxidant and associated enzymes such as peroxidases and transferases [41]. The chemical structure of GSH determines its potential functions and its broad distribution among all living organisms reflects

elles.

52 New Discoveries in Embryology

**Figure 3.** Compartmentalization of antioxidant system

Five isoforms of glutathione peroxidase exist in the body: GPx1, GPx2, GPx3, GPx4, and GPx5. GPx1 is the cytosolic isoform that is widely distributed in tissues, while GPx2 encodes a gastrointestinal form with no specific function; GPx3 is present in plasma and epididymal fluid. GPx 4 specifically detoxifies phospholipid hydroperoxide within biological membranes. Free glutathione exists in vivo mostly as two forms, reduced (GSH) and oxidized (glutathione disulfide; GSSG). GPx5 is found in the epididymis [39].

Superoxide dismutase (SOD): Other enzymes directly detoxify ROS. SOD reacts with super‐ oxide anion radicals to form oxygen and H2O2. The enzyme SOD exists as three isoenzymes: SOD 1, SOD 2, and SOD 3. SOD 1 contains Cu and zinc (Zn) (Cu, Zn-SOD) as metal co-factors and is located in the cytosol. SOD 2 (Mn-SOD) is a mitochondrial isoform containing manga‐ nese (Mn), and SOD 3 encodes the extracellular form (ECSOD). SOD 3 is structurally similar to Cu, Zn-SOD, as it contains Cu and Zn as cofactors [45, 46].

Catalase (CAT) is a heme-containing homotetrameric protein. CAT can decompose hydrogen peroxide (H2O2) in reactions catalyzed by two different modes of enzymatic activity: the catalatic mode of activity (2H2O2 → O2 + 2H2O) and the peroxidatic mode of activity (H2O2 + AH2 → A + 2H2O). Although several substrates such as methanol and ethanol can be oxidized by the peroxidation reaction, the physiological significance of this catalase function is not understood. Decomposition of H2O2 by the catalatic activity of catalase follows the fashion of a first-order reaction, and its rate is dependent on the concentration of H2O2. In fact, catalase belongs to the group of enzymes that catalyze reactions at a rate near kinetic perfection; the reaction rate is only limited by the rate at which the enzyme collides with the substrate. Catalase is ubiquitously present in all prokaryotes and eukaryotes. With the exception of erythrocytes, it is predominantly located in peroxisomes of all types of mammalian cells where H2O2 is generated by various oxidases. However, a certain amount of catalase has also been found in mitochondria of rat heart. Since H2O2 serves as a substrate for Fenton reaction to generate the highly reactive hydroxyl radical, catalase is believed to play a role in cellular antioxidant defense mechanisms by limiting the accumulation of H2O2 [47-49].

The non-enzymatic antioxidants consist of dietary supplements and synthetic antioxidants such as vitamin C, GSH, taurine, hypotaurine, vitamin E, Zn, selenium (Se), betacarotene, and carotene [41]. Vitamin C (ascorbic acid) is a known redox catalyst that can reduce and neu‐ tralize ROS. Its reduced form is maintained through reactions with GSH and can be catalyzed by protein disulfide isomerase and glutaredoxins. Glutathione is a peptide found in most forms of aerobic life as it is made in the cytosol from cysteine, glutamate, and glycine [42]; it is also the major nonenzymatic antioxidant found in oocytes and embryos. Its antioxidant properties stem from the thiol group of its cysteine component, which is a reducing agent that allows it to be reversibly oxidized and reduced to its stable form [42]. Levels of GSH are regulated by its formation de-novo, which is catalyzed by the enzymes gamma-GCS and glutathione synthetase [4, 11]. Glutathione participates in reactions, including the formation of glutathione disulfide, which is transformed back to GSH by glutathione reductase at the expense of NADPH [17].

Cysteine and cysteamine (CSH) increase the GSH content of the oocyte. Cysteamine also acts as a scavenger and is an antioxidant essential for the maintenance ofhigh GSH levels. Fur‐ thermore, CSH can be converted to another antioxidant, hypotaurine [43, 44].

The concentrations of many amino acids, including taurine, fluctuate considerably during folliculogenesis. Taurine and hypotaurine are scavengers that help maintain redox homeosta‐ sis in gametes. Both neutralize lipid peroxidation products, and hypotaurine further neutral‐ izes hydroxyl radicals [44].

Like GSH, the Thioredoxin (Trx) system regulates gene functions and coordinates various enzyme activities. It detoxifies H2O2 and converts it to its reduced state via Trx reductase [45]. Normally, Trx is bound to apoptosis-regulating signal kinase (ASK) 1, rendering it inactive. However, when the thiol group of Trx is oxidized by the SO anion, ASK1 detaches from Trx and becomes active leading to enhanced apoptosis. ASK1 can also be activated by exposure to H2O2 or hypoxiareoxygenation, and inhibited by vitamins C and E. The Trx system also plays a role in female reproduction and fetal development by being involved in cell growth, differentiation, and death. Incorrect protein folding and formation of disulfide bonds can occur through H+ ion release from the thiol group of cysteine, leading to disordered protein function, aggregation, and apoptosis [2].

Vitamin E (α-tocopherol) is a lipid soluble vitamin with antioxidant activity. It consists of eight tocopherols and tocotrienols. It plays a major role in antioxidant activities because it reacts with lipid radicals produced during lipid peroxidation [42]. This reaction produces oxidized α-tocopheroxyl radicals that can be transformed back to the active reduced form by reacting with other antioxidants like ascorbate, retinol, or ubiquinol.

The hormone melatonin is an antioxidant that, unlike vitamins C and E and GSH, is produced by the human body. In contrast to other antioxidants, however, melatonin cannot undergo redox cycling; once it is oxidized, melatonin is unable to return to its reduced state because it forms stable end-products after the reaction occurs (see below for functions).

### **5. Commonly used markers of ROS-induced modification of cellular components**

The non-enzymatic antioxidants consist of dietary supplements and synthetic antioxidants such as vitamin C, GSH, taurine, hypotaurine, vitamin E, Zn, selenium (Se), betacarotene, and carotene [41]. Vitamin C (ascorbic acid) is a known redox catalyst that can reduce and neu‐ tralize ROS. Its reduced form is maintained through reactions with GSH and can be catalyzed by protein disulfide isomerase and glutaredoxins. Glutathione is a peptide found in most forms of aerobic life as it is made in the cytosol from cysteine, glutamate, and glycine [42]; it is also the major nonenzymatic antioxidant found in oocytes and embryos. Its antioxidant properties stem from the thiol group of its cysteine component, which is a reducing agent that allows it to be reversibly oxidized and reduced to its stable form [42]. Levels of GSH are regulated by its formation de-novo, which is catalyzed by the enzymes gamma-GCS and glutathione synthetase [4, 11]. Glutathione participates in reactions, including the formation of glutathione disulfide, which is transformed back to GSH by glutathione reductase at the expense of

Cysteine and cysteamine (CSH) increase the GSH content of the oocyte. Cysteamine also acts as a scavenger and is an antioxidant essential for the maintenance ofhigh GSH levels. Fur‐

The concentrations of many amino acids, including taurine, fluctuate considerably during folliculogenesis. Taurine and hypotaurine are scavengers that help maintain redox homeosta‐ sis in gametes. Both neutralize lipid peroxidation products, and hypotaurine further neutral‐

Like GSH, the Thioredoxin (Trx) system regulates gene functions and coordinates various enzyme activities. It detoxifies H2O2 and converts it to its reduced state via Trx reductase [45]. Normally, Trx is bound to apoptosis-regulating signal kinase (ASK) 1, rendering it inactive. However, when the thiol group of Trx is oxidized by the SO anion, ASK1 detaches from Trx and becomes active leading to enhanced apoptosis. ASK1 can also be activated by exposure to H2O2 or hypoxiareoxygenation, and inhibited by vitamins C and E. The Trx system also plays a role in female reproduction and fetal development by being involved in cell growth, differentiation, and death. Incorrect protein folding and formation of disulfide bonds can occur

Vitamin E (α-tocopherol) is a lipid soluble vitamin with antioxidant activity. It consists of eight tocopherols and tocotrienols. It plays a major role in antioxidant activities because it reacts with lipid radicals produced during lipid peroxidation [42]. This reaction produces oxidized α-tocopheroxyl radicals that can be transformed back to the active reduced form by reacting

The hormone melatonin is an antioxidant that, unlike vitamins C and E and GSH, is produced by the human body. In contrast to other antioxidants, however, melatonin cannot undergo redox cycling; once it is oxidized, melatonin is unable to return to its reduced state because it

forms stable end-products after the reaction occurs (see below for functions).

ion release from the thiol group of cysteine, leading to disordered protein function,

thermore, CSH can be converted to another antioxidant, hypotaurine [43, 44].

NADPH [17].

54 New Discoveries in Embryology

through H+

izes hydroxyl radicals [44].

aggregation, and apoptosis [2].

with other antioxidants like ascorbate, retinol, or ubiquinol.

It seems that despite their high chemical reactivity most generated ROS do not lead to serious negative physiological consequences for organisms. That is mainly due to the action of highly efficient systems of ROS neutralization operating in concert with reparation and elimination of ROS-modified molecules always exists, that may be called the basal steady-state (stationary) level [37, 50]. Reactive oxygen species can modify most types of biomolecules including proteins, lipids, carbohydrates, nucleic acids, metabolic intermediates, etc. It is widely accepted that the use of only one type of modification to assess oxidative damage during oxidative stress is not sufficient. That is due to the different sensitivity, dynamics, and nature of ROS-promoted modifications. Instead, in order to evaluate the intensity of ROS-involving processes, several approaches for the evaluation of particular oxidatively modified molecules have been selected. They reflect the level of products of interaction between ROS and cellular components of different natures. "Classically", several essential markers are used. They are: (*i*) for lipids – the formation of malonic dialdehyde (MDA), isopsoralens, and lipid peroxides; (*ii*) for proteins – protein carbonyl groups; and (*iii*) for DNA – 8-oxoguanine. Malonic dialde‐ hyde is commonly measured via its reaction with thiobarbituric acid (TBA). However, this reaction is not specific and many other compounds react with TBA under the assay conditions. The array of products formed is collectively called thiobarbituric acid reactive substances (TBARS) to reflect this low specificity. Certain amino acids, carbohydrates, aldehydes and other compounds interfere with the reaction measurement and, therefore, this method should be used with precaution and discussed taking into account the highlighted issues [50]. In the last decade, an HPLC technique was applied to evaluate MDA levels and this method, along with immunochemical identification [51] can now be recommended as more reliable than the TBARS assay. There are also many other approaches to evaluate the intensity of ROS induced lipid peroxidation and the measurement of lipid peroxides [51], 4-hydroxynonenal [52] are just some of them. Selection of methods depends on many things, particularly tools available [33]. Probably the most popular method for detection of ROS-modified proteins is the one based on the formation of additional carbonyl groups with their visualization due to their interaction with 2, 4-dinitrophenylhydrazine [53]. The hydrazones formed are measured spectrophotometrically. Specific antibodies that interact with carbonyl groups on proteins [54] have also been developed. In some cases, there is also the possibility to evaluate the amount of dityrosines and other products of free radical induced oxidation of proteins. Oxidation of nucleic acids also forms an array of products, but in this case there are some favorites that are relatively easy to quantify. These are mainly oxidatively modified guanine derivatives, of which 8-hydroxyguanine (8-OHG) is the most commonly used, but 8-oxo-7, 8- dihydro-2′ deoxyguanosine (8-oxodG) and 8-oxo-7, 8-dihydroguanine (8-oxoGua) can also be measured. Certainly, there are many more different markers of ROS-induced modification of cellular constituents, but those listed here are the most widely used and applied approaches.

### **6. Influence of ROS on reproductive functions**

ROS affect multiple physiological processes in reproduction and fertility, from oocyte matu‐ ration to fertilization, embryo development and pregnancy. Several studies indicate that follicular atresia in mammalian species due to the accumulation of toxic metabolites often results from oxidative stress. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Conversely, increase of ROS levels is primarily responsible for the alteration of macromole‐ cules, such as lipids, proteins and nucleic acids, that lead to significant damage of cell structures and thereby cause oxidative stress. To prevent damage due to ROS, cells possess a number of nonenzymatic and enzymatic antioxidants. Nonenzymatic antioxidant include Vitamin C, glutathione, cysteamine, vitamin E. Enzymatic antioxidants consist of superoxide dismutases (MnSOD and Cu/ZnSOD, which are in the mitochondria and cytosol, respectively), that convert superoxide into hydrogen peroxide; glutathione peroxidase (GPX) and catalase (CAT) which neutralize hydrogen peroxide. Intracellular homeostasis is ensured by the complex interactions between pro-oxidants and antioxidants.

This chapter describes gathering evidence that oxidative stress is involved in ovarian physiopathology caused by diverse stimuli. There is strong evidence that ROS are involved in initiation of apoptosis in antral follicles caused by several chemical and physical agents, in the fluid follicular environment, influencing the folliculogenesis and the steroidogenesis. Al‐ though less attention has been focused on the roles of ROS in primordial and primary follicle death, several studies have shown protective effects of antioxidants and/or evidence of oxidative damage, suggesting that ROS may play a role in these smaller follicles as well. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity. It has been investigated the role of antioxidant enzymes, such as catalase, glutathione peroxidase and the SOD isoforms in maintaining low levels of oxidative stress.

The literature provides some evidence of oxidative stress influencing the entire reproductive cycle. OS plays a role in multiple physiological processes from oocyte maturation to fertiliza‐ tion and embryo development. An increasing number of published studies have pointed towards increased importance of the role of OS in female reproduction. Of course, there is much to learn about this topic, whereby it cannot be underestimated.

### **7. Role of ROS in folliculogenesis, ovulation, and corpus luteum function**

The ROS should not always be coupled with negative effects [56]. Accumulating data have recently shown that reactive oxygen species can regulate cell function by controlling produc‐ tion or the activation of substances that have biological activities.

Numerous genes related to inflammation are induced in preovulatory follicles by the LH surge. The analogy of ovulation with an acute inflammation may suggest a role for ROS along this process. Because ROS are massively generated during the inflammatory process hypothesized that ROS could be involved in the signaling cascade leading to ovulation. The findings were that H2O2 mimicked the effect of LH, bringing about an extensive mucification/expansion of the follicle-enclosed cumulus–oocyte complexes; impaired progesterone production was observed in isolated follicles incubated with LH in the presence of antioxidant agents; furthermore, LH-stimulated up-regulation of genes, the expression of which is crucial for ovulation, was substantially attenuated upon ROS ablation. Together, these results provide evidence that ovarian production of ROS is an essential for preovulatory signaling events, most probably transiently triggered by LH [56].

**6. Influence of ROS on reproductive functions**

56 New Discoveries in Embryology

interactions between pro-oxidants and antioxidants.

in maintaining low levels of oxidative stress.

ROS affect multiple physiological processes in reproduction and fertility, from oocyte matu‐ ration to fertilization, embryo development and pregnancy. Several studies indicate that follicular atresia in mammalian species due to the accumulation of toxic metabolites often results from oxidative stress. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Conversely, increase of ROS levels is primarily responsible for the alteration of macromole‐ cules, such as lipids, proteins and nucleic acids, that lead to significant damage of cell structures and thereby cause oxidative stress. To prevent damage due to ROS, cells possess a number of nonenzymatic and enzymatic antioxidants. Nonenzymatic antioxidant include Vitamin C, glutathione, cysteamine, vitamin E. Enzymatic antioxidants consist of superoxide dismutases (MnSOD and Cu/ZnSOD, which are in the mitochondria and cytosol, respectively), that convert superoxide into hydrogen peroxide; glutathione peroxidase (GPX) and catalase (CAT) which neutralize hydrogen peroxide. Intracellular homeostasis is ensured by the complex

This chapter describes gathering evidence that oxidative stress is involved in ovarian physiopathology caused by diverse stimuli. There is strong evidence that ROS are involved in initiation of apoptosis in antral follicles caused by several chemical and physical agents, in the fluid follicular environment, influencing the folliculogenesis and the steroidogenesis. Al‐ though less attention has been focused on the roles of ROS in primordial and primary follicle death, several studies have shown protective effects of antioxidants and/or evidence of oxidative damage, suggesting that ROS may play a role in these smaller follicles as well. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity. It has been investigated the role of antioxidant enzymes, such as catalase, glutathione peroxidase and the SOD isoforms

The literature provides some evidence of oxidative stress influencing the entire reproductive cycle. OS plays a role in multiple physiological processes from oocyte maturation to fertiliza‐ tion and embryo development. An increasing number of published studies have pointed towards increased importance of the role of OS in female reproduction. Of course, there is

**7. Role of ROS in folliculogenesis, ovulation, and corpus luteum function**

The ROS should not always be coupled with negative effects [56]. Accumulating data have recently shown that reactive oxygen species can regulate cell function by controlling produc‐

much to learn about this topic, whereby it cannot be underestimated.

tion or the activation of substances that have biological activities.

The increase in steroid production in the growing follicle causes an increase in P450, resulting in ROS formation. Reactive oxygen species produced by the pre-ovulatory follicle are consid‐ ered important inducers for ovulation. Oxygen deprivation stimulates follicular angiogenesis, which is important for adequate growth and development of the ovarian follicle. Follicular ROS promotes apoptosis, whereas GSH and follicular stimulating hormone (FSH) counter‐ balance this action in the growing follicle. Estrogen increases in response to FSH, triggering the generation of catalase in the dominant follicle, and thus avoiding apoptosis [26].

In ovaries, the corpus luteum is formed after ovulation and produces progesterone, which is necessary for the establishment and maintenance of pregnancy. When pregnancy occurs, the rescue of the corpus luteum and subsequent progesterone production are important for the maintenance of pregnancy. In contrast, when pregnancy does not occur after ovulation, the decline of progesterone production is important for the follicle development of the next reproductive cycle. The chance of conception occurring as soon as possible and as often as possible depends on how rapidly progesterone production declines. Therefore, the strategy for reproduction in the ovary is the rapid rescue of the corpus luteum when pregnancy occurs, and the rapid termination of the corpus luteum function when pregnancy does not occur after ovulation. Corpus luteum regression is defined as that the corpus luteum declines in function, decreases in volume, and thereafter disappears from the ovary. Corpus luteum regression consists of two stages of regression, functional luteolysis and structural luteolysis. Structural luteolysis is defined as structural involution of the corpus luteum, and is clearly distinguished from functional luteolysis which is characterized by depletion of progesterone production without structural changes such as loss of luteal cells and blood vessels. Rapid decline in progesterone production is important for follicle growth in the next reproductive cycle. It is therefore of interest to study the mechanism of functional luteolysis. ROS and SOD are involved in functional luteolysis. ROS are produced in the corpus luteum [26]. There are several potential sources of ROS in the corpus luteum. Macrophages and neutrophils, that are clear sources of reactive oxygen species, are well documented as residing in the corpus luteum [57-61] The increase in ROS in the corpus luteum is involved in functional luteolysis. The decrease in Cu, Zn-SOD expression could be one of the causes for the increase in reactive oxygen species in the regressing corpus luteum. It seems there is another possible mechanism able to increase ROS. PGF2α has been well recognized as a luteolysin since it increases in the corpus luteum during the regression phase [62] and inhibits the production of progesterone by luteal cells. A number of reports have shown so far that the inhibitory effect of PGF2α on progesterone production by the corpus luteum is, in part, mediated through the increase of ROS [63, 64]. ROS can activate phospholipase A2 activity and cyclooxygenase-2 expression in the corpus luteum which are key enzymes for PGF2α synthesis. Thus, there seems to be a close interrelation between PGF2α and ROS [65, 66].

Steroidogenic cells are also potential sources of reactive oxygen species because reactive oxygen species are generated as byproducts of normal metabolism. Intracellular sources of ROS include mitochondrial electron transport, endoplasmic reticulum, nuclear membrane electron transport systems and plasma membranes [67]. There is a significant co-relationship between Cu, Zn-SOD activities and serum progesterone concentrations. In contrast, lipid peroxide levels increase in the corpus luteum during the regression phase in the both rat models and show an opposite change from serum progesterone concentrations [68, 69]. Reactive oxygen species generated normally during steroidogenesis restrict the capacity of the corpus luteum to produce progesterone [70]. In pregnancy, the decrease in Cu, Zn-SOD expression causes the inhibition of progesterone production via the increase in ROS. Therefore, the increase in ability to scavenge ROS may be associated with the maintenance of luteal cell integrity and prolonged life span of the corpus luteum [71]. In other animals, such bovines, SOD and CAT have been reported to be correlated with progesterone production by the corpus luteum [72] It is plausible that the luteotropic substances, usually synthesized by placenta during pregnancy, stimulate the expression of molecules that protect luteal cells from ROS. Finally, the increase in Cu, Zn-SOD by placental luteotropins is an important mechanism to rescue the corpus luteum and maintain progesterone production [73].

Aerobic metabolism utilizing oxygen is essential for energy requirements of the gametes, and the free radicals play a significant role in physiological processes within the ovary. Many studies have demonstrated involvement of ROS in the follicular-fluid environment, folliculo‐ genesis, and steroidogenesis [74]. The immunohistochemical distribution of the copper-zinc superoxide dismutase (Cu, Zn-SOD) in the human ovary was given by [74]. They found, for the first time, that the gestational corpus luteum, theca and granulosa lutein cells showed intensive and moderate staining activity, respectively, to Cu, Zn-SOD. Furthermore, they suggested that, as SOD catalyses the dismutation reaction of superoxide anion radicals, the theca interna cells play an important role in the protection of the developing oocyte from oxygen radicals by acting as a blood-follicular barrier during follicle maturation, [76] under‐ lined the presence of manganese superoxide dismutase (Mn-SOD) and Cu, Zn-SOD in human ovaries and fallopian tubes, with different localizations and actions. The superoxide radical-SOD system might play an important role in ovulation and in the luteal function of the human ovary in the human ovary and fallopian tube, and to examine the role of superoxide radicals and SODs in the human ovulatory process. These enzymes can be considered as markers of cytoplasmic maturation [77].

Culture of small and large (preovulatory) antral rat follicles without gonadotropin support leads to apoptotic death within 24 h, while FSH suppresses apoptosis [78].To investigate if oxidative stress plays a role in granulosa cell apoptosis during follicular atresia in the immature rat ovary, healthy antral follicles obtained from rats were in the absence or presence of FSH, SOD, ascorbic acid (a free radical scavenger), N-acetyl-L-cysteine (a free radical scavenger and stimulator of endogenous glutathione peroxidase activity), or CAT. The results showed that each antioxidant was able to protect against apoptosis in rat large antral follicles cultured without gonadotropin support [79].

able to increase ROS. PGF2α has been well recognized as a luteolysin since it increases in the corpus luteum during the regression phase [62] and inhibits the production of progesterone by luteal cells. A number of reports have shown so far that the inhibitory effect of PGF2α on progesterone production by the corpus luteum is, in part, mediated through the increase of ROS [63, 64]. ROS can activate phospholipase A2 activity and cyclooxygenase-2 expression in the corpus luteum which are key enzymes for PGF2α synthesis. Thus, there seems to be a close

Steroidogenic cells are also potential sources of reactive oxygen species because reactive oxygen species are generated as byproducts of normal metabolism. Intracellular sources of ROS include mitochondrial electron transport, endoplasmic reticulum, nuclear membrane electron transport systems and plasma membranes [67]. There is a significant co-relationship between Cu, Zn-SOD activities and serum progesterone concentrations. In contrast, lipid peroxide levels increase in the corpus luteum during the regression phase in the both rat models and show an opposite change from serum progesterone concentrations [68, 69]. Reactive oxygen species generated normally during steroidogenesis restrict the capacity of the corpus luteum to produce progesterone [70]. In pregnancy, the decrease in Cu, Zn-SOD expression causes the inhibition of progesterone production via the increase in ROS. Therefore, the increase in ability to scavenge ROS may be associated with the maintenance of luteal cell integrity and prolonged life span of the corpus luteum [71]. In other animals, such bovines, SOD and CAT have been reported to be correlated with progesterone production by the corpus luteum [72] It is plausible that the luteotropic substances, usually synthesized by placenta during pregnancy, stimulate the expression of molecules that protect luteal cells from ROS. Finally, the increase in Cu, Zn-SOD by placental luteotropins is an important mechanism to

Aerobic metabolism utilizing oxygen is essential for energy requirements of the gametes, and the free radicals play a significant role in physiological processes within the ovary. Many studies have demonstrated involvement of ROS in the follicular-fluid environment, folliculo‐ genesis, and steroidogenesis [74]. The immunohistochemical distribution of the copper-zinc superoxide dismutase (Cu, Zn-SOD) in the human ovary was given by [74]. They found, for the first time, that the gestational corpus luteum, theca and granulosa lutein cells showed intensive and moderate staining activity, respectively, to Cu, Zn-SOD. Furthermore, they suggested that, as SOD catalyses the dismutation reaction of superoxide anion radicals, the theca interna cells play an important role in the protection of the developing oocyte from oxygen radicals by acting as a blood-follicular barrier during follicle maturation, [76] under‐ lined the presence of manganese superoxide dismutase (Mn-SOD) and Cu, Zn-SOD in human ovaries and fallopian tubes, with different localizations and actions. The superoxide radical-SOD system might play an important role in ovulation and in the luteal function of the human ovary in the human ovary and fallopian tube, and to examine the role of superoxide radicals and SODs in the human ovulatory process. These enzymes can be considered as markers of

rescue the corpus luteum and maintain progesterone production [73].

cytoplasmic maturation [77].

interrelation between PGF2α and ROS [65, 66].

58 New Discoveries in Embryology

Markers of peroxidation were measured in follicular fluids and sera of women attending an in vitro fertilization (IVF), to assess the pro or anti oxidative status and the effects of the administration of antioxidants. The substances in follicular fluid were all significantly lower than those in serum, both in the presence or absence of antioxidants. In conclusion, the intensity of peroxidation in the Graafian follicle is much lower than that in serum. This gradient is the result of the lower rate of initiation of peroxidation in the follicular fluid due to, probably, the presence of efficient antioxidant defense systems in the direct milieu of the oocyte before ovulation [80].

The role of ROS and antioxidant enzymes was provided using immunohistochemical locali‐ zation, mRNA expression, and thiobarbituric acid methods that suggested a complex role in ovulation and luteal function in the human ovary [80]. Oxidative stress has been shown to affect the midluteal corpus luteum and steroidogenic capacity both in vitro and in vivo. In a very interesting study, using corpora lutea collected from pregnant and nonpregnant patients, it was observed that during normal situations, Zn-SOD expression parallels the levels of progesterone, with a rise from early luteal to midluteal phase and decrease during regression of the corpus luteum. The mRNA expression, however, of Cu, Zn-SOD in the corpus luteum during pregnancy was much higher than those of midcycle corpora lutea. This factor enhanced SOD expression during pregnancy, possibly caused by increased human chorionic gonado‐ tropin (HCG) levels, and may be the cause of apoptosis of the corpora lutea. Similarly, the antioxidant enzymes glutathione peroxidase and MnSOD are considered the markers for cytoplasmic maturation, as these are expressed only in metaphase II oocytes [6]. Decreased developmental potential of oocytes from poorly vascularized follicles has also been attributed to low intrafollicular oxygenation [8]. Studies demonstrate intensified lipid peroxidation in the preovulatory Graafian follicle and that glutathione peroxidase may help in maintaining low levels of hydroperoxides inside follicle, suggesting an important role of oxidative stress in ovarian function. Oxidative stress and inflammatory process have roles in the pathophysi‐ ology of polycystic ovarian disease and drugs such as Rosiglitazone maybe effective by decreasing the levels of oxidative stress [81].

Two groups have developed Cu, Zn-SOD null mice, and both groups reported that the female mice were subfertile; however, the mechanistic basis for the reduced fertility of female Cu, Zn-SOD null mice remains unclear. [82] reported that ovaries of adult female Cu, Zn-SOD null mice had reduced numbers of preovulatory follicles and corpora lutea. They concluded that these mice were subfertile because of a defect in late follicular development or ovulation. In contrast, [83] reported that Mn-ZnSOD null female mice had normal ovarian histology and ovulated similar numbers of ova during a natural estrous cycle but displayed increased postimplantation embryonic lethality. Perhaps the different genetic backgrounds of these two Cu, Zn-SOD knockout models accounts for these different findings. A study by [84] on copper chaperone for superoxide dismutase null mice, which have decreased ability to incorporate copper into Mn-ZnSOD, found a similar phenotype as [85], with abnormal development of antral follicles and no corpora lutea. Taken together, the evidence seems to support a role for Cu-ZnSOD in antral follicle development. Cu, Zn-SOD knockout is lethal prior to puberty. However, transplantation of ovaries from Mn-SOD knockout juvenile mice to the ovarian bursa of wild-type mice, in which the ipsilateral ovaries had been removed and the contrala‐ teral oviducts had been cut, resulted in all stages of follicular development, ovulation, and fertility, suggesting that this enzyme is not critical for ovarian function.

Superoxide, hydrogen peroxide and lipid peroxides are generated in luteal tissue during natural and prostaglandin-induced regression in the rat, and this response is associated with reversible depletion of ascorbic acid. ROS immediately uncouple the luteinizing hormone receptor from adenylate cyclase and inhibit steroidogenesis by interrupting transmitochon‐ drial cholesterol transport. The cellular origin of oxygen radicals in regressing corpora lutea is predominantly from resident and infiltrated leukocytes, especially neutrophils. ROS are also produced within the follicle at ovulation and, as the corpus luteum, leukocytes are the major source of these products. Antioxidants block the resumption of meiosis, whereas the genera‐ tion of reactive oxygen induces oocyte maturation in the follicle. Although oxygen radicals may serve important physiologic roles within the ovary, the cyclic production of these damaging agents over years may lead to an increased cumulative risk of ovarian pathology that would probably be exacerbated under conditions of reduced antioxidant status [87].

Melatonin appears to have some kinds of functions at different stages of follicle development, oocyte maturation, and luteal stage. Melatonin concentration in the growing follicle may be an important factor in avoiding atresia, because melatonin in the follicular fluid reduces apoptosis of critical cells. Melatonin also has protective actions during oocyte maturation reducing intrafollicular oxidative damage. An association between melatonin concentrations in follicular fluid and oocyte quality has been reported In the ovarian follicle, melatonin impacts the function of numerous cells, especially granulosa cells and the ovum (oocyte). The actions of melatonin in these cells are mediated via membrane receptors and also possibly via binding sites in the nucleus and in the cytosol. In addition to its receptor-mediated actions, melatonin also functions as a direct free radical scavenger to reduce oxidative stress at the level of the ovary; this beneficial action is carried out without an interaction with a receptor. Additional antioxidant functions of melatonin are achieved when the indole stimulates enzymes which metabolize free radicals to less toxic products. The antioxidative enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in thecal cells, granulosa cells and in the follicular fluid. Via these actions, melatonin reduces free radical damage, which would be especially bad for the ovum, and maintains these elements in an optimally functional state. The origin of melatonin in the follicular fluid is the blood and from its local synthesis in granulosa cells [87-89].

### **8. Assisted Reproductive Techniques (ART) and ROS**

Assisted reproductive techniques (ART) are advanced technological procedures, which are the treatments of choice in many cases of female and male infertility or assisted fertilization, included the use of medical techniques, such as drug therapy, artificial insemination, or in vitro fertilization, to enhance fertility. Expanded ART include any directed action taken by humans to enhance reproduction in animals, both through 1) Assisted reproduction with a technical component (mostly mammals), 2) assisted reproduction using various forms of population management. The two are not mutually exclusive.

ART include:

ovulated similar numbers of ova during a natural estrous cycle but displayed increased postimplantation embryonic lethality. Perhaps the different genetic backgrounds of these two Cu, Zn-SOD knockout models accounts for these different findings. A study by [84] on copper chaperone for superoxide dismutase null mice, which have decreased ability to incorporate copper into Mn-ZnSOD, found a similar phenotype as [85], with abnormal development of antral follicles and no corpora lutea. Taken together, the evidence seems to support a role for Cu-ZnSOD in antral follicle development. Cu, Zn-SOD knockout is lethal prior to puberty. However, transplantation of ovaries from Mn-SOD knockout juvenile mice to the ovarian bursa of wild-type mice, in which the ipsilateral ovaries had been removed and the contrala‐ teral oviducts had been cut, resulted in all stages of follicular development, ovulation, and

Superoxide, hydrogen peroxide and lipid peroxides are generated in luteal tissue during natural and prostaglandin-induced regression in the rat, and this response is associated with reversible depletion of ascorbic acid. ROS immediately uncouple the luteinizing hormone receptor from adenylate cyclase and inhibit steroidogenesis by interrupting transmitochon‐ drial cholesterol transport. The cellular origin of oxygen radicals in regressing corpora lutea is predominantly from resident and infiltrated leukocytes, especially neutrophils. ROS are also produced within the follicle at ovulation and, as the corpus luteum, leukocytes are the major source of these products. Antioxidants block the resumption of meiosis, whereas the genera‐ tion of reactive oxygen induces oocyte maturation in the follicle. Although oxygen radicals may serve important physiologic roles within the ovary, the cyclic production of these damaging agents over years may lead to an increased cumulative risk of ovarian pathology that would probably be exacerbated under conditions of reduced antioxidant status [87].

Melatonin appears to have some kinds of functions at different stages of follicle development, oocyte maturation, and luteal stage. Melatonin concentration in the growing follicle may be an important factor in avoiding atresia, because melatonin in the follicular fluid reduces apoptosis of critical cells. Melatonin also has protective actions during oocyte maturation reducing intrafollicular oxidative damage. An association between melatonin concentrations in follicular fluid and oocyte quality has been reported In the ovarian follicle, melatonin impacts the function of numerous cells, especially granulosa cells and the ovum (oocyte). The actions of melatonin in these cells are mediated via membrane receptors and also possibly via binding sites in the nucleus and in the cytosol. In addition to its receptor-mediated actions, melatonin also functions as a direct free radical scavenger to reduce oxidative stress at the level of the ovary; this beneficial action is carried out without an interaction with a receptor. Additional antioxidant functions of melatonin are achieved when the indole stimulates enzymes which metabolize free radicals to less toxic products. The antioxidative enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in thecal cells, granulosa cells and in the follicular fluid. Via these actions, melatonin reduces free radical damage, which would be especially bad for the ovum, and maintains these elements in an optimally functional state. The origin of melatonin in the follicular fluid is the blood and

fertility, suggesting that this enzyme is not critical for ovarian function.

60 New Discoveries in Embryology

from its local synthesis in granulosa cells [87-89].


They function, in humans, as an alternative to overcome causative factors of infertility, such as endometriosis, tubal factor infertility, male factor infertility. They can be used in the veterinary field also [90]. ART, in fact, were recently accepted into the programs for the safeguard of endangered species from extinction [90-93]. In a feasible program it is necessary proceed in the following five steps: 1) Technique development in a domestic animal counter‐ part, if available; 2) characterization of species-specific reproductive biology in a targeted nondomestic animal; 3) assessment of technique feasibility for producing offspring; 4) demonstration of adequate efficiency for applied usage; 5) application of new tool for popu‐ lation management [90] Figs 4, 5, and 6 show cumulus oocyte complexes (COCs) from mare explanted ovaries: these tools are employed in ART to have genetic improvement in horses,

Oxidative stress is involved in ovarian physio-pathology caused by diverse stimuli caused by several chemical and physical agents: ROS are involved in initiation of apoptosis in antral follicles in the fluid follicular environment, influencing the folliculogenesis and the steroido‐ genesis. ROS may play a role in these smaller follicles as well. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity. It has been investigated the role of antioxidant enzymes, such as catalase, glutathione peroxidase and the SOD isoforms in maintaining low levels of oxidative stress [46]. It was demonstrated for the first time by [94] that high oxygen concen‐ tration compromises nuclear maturation rates and worsens the oxidative stress during in vitro maturation (IVM) of canine oocytes.

Incubated oocytes showed severely high quantities of superoxide dismutase (SOD), gluta‐ thione reductase (GSR), glutathione peroxidase (GPX1) and catalase (CAT) mRNA and this effect results in a protective mechanism against oxidative stress [95].

[45] studied the effect of ovary transport media supplementation with SOD on ovarian cell viability and apoptosis and in vitro embryo production (IVEP). They proposed, as mechanism of action, the intervention of SOD in inactivating the atmospheric O2, potential deleterious precursor of free radicals.

With IVF, sperm-oocyte interaction occurs in culture media, leading to fertilization [32]. Reactive oxygen species may develop as a consequence of increased oocyte number per dish, spermatozoa, and cumulus cell mass. Cumulus cells demonstrate higher antioxidant activity at the beginning of culture than denuded oocytes do [96]. In ICSI, a single sperm is injected into an oocyte's cytoplasm [142]. It bypasses natural selection, thus allowing for the injection of damaged spermatozoon into the oocyte. Alternatively, the IVF process prevents fertilization by DNA-damaged spermatozoa [97].

Recently, OS has been identified as an important factor in ART success. Oocyte metabolism and a lack of antioxidants combined with the follicular and oviductal fluid of the embryo causes an increase in ROS levels [384]. Follicular fluid is the net result of both the transfer of plasma constituents to follicles and the secretory activity of granulose and theca cells [385]. The oocyte develops within the FF environment and this intimately affects the quality of oocytes and their interaction with sperm, thus affecting implantation and embryonic devel‐ opment [98]. Oxidative stress contributes to oocyte quality, and its degree can be assessed by biomarkers of lipid peroxidation [99]. The effects of OS may be may be further altered by environmental factors. A hyperoxic environment augments SO radical levels by promoting enzyme activity. Particularly in IVF, increased incubation time heightens exposure to O2 concentration [100]. As in biological systems, metallic cations act as exogenous sources of OS by stimulating ROS formation in ART culture media, and metal chelators such as EDTA and transferrin can ameliorate the production of ROS [43]. Furthermore, visible light can cause ROS formation, thereby damaging DNA [101]. Fertilization success in ART is determined by the quality of spermatozoa involved [32]. Although ROS contribute to normal sperm functions such as oocyte fusion, capacitation, and acrosome reaction, OS produced by spermatozoa may provoke oxidative damage to the oocyte, decreasing the likelihood for fertilization [81].

The in vitro environment exposes gametes and embryos to an excess of ROS with the absence of enzymatic antioxidant protection normally present during in vivo fertilization and preg‐ nancy. Free radicals are thought to act as determinants in reproductive outcomes

due to their effects on oocytes, sperm, and embryos [95]. Oxidative stress disturbs human oocyte intracellular Ca2+ homeostasis as well as oocyte maturation and fertilization. During ovulation, ROS are produced within the follicles, however, the excessive production of ROS may increase the risk for poor oocyte quality since oxidative stimulation promotes oocyte maturation and wall rupture within the follicle [390]. A physiologic amount of ROS in follicular fluid is indicative of a healthy developing oocyte [102]. In vitro fertilization can disturb the oxidant-antioxidant balance, rendering the culture media less protected against oxidation. The adverse effects of sustained OS and resulting loss of oocyte antioxidant content were shown to be improved by adding lipophilic and hydrosoluble antioxidants to the culture media to lessen OS [103]. Oral vitamin and mineral supplementation have been shown to increase serum concentrations of GSH and vitamins C and E; these antioxidants have been suggested to play a significant role in IVF outcomes [104].

tration compromises nuclear maturation rates and worsens the oxidative stress during in vitro

Incubated oocytes showed severely high quantities of superoxide dismutase (SOD), gluta‐ thione reductase (GSR), glutathione peroxidase (GPX1) and catalase (CAT) mRNA and this

[45] studied the effect of ovary transport media supplementation with SOD on ovarian cell viability and apoptosis and in vitro embryo production (IVEP). They proposed, as mechanism of action, the intervention of SOD in inactivating the atmospheric O2, potential deleterious

With IVF, sperm-oocyte interaction occurs in culture media, leading to fertilization [32]. Reactive oxygen species may develop as a consequence of increased oocyte number per dish, spermatozoa, and cumulus cell mass. Cumulus cells demonstrate higher antioxidant activity at the beginning of culture than denuded oocytes do [96]. In ICSI, a single sperm is injected into an oocyte's cytoplasm [142]. It bypasses natural selection, thus allowing for the injection of damaged spermatozoon into the oocyte. Alternatively, the IVF process prevents fertilization

Recently, OS has been identified as an important factor in ART success. Oocyte metabolism and a lack of antioxidants combined with the follicular and oviductal fluid of the embryo causes an increase in ROS levels [384]. Follicular fluid is the net result of both the transfer of plasma constituents to follicles and the secretory activity of granulose and theca cells [385]. The oocyte develops within the FF environment and this intimately affects the quality of oocytes and their interaction with sperm, thus affecting implantation and embryonic devel‐ opment [98]. Oxidative stress contributes to oocyte quality, and its degree can be assessed by biomarkers of lipid peroxidation [99]. The effects of OS may be may be further altered by environmental factors. A hyperoxic environment augments SO radical levels by promoting enzyme activity. Particularly in IVF, increased incubation time heightens exposure to O2 concentration [100]. As in biological systems, metallic cations act as exogenous sources of OS by stimulating ROS formation in ART culture media, and metal chelators such as EDTA and transferrin can ameliorate the production of ROS [43]. Furthermore, visible light can cause ROS formation, thereby damaging DNA [101]. Fertilization success in ART is determined by the quality of spermatozoa involved [32]. Although ROS contribute to normal sperm functions such as oocyte fusion, capacitation, and acrosome reaction, OS produced by spermatozoa may provoke oxidative damage to the oocyte, decreasing the likelihood for fertilization [81].

The in vitro environment exposes gametes and embryos to an excess of ROS with the absence of enzymatic antioxidant protection normally present during in vivo fertilization and preg‐

due to their effects on oocytes, sperm, and embryos [95]. Oxidative stress disturbs human oocyte intracellular Ca2+ homeostasis as well as oocyte maturation and fertilization. During ovulation, ROS are produced within the follicles, however, the excessive production of ROS may increase the risk for poor oocyte quality since oxidative stimulation promotes oocyte maturation and wall rupture within the follicle [390]. A physiologic amount of ROS in follicular

nancy. Free radicals are thought to act as determinants in reproductive outcomes

effect results in a protective mechanism against oxidative stress [95].

maturation (IVM) of canine oocytes.

by DNA-damaged spermatozoa [97].

precursor of free radicals.

62 New Discoveries in Embryology

Much research on IVEP has focused on the damaging effects of an oxidative environment and the inherent creation of reactive oxygen species that may impair embryo development. There are evidences that endoplasmic reticulum (RE) is significantly less reducing, consequently, excessive supplementation of reducing agents in media to offset oxidative damage has resulted in controversial outcomes as slight redox imbalances are detrimental for embryo development [73]. Conversely, an excess of ROS produced without sufficient antioxidant protection may lead to disequilibrium of the redox balance versus oxidative stress characterized by damaging DNA, RNA, protein and lipids [74]. Studies have been performed under high and low oxygen tension conditions and have resulted in controversial findings. Studies using antioxidants on swine model, indicated that the effect of the combination of GSH, β-ME and cysteine on embryo development. Treatment groups had a greater number of developing embryos than the control and the favorable result depended on the high O2 culture conditions were used [105].

In contrast, guaiazulene (a component of various chamomile species with antioxidant prop‐ erties) had no positive effect on embryo development under low oxygen tension (5 % O2) [106]. Furthermore, [94] found that low oxygen gas composition improves nuclear maturation rates and alleviates the oxidative stress for canine oocytes during in vitro maturation.

**Figure 4.** Cumulus Oocyte Complexes (COCs) of Pre Antral Follicle from explanted mare ovaries. Ooplama bipolarisa‐ tion with a dark and a clear portion (ptical microscope, 100x)

**Figure 5.** Cumulus Oocyte Complexes (COCs) of Pre Antral Follicle from explanted mare ovaries. COC stained with 5 carboxyfluorescein diacetate (cFDA) and trypan blue (with unviable cumulus cells and viable oocyte) (optical micro‐ scope 200x).

**Figure 6.** Cumulus Oocyte Complexes (COCs) of Pre Antral Follicle from explanted mare ovaries. Viable COC stained with 5-carboxyfluorescein diacetate (cFDA) and trypan blue (optical microscope 200x)

### **9. Conclusions**

Oxidative stress has been extensively studied for about four decades. Substantial progress has been achieved to date – from descriptive characterization of this process to delineation of molecular mechanisms underlining adaptive responses and targeted manipulations of expected responses. In recent years, the importance of ROS synthesis in ovarian functions has been established also. Several data have recently shown that reactive oxygen species can regulate cell function by controlling production or the activation of substances that have biological activities. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Converse‐ ly, increase of ROS levels is primarily responsible for the alteration of macromolecules, such as lipids, proteins and nucleic acids, that lead to significant damage of cell structures and thereby cause oxidative stress. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality after early life exposure to several toxicants. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity. To prevent damage due to ROS, cells possess a number of nonenzymatic and enzymatic antioxidants. that include Vitamin C, glutathione, cysteamine, vitamin E, superoxide dismutases (SOD1, SOD2, and SOD3), glutathione peroxidase, and catalase. Intracellular homeostasis is ensured by the complex interactions between pro-oxidants and antioxidants. The bulk of evidence in support of therapeutic effects of antioxidants to date, has been observed through experimental studies on animals and humans ART, whose aim is depth knowledge of human reproductive functions, conservation of species in danger of extinction, and acceleration of life cycles using reproduc‐ tion for purposes of genetic and productive.

In the future, the hope is to clarify the efficacy of antioxidants as potential therapies for infertility and in ART the use of specific antioxidants to improve multiple physiological processes from oocyte maturation to fertilization, embryo development and pregnancy.

### **Author details**

**Figure 5.** Cumulus Oocyte Complexes (COCs) of Pre Antral Follicle from explanted mare ovaries. COC stained with 5 carboxyfluorescein diacetate (cFDA) and trypan blue (with unviable cumulus cells and viable oocyte) (optical micro‐

**Figure 6.** Cumulus Oocyte Complexes (COCs) of Pre Antral Follicle from explanted mare ovaries. Viable COC stained

Oxidative stress has been extensively studied for about four decades. Substantial progress has been achieved to date – from descriptive characterization of this process to delineation of

with 5-carboxyfluorescein diacetate (cFDA) and trypan blue (optical microscope 200x)

scope 200x).

64 New Discoveries in Embryology

**9. Conclusions**

Francesca Ciani\* , Natascia Cocchia, Danila d'Angelo and Simona Tafuri

\*Address all correspondence to: ciani@unina.it

Department of Veterinary Medicine and Animal Productions – University of Naples Federi‐ co II, Naples, Italy

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## **Embryo Implantation**
