Preface

We are pleased to present our new book *Reproductive Biology and Technology in Animals*. This book has been designed to provide innovative information on new reproductive technologies in different animal species, including sheep, gilts, pigs, and other invertebrates. It is intended for academics, scientists, and veterinarians to update and deepen their knowledge of the physiology of reproduction. Therefore, the motivation to publish a comprehensive volume on advances in biotechnology arose from the growing awareness of recent advances in sperm management, oocyte characteristics, various genomic aspects related to somatic cell nuclear transfer, and the microarchitecture of the female reproductive system in these animal species. Other aspects have also been considered that refer to the reproductive cycles present in gastropods and fish. This volume is the culmination of the efforts of several researchers, scientists, and scholars from around the world who are well known and respected in the various frontiers of research into the physiology of reproduction. We sincerely believe that the book will prove to be a useful contribution not only to science but also to the general public. We hope that this book will be a useful source of information from both a scientific and practical point of view. We are grateful to all the contributing authors and to all the members of the international review panel who helped us enormously with their contributions, time, critical thinking, and suggestions in producing this edited and peer-reviewed volume. The editors are also grateful to IntechOpen and its team members for the opportunity to publish this book. Finally, we thank our family members for their love, support, encouragement, and patience throughout the period of this work.

**II**

**Chapter 7 113**

*by Verónica Mitsui Saito-Quezada, Esther Uría-Galicia, José Luis Gómez-Márquez,*

Reproductive Cycle of *Hexaplex princeps* (Broderip, 1833)

*and Isaías Hazarmabeth Salgado-Ugarte*

*Ana Bertha Villaseñor-Martínez, Ma. De Lourdes Jiménez-Badillo*

#### **Juan Carlos Gardon Poggi**

Department of Animal Medicine and Surgery, University Catholic of Valencia "San Vicente Mártir", Valencia, Spain

#### **Katy Satué Ambrojo**

Faculty of Veterinary, Department of Animal Medicine and Surgery, University of CEU-Cardenal Herrera, Valencia, Spain

Section 1

Mammals

**1**

Section 1 Mammals

**Chapter 1**

**Abstract**

**1. Introduction**

until birth [1].

**3**

**2. Microstructure of cortex of the ovary**

Micro-architecture of the Female

The female reproductive system consists of the ovary, oviduct, uterus, and vagina. This chapter will discuss how these organs look like under the microscope and what types of ultrastructural tissues are present in it, how the shape and physiology of the tissues/cells change with the physiological activities including reproductive cycles, what type of alterations occurs in the ovary during ovulation and how its follicle and epithelium differ, and how the ovulation takes place. The chapter will also elaborate how the lining epithelium and the tract mucosa facilitate the fertilized ovum and conceptus. Also, the chapter is highlighting the architectural changes within the mucosa of the uterus during and after pregnancy and type

**Keywords:** ovary, reproduction, microstructures, cortex, mucosa, zygote, sperm

the follicles especially the graafian follicle including mature ova, and the

It is important to know the microstructure of the female reproductive system. In this chapter those microarchitectures are highlighted which played an important role in theriogenology right from release to fertilization and care of embryo and infants. The micro-architecture of the ovarian cortex, the microscopic structures of

histomorphology of the fimbriae and oviduct and its function in ova transportation, zygote transformation, and embryo implantation were highlighted. The microstructures of various microscopic layers of the uterus and its role in reproduction were addressed. The structure and function of the cervix and vulva and its role in reproduction are mentioned. Also, the most suitable type of ova and sperm for fertilization was also mentioned. The whole reproductive system is concerned with the production and transport of the ovum, facilitation in the transport of spermatozoa, the fusion of both gametes, and the accommodation of the embryo and fetus

The cortex is a wide peripheral part of the ovary that contains follicles in various stages [2]. The surface of the ovary is covered by a thin layer of cuboidal cells, also called as germinal epithelium. Next to the germinal epithelium in the inferior side is

a thick layer of connective tissue (CT) called the tunica albuginea. And the

of ovary and spermatozoa that is most suitable for fertilization.

Reproductive System

*Arbab Sikandar and Muhammad Ali*

#### **Chapter 1**

## Micro-architecture of the Female Reproductive System

*Arbab Sikandar and Muhammad Ali*

#### **Abstract**

The female reproductive system consists of the ovary, oviduct, uterus, and vagina. This chapter will discuss how these organs look like under the microscope and what types of ultrastructural tissues are present in it, how the shape and physiology of the tissues/cells change with the physiological activities including reproductive cycles, what type of alterations occurs in the ovary during ovulation and how its follicle and epithelium differ, and how the ovulation takes place. The chapter will also elaborate how the lining epithelium and the tract mucosa facilitate the fertilized ovum and conceptus. Also, the chapter is highlighting the architectural changes within the mucosa of the uterus during and after pregnancy and type of ovary and spermatozoa that is most suitable for fertilization.

**Keywords:** ovary, reproduction, microstructures, cortex, mucosa, zygote, sperm

#### **1. Introduction**

It is important to know the microstructure of the female reproductive system. In this chapter those microarchitectures are highlighted which played an important role in theriogenology right from release to fertilization and care of embryo and infants. The micro-architecture of the ovarian cortex, the microscopic structures of the follicles especially the graafian follicle including mature ova, and the histomorphology of the fimbriae and oviduct and its function in ova transportation, zygote transformation, and embryo implantation were highlighted. The microstructures of various microscopic layers of the uterus and its role in reproduction were addressed. The structure and function of the cervix and vulva and its role in reproduction are mentioned. Also, the most suitable type of ova and sperm for fertilization was also mentioned. The whole reproductive system is concerned with the production and transport of the ovum, facilitation in the transport of spermatozoa, the fusion of both gametes, and the accommodation of the embryo and fetus until birth [1].

#### **2. Microstructure of cortex of the ovary**

The cortex is a wide peripheral part of the ovary that contains follicles in various stages [2]. The surface of the ovary is covered by a thin layer of cuboidal cells, also called as germinal epithelium. Next to the germinal epithelium in the inferior side is a thick layer of connective tissue (CT) called the tunica albuginea. And the

remaining portion of the ovarian cortex is covered by the CT which contains the primordial follicles, surrounded by a flat follicular cell (squamous shaped). Another follicle a bit larger is called a primary follicle, internally lined by the simple and stratified cuboidal epithelium termed as granulosa cells. In the connective tissue of the cortex of the ovary, many blood vessels surround the developing follicles. The granulosa cells are more prominent in the larger follicle called as secondary follicle or also called as antral follicle [3]. The antrum is the cavity formation within the follicle, which splits the granulosa cells into two layers. The fluid within the antrum is called as liquor folliculi. A single large ovum is present in the follicle, and a single layer of granulosa cells which surrounds the oocyte is known as corona radiata [4]. There are acellular glycoprotein layers known as zona pellucida between the corona radiata and oocyte. The zona pellucida which is 3–5 μm in thickness is produced by the ova and the granulosa cells. Within the antrum the ova are anchored by a compact mass of cells called as cumulus oophorus. After releasing the ova, the granulosa cells are disorganized within the antrum with pyknotic nuclei (fragmentation of nuclei) in the remaining follicles. This condition of the inactive follicles is called as atretic follicle [5].

luteinizing hormone (LH). Theca interna is highly vascularized, and its function is to deliver hormone, nutrients, vitamins, and cofactors, which are necessary for

In the graafian follicle, the granulosa cells and oocyte have a specific shape and

It is rich in hyaluronan and proteoglycans. It helps in follicle ruptures. Ovulation

occurs under the influence of follicle-stimulating hormone (FSH) and LH [7]. During ovulation the ova are in the metaphase stage of the second meiotic division.

**4. Microstructure and physiology of the oviduct and how it facilitates**

The oviduct has several layers including the mucosa, muscular layer, and a connective serosa. The size of these layers depends on different regions of the oviduct. The myosalpinx is comprised of smooth muscle; it is thin in the region of the ampulla and multilayered thick in the region of the isthmus. The endosalpinx is a term used for a mucosa, which is lined with simple columnar ciliated epithelium [8]. In the ampulla, the lining ciliated cells are numerous compared with that of secretory cells. The lining mucosa forms folds upward that fill the tubular lumen [9]. Due to the abundance of upward-directed folds, the lining mucosa of the ampulla increased the surface area, assuming excessive ability for the metabolic conversation between the epithelial, luminal, and the underlining vascular compartments. The mucosa of the isthmus has a smaller number of folds, while the

Sperm and oocyte will not reach the oviduct at the same time. In some animals the oocyte reaches first, and, in some, sperm approaches initially to the site of fertilization. The movement of sperm toward the isthmus is promoted by the ciliary movement of the lining epithelium. The spermatozoa are stored and remain active in the oviduct for up to 72 hours, but in some species the time is extended for a month like in bats. The fallopian tube contains fluid secreted by the glandular epithelium, which has a positive impact on the viability of sperm and provides nutrition to the oocyte [8]. This fluid helps in the maturation of the oocyte in the oviduct. Sperm adhere to the epithelial wall of the fallopian tube which preserves

position [6]. There are four different types of granulosa cell layers:

growth of the oocyte and granulosa cells [4].

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

• The outermost is membrana granulosa.

• The intermediate is cumulus oophorus.

• Just opposed to oocyte is corona radiata.

**the sperm, ovum, and early-stage embryo**

abundant number of secretory cells is present there.

**3.3 Granulosa cells and oocyte**

• The inner most is periantral.

**4.1 Microstructure of the oviduct**

**4.2 Function of the oviduct**

**5**

**3.4 Follicular fluid**

#### **3. Microstructure modulation in graafian follicle**

A graafian follicle consists of a large cavity called antrum having fluid termed as follicular fluid. Graafian follicle is also known as antral follicle [3]. Before ovulation, secondary follicle undergoes first mitotic division, and as a result the graafian follicle is formed which has two N haploid chromosomes. The characteristic structural feature of graafian follicle is follicular antrum in which granulosa cell and oocyte are present. Antral follicle is a three-dimensional structure having a cavity (antrum) which is surrounded by different types of cells [6]. The antral follicle has six different histologic components which are as follows:

#### **3.1 Theca externa**

Theca externa consists of smooth muscle cells that are innervated by the autonomic nerve. The functional importance of theca externa is still unclear, but there is evidence that changes in the contractile activity of theca externa are involved in atresia and ovulation [5].

#### **3.2 Theca interna**

It is consisting of different cells which are present in the matrix of loose connective tissues and blood vessels. The function of theca interna cells is regulated by luteinizing hormone (LH). Theca interna is highly vascularized, and its function is to deliver hormone, nutrients, vitamins, and cofactors, which are necessary for growth of the oocyte and granulosa cells [4].

#### **3.3 Granulosa cells and oocyte**

remaining portion of the ovarian cortex is covered by the CT which contains the primordial follicles, surrounded by a flat follicular cell (squamous shaped). Another follicle a bit larger is called a primary follicle, internally lined by the simple and stratified cuboidal epithelium termed as granulosa cells. In the connective tissue of the cortex of the ovary, many blood vessels surround the developing follicles. The granulosa cells are more prominent in the larger follicle called as secondary follicle or also called as antral follicle [3]. The antrum is the cavity formation within the follicle, which splits the granulosa cells into two layers. The fluid within the antrum is called as liquor folliculi. A single large ovum is present in the follicle, and a single layer of granulosa cells which surrounds the oocyte is known as corona radiata [4]. There are acellular glycoprotein layers known as zona pellucida between the corona radiata and oocyte. The zona pellucida which is 3–5 μm in thickness is produced by the ova and the granulosa cells. Within the antrum the ova are anchored by a compact mass of cells called as cumulus oophorus. After releasing the ova, the granulosa cells are disorganized within the antrum with pyknotic nuclei (fragmentation of nuclei) in the remaining follicles. This condition of the inactive follicles is

A graafian follicle consists of a large cavity called antrum having fluid termed as follicular fluid. Graafian follicle is also known as antral follicle [3]. Before ovulation, secondary follicle undergoes first mitotic division, and as a result the graafian follicle is formed which has two N haploid chromosomes. The characteristic structural feature of graafian follicle is follicular antrum in which granulosa cell and oocyte are present. Antral follicle is a three-dimensional structure having a cavity (antrum) which is surrounded by different types of cells [6]. The antral follicle has

Theca externa consists of smooth muscle cells that are innervated by the autonomic nerve. The functional importance of theca externa is still unclear, but there is evidence that changes in the contractile activity of theca externa are involved in

It is consisting of different cells which are present in the matrix of loose connective tissues and blood vessels. The function of theca interna cells is regulated by

called as atretic follicle [5].

*Reproductive Biology and Technology in Animals*

• The theca externa

• The theca interna

• The basal lamina

• The granulosa cell

• The follicular fluid

atresia and ovulation [5].

• The oocyte

**3.1 Theca externa**

**3.2 Theca interna**

**4**

**3. Microstructure modulation in graafian follicle**

six different histologic components which are as follows:

In the graafian follicle, the granulosa cells and oocyte have a specific shape and position [6]. There are four different types of granulosa cell layers:

#### **3.4 Follicular fluid**

It is rich in hyaluronan and proteoglycans. It helps in follicle ruptures. Ovulation occurs under the influence of follicle-stimulating hormone (FSH) and LH [7]. During ovulation the ova are in the metaphase stage of the second meiotic division.

#### **4. Microstructure and physiology of the oviduct and how it facilitates the sperm, ovum, and early-stage embryo**

#### **4.1 Microstructure of the oviduct**

The oviduct has several layers including the mucosa, muscular layer, and a connective serosa. The size of these layers depends on different regions of the oviduct. The myosalpinx is comprised of smooth muscle; it is thin in the region of the ampulla and multilayered thick in the region of the isthmus. The endosalpinx is a term used for a mucosa, which is lined with simple columnar ciliated epithelium [8]. In the ampulla, the lining ciliated cells are numerous compared with that of secretory cells. The lining mucosa forms folds upward that fill the tubular lumen [9]. Due to the abundance of upward-directed folds, the lining mucosa of the ampulla increased the surface area, assuming excessive ability for the metabolic conversation between the epithelial, luminal, and the underlining vascular compartments. The mucosa of the isthmus has a smaller number of folds, while the abundant number of secretory cells is present there.

#### **4.2 Function of the oviduct**

Sperm and oocyte will not reach the oviduct at the same time. In some animals the oocyte reaches first, and, in some, sperm approaches initially to the site of fertilization. The movement of sperm toward the isthmus is promoted by the ciliary movement of the lining epithelium. The spermatozoa are stored and remain active in the oviduct for up to 72 hours, but in some species the time is extended for a month like in bats. The fallopian tube contains fluid secreted by the glandular epithelium, which has a positive impact on the viability of sperm and provides nutrition to the oocyte [8]. This fluid helps in the maturation of the oocyte in the oviduct. Sperm adhere to the epithelial wall of the fallopian tube which preserves

its viability and stops the premature capacitation. This enhances the chances of fertilization [1].

**5.2 Process of fertilization within the fallopian tube**

[11], which results in the formation of zygote.

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

**reproduction**

**6.1 Mucosa**

**6.2 Submucosa**

**7**

At the site of ampulla-isthmus junction, the capacitated and hyperactive spermatozoa crossed the layer of corona radiata and pierced the glycoprotein layer of zona pellucida. Afterward the fusion of the spermatozoa with the ova takes place

If we see the cross section of the uterus, it consists of four layers including the innermost mucosa, the submucosa, the muscularis, and the outermost serosa [9].

It is the innermost layer facing the lumen. It is lined with secretary layers of columnar epithelium. The embryo after a series of changes including zygote, twocell (blastomeres) stage, morula (16-cell stage), blastula (inner cell mass leads to embryo, and the outer cell mass leads to the formation of the placenta, with the formation of fluid filled cavity), and gastrula (where formation of three characteristic embryonic germ layers occurs) is implanted in the endometrial mucosa with a layer of trophoblast [1]. The trophoblastic cell layers are proliferated in the lamina propria (uterine stroma) forming lacunae. The lacunae form an open connection with the maternal sinusoid in the uterine stroma, which results in the establishment of the uteroplacental circulation. The endometrial glands develop from the mucosa and invaginate inward into the submucosa adopting a coiled shape. The endometrial

glands secrete PGF2 alpha at a critical time during estrous cycle that causes

helps in pushing the baby during parturition [8].

• Transportation of the secretary products

• Transportation of the gametes (sperm and ova)

luteolysis of corpus luteum if the animal is not pregnant [12]. The endometrium of ruminants has caruncles that are highly vascularized and provide a maternal portion of the placenta if attachment of the embryo occurs. While these caruncles are absent in sow and mare, instead endometrial folds are present. These endometrial folds provide uterine surface for the attachment of the placenta. So, the uterus is the place where attachment and nourishment of the fertilized ova take place, and it

After the mucosa, a layer of submucosa is present. It houses a rich blood supply, nerve endings, and the lymphatics in addition to the rich supply of mononuclear cells. It has a role in providing nutrition and maintaining coordination and has supporting effect to the mucosa [9]. The muscularis is present beneath the submucosa and consists of double arranged layers of smooth muscles like longitudinal muscles (outer layer) and inner circular muscles (inner layer). A very prominent third layer of smooth muscle in oblique arrangement can also be seen in the gravid uterus. These layers played a very important function by providing the uterus with the ability to contract, and that contraction is very important on the following basis:

**6. Histological layers, physiology of the uterus, and its role in**

Fertilization of the ovum results in the formation of zygotes. After all the embryonic changes in the zygote, the later embryo is transferred along the fallopian tube. Some hormone also helps in the transportation of the embryo. The production of the estradiol (E2) by the embryo also helps in its transportation. In the mare, the secretion of prostaglandin (PGE2) during the first week acts on the oviduct locally and causes the transfer of the embryo to the posterior region of the uterus. The 4- to 8-cell stage embryos in hamster secrete platelet-activating factor (PAF). The later factor also acts on the oviduct locally, resulting in the transfer of the embryo toward the uterine horns. It is important to note that in the hamster and the mare, the main factor, decisive whether and when the fertilized ovum will drive toward the uterine horns or not, is based on the condition of the fertilized eggs.

#### **4.3 Transportation of oocyte**

During ovulation the fimbriae attached to the surface of graafian follicle and sweep the ova further anterior toward the ampulla [8]. The coordinated contraction of the myosalpinx along the length of the fallopian tube and the ciliary beats of the epithelial cells results in the transportation of the oocyte to the site of fertilization [9].

#### **5. Microstructures and physiology of fimbriae**

Fimbriae are finger-like growth processes that are present at the terminal of each fallopian tube. They increase the surface area of the infundibulum and cause it to slip over the surface of the ovary when ovulation occurs. This activity of the fimbriae increases the possibility that the oozed-out oocyte will be captured after ovulation from the graafian follicle and transported through a wider opening called ostium into the ampulla of the oviduct [9]. Above the mucus membrane of the fallopian tube, there are three layers of tissues, viz., the innermost layer consists of spirally arranged fibers, the middle layer consists of fibers with circular arrangement, and the outermost sheath has longitudinally arranged fibers that end in fimbriae near ovaries, forming a funnel-shaped depository structure called the infundibulum. One fimbria out of all fimbriae is long enough to reach the external surface of the ovary, which is called as fimbria ovarica [10]. The fallopian tube at this point is lined by small epithelial cells with small, slender hair-like cilia pulsating unidirectionally inside the fallopian tube to guide and direct the ovum from the ovary to the uterus [9].

#### **5.1 Role of fimbriae in directing the ova to area of fertilization**

As there is no direct connection between ovaries and the oviduct, the egg is transported to the uterus in peritoneal fluid produced by fimbriae on the terminal of tube's opening. The specialized granulosa cells of oocyte (cumulus oophorous) at ovulation are sticky and help in adhering to the surface of fimbriae. After this the fimbriae through muscular control create negative pressure that picks up oocyte and moves it to the fallopian tube [9]. There are also some hormonal changes that control the picking up of oocyte [7].

#### **5.2 Process of fertilization within the fallopian tube**

At the site of ampulla-isthmus junction, the capacitated and hyperactive spermatozoa crossed the layer of corona radiata and pierced the glycoprotein layer of zona pellucida. Afterward the fusion of the spermatozoa with the ova takes place [11], which results in the formation of zygote.

#### **6. Histological layers, physiology of the uterus, and its role in reproduction**

If we see the cross section of the uterus, it consists of four layers including the innermost mucosa, the submucosa, the muscularis, and the outermost serosa [9].

#### **6.1 Mucosa**

its viability and stops the premature capacitation. This enhances the chances

tube. Some hormone also helps in the transportation of the embryo. The

Fertilization of the ovum results in the formation of zygotes. After all the embryonic changes in the zygote, the later embryo is transferred along the fallopian

production of the estradiol (E2) by the embryo also helps in its transportation. In the mare, the secretion of prostaglandin (PGE2) during the first week acts on the oviduct locally and causes the transfer of the embryo to the posterior region of the uterus. The 4- to 8-cell stage embryos in hamster secrete platelet-activating factor (PAF). The later factor also acts on the oviduct locally, resulting in the transfer of the embryo toward the uterine horns. It is important to note that in the hamster and the mare, the main factor, decisive whether and when the fertilized ovum will drive toward the uterine horns or not, is based on the condition of the

During ovulation the fimbriae attached to the surface of graafian follicle and

contraction of the myosalpinx along the length of the fallopian tube and the ciliary beats of the epithelial cells results in the transportation of the oocyte to the site of

Fimbriae are finger-like growth processes that are present at the terminal of each fallopian tube. They increase the surface area of the infundibulum and cause it to slip over the surface of the ovary when ovulation occurs. This activity of the fimbriae increases the possibility that the oozed-out oocyte will be captured after ovulation from the graafian follicle and transported through a wider opening called ostium into the ampulla of the oviduct [9]. Above the mucus membrane of the fallopian tube, there are three layers of tissues, viz., the innermost layer consists of spirally arranged fibers, the middle layer consists of fibers with circular arrangement, and the outermost sheath has longitudinally arranged fibers that end in fimbriae near ovaries, forming a funnel-shaped depository structure called the infundibulum. One fimbria out of all fimbriae is long enough to reach the external surface of the ovary, which is called as fimbria ovarica [10]. The fallopian tube at this point is lined by small epithelial cells with small, slender hair-like cilia pulsating unidirectionally inside the fallopian tube to guide and direct the ovum from the

sweep the ova further anterior toward the ampulla [8]. The coordinated

**5. Microstructures and physiology of fimbriae**

**5.1 Role of fimbriae in directing the ova to area of fertilization**

As there is no direct connection between ovaries and the oviduct, the egg is transported to the uterus in peritoneal fluid produced by fimbriae on the terminal of tube's opening. The specialized granulosa cells of oocyte (cumulus oophorous) at ovulation are sticky and help in adhering to the surface of fimbriae. After this the fimbriae through muscular control create negative pressure that picks up oocyte and moves it to the fallopian tube [9]. There are also some hormonal changes that

of fertilization [1].

*Reproductive Biology and Technology in Animals*

fertilized eggs.

fertilization [9].

ovary to the uterus [9].

**6**

control the picking up of oocyte [7].

**4.3 Transportation of oocyte**

It is the innermost layer facing the lumen. It is lined with secretary layers of columnar epithelium. The embryo after a series of changes including zygote, twocell (blastomeres) stage, morula (16-cell stage), blastula (inner cell mass leads to embryo, and the outer cell mass leads to the formation of the placenta, with the formation of fluid filled cavity), and gastrula (where formation of three characteristic embryonic germ layers occurs) is implanted in the endometrial mucosa with a layer of trophoblast [1]. The trophoblastic cell layers are proliferated in the lamina propria (uterine stroma) forming lacunae. The lacunae form an open connection with the maternal sinusoid in the uterine stroma, which results in the establishment of the uteroplacental circulation. The endometrial glands develop from the mucosa and invaginate inward into the submucosa adopting a coiled shape. The endometrial glands secrete PGF2 alpha at a critical time during estrous cycle that causes luteolysis of corpus luteum if the animal is not pregnant [12]. The endometrium of ruminants has caruncles that are highly vascularized and provide a maternal portion of the placenta if attachment of the embryo occurs. While these caruncles are absent in sow and mare, instead endometrial folds are present. These endometrial folds provide uterine surface for the attachment of the placenta. So, the uterus is the place where attachment and nourishment of the fertilized ova take place, and it helps in pushing the baby during parturition [8].

#### **6.2 Submucosa**

After the mucosa, a layer of submucosa is present. It houses a rich blood supply, nerve endings, and the lymphatics in addition to the rich supply of mononuclear cells. It has a role in providing nutrition and maintaining coordination and has supporting effect to the mucosa [9]. The muscularis is present beneath the submucosa and consists of double arranged layers of smooth muscles like longitudinal muscles (outer layer) and inner circular muscles (inner layer). A very prominent third layer of smooth muscle in oblique arrangement can also be seen in the gravid uterus. These layers played a very important function by providing the uterus with the ability to contract, and that contraction is very important on the following basis:

It also has a key role in the expulsion of the fetus and fetal membranes at the time of parturition.

#### **6.3 Serosa**

It is the outer covering of the uterus that consists of a single layer of squamous cells.

**8. Gross and microscopic anatomy of the vulva and its role in**

present in the vulvar region of the female reproductive system:

• Apocrine glands (scent glands)

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

• Eccrine glands (sweat glands)

**8.1 Microscopic anatomy of vulva**

**8.2 Role of the vulva in reproduction**

• Enabling sperm to enter in the body of female

• Providing the sexual pleasure

The vulva is the most outer part of the female reproductive track immediately external to the vaginae. It is composed of various structures, including the mons pubis, clitoris, labia minora, labia majora, vulvar vestibule, vestibulovaginal bulbs, urethral meatus, hymen, and Bartholin and Skene glands and ducts. Bartholin glands are like the bulbourethral glands in male.  The hymen is the stratified squamous nonkeratinized epithelium. The lateral vulvar surfaces are made up of labia majora and are comprised of fibrous and adipose folds. The labia minora also consists of connective tissue in two folds containing few (if any) adipose tissues. The labia minora is bifurcated anteriorly. The vestibule is the zone between Hart's line and the hymen, which consists of stratified squamous nonkeratinized epithelium [9]. The following are the glands

Vaginal lining epithelium is stratified squamous and nonkeratinized, whereas the epithelium of the labia majora is stratified squamous and keratinized. Microscopic layers of the vulva are the mucosa, submucosa, muscularis, and serosa [14, 15]. Glands are present in the submucosal layer and the innermost layer is the

Being a gateway to the uterus, its primary role is to offer protection by closing the labia [16]. The external uterine orifice is protected by the vulva supported by thick large lips of labia majora along with small lips of labia minora. The urethra also opens in the vulva that is known as the urethral meatus, thus performing a function of urine passage. It also gives the pathway for sperm for entry in the body. Sometimes due to the folds and the moisture, the fungal infection may occur. The area between the opening of the vagina and the anus, below the labia majora, is called the perineum [12]. By observing the vulva, we can detect if the female is in estrous or not. If the vulva is thick and edematous, it is in the heat, and if not the female is not in the heat; this is due to the estrogen level in the female body. The penis like clitoris detects the nerve stimuli and performs the following

• Protecting the internal genital organs from infection causing organisms

**reproduction**

• Skene glands

mucosa.

functions:

**9**

• Sebaceous glands

#### **7. Gross and microscopic structures and functions of the cervix**

The whole reproductive tract is tubular except the ovary. The cervix is a thickwalled tubular structure where mucosa-submucosa-derived folds or rings are present, but their number varies from species to species.  In cow and ewe, rings/folds are having interlocking finger-like projections. In pigs, rings interdigitate so interdigitation is seen in them. The cervix is soft in mare due to the presence of loose folds of the mucosa.  The primary function of the cervix is lubrication of the canal which is required during copulation [8]. Lubrication is by two types of secretions, e.g., the sulfomucin which is viscous (mobility is directed toward exterior (vagina) and inhibits sperm transport in the uterus), and the sialomucins which facilitate the sperm to move into the uterus. The cervix isolates the conceptus inside the uterus from the outer environment by the formation of cervical seal under the action of progesterone which thickens the mucus produced in the cervix and glues the folding together to prevent entry of microbes during pregnancy.

Like other tubular organs, it consists of four tunics/laminae, i.e., mucosa, submucosa, muscularis, and serosa/adventitia [9]. The mucosal tunics of the endocervix are lined by simple columnar epithelium along with the presence of mucus-producing cells called goblet cells. In some animal species, a few columnar cells are found ciliated and in simple tubular glands may be seen in the ruminant's mucosa. In the case of pigs, up to 90% of the mucosal lining epithelium is stratified squamous. Most of the ectocervical portion of the cervical mucosa is lined by the stratified squamous epithelium. Mucosa and the submucosa form folds into the cervical lumen. These folds vary in height, width, and thickness from species to species. The lamina propria of the mucosa and submucosa combines to form propria-submucosa. This propriasubmucosa is heavily infiltrated by dense irregular connective tissue and supply of rich blood vessels extended deep in the submucosa. The muscularis layer is composed of two smooth muscle layers. The inner layer is circular layer and the outer layer is longitudinal in arrangements. The myenteric plexus is present in the muscular layers. Some elastic fibers are also observed in this layer. Serosa/adventitia is a loose connective tissue layer present in the outer surface. This layer is surrounded by the mesothelium which is a layer of simple squamous epithelium. Gartner's ducts may be seen in serosal layer unilateral or bilateral.

#### **7.1 Functions of the cervix**

During estrus stage of estrous cycle, the mucosa is responsible for mucus secretion. Dense irregular connective tissue becomes edematous and forms areolar structure, i.e., loose connective tissue. Circular muscular layer along with elastic fiber is responsible for the involution of the cervix after parturition [8].

#### **8. Gross and microscopic anatomy of the vulva and its role in reproduction**

The vulva is the most outer part of the female reproductive track immediately external to the vaginae. It is composed of various structures, including the mons pubis, clitoris, labia minora, labia majora, vulvar vestibule, vestibulovaginal bulbs, urethral meatus, hymen, and Bartholin and Skene glands and ducts. Bartholin glands are like the bulbourethral glands in male.  The hymen is the stratified squamous nonkeratinized epithelium. The lateral vulvar surfaces are made up of labia majora and are comprised of fibrous and adipose folds. The labia minora also consists of connective tissue in two folds containing few (if any) adipose tissues. The labia minora is bifurcated anteriorly. The vestibule is the zone between Hart's line and the hymen, which consists of stratified squamous nonkeratinized epithelium [9]. The following are the glands present in the vulvar region of the female reproductive system:

• Movement of early embryo to the appropriate location

*Reproductive Biology and Technology in Animals*

• Maintaining and nourishment of the embryo and fetus

time of parturition.

entry of microbes during pregnancy.

in serosal layer unilateral or bilateral.

**7.1 Functions of the cervix**

**8**

**6.3 Serosa**

It also has a key role in the expulsion of the fetus and fetal membranes at the

It is the outer covering of the uterus that consists of a single layer of squamous cells.

The whole reproductive tract is tubular except the ovary. The cervix is a thickwalled tubular structure where mucosa-submucosa-derived folds or rings are present, but their number varies from species to species.  In cow and ewe, rings/folds are having interlocking finger-like projections. In pigs, rings interdigitate so interdigitation is seen in them. The cervix is soft in mare due to the presence of loose folds of the mucosa.  The primary function of the cervix is lubrication of the canal which is required during copulation [8]. Lubrication is by two types of secretions, e.g., the sulfomucin which is viscous (mobility is directed toward exterior (vagina) and inhibits sperm transport in the uterus), and the sialomucins which facilitate the sperm to move into the uterus. The cervix isolates the conceptus inside the uterus from the outer environment by the formation of cervical seal under the action of progesterone which thickens the mucus produced in the cervix and glues the folding together to prevent

Like other tubular organs, it consists of four tunics/laminae, i.e., mucosa, submucosa, muscularis, and serosa/adventitia [9]. The mucosal tunics of the endocervix are lined by simple columnar epithelium along with the presence of mucus-producing cells called goblet cells. In some animal species, a few columnar cells are found ciliated and in simple tubular glands may be seen in the ruminant's mucosa. In the case of pigs, up to 90% of the mucosal lining epithelium is stratified squamous. Most of the ectocervical portion of the cervical mucosa is lined by the stratified squamous epithelium. Mucosa and the submucosa form folds into the cervical lumen. These folds vary in height, width, and thickness from species to species. The lamina propria of the mucosa and submucosa combines to form propria-submucosa. This propriasubmucosa is heavily infiltrated by dense irregular connective tissue and supply of rich blood vessels extended deep in the submucosa. The muscularis layer is composed of two smooth muscle layers. The inner layer is circular layer and the outer layer is longitudinal in arrangements. The myenteric plexus is present in the muscular layers. Some elastic fibers are also observed in this layer. Serosa/adventitia is a loose connective tissue layer present in the outer surface. This layer is surrounded by the mesothelium which is a layer of simple squamous epithelium. Gartner's ducts may be seen

During estrus stage of estrous cycle, the mucosa is responsible for mucus secre-

tion. Dense irregular connective tissue becomes edematous and forms areolar structure, i.e., loose connective tissue. Circular muscular layer along with elastic

fiber is responsible for the involution of the cervix after parturition [8].

**7. Gross and microscopic structures and functions of the cervix**

#### **8.1 Microscopic anatomy of vulva**

Vaginal lining epithelium is stratified squamous and nonkeratinized, whereas the epithelium of the labia majora is stratified squamous and keratinized. Microscopic layers of the vulva are the mucosa, submucosa, muscularis, and serosa [14, 15]. Glands are present in the submucosal layer and the innermost layer is the mucosa.

#### **8.2 Role of the vulva in reproduction**

Being a gateway to the uterus, its primary role is to offer protection by closing the labia [16]. The external uterine orifice is protected by the vulva supported by thick large lips of labia majora along with small lips of labia minora. The urethra also opens in the vulva that is known as the urethral meatus, thus performing a function of urine passage. It also gives the pathway for sperm for entry in the body. Sometimes due to the folds and the moisture, the fungal infection may occur. The area between the opening of the vagina and the anus, below the labia majora, is called the perineum [12]. By observing the vulva, we can detect if the female is in estrous or not. If the vulva is thick and edematous, it is in the heat, and if not the female is not in the heat; this is due to the estrogen level in the female body.  Type of sperm which is most favorable for fertilization**

zona pellucida is the innermost thick transparent glycoprotein membranes surrounding the plasma membranes. With a maturation of oocyte, multiple granulosal cells enlarge called cumulus oophorus [3]. This oocyte and granulosal cells form complex known as cumulus oocyte complex (COC). The cumulus of mature COC adheres to the surface of fimbriae as sticky substances. After the sex hormones signal the fimbriae, it contracts and releases oocyte. After release, oocyte has only 24–28 hours to fuse with sperm; otherwise, it is lost. Oocyte releases some chemicals that attract the sperm, and the sperm tries to penetrate two protective layers of oocyte. First it burrows to the cell of corona radiata and then move toward zona pellucida layer. Zona pellucida is composed of four glycoproteins ZP1, ZP2, ZP3, and ZP4. The primary ligand of sperm oocyte binding is ZP3 and ZP4. Both induce acrosomal reaction of sperm to complete fusion reaction and form a zygote [18].

These factors make the ovum gamete most favorable for fertilization.

mucosa with the fertilized ovum and conceptus.

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

hereby acknowledged in helping me in the write-up.

\* and Muhammad Ali2

\*Address all correspondence to<EMAIL_ADDRESS>
The microarchitectural examination of the reproductive organs at microscopic level is very much important to recognize its structure and function during reproductive physiology. Furthermore, this chapter highlighted the dealing of the lining

The author would like to acknowledge the efforts of Prof. Dr. Ashiq Hussain Cheema for facilitating and guiding. Furthermore, Jr. lab attendant Mr. Saqib Ali, is

1 Sub-campus, Jhang, University of Veterinary and Animal Sciences, Lahore,

2 University College of Veterinary and Animal Sciences, The Islamia University of

© 2019 The Author(s). Licensee IntechOpen. 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,

**11. Conclusion**

**Acknowledgements**

**Author details**

Arbab Sikandar<sup>1</sup>

Bahawalpur, Pakistan

<EMAIL_ADDRESS>
provided the original work is properly cited.

Pakistan

**11**

As we know around 250 million sperm cells enter the female external genitalia, but just a few thousands can enter the fallopian tube, and only a single sperm will fertilize the ova [14]. Several problems and barriers came in the pathway of the spermatozoa to touch the final goal. These range from the low pH in and around the vagina, the mucus of the cervix, the narrowness of the uterotubal junction (the entrance of the cervix), the WBCs of the immune system which treat the spermatozoa as a foreign entity to destroy, cell-to-cell interactions, gene expression, phenotypic sperm traits, sperm motility defects, DNA status, lack of capacitation or morphological normality, and failure of abnormal spermatozoa to reach to the site of fertilization. The wall of seminiferous tubules which are the coiled structure is responsible to produce the sperm. For a spermatozoon, around 28–42 days is lapsed to cross the male reproductive system. In the female reproductive tract, sperm undergo changes that help in fertilization called activation and capacitation, but all sperm are not capacitated at the same time; therefore, all sperm are not able to fertilize the cells [16]. However, several mechanisms that aid this process are good motility, adequate morphology, and normal DNA status of the cell. A sperm which has normal head, nucleus, and tail and has a moderate motility is considerable to fertilize the egg [17]. The sperm are guided upon their journey by the chemical and the temperature signals. A sperm reservoir in the fallopian tubes, where sperms bind to the epithelial lining (columnar in shape) of the tubes, reduces the chance of fertilization by multiple sperm. During ovulation, the sperm are hyperactivated to help them to the penetration of the mucus in the fallopian tubes and the outer coating of the egg. Sperm outer membrane fuses with egg outer membrane to facilitate fertilization. Acrosomal reaction occurs in the sperm head once the spermatozoa reached the ova. Acrosin enzyme helps in acrosomal reaction. Hyaluronic acid enzyme has an important role in the permeability and motility of sperms and their interactions with gametes. Formation of a functional reservoir through epithelial binding of sperm to the oviductal isthmus reduces the likelihood of the polyspermic fertilization. Motility or hyperactivation helps sperm in penetrating the mucus in the fallopian tubes and the cumulus oophorus (corona radiata), and the acrosome exocytosis may assist penetration of oocyte zona pellucida that proceeds the fusion within oocyte plasma membrane. Zona pellucida then undergoes biochemical changes so that further sperm cannot penetrate the cell called zona block [17]. In addition to alteration of zona pellucida, the cortical reaction reduces the ability of oocyte plasma membrane to fuse with additional spermatozoa, thus causing vitelline block. Both zona block and vitelline block prevent polyspermy.

#### **10. The type of ova that is most favorable for fertilization**

Fertilization is a process in which male and female gametes fuse to form a zygote. Ovum is the female gamete, and its selection is the criteria of fertilization. There are many factors that are responsible for its selection including follicular dominance, cumulus oophorus formation, cumulus oocyte complex formation, fimbrial supportive structure, and zona pellucidal layer that enable sperm to fuse with ovum.

Follicular dominance is based on E2 production. The follicle, which has greater ability of E2 production, has greater capability of its dominance. Dominancy is responsible for onward ovulation. Ovulated egg is surrounded by two protective layers [9]. Corona radiata is the outermost layer containing granulosal cells, and

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

zona pellucida is the innermost thick transparent glycoprotein membranes surrounding the plasma membranes. With a maturation of oocyte, multiple granulosal cells enlarge called cumulus oophorus [3]. This oocyte and granulosal cells form complex known as cumulus oocyte complex (COC). The cumulus of mature COC adheres to the surface of fimbriae as sticky substances. After the sex hormones signal the fimbriae, it contracts and releases oocyte. After release, oocyte has only 24–28 hours to fuse with sperm; otherwise, it is lost. Oocyte releases some chemicals that attract the sperm, and the sperm tries to penetrate two protective layers of oocyte. First it burrows to the cell of corona radiata and then move toward zona pellucida layer. Zona pellucida is composed of four glycoproteins ZP1, ZP2, ZP3, and ZP4. The primary ligand of sperm oocyte binding is ZP3 and ZP4. Both induce acrosomal reaction of sperm to complete fusion reaction and form a zygote [18]. These factors make the ovum gamete most favorable for fertilization.

#### **11. Conclusion**

**9. Type of sperm which is most favorable for fertilization**

*Reproductive Biology and Technology in Animals*

**10. The type of ova that is most favorable for fertilization**

with ovum.

**10**

Fertilization is a process in which male and female gametes fuse to form a zygote. Ovum is the female gamete, and its selection is the criteria of fertilization. There are many factors that are responsible for its selection including follicular dominance, cumulus oophorus formation, cumulus oocyte complex formation, fimbrial supportive structure, and zona pellucidal layer that enable sperm to fuse

Follicular dominance is based on E2 production. The follicle, which has greater ability of E2 production, has greater capability of its dominance. Dominancy is responsible for onward ovulation. Ovulated egg is surrounded by two protective layers [9]. Corona radiata is the outermost layer containing granulosal cells, and

As we know around 250 million sperm cells enter the female external genitalia, but just a few thousands can enter the fallopian tube, and only a single sperm will fertilize the ova [14]. Several problems and barriers came in the pathway of the spermatozoa to touch the final goal. These range from the low pH in and around the vagina, the mucus of the cervix, the narrowness of the uterotubal junction (the entrance of the cervix), the WBCs of the immune system which treat the spermatozoa as a foreign entity to destroy, cell-to-cell interactions, gene expression, phenotypic sperm traits, sperm motility defects, DNA status, lack of capacitation or morphological normality, and failure of abnormal spermatozoa to reach to the site of fertilization. The wall of seminiferous tubules which are the coiled structure is responsible to produce the sperm. For a spermatozoon, around 28–42 days is lapsed to cross the male reproductive system. In the female reproductive tract, sperm undergo changes that help in fertilization called activation and capacitation, but all sperm are not capacitated at the same time; therefore, all sperm are not able to fertilize the cells [16]. However, several mechanisms that aid this process are good motility, adequate morphology, and normal DNA status of the cell. A sperm which has normal head, nucleus, and tail and has a moderate motility is considerable to fertilize the egg [17]. The sperm are guided upon their journey by the chemical and the temperature signals. A sperm reservoir in the fallopian tubes, where sperms bind to the epithelial lining (columnar in shape) of the tubes, reduces the chance of fertilization by multiple sperm. During ovulation, the sperm are hyperactivated to help them to the penetration of the mucus in the fallopian tubes and the outer coating of the egg. Sperm outer membrane fuses with egg outer membrane to facilitate fertilization. Acrosomal reaction occurs in the sperm head once the spermatozoa reached the ova. Acrosin enzyme helps in acrosomal reaction. Hyaluronic acid enzyme has an important role in the permeability and motility of sperms and their interactions with gametes. Formation of a functional reservoir through epithelial binding of sperm to the oviductal isthmus reduces the likelihood of the polyspermic fertilization. Motility or hyperactivation helps sperm in penetrating the mucus in the fallopian tubes and the cumulus oophorus (corona radiata), and the acrosome exocytosis may assist penetration of oocyte zona pellucida that proceeds the fusion within oocyte plasma membrane. Zona pellucida then undergoes biochemical changes so that further sperm cannot penetrate the cell called zona block [17]. In addition to alteration of zona pellucida, the cortical reaction reduces the ability of oocyte plasma membrane to fuse with additional spermatozoa, thus causing vitelline block. Both zona block and vitelline block prevent polyspermy.

The microarchitectural examination of the reproductive organs at microscopic level is very much important to recognize its structure and function during reproductive physiology. Furthermore, this chapter highlighted the dealing of the lining mucosa with the fertilized ovum and conceptus.

#### **Acknowledgements**

The author would like to acknowledge the efforts of Prof. Dr. Ashiq Hussain Cheema for facilitating and guiding. Furthermore, Jr. lab attendant Mr. Saqib Ali, is hereby acknowledged in helping me in the write-up.

#### **Author details**

Arbab Sikandar<sup>1</sup> \* and Muhammad Ali2

1 Sub-campus, Jhang, University of Veterinary and Animal Sciences, Lahore, Pakistan

2 University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

\*Address all correspondence to<EMAIL_ADDRESS><EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. 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.

### **References**

[1] Arrotéia KF, Garcia PV, Barbieri MF, Justino ML, Pereira LAV. The epididymis: Embryology, structure, function and its role in fertilization and infertility. In: Pereira LAV, ed. Embryology-Updates and Highlights on Classic Topics. Rijeka, Croatia: IntechOpen; 2012. DOI: 10.5772/2142

[2] Osvaldo-Decima L. Smooth muscle in the ovary of the rat and monkey. Journal of Ultrastructure Research. 1970;**30** (1–2):218-237

[3] Fortune JE. Ovarian follicular growth and development in mammals. Biology of Reproduction. 1994;**50**(2):225-232

[4] Hyttel PFTCH, Fair T, Callesen H, Greve T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology. 1997;**47**(1):23-32

[5] Saidapur SK. Follicular atresia in the ovaries of nonmammalian vertebrates. In: International Review of Cytology. Vol. 54. New York: Academic Press; 1978. pp. 225-244

[6] Rodgers RJ, Irving-Rodgers HF. Morphological classification of bovine ovarian follicles. Reproduction. 2010; **139**(2):309

[7] Wiley C, Jahnke M, Redifer C, Gunn PJ, Dohlman T. Effects of endogenous progesterone during ovarian follicle superstimulation on embryo quality and quantity in beef cows. Theriogenology. 2019;**129**:54-60

[8] Padubidri VG, Daftary SN, editors. Shaw's Textbook of Gynecology-EBOOK. Elsevier, India: Elsevier Health Sciences; 2018

[9] Boyd KL, Muehlenbachs A, Rendi MH, Garcia RL, Gibson-Corley KN. Female reproductive system. In: Comparative Anatomy and Histology. London, United Kingdom: Academic Press; 2018. pp. 303-334

[10] Guan J, Watrelot A. Fallopian subtle pathology. Best Practice and Research Clinical Obstetrics and Gynaecology. 2019. https://doi.org/10.1016/j.bpobgyn. 2018.12.012

[11] Carlson BM. Human Embryology and Developmental Biology E-Book. New York: Elsevier Health Sciences; 2018

[12] Bazer FW, Burghardt RC, Johnson GA, Spencer TE, Wu G. Mechanisms for the establishment and maintenance of pregnancy: Synergies from scientific collaborations.  Differentiation. 2018;**103**:74-85

[14] Vuarin P, Hingrat Y, Lesobre L, Jalme MS, Lacroix F, Sorci G. Sperm competition accentuates selection on ejaculate attributes. Biology Letters. 2019;**15**(3):20180889

[15] Kumar V. Histological, histochemical and scanning electron microscopic studies on the oviduct and uterus of jaffarabadi buffalo (*Bubalus bubalis*) during follicular and luteal phases 2514 (doctoral dissertation). Junagadh: JAU; 2018

[16] Lucas D, Fox J. The psychology of human sexuality. The psychology of human sexuality. In: Noba Textbook Series: Psychology. Champaign, IL: DEF Publishers; 2018

[17] Lehmann R. Matchmaking molecule for egg and sperm. Science. 2018; **361**(6406):974-975

[18] Hirohashi N, Yanagimachi R. Sperm acrosome reaction: Its site and role in fertilization. Biology of Reproduction. 2018

**13**

**Chapter 2**

**Abstract**

High Surveillance

*François J. Richard*

developmental competence.

**1. Introduction**

Oocyte Meiotic Resumption under

Germinal vesicle breakdown (GVBD) is the hallmark of oocyte meiotic resump-

tion. It occurs under minimal stimulation during in vitro maturation (IVM). Several factors have been described to be involved in the inhibition of oocyte meiotic resumption such as purine derivatives. This study was assessing whether adenosinergic and guanosinergic systems are functional and participating in the inhibition of oocyte maturation. The objectives of the present study were to evaluate the effect of two purines, adenosine (ADO) and guanosine (GUO), on in vitro oocyte meiotic resumption, cumulus cell expansion, and gap junction communication. Both ADO and GUO significantly inhibited GVBD oocytes. The inhibitory effect lasted 24 hours and was reversible for meiotic resumption and cumulus cell expansion. Both ADO and GUO increased gap junction communication in cumulus cells. Equine chorionic gonadotropin (eCG) and the adenylyl cyclase stimulator, forskolin (FK), were both supportive of ADO and GUO inhibitory effect. The results are suggesting both adenosinergic and guanosinergic systems efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to explore in order to improve oocyte

**Keywords:** oocyte, purine, adenosine, guanosine, meiotic resumption

Oocyte meiosis begins during fetal development in large animals such as in cow, sow, and ewe. Once the sub-phases of the first prophase are completed, the oocyte meiosis stops at the dictyate stage. At this point, crossing over is a past event and chromatin is accessible for transcription. This G2/M phase transition of the cell cycle characterizes mammalian oocytes. The female gamete bears 4n chromosomes as long as the ovulatory LH peak generates its effect on the preovulatory follicle to induce oocyte meiotic resumption. Dysregulation of the oocyte cell cycle induced by c-MOS proto-oncogene after gene null mutation caused parthenogenic development of the oocyte and explained female mouse infertility [1]. This phenotype illustrates how important it is to appropriately control oocyte meiotic resumption. Oocyte meiotic resumption is a highly important physiological event for species survival since it refers to a successful reproduction by appropriately preparing the female gamete. This unique cell division has to occur at the right time and imply high surveillance. From an evolutionary point of view, the number of female gametes produced went from a large number, such as in frog, to a small number in mammals. Although several thousands of oocytes are found in the ovaries, only a

#### **Chapter 2**

**References**

(1–2):218-237

[1] Arrotéia KF, Garcia PV, Barbieri MF,

*Reproductive Biology and Technology in Animals*

[10] Guan J, Watrelot A. Fallopian subtle pathology. Best Practice and Research Clinical Obstetrics and Gynaecology. 2019. https://doi.org/10.1016/j.bpobgyn.

[11] Carlson BM. Human Embryology and Developmental Biology E-Book. New York: Elsevier Health Sciences; 2018

[12] Bazer FW, Burghardt RC, Johnson GA, Spencer TE, Wu G. Mechanisms for the establishment and maintenance of pregnancy: Synergies from scientific collaborations. Biology of Reproduction.

[13] Baskin L, Shen J, Sinclair A, Cao M, Liu X, Liu G, et al. Development of the

[14] Vuarin P, Hingrat Y, Lesobre L, Jalme MS, Lacroix F, Sorci G. Sperm competition accentuates selection on ejaculate attributes. Biology Letters.

[15] Kumar V. Histological, histochemical and scanning electron microscopic studies on the oviduct and uterus of jaffarabadi buffalo (*Bubalus bubalis*) during follicular

and luteal phases 2514 (doctoral dissertation). Junagadh: JAU; 2018

[16] Lucas D, Fox J. The psychology of human sexuality. The psychology of human sexuality. In: Noba Textbook Series: Psychology. Champaign, IL: DEF

[17] Lehmann R. Matchmaking molecule for egg and sperm. Science. 2018;

[18] Hirohashi N, Yanagimachi R. Sperm acrosome reaction: Its site and role in fertilization.  Differentiation. 2018;**103**:74-85

2019;**15**(3):20180889

Publishers; 2018

**361**(6406):974-975

2018

[2] Osvaldo-Decima L. Smooth muscle in the ovary of the rat and monkey. Journal of Ultrastructure Research. 1970;**30**

[3] Fortune JE. Ovarian follicular growth and development in mammals. Biology of Reproduction. 1994;**50**(2):225-232

[4] Hyttel PFTCH, Fair T, Callesen H, Greve T. Oocyte growth, capacitation

[5] Saidapur SK. Follicular atresia in the ovaries of nonmammalian vertebrates. In: International Review of Cytology. Vol. 54. New York: Academic Press;

[6] Rodgers RJ, Irving-Rodgers HF. Morphological classification of bovine ovarian follicles. Reproduction. 2010;

[7] Wiley C, Jahnke M, Redifer C, Gunn PJ, Dohlman T. Effects of endogenous progesterone during ovarian follicle superstimulation on embryo quality and quantity in beef cows. Theriogenology.

[8] Padubidri VG, Daftary SN, editors. Shaw's Textbook of Gynecology-EBOOK. Elsevier, India: Elsevier Health

[9] Boyd KL, Muehlenbachs A, Rendi MH, Garcia RL, Gibson-Corley KN. Female reproductive system. In:

Histology. London, United Kingdom: Academic Press; 2018. pp. 303-334

Comparative Anatomy and

and final maturation in cattle. Theriogenology. 1997;**47**(1):23-32

1978. pp. 225-244

**139**(2):309

2019;**129**:54-60

Sciences; 2018

**12**

Justino ML, Pereira LAV. The epididymis: Embryology, structure, function and its role in fertilization and

infertility. In: Pereira LAV, ed. Embryology-Updates and Highlights on Classic Topics. Rijeka, Croatia: IntechOpen; 2012. DOI: 10.5772/2142

## Oocyte Meiotic Resumption under High Surveillance

*François J. Richard*

#### **Abstract**

Germinal vesicle breakdown (GVBD) is the hallmark of oocyte meiotic resumption. It occurs under minimal stimulation during in vitro maturation (IVM). Several factors have been described to be involved in the inhibition of oocyte meiotic resumption such as purine derivatives. This study was assessing whether adenosinergic and guanosinergic systems are functional and participating in the inhibition of oocyte maturation. The objectives of the present study were to evaluate the effect of two purines, adenosine (ADO) and guanosine (GUO), on in vitro oocyte meiotic resumption, cumulus cell expansion, and gap junction communication. Both ADO and GUO significantly inhibited GVBD oocytes. The inhibitory effect lasted 24 hours and was reversible for meiotic resumption and cumulus cell expansion. Both ADO and GUO increased gap junction communication in cumulus cells. Equine chorionic gonadotropin (eCG) and the adenylyl cyclase stimulator, forskolin (FK), were both supportive of ADO and GUO inhibitory effect. The results are suggesting both adenosinergic and guanosinergic systems efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to explore in order to improve oocyte developmental competence.

**Keywords:** oocyte, purine, adenosine, guanosine, meiotic resumption

#### **1. Introduction**

Oocyte meiosis begins during fetal development in large animals such as in cow, sow, and ewe. Once the sub-phases of the first prophase are completed, the oocyte meiosis stops at the dictyate stage. At this point, crossing over is a past event and chromatin is accessible for transcription. This G2/M phase transition of the cell cycle characterizes mammalian oocytes. The female gamete bears 4n chromosomes as long as the ovulatory LH peak generates its effect on the preovulatory follicle to induce oocyte meiotic resumption. Dysregulation of the oocyte cell cycle induced by c-MOS proto-oncogene after gene null mutation caused parthenogenic development of the oocyte and explained female mouse infertility [1]. This phenotype illustrates how important it is to appropriately control oocyte meiotic resumption.

Oocyte meiotic resumption is a highly important physiological event for species survival since it refers to a successful reproduction by appropriately preparing the female gamete. This unique cell division has to occur at the right time and imply high surveillance. From an evolutionary point of view, the number of female gametes produced went from a large number, such as in frog, to a small number in mammals. Although several thousands of oocytes are found in the ovaries, only a

small percentage is ovulated, and even less are fertilized. Considering this selective restriction, a framework of the meiotic resumption process has developed. Throughout the evolutionary process, mechanisms have been added to ensure a very precise control of this crucial event related to species survival, which is the final phase of gamete preparation for fertilization. Interestingly, the role of EGF-like peptides fully fits with this notion. It is well-known that the LH peak induces oocyte meiotic resumption in the preovulatory follicles. However, the EGF-like peptides are also active participants in the ovulatory process, meiotic resumption, and cumulus cell expansion, clearly supporting an add-up to the LH surge. Going back to the control of oocyte meiotic resumption, although the contribution of cAMP and cGMP is well described, it is obvious that other mechanisms may still be involved and be discovered.

It has long been known that adenosine (ADO) is a molecule playing an important role in various physiological systems such as in the central nervous system and cardiac function [2]. On the other hand, there are very few studies on the role of ADO in mammalian ovarian follicle. ADO is known to act on specific receptors, to cross plasma membrane using transporters, and to be generated from functional catabolism by extracellular enzymes, a system called adenosinergic [3]. On the other hand, guanosine (GUO) has not been as popular in research. However, in recent years a new interest on GUO has revealed its importance in the effect of ADO on the functioning of the central nervous system [4]. Although no specific receptor for GUO has been yet identified, its physiological impact leaves no doubt. GUO has neuroprotective effects, it diminishes the apoptotic effects observed in Parkinson's disease, and it also has a protective role during a challenge with glutamate, during mitochondrial stress, and during ischemia [4–6]. Because GUO can also be generated by a functional catabolism using extracellular enzymes, these results support the existence of a so-called guanosinergic system.

ADO has also been identified in the follicular fluid with several other purine derivatives. Among these derivatives, hypoxanthine is the compound that has attracted the most studies in last three decades. In mice, ADO improves the inhibitory effect of hypoxanthine on the resumption of meiosis but has no effect when used alone, even at a dose of 5 mM [7]. In the rat, the effect of ADO is also minimal [8]. In cattle, ADO used at 200 μM slowed meiotic resumption [9]. There is one study reporting that it has not been able to measure GUO in follicular fluid [10]. In contrast, GUO showed a very potent effect on the inhibition of meiotic resumption in mice [10] and rat [8].

This study is proposing to assess whether an adenosinergic and guanosinergic system are functional and participating in the inhibition of oocyte meiotic resumption. Specifically, the research presented here aims to study the involvement of ADO and GUO in the physiology of the ovarian follicle by targeting their effect on in vitro meiotic resumption using swine as the animal model.

#### **2. Material and methods**

#### **2.1 Chemicals**

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The adenylyl cyclase activator, forskolin (FK), was prepared as a millimolar stock solution and stored at −20°C as already described [11]. ADO and GUO were prepared from the stock powder directly in the culture medium on the day of the experiment. 8-Bromoadenosine (8-BrADO) and 8-Bromoguanosine (8-BrGUO) were dissolved in DMSO, and a DMSO control was run simultaneously.

**15**

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

solution having antibiotics and antimycotics at 37°C.

**2.4 Recovery of cumulus-oocyte complexes (COC)**

tion of the oocytes.

**2.2 Ovary collections**

**2.3 Maturation medium**

−20°C until used [11].

with 100% humidity.

**2.5 Selecting COC and denuding oocytes**

geneous cytoplasm were selected.

**2.6 Assessment of oocyte nuclear maturation stage**

The chemicals were added to the maturation medium a few hours prior to the addi-

As previously described, prepubertal gilt ovaries were collected from a local slaughterhouse [12]. In brief, they were placed in saline (0.9% NaCl containing antibiotics and antimycotics, 100,000 IU/L penicillin G, 100 mg/L streptomycin, 250 μg/L amphotericin B) and kept at 37°C. They were rinsed once in a fresh saline

Oocytes were matured in BSA-free North Carolina State University 23 (NCSU) medium [13] supplemented with 25 μM β-mercaptoethanol (Bio-Rad, Hercules, CA, USA), 0.1 mg/mL cysteine, 10% (v/v) porcine follicular fluid (PFF), and gonadotropins (2.5 IU/well for hCG [APL, Ayerst Laboratories Inc., Philadelphia, PA, USA] and 2.5 IU/well for eCG [Folligon, Intervet, Whitby, ON, Canada]) [12]. PFF was collected from follicles of 2–6 mm in diameter. After centrifugation (1500×*g*, 30 minutes), the supernatant was filtered (0.8 and 0.45 μm) and stored at

Cumulus-oocyte complexes were collected from follicles of 2–6 mm in diameter. They were aspirated with a 10-mL syringe and an 18G needle [11]. The follicular contents were pooled in 50-mL conical tubes (Falcon, Franklin Lakes, NJ, USA). The pellet was washed twice with HEPES-buffered Tyrode's medium containing 0.01% (w/v) polyvinyl alcohol (PVA-TLH) [14]. The COC were recovered under a stereomicroscope and transferred to a petri dish containing PVA-TLH. The COC were washed three times with PVA-TLH and then subjected to their respective treatments. Groups of 20–30 COC were placed in the wells of four-well multi-dishes (Nunc, Roskilde, Denmark) containing 500 μL of maturation medium. The COC were cultured at 38.5°C, 5% CO2 in 95% air atmosphere

The criteria of selection were COC with a minimum of three layers of clear and compact cumulus cells which surrounded the oocyte [12]. Those with dark, pyknotic, or expanded cumulus cells, and those containing oocytes with a very clear cytoplasm or of small diameter were rejected. The oocytes were denuded of their cumulus cells by drawing several times the COC into a pipette using PVA-TLH. Once denuded, the oocytes were rinsed in PVA-TLH, and those with a homo-

The oocyte nuclear maturation stage was evaluated following a 48-hour fixation period in a solution of ethanol and acetic acid (3:1). Using a phase contrast microscope at 100 and 400× magnification immediately after staining with 1% aceto-orcein [15] allows us to assess oocyte nuclear maturation stage. Those having a nuclear membrane were considered at the germinal vesicle (GV) stage, whereas

The chemicals were added to the maturation medium a few hours prior to the addition of the oocytes.

### **2.2 Ovary collections**

*Reproductive Biology and Technology in Animals*

the existence of a so-called guanosinergic system.

in vitro meiotic resumption using swine as the animal model.

and be discovered.

in mice [10] and rat [8].

**2. Material and methods**

**2.1 Chemicals**

small percentage is ovulated, and even less are fertilized. Considering this selective restriction, a framework of the meiotic resumption process has developed. Throughout the evolutionary process, mechanisms have been added to ensure a very precise control of this crucial event related to species survival, which is the final phase of gamete preparation for fertilization. Interestingly, the role of EGF-like peptides fully fits with this notion. It is well-known that the LH peak induces oocyte meiotic resumption in the preovulatory follicles. However, the EGF-like peptides are also active participants in the ovulatory process, meiotic resumption, and cumulus cell expansion, clearly supporting an add-up to the LH surge. Going back to the control of oocyte meiotic resumption, although the contribution of cAMP and cGMP is well described, it is obvious that other mechanisms may still be involved

It has long been known that adenosine (ADO) is a molecule playing an important role in various physiological systems such as in the central nervous system and cardiac function [2]. On the other hand, there are very few studies on the role of ADO in mammalian ovarian follicle. ADO is known to act on specific receptors, to cross plasma membrane using transporters, and to be generated from functional catabolism by extracellular enzymes, a system called adenosinergic [3]. On the other hand, guanosine (GUO) has not been as popular in research. However, in recent years a new interest on GUO has revealed its importance in the effect of ADO on the functioning of the central nervous system [4]. Although no specific receptor for GUO has been yet identified, its physiological impact leaves no doubt. GUO has neuroprotective effects, it diminishes the apoptotic effects observed in Parkinson's disease, and it also has a protective role during a challenge with glutamate, during mitochondrial stress, and during ischemia [4–6]. Because GUO can also be generated by a functional catabolism using extracellular enzymes, these results support

ADO has also been identified in the follicular fluid with several other purine derivatives. Among these derivatives, hypoxanthine is the compound that has attracted the most studies in last three decades. In mice, ADO improves the inhibitory effect of hypoxanthine on the resumption of meiosis but has no effect when used alone, even at a dose of 5 mM [7]. In the rat, the effect of ADO is also minimal [8]. In cattle, ADO used at 200 μM slowed meiotic resumption [9]. There is one study reporting that it has not been able to measure GUO in follicular fluid [10]. In contrast, GUO showed a very potent effect on the inhibition of meiotic resumption

This study is proposing to assess whether an adenosinergic and guanosinergic system are functional and participating in the inhibition of oocyte meiotic resumption. Specifically, the research presented here aims to study the involvement of ADO and GUO in the physiology of the ovarian follicle by targeting their effect on

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The adenylyl cyclase activator, forskolin (FK), was prepared as a millimolar stock solution and stored at −20°C as already described [11]. ADO and GUO were prepared from the stock powder directly in the culture medium on the day of the experiment. 8-Bromoadenosine (8-BrADO) and 8-Bromoguanosine (8-BrGUO) were dissolved in DMSO, and a DMSO control was run simultaneously.

**14**

As previously described, prepubertal gilt ovaries were collected from a local slaughterhouse [12]. In brief, they were placed in saline (0.9% NaCl containing antibiotics and antimycotics, 100,000 IU/L penicillin G, 100 mg/L streptomycin, 250 μg/L amphotericin B) and kept at 37°C. They were rinsed once in a fresh saline solution having antibiotics and antimycotics at 37°C.

#### **2.3 Maturation medium**

Oocytes were matured in BSA-free North Carolina State University 23 (NCSU) medium [13] supplemented with 25 μM β-mercaptoethanol (Bio-Rad, Hercules, CA, USA), 0.1 mg/mL cysteine, 10% (v/v) porcine follicular fluid (PFF), and gonadotropins (2.5 IU/well for hCG [APL, Ayerst Laboratories Inc., Philadelphia, PA, USA] and 2.5 IU/well for eCG [Folligon, Intervet, Whitby, ON, Canada]) [12]. PFF was collected from follicles of 2–6 mm in diameter. After centrifugation (1500×*g*, 30 minutes), the supernatant was filtered (0.8 and 0.45 μm) and stored at −20°C until used [11].

#### **2.4 Recovery of cumulus-oocyte complexes (COC)**

Cumulus-oocyte complexes were collected from follicles of 2–6 mm in diameter. They were aspirated with a 10-mL syringe and an 18G needle [11]. The follicular contents were pooled in 50-mL conical tubes (Falcon, Franklin Lakes, NJ, USA). The pellet was washed twice with HEPES-buffered Tyrode's medium containing 0.01% (w/v) polyvinyl alcohol (PVA-TLH) [14]. The COC were recovered under a stereomicroscope and transferred to a petri dish containing PVA-TLH. The COC were washed three times with PVA-TLH and then subjected to their respective treatments. Groups of 20–30 COC were placed in the wells of four-well multi-dishes (Nunc, Roskilde, Denmark) containing 500 μL of maturation medium. The COC were cultured at 38.5°C, 5% CO2 in 95% air atmosphere with 100% humidity.

#### **2.5 Selecting COC and denuding oocytes**

The criteria of selection were COC with a minimum of three layers of clear and compact cumulus cells which surrounded the oocyte [12]. Those with dark, pyknotic, or expanded cumulus cells, and those containing oocytes with a very clear cytoplasm or of small diameter were rejected. The oocytes were denuded of their cumulus cells by drawing several times the COC into a pipette using PVA-TLH. Once denuded, the oocytes were rinsed in PVA-TLH, and those with a homogeneous cytoplasm were selected.

#### **2.6 Assessment of oocyte nuclear maturation stage**

The oocyte nuclear maturation stage was evaluated following a 48-hour fixation period in a solution of ethanol and acetic acid (3:1). Using a phase contrast microscope at 100 and 400× magnification immediately after staining with 1% aceto-orcein [15] allows us to assess oocyte nuclear maturation stage. Those having a nuclear membrane were considered at the germinal vesicle (GV) stage, whereas

those without a nuclear membrane were considered to have resumed meiosis. The oocytes were considered mature when they were in anaphase I, telophase I, and metaphase II.

#### **2.7 Cumulus-cumulus gap-FRAP assay to measure gap junction communications (GJC)**

After 4 hours of in vitro culture, COC were loaded with calcein-AM (39,69-di(*O*-acetyl)-29,79-bis(*N*,*N*-bis(carboxymethyl) amino methyl)-fluorescein) and tetra(acetoxymethyl ester) (Molecular Probes C-3100) in IVM medium containing 0.1 mg/mL PVA [16]. After 20 minutes at 38.5°C, the live COC were mounted on glass slides in the PVA-containing IVM medium. Fluorescence recovery after photobleaching (FRAP) assays were conducted using Nikon Eclipse TE2000-E inverted confocal microscope. The bleaching was performed for 5 minutes using laser pulses on a limited region of cumulus cells observed at a magnification of 90×. The COC were photographed at 60× before bleaching and every 3 minutes thereafter for 12 minutes. Fluorescence intensity was quantified using ImageJ software (National Institutes of Health, USA). A relative fluorescence value was achieved by dividing the raw fluorescence measurement in the bleached area by the mean fluorescence in two adjacent regions. This value was further divided by the fluorescence value of a region at the opposite end of the COC to correct for unintended bleaching caused by the laser excitation.

#### **2.8 Statistical analyses**

All values are presented with their corresponding SEM, and the number of replicates is indicated for each experiment (at least three). The data were analyzed by one-way ANOVA using GraphPad Prism v7.02 for Windows (GraphPad Software, San Diego, CA, USA). When ANOVA indicated a significant effect of treatment (*P* < 0.05), individual treatment differences were compared using Bonferroni's multiple comparison post-hoc test. Significant effects were identified by \* or different letters.

#### **3. Results**

In our hands, prior to in vitro maturation, more than 95% of swine oocytes have not started their nuclear maturation, that is, their meiotic resumption (<5% of GVDB oocytes), and cumulus cells have not expanded yet [12].

#### **3.1 Adenosine inhibits oocyte meiotic resumption**

The first experiment is aiming to assess ADO on inhibition of oocyte GVBD. When COC are cultured for 24 hours in control treatment, more than 94.3 ± 3.0% of the oocytes were in GVBD, that is, oocyte meiotic resumption took place (**Figure 1A**). The inhibitory effect of ADO was dose-dependent with only 13.9 ± 2.1% in GVBD after an exposure to ADO at 2.0 mM for 24 hours (**Figure 1A**). The EC50 of ADO on the percentage of GVBD oocyte is calculated as already described [11] and determined to be 1.3 mM. In addition, at 2.0 mM ADO inhibited cumulus cell expansion (**Figure 1B5**) compared with the control (**Figure 1B1**). When cultured for 24 hours as oocytes denuded of their cumulus cells (DO), ADO (2.0 mM) did not significantly inhibit oocyte GVBD compared with the control conditions (**Figure 1C**). These results support an inhibitory effect of ADO on both oocyte meiotic resumption and cumulus expansion when cultured as COC but not as DO.

**17**

**Figure 1.**

adenosine, 8-BrADO.

**3.3 Reversibility of GVBD inhibition**

*0.5 mM, (4) ADO 1.0 mM, and (5) ADO 2.0 mM.*

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

**3.2 Cell-permeable analog of adenosine (8-BrADO) inhibits GVBD**

The following experiment is performed to assess a membrane-permeable analog of ADO, 8-BrADO (mimicking the intracellular effect). When COC are cultured for 24 hours, 12.2 ± 2.7% of the oocytes were in GVBD when treated with 2.0 mM ADO and 15.9 ± 0.8% when treated with 2.0 mM 8-BrADO (**Figure 2A**). These two treatments are significantly different to the control treatment (88.7 ± 2.9%; **Figure 2A**). The inhibition of cumulus cell expansion is also observed in both treatments (**Figure 2B2** and **B3**). These results support that the inhibitory effect of ADO may be mimicked by the cell-permeable analog of

*Dose-response of ADO on both (A) the percentage of GVBD oocyte and (B) cumulus cell expansion. (C) Effect of ADO (2.0 mM) on the percentage of denuded oocytes in GVBD. Data are presented as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In (B) treatments were (1) Ct, (2) ADO 0.25 mM, (3) ADO*

To assess the reversibility, the test compounds (ADO and 8-BrADO) are washed

out, and the COC are cultured under control conditions for a second 24-hour

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

#### **Figure 1.**

*Reproductive Biology and Technology in Animals*

**communications (GJC)**

caused by the laser excitation.

**2.8 Statistical analyses**

**3. Results**

metaphase II.

those without a nuclear membrane were considered to have resumed meiosis. The oocytes were considered mature when they were in anaphase I, telophase I, and

**2.7 Cumulus-cumulus gap-FRAP assay to measure gap junction**

After 4 hours of in vitro culture, COC were loaded with calcein-AM (39,69-di(*O*-acetyl)-29,79-bis(*N*,*N*-bis(carboxymethyl) amino methyl)-fluorescein) and tetra(acetoxymethyl ester) (Molecular Probes C-3100) in IVM medium containing 0.1 mg/mL PVA [16]. After 20 minutes at 38.5°C, the live COC were mounted on glass slides in the PVA-containing IVM medium. Fluorescence recovery after photobleaching (FRAP) assays were conducted using Nikon Eclipse TE2000-E inverted confocal microscope. The bleaching was performed for 5 minutes using laser pulses on a limited region of cumulus cells observed at a magnification of 90×. The COC were photographed at 60× before bleaching and every 3 minutes thereafter for 12 minutes. Fluorescence intensity was quantified using ImageJ software (National Institutes of Health, USA). A relative fluorescence value was achieved by dividing the raw fluorescence measurement in the bleached area by the mean fluorescence in two adjacent regions. This value was further divided by the fluorescence value of a region at the opposite end of the COC to correct for unintended bleaching

All values are presented with their corresponding SEM, and the number of replicates is indicated for each experiment (at least three). The data were analyzed by one-way ANOVA using GraphPad Prism v7.02 for Windows (GraphPad Software, San Diego, CA, USA). When ANOVA indicated a significant effect of treatment (*P* < 0.05), individual treatment differences were compared using Bonferroni's multiple compari-

In our hands, prior to in vitro maturation, more than 95% of swine oocytes have

not started their nuclear maturation, that is, their meiotic resumption (<5% of

The first experiment is aiming to assess ADO on inhibition of oocyte GVBD. When COC are cultured for 24 hours in control treatment, more than 94.3 ± 3.0% of the oocytes were in GVBD, that is, oocyte meiotic resumption took place (**Figure 1A**). The inhibitory effect of ADO was dose-dependent with only 13.9 ± 2.1% in GVBD after an exposure to ADO at 2.0 mM for 24 hours (**Figure 1A**).

The EC50 of ADO on the percentage of GVBD oocyte is calculated as already described [11] and determined to be 1.3 mM. In addition, at 2.0 mM ADO inhibited cumulus cell expansion (**Figure 1B5**) compared with the control (**Figure 1B1**). When cultured for 24 hours as oocytes denuded of their cumulus cells (DO), ADO (2.0 mM) did not significantly inhibit oocyte GVBD compared with the control conditions (**Figure 1C**). These results support an inhibitory effect of ADO on both oocyte meiotic resumption and cumulus expansion when cultured as COC but not as DO.

GVDB oocytes), and cumulus cells have not expanded yet [12].

**3.1 Adenosine inhibits oocyte meiotic resumption**

son post-hoc test. Significant effects were identified by \* or different letters.

**16**

*Dose-response of ADO on both (A) the percentage of GVBD oocyte and (B) cumulus cell expansion. (C) Effect of ADO (2.0 mM) on the percentage of denuded oocytes in GVBD. Data are presented as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In (B) treatments were (1) Ct, (2) ADO 0.25 mM, (3) ADO 0.5 mM, (4) ADO 1.0 mM, and (5) ADO 2.0 mM.*

#### **3.2 Cell-permeable analog of adenosine (8-BrADO) inhibits GVBD**

The following experiment is performed to assess a membrane-permeable analog of ADO, 8-BrADO (mimicking the intracellular effect). When COC are cultured for 24 hours, 12.2 ± 2.7% of the oocytes were in GVBD when treated with 2.0 mM ADO and 15.9 ± 0.8% when treated with 2.0 mM 8-BrADO (**Figure 2A**). These two treatments are significantly different to the control treatment (88.7 ± 2.9%; **Figure 2A**). The inhibition of cumulus cell expansion is also observed in both treatments (**Figure 2B2** and **B3**). These results support that the inhibitory effect of ADO may be mimicked by the cell-permeable analog of adenosine, 8-BrADO.

#### **3.3 Reversibility of GVBD inhibition**

To assess the reversibility, the test compounds (ADO and 8-BrADO) are washed out, and the COC are cultured under control conditions for a second 24-hour

#### **Figure 2.**

*Comparison of the effect of ADO (2.0 mM) with the cell-permeable analog 8-BrADO (2.0 mM) on both the percentage of GVBD oocytes and cumulus cell expansion after (A–B) 24 hours and (C–D) reversibility of 24 hours (Rev). DMSO treatment at 0.1% is the control for 8-BrADO. (C) Reversibility: COC were first cultured for 24 hours according to treatment and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ±SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of treatment at P < 0.05 is shown by different letters and \*. In (B), treatments were (1) Ct, (2) ADO, and (3) 8-BrADO. In (D) treatments were (1) Ct, (2) ADO, (3) DMSO, and (4) 8-BrADO.*

period. The ADO-treated oocytes resumed meiosis showing no statistical difference with the control treatment, whereas a significant lower percentage of GVBD oocytes is observed following treatment with 8-BrADO (**Figure 2C**). Cumulus cell expansion is compromised by 8-BrADO (**Figure 2D4**), supporting an impairment of oocyte maturation (nuclear maturation and cumulus cell expansion) when using the halogenate compound. However, cumulus cells are expanding following reversibility treatment of the ADO-treated COC (**Figure 2D2**), supporting the reversibility of the ADO inhibitory effect. The appropriate controls are presented in **Figure 2D1** and **D3**. These data are supportive of an adenosinergic system involved in the control of oocyte meiotic resumption.

**19**

**Figure 3.**

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

**assay**

**3.4 Gap junction communications measured by cumulus-cumulus gap-FRAP**

tion [17, 18], the aim of the following experiment was to measure the impact of

Since gap junction communications are highly regulated during in vitro matura-

*Effect of ADO (A) on gap junction communication, (B) according to hormonal supplementation, and (C) according to FK supplementation. (A) The effect of ADO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The COC were prepared using calcein-AM as fluorescent probe. The data were plotted as relative intensity and presented as the mean ±SEM of a minimum of three replicates. ADO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (B) The effect of ADO according to hormonal supplementation on the percentage of GVBD oocyte after 24 hours of in vitro maturation. ADO (2.0 mM) was added either with eCG or hCG or both eCG and hCG. Data are expressed as the mean ± SEM of a minimum of three replicates. Statistically significant effect of the treatment at P < 0.05 is shown by \*. (C) The effect of ADO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses.*

*Statistically significant effect of the treatment at P < 0.05 is shown by \*.*

*Reproductive Biology and Technology in Animals*

**18**

**Figure 2.**

in the control of oocyte meiotic resumption.

period. The ADO-treated oocytes resumed meiosis showing no statistical difference with the control treatment, whereas a significant lower percentage of GVBD oocytes is observed following treatment with 8-BrADO (**Figure 2C**). Cumulus cell expansion is compromised by 8-BrADO (**Figure 2D4**), supporting an impairment of oocyte maturation (nuclear maturation and cumulus cell expansion) when using the halogenate compound. However, cumulus cells are expanding following reversibility treatment of the ADO-treated COC (**Figure 2D2**), supporting the reversibility of the ADO inhibitory effect. The appropriate controls are presented in **Figure 2D1** and **D3**. These data are supportive of an adenosinergic system involved

*ADO, and (3) 8-BrADO. In (D) treatments were (1) Ct, (2) ADO, (3) DMSO, and (4) 8-BrADO.*

*Comparison of the effect of ADO (2.0 mM) with the cell-permeable analog 8-BrADO (2.0 mM) on both the percentage of GVBD oocytes and cumulus cell expansion after (A–B) 24 hours and (C–D) reversibility of 24 hours (Rev). DMSO treatment at 0.1% is the control for 8-BrADO. (C) Reversibility: COC were first cultured for 24 hours according to treatment and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ±SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of treatment at P < 0.05 is shown by different letters and \*. In (B), treatments were (1) Ct, (2)*

#### **3.4 Gap junction communications measured by cumulus-cumulus gap-FRAP assay**

Since gap junction communications are highly regulated during in vitro maturation [17, 18], the aim of the following experiment was to measure the impact of

#### **Figure 3.**

*Effect of ADO (A) on gap junction communication, (B) according to hormonal supplementation, and (C) according to FK supplementation. (A) The effect of ADO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The COC were prepared using calcein-AM as fluorescent probe. The data were plotted as relative intensity and presented as the mean ±SEM of a minimum of three replicates. ADO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (B) The effect of ADO according to hormonal supplementation on the percentage of GVBD oocyte after 24 hours of in vitro maturation. ADO (2.0 mM) was added either with eCG or hCG or both eCG and hCG. Data are expressed as the mean ± SEM of a minimum of three replicates. Statistically significant effect of the treatment at P < 0.05 is shown by \*. (C) The effect of ADO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*.*

ADO treatment on gap junction communications measured by cumulus-cumulus gap-FRAP assay using calcein-AM as already described [12, 16]. Gap junction communication in cumulus cells was assessed after a 4-hour incubation in the presence of ADO (2.0 mM). The results show that gap junction communications between cumulus cells is increased following ADO treatment compared with the control treatment (**Figure 3A**). These results support that ADO maintains functional gap junctional communications between cumulus cells.

#### **3.5 The contribution of eCG in adenosine treatment**

The goal of the following experiment was to assess the contribution of gonadotropins in ADO-treated COC. The effect of ADO in inhibiting GVBD oocytes is observed in the presence of either eCG or hCG, or both in combination (**Figure 3B**). Comparison of ADO treatment supplemented with these hormones revealed no significant difference between eCG and the combination of eCG and hCG. However, a significant decrease in the percentage of GVBD was measured

#### **Figure 4.**

*The effect of GUO (A) on the percentage of GVBD oocytes, (B) on cumulus cell expansion, (C) on reversibility, (D) on gap junction communication, and (E) according to FK supplementation. (A–B) Comparison of the effect of GUO (2.0 mM) and 8-BrGUO (2.0 mM) after 24 hours. (C) Reversibility: COC were first cultured for 24 hours in the presence of 2.0 mM of GUO and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (D) The effect of GUO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The data were plotted as relative intensity and presented as the mean ± SEM of a minimum of three replicates. GUO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (E) The effect of GUO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In (B), treatments were (1) Ct, (2) GUO, and (3) 8-BrGUO.*

**21**

**Figure 5.**

*(4) ADO 2.0 mM and GUO 2.0 mM.*

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

**3.6 Adenylyl cyclase activator (forskolin)**

adenosinergic system.

between hCG alone and the combination of hCG and eCG (**Figure 3B**), showing the significant contribution of eCG to ADO-inhibiting GVBD, yet supporting an

Since eCG contributes to ADO inhibitory effect, the following experiment was to evaluate the involvement of cAMP while using the adenylyl cyclase activator, forskolin. On itself, forskolin is significantly decreasing the percentage of GVBD oocytes (**Figure 3C**). ADO was significantly inhibiting GVBD both in presence of eCG-hCG and forskolin (**Figure 3C**). However, forskolin was not significantly

*The effect of supplementing both ADO and GUO (2.0 mM each) on (A) the percentage of oocyte meiotic resumption and (B) cumulus cell expansion. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (B) The treatments were (1) Ct, (2) ADO 2.0 mM, (3) GUO 2.0 mM,*

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

between hCG alone and the combination of hCG and eCG (**Figure 3B**), showing the significant contribution of eCG to ADO-inhibiting GVBD, yet supporting an adenosinergic system.

#### **3.6 Adenylyl cyclase activator (forskolin)**

Since eCG contributes to ADO inhibitory effect, the following experiment was to evaluate the involvement of cAMP while using the adenylyl cyclase activator, forskolin. On itself, forskolin is significantly decreasing the percentage of GVBD oocytes (**Figure 3C**). ADO was significantly inhibiting GVBD both in presence of eCG-hCG and forskolin (**Figure 3C**). However, forskolin was not significantly

*Reproductive Biology and Technology in Animals*

junctional communications between cumulus cells.

**3.5 The contribution of eCG in adenosine treatment**

ADO treatment on gap junction communications measured by cumulus-cumulus gap-FRAP assay using calcein-AM as already described [12, 16]. Gap junction communication in cumulus cells was assessed after a 4-hour incubation in the presence of ADO (2.0 mM). The results show that gap junction communications between cumulus cells is increased following ADO treatment compared with the control treatment (**Figure 3A**). These results support that ADO maintains functional gap

The goal of the following experiment was to assess the contribution of gonadotropins in ADO-treated COC. The effect of ADO in inhibiting GVBD oocytes is observed in the presence of either eCG or hCG, or both in combination (**Figure 3B**). Comparison of ADO treatment supplemented with these hormones revealed no significant difference between eCG and the combination of eCG and hCG. However, a significant decrease in the percentage of GVBD was measured

*The effect of GUO (A) on the percentage of GVBD oocytes, (B) on cumulus cell expansion, (C) on reversibility, (D) on gap junction communication, and (E) according to FK supplementation. (A–B) Comparison of the effect of GUO (2.0 mM) and 8-BrGUO (2.0 mM) after 24 hours. (C) Reversibility: COC were first cultured for 24 hours in the presence of 2.0 mM of GUO and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (D) The effect of GUO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The data were plotted as relative intensity and presented as the mean ± SEM of a minimum of three replicates. GUO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (E) The effect of GUO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In*

*(B), treatments were (1) Ct, (2) GUO, and (3) 8-BrGUO.*

**20**

**Figure 4.**

#### **Figure 5.**

*The effect of supplementing both ADO and GUO (2.0 mM each) on (A) the percentage of oocyte meiotic resumption and (B) cumulus cell expansion. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (B) The treatments were (1) Ct, (2) ADO 2.0 mM, (3) GUO 2.0 mM, (4) ADO 2.0 mM and GUO 2.0 mM.*

changing the percentage of GVBD compared with eCG-hCG (**Figure 3C**). The results are proposing the contribution of cAMP to ADO-inhibiting GVBD.

#### **3.7 Guanosine inhibits oocyte meiotic resumption**

High-performance liquid chromatography was used to detect the presence of GUO in porcine follicular fluid from follicles of 2–6 mm in diameter (data not shown). Since GUO was detected in PFF, we undertook to assess whether GUO could play a role in inhibiting GVBD. **Figure 4** shows the significant effect of GUO in inhibiting both GVBD and cumulus expansion after 24 hours, highlighting the contribution of GUO in oocyte maturation. However, the membrane-permeable analog 8-BrGUO did not significantly inhibit GVBD (**Figure 4A**). Cumulus cell expansion was observed (**Figure 4B3**), supporting the inefficacy of 8-BrGUO in inhibiting oocyte GVBD and cumulus cell expansion. The reversibility treatment showed that GUO-treated COC resumed meiosis without any statistical significance compared with the control treatment (**Figure 4C**), supporting the reversibility of GUO inhibitory effect on GVBD. As ADO, GUO was also significantly increasing gap junction communication as measured by gap-FRAP assay (**Figure 4D**). Finally, forskolin was not significantly improving the inhibitory effect of GUO compared to eCG-hCG supplementation as measured on the percentage of GVBD oocytes (**Figure 4E**). GUO was also reversibly inhibiting oocyte GVBD, thus supporting a guanosinergic system.

#### **3.8 The effect of using both ADO and GUO**

The final experiment evaluated the effect of supplementing both ADO and GUO at 2.0 mM each on GVBD oocytes using COC (**Figure 5A**). The results showed that ADO, GUO, and both ADO and GUO were all significantly decreasing GVDB percentage compared with the control treatment. Although a significant effect of adding ADO with GUO was observed when compared with GUO (**Figure 5A**), the impact on the percentage went from 28.6 ± 1.4% for GUO to 11.8 ± 2.5% for ADO and GUO. Cumulus cell expansion was inhibited by ADO (**Figure 5B2**), GUO (**Figure 5B3**), and the combination of ADO and GUO (**Figure 5B4**) when compared with the control (**Figure 5B1**).

#### **4. Discussion**

The present study showed that both ADO and GUO are efficient in reversibly inhibiting swine oocyte GVBD (**Figures 1** and **4**). Cumulus cell expansion is significantly inhibited after 24 hours in presence to either ADO or GUO (**Figures 1** and **4**), supporting that these two purine nucleosides inhibit oocyte maturation with low GVBD percentages and no cumulus cell expansion. Both ADO and GUO also increased GJC in cumulus cells (**Figures 3** and **4**). eCG and FK are both supporting the two purines' inhibitory effect (**Figures 3** and **4**). The data suggest that the inhibitory effect of these two purines on GVBD strengthens the involvement of both adenosinergic and guanosinergic systems in meiotic resumption.

Several years ago it was clearly shown that hypoxanthine, a purine derivative, was an important component of a low molecular weight fraction from porcine follicular fluid and efficient at inhibiting oocyte GVBD [19]. It was also reported that this fraction did not exclusively contain hypoxanthine [10]. Although hypoxanthine was efficient in the mouse and rat, the question was still not fully assessed regarding the efficacy of ADO and GUO on swine COC with respect to the inhibition of oocyte meiotic resumption.

**23**

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

From a broader perspective, it is known that ADO is involved in several biological functions such as nucleotide biosynthesis and cellular energy metabolism [19]. The cellular uptake of ADO played by two classes of nucleotide transporters (SLC28 and SLC29) regulates these biological functions. Inside the cell, ADO is rapidly metabolized and either converted to inosine or adenosine monophosphate through adenosine deaminase or adenosine kinase, respectively [20]. Alternatively, extracellular ADO may serve as a signaling molecule, which activates adenosine receptors (ADORA) on the cell membrane surface. Four different seven-transmembrane domain receptors have been described [21]. It has been reported that a low dose of ADO (0.2 mM) produced a transient delay in bovine oocyte GVBD [9]. After 21 hours in culture, neither ADO nor hypoxanthine resulted in an efficient inhibition of oocyte GVBD [9]. In the mouse, 4.0 mM of hypoxanthine was clearly efficient at inhibiting oocyte GVBD, while the use of ADO by itself did not produce a significant inhibition [10]. However, the efficacy was enhanced when ADO was used in combination with hypoxanthine [7]. This was also observed when ADO was used together with FSH [22], forskolin [23], or cAMP analogs such as 8-bromocAMP [8]. Although these results have been provided to support that adenosine uptake and metabolism contribute to the inhibition of GVBD [24], ample evidence shows that functional adenosine receptors are present on ovarian cells [25]. The measured concentration of ADO in murine follicular fluid was between 0.38 and 0.68 mM [7]. In the present study, the inhibitory effect on GVBD was dosedependent with an IC50 of 1.3 mM (**Figure 1**). Using 2.0 mM, ADO was efficient at reversibly inhibiting swine oocyte GVBD and cumulus cell expansion (**Figure 1**). Although hypoxanthine and ADO have been measured in PFF [10], GUO has never been reported. In the present study, GUO has been found in PFF from 2 to 6 mm diameter follicles (data not shown). While limited information is available in the literature regarding GUO, it has been described as having important functions as an intercellular messenger especially in the central nervous system [4]. The mechanism underlying the neuroprotective properties of GUO is still not fully understood. One working hypothesis is that GUO may exert its biological effect by synchronizing distinct signaling pathways that may be related to the activation of purinergic receptor and specific G-protein binding sites [26–28]. In this study, 2.0 mM of GUO is reversibly inhibiting GVBD and cumulus cell expansion (**Figure 5**). In the mouse, GUO (1.0 mM) was reported to inhibit GVBD, while the same concentration of either hypoxanthine or ADO was inefficient [10]. In rats, the reported order of potency of these nucleosides was GUO > hypoxanthine > ADO [8]. In the mouse, there is an assumption based on a synergistic effect of ADO and GUO on the inhibition oocyte GVBD [7]. In the present study, the combination at 2.0 mM significantly increased the effect of GUO (**Figure 5**). Thus, the inhibitory effect of the combination of both ADO and GUO are somewhat additive according to the concentration used. Although GUO is proposed to activate a specific G-protein-coupled receptor with the involvement of P1 receptor [29], it has been recently reported that GUO functions as an extracellular signaling molecule without the need for GUO receptors [30]. In vascular smooth muscle cells, extracellular GUO regulated extracellular ADO [30]. This proposed GUO-ADO mechanism further regulated cell proliferation in vitro [31] and decreased inflammation in vivo

[32], supporting the additive inhibitory effect observed on oocyte GVBD.

We recently demonstrated the regulation of gap junction communications between cumulus cells during in vitro maturation [16–18]. An increase in gap junction communication was evident after 4 hours of in vitro culture [17, 18]. This observation provides an indication of how cumulus cells respond to the treatment and not only looking at cumulus cell expansion. In the present study, the effect of both treatments, ADO and GUO, was to increase gap junction communications

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

*Reproductive Biology and Technology in Animals*

guanosinergic system.

**4. Discussion**

**3.8 The effect of using both ADO and GUO**

pared with the control (**Figure 5B1**).

**3.7 Guanosine inhibits oocyte meiotic resumption**

changing the percentage of GVBD compared with eCG-hCG (**Figure 3C**). The results are proposing the contribution of cAMP to ADO-inhibiting GVBD.

High-performance liquid chromatography was used to detect the presence of GUO in porcine follicular fluid from follicles of 2–6 mm in diameter (data not shown). Since GUO was detected in PFF, we undertook to assess whether GUO could play a role in inhibiting GVBD. **Figure 4** shows the significant effect of GUO in inhibiting both GVBD and cumulus expansion after 24 hours, highlighting the contribution of GUO in oocyte maturation. However, the membrane-permeable analog 8-BrGUO did not significantly inhibit GVBD (**Figure 4A**). Cumulus cell expansion was observed (**Figure 4B3**), supporting the inefficacy of 8-BrGUO in inhibiting oocyte GVBD and cumulus cell expansion. The reversibility treatment showed that GUO-treated COC resumed meiosis without any statistical significance compared with the control treatment (**Figure 4C**), supporting the reversibility of GUO inhibitory effect on GVBD. As ADO, GUO was also significantly increasing gap junction communication as measured by gap-FRAP assay (**Figure 4D**). Finally, forskolin was not significantly improving the inhibitory effect of GUO compared to eCG-hCG supplementation as measured on the percentage of GVBD oocytes (**Figure 4E**). GUO was also reversibly inhibiting oocyte GVBD, thus supporting a

The final experiment evaluated the effect of supplementing both ADO and GUO at 2.0 mM each on GVBD oocytes using COC (**Figure 5A**). The results showed that ADO, GUO, and both ADO and GUO were all significantly decreasing GVDB percentage compared with the control treatment. Although a significant effect of adding ADO with GUO was observed when compared with GUO (**Figure 5A**), the impact on the percentage went from 28.6 ± 1.4% for GUO to 11.8 ± 2.5% for ADO and GUO. Cumulus cell expansion was inhibited by ADO (**Figure 5B2**), GUO (**Figure 5B3**), and the combination of ADO and GUO (**Figure 5B4**) when com-

The present study showed that both ADO and GUO are efficient in reversibly inhibiting swine oocyte GVBD (**Figures 1** and **4**). Cumulus cell expansion is significantly inhibited after 24 hours in presence to either ADO or GUO (**Figures 1** and **4**), supporting that these two purine nucleosides inhibit oocyte maturation with low GVBD percentages and no cumulus cell expansion. Both ADO and GUO also increased GJC in cumulus cells (**Figures 3** and **4**). eCG and FK are both supporting the two purines' inhibitory effect (**Figures 3** and **4**). The data suggest that the inhibitory effect of these two purines on GVBD strengthens the involvement of

Several years ago it was clearly shown that hypoxanthine, a purine derivative, was an important component of a low molecular weight fraction from porcine follicular fluid and efficient at inhibiting oocyte GVBD [19]. It was also reported that this fraction did not exclusively contain hypoxanthine [10]. Although hypoxanthine was efficient in the mouse and rat, the question was still not fully assessed regarding the efficacy of ADO and GUO on swine COC with respect to the inhibition of oocyte meiotic resumption.

both adenosinergic and guanosinergic systems in meiotic resumption.

**22**

From a broader perspective, it is known that ADO is involved in several biological functions such as nucleotide biosynthesis and cellular energy metabolism [19]. The cellular uptake of ADO played by two classes of nucleotide transporters (SLC28 and SLC29) regulates these biological functions. Inside the cell, ADO is rapidly metabolized and either converted to inosine or adenosine monophosphate through adenosine deaminase or adenosine kinase, respectively [20]. Alternatively, extracellular ADO may serve as a signaling molecule, which activates adenosine receptors (ADORA) on the cell membrane surface. Four different seven-transmembrane domain receptors have been described [21]. It has been reported that a low dose of ADO (0.2 mM) produced a transient delay in bovine oocyte GVBD [9]. After 21 hours in culture, neither ADO nor hypoxanthine resulted in an efficient inhibition of oocyte GVBD [9]. In the mouse, 4.0 mM of hypoxanthine was clearly efficient at inhibiting oocyte GVBD, while the use of ADO by itself did not produce a significant inhibition [10]. However, the efficacy was enhanced when ADO was used in combination with hypoxanthine [7]. This was also observed when ADO was used together with FSH [22], forskolin [23], or cAMP analogs such as 8-bromocAMP [8]. Although these results have been provided to support that adenosine uptake and metabolism contribute to the inhibition of GVBD [24], ample evidence shows that functional adenosine receptors are present on ovarian cells [25]. The measured concentration of ADO in murine follicular fluid was between 0.38 and 0.68 mM [7]. In the present study, the inhibitory effect on GVBD was dosedependent with an IC50 of 1.3 mM (**Figure 1**). Using 2.0 mM, ADO was efficient at reversibly inhibiting swine oocyte GVBD and cumulus cell expansion (**Figure 1**).

Although hypoxanthine and ADO have been measured in PFF [10], GUO has never been reported. In the present study, GUO has been found in PFF from 2 to 6 mm diameter follicles (data not shown). While limited information is available in the literature regarding GUO, it has been described as having important functions as an intercellular messenger especially in the central nervous system [4]. The mechanism underlying the neuroprotective properties of GUO is still not fully understood. One working hypothesis is that GUO may exert its biological effect by synchronizing distinct signaling pathways that may be related to the activation of purinergic receptor and specific G-protein binding sites [26–28]. In this study, 2.0 mM of GUO is reversibly inhibiting GVBD and cumulus cell expansion (**Figure 5**). In the mouse, GUO (1.0 mM) was reported to inhibit GVBD, while the same concentration of either hypoxanthine or ADO was inefficient [10]. In rats, the reported order of potency of these nucleosides was GUO > hypoxanthine > ADO [8]. In the mouse, there is an assumption based on a synergistic effect of ADO and GUO on the inhibition oocyte GVBD [7]. In the present study, the combination at 2.0 mM significantly increased the effect of GUO (**Figure 5**). Thus, the inhibitory effect of the combination of both ADO and GUO are somewhat additive according to the concentration used. Although GUO is proposed to activate a specific G-protein-coupled receptor with the involvement of P1 receptor [29], it has been recently reported that GUO functions as an extracellular signaling molecule without the need for GUO receptors [30]. In vascular smooth muscle cells, extracellular GUO regulated extracellular ADO [30]. This proposed GUO-ADO mechanism further regulated cell proliferation in vitro [31] and decreased inflammation in vivo [32], supporting the additive inhibitory effect observed on oocyte GVBD.

We recently demonstrated the regulation of gap junction communications between cumulus cells during in vitro maturation [16–18]. An increase in gap junction communication was evident after 4 hours of in vitro culture [17, 18]. This observation provides an indication of how cumulus cells respond to the treatment and not only looking at cumulus cell expansion. In the present study, the effect of both treatments, ADO and GUO, was to increase gap junction communications

compared with the control (**Figures 3** and **4**). Since gap junction communications are known to play a primordial role in oocyte maturation [33, 34], this result supports a clear impact of the treatments on cumulus cell functions that may be beneficial for the oocyte.

This study also provides evidence of hormonal supplementation impacting the inhibition of GVBD oocytes by ADO (**Figure 3**). This ADO-mediated inhibition of GVBD oocytes is improved according to the supplementation. Although it is not the purpose of this study to understand how this effect is transduced into the cells, eCG clearly improved the effect of ADO in the presence of hCG. This FSH-type stimulation seemed to be sufficient since the effect of eCG alone was not significantly different from that of eCG and hCG (**Figure 3**). As it is well-known that cumulus cells have an efficient response to FSH [35, 36], the inhibitory effect of ADO is significantly increased by FSH. This effect has been reported in the rat where the percentage of GVBD oocyte treated with ADO was decreased in the presence of FSH [22]. In addition, the FSH-induced granulosa cell differentiation was reduced by ADO [37], supporting the involvement of ADO in FSH response.

Although different forms of adenylyl cyclase have been characterized in oocytes and in cumulus cells [38], forskolin, a known adenylyl cyclase activator, makes a significant contribution to the inhibition of spontaneous maturation in several species as observed for rat [39], bovine [40], and porcine [11] oocytes. In the present study, forskolin inhibited oocyte GVBD while treated with ADO (**Figure 3C**). Similar results were obtained in the presence of GUO (**Figure4E**). These results support that constant stimulation of adenylyl cyclase, which increases the intracellular concentration of cAMP [40], promoted the inhibitory effects of ADO and GUO. In this regard, the results are proposing the contribution of cAMP to both ADO- and GUO-inhibiting GVBD.

The effect of the two purines goes well with the current working model of inhibition of oocyte GVBD involving C-type natriuretic peptide (CNP) as an oocytemeiosis-inhibiting peptide [41, 42]. CNP is involved in inhibiting oocyte GVBD [41, 43, 44]. CNP produced by granulosa cells is a ligand for NPR2, a member of guanylyl cyclase receptor family. NPR2 stimulation by CNP increased intracellular concentration of cGMP, inhibited oocyte phosphodiesterase type 3A, and thus maintained high intra-oocyte concentration of cAMP. The contribution of both purine nucleosides supports adenosinergic and guanosinergic system in the inhibition of oocyte meiotic resumption.

#### **5. Conclusion**

In conclusion, this study puts forward the contribution of ADO and GUO as inhibitory for oocyte GVBD in vitro suggesting that both adenosinergic and guanosinergic systems are efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to explore in order to improve oocyte developmental competence.

Finally, it should be emphasized that the signaling involved in oocyte meiotic resumption may be modulated through the contribution of different pathways. The adenosinergic and guanosinergic systems of which we have presented the contribution illustrate this situation for meiotic resumption. It is also to be expected that other studies will pave the way for additional contributions. For example, a preliminary study from our lab revealed that, as demonstrated in bovine [45], porcine theca cells secreted efficient factors involved in oocyte meiotic resumption. Without knowing these secreted elements, this result highlights that oocyte meiotic resumption is under the control of the ovarian follicular cells. Meiotic resumption is thus under high surveillance!

**25**

**Author details**

François J. Richard

Centre de recherche en reproduction, développement et santé intergénérationnelle, Département des Sciences Animales, Faculté des sciences de l'agriculture et de

© 2019 The Author(s). Licensee IntechOpen. 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,

l'alimentation, Université Laval, Québec, Québec, Canada

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

for his contribution to the experimental work.

The author has no conflict of interest to declare.

This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-04774). Appreciation is extended to Mario Mayes

**Acknowledgements**

**Conflict of interest**

### **Acknowledgements**

*Reproductive Biology and Technology in Animals*

beneficial for the oocyte.

compared with the control (**Figures 3** and **4**). Since gap junction communications are known to play a primordial role in oocyte maturation [33, 34], this result supports a clear impact of the treatments on cumulus cell functions that may be

by ADO [37], supporting the involvement of ADO in FSH response.

the contribution of cAMP to both ADO- and GUO-inhibiting GVBD.

explore in order to improve oocyte developmental competence.

tion of oocyte meiotic resumption.

thus under high surveillance!

**5. Conclusion**

This study also provides evidence of hormonal supplementation impacting the inhibition of GVBD oocytes by ADO (**Figure 3**). This ADO-mediated inhibition of GVBD oocytes is improved according to the supplementation. Although it is not the purpose of this study to understand how this effect is transduced into the cells, eCG clearly improved the effect of ADO in the presence of hCG. This FSH-type stimulation seemed to be sufficient since the effect of eCG alone was not significantly different from that of eCG and hCG (**Figure 3**). As it is well-known that cumulus cells have an efficient response to FSH [35, 36], the inhibitory effect of ADO is significantly increased by FSH. This effect has been reported in the rat where the percentage of GVBD oocyte treated with ADO was decreased in the presence of FSH [22]. In addition, the FSH-induced granulosa cell differentiation was reduced

Although different forms of adenylyl cyclase have been characterized in oocytes and in cumulus cells [38], forskolin, a known adenylyl cyclase activator, makes a significant contribution to the inhibition of spontaneous maturation in several species as observed for rat [39], bovine [40], and porcine [11] oocytes. In the present study, forskolin inhibited oocyte GVBD while treated with ADO (**Figure 3C**). Similar results were obtained in the presence of GUO (**Figure4E**). These results support that constant stimulation of adenylyl cyclase, which increases the intracellular concentration of cAMP [40], promoted the inhibitory effects of ADO and GUO. In this regard, the results are proposing

The effect of the two purines goes well with the current working model of inhibition of oocyte GVBD involving C-type natriuretic peptide (CNP) as an oocytemeiosis-inhibiting peptide [41, 42]. CNP is involved in inhibiting oocyte GVBD [41, 43, 44]. CNP produced by granulosa cells is a ligand for NPR2, a member of guanylyl cyclase receptor family. NPR2 stimulation by CNP increased intracellular concentration of cGMP, inhibited oocyte phosphodiesterase type 3A, and thus maintained high intra-oocyte concentration of cAMP. The contribution of both purine nucleosides supports adenosinergic and guanosinergic system in the inhibi-

In conclusion, this study puts forward the contribution of ADO and GUO as inhibitory for oocyte GVBD in vitro suggesting that both adenosinergic and guanosinergic systems are efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to

Finally, it should be emphasized that the signaling involved in oocyte meiotic resumption may be modulated through the contribution of different pathways. The adenosinergic and guanosinergic systems of which we have presented the contribution illustrate this situation for meiotic resumption. It is also to be expected that other studies will pave the way for additional contributions. For example, a preliminary study from our lab revealed that, as demonstrated in bovine [45], porcine theca cells secreted efficient factors involved in oocyte meiotic resumption. Without knowing these secreted elements, this result highlights that oocyte meiotic resumption is under the control of the ovarian follicular cells. Meiotic resumption is

**24**

This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-04774). Appreciation is extended to Mario Mayes for his contribution to the experimental work.

### **Conflict of interest**

The author has no conflict of interest to declare.

### **Author details**