Abstract

Purinergic signaling is a sophisticated system of elements in which ATP and related molecules function as intercellular messengers. When ATP is released into the extracellular space, it activates specific receptors that belong to the P2 family. In parallel, ectonucleotidases transform ATP in its dephosphorylated metabolites including adenosine, which stimulates P1 receptors. The activity of both receptors influences various cellular processes. Moreover, metabolic conditions are concatenated with purine signaling to conform a dynamic and continuous informational network. The role of purinergic signaling in ovarian cells has been investigated, for instance, it is known that cells conforming the follicle express functional receptors that modulate basic cellular process such as proliferation, induction of apoptotic cell death, and steroidogenesis. In this chapter, we review contemporary information on purinergic action in ovarian cell physiology and state its relevance in this field.

Keywords: purinergic signaling, ovary, granulosa, theca, OSE

## 1. Introduction

The ovary is a complex cell system where folliculogenesis and steroid hormone synthesis take place. These processes involve dynamic changes in the cellular populations of this tissue and highly precise mechanisms of regulation. Folliculogenesis requires a complex coordination of three stages: recruitment, which is the initial growth of a group of follicles from the reserve pool; selection, during which a subgroup from this pool of recruited follicles survives and grows, while the remainders suffer atresia; and dominance, in which the follicles that will be ovulated reach the preovulatory stage and increase their size, while the subordinate follicles arrest their growth. It is well known that the initial stages of folliculogenesis are independent of gonadotropins, whereas the advanced stages depend on these hormones. At the same time, selection and dominance stages implicate the elimination of subordinated follicles by apoptosis; thus, a coordinated set of events directs growth and surveillance of some follicles and disappearance of others with extreme precision [1].

Folliculogenesis involves constant rearrangement in ovarian cellular architecture. A primordial follicle is formed by an oocyte surrounded by a layer of

squamous epithelial cells and is arrested in the diplotene phase of meiosis I. When it is recruited and enters the growing stage, granulosa cells change their shape from flat to cuboid, starting their proliferation; this entity is known as primary follicle. The granulosa layer continues proliferating to adopt a stratified epithelium; then, the theca layer surrounding the follicle emerges. This layer is innervated and vascularized conforming a secondary follicle. Inside the follicle, three to four cavities filled with fluid are formed; the cavities fuse and form the antrum. This stage is known as antral follicle. Antrum formation implicates the emergence of two granulosa populations: cumulus cells in contact with the oocyte and mural cells attached to the follicular theca. These follicles grow until reaching the preovulatory stage [1]. Changes in follicle development are orchestrated by diverse cellular messengers (reviewed in [2]).

Moreover, synthesis and secretion of ovarian steroid hormones are coordinated by the somatic components of the follicle; thus, cholesterol and acetate uptake from the blood by the theca layer allows the synthesis of androgens that are aromatized into estrogens in the granulosa cells [3]. This process is finely regulated by the coordinated action of intraovarian and endocrine components.

All these dynamic processes are organized at distinct levels by endocrine, nervous, and autocrine-paracrine mechanisms acting with high systematic precision. Recent findings suggest that purinergic signaling participates in the control of cellular process in the follicle cells, making it a new player in the network of signals regulating the cellular biology of ovarian physiology. Given that follicle cell types have defined roles and are typical for each follicular growth stage, receptor expression in each of these cell types suggests a potential physiological role; thus, we organized the information of this chapter to cover the role played by purinergic receptors in ovarian physiology.

### 2. Purinergic system

From a chemical perspective, purines are defined as nitrogen-containing heterocyclic aromatic compounds formed by the fusion of a pyrimidine and an imidazole ring (in total, five carbon and four nitrogen atoms). The purine molecule can be associated with an amino group, such as adenine (6-amino purine), or with amino and keto groups, such as guanine (2-amino-6-oxy purine). The existence of functional groups that are weakly acidic next to a system of conjugated dienes favors the relocation of a proton along the molecule, allowing the formation of constitutional isomers called tautomers. The principal tautomeric equilibriums in purine molecules are amino (predominant)-imino forms and lactam (predominant)-lactim forms [4].

Figure 1.

derivatives.

27

Purines in cellular signaling and metabolic networks. The figure depicts two cells in communication using purines as signaling molecules. Released ATP is recognized by two types of receptors (P2X, ionotropic, and P2Y, metabotropic). ATP can be hydrolyzed by different ectonucleotidases (such as CD39 and CD73) to be transformed in less phosphorylated nucleotides (ADP and AMP) and eventually to the nucleoside adenosine (ADO). The receptors that mediate ADO's actions are all metabotropic. Thus, purinergic signaling is the result of the types of receptors present in the cellular system, as well as the variable proportion of adenine nucleotides and ADO that result from the enzymatic activities of the expressed ectonucleotidases. There are membrane transporters with the capacity to transfer ADO to the cellular inside. Within the cell, ADO can be transformed in multiple metabolites and act as modulator of strategic metabolic pathways. ADO is catabolized by adenosine deaminase (ADA) resulting in a set of purines that culminates with uric acid (a mild antioxidant) with

adenosine kinase, ADO can turn into a nucleotide (AMP) that is a modulator of the AMPK, which is an important enzyme to regulate energy homeostasis. AMP can be further phosphorylated to ADP and ATP. Eventually, ATP can go to the extracellular space to fulfill its messenger role by exocytosis or by specialized channels (pannexin and connexin hemichannels). The proportion of adenine nucleotides (energy charge) is an important modulator of the equilibrium between catabolic and anabolic reactions. It has been reported that ADO is able to increase the cellular energy charge in the liver. When ADO binds homocysteine to form S-adenosylhomocysteine (SAH), it can also influence the transmethylation and transsulfuration pathways. SAH is usually formed when the methylating agent S-adenosylmethionine (SAM) transfers a methyl moiety to a given substrate. Interestingly, SAM can also participate in the synthesis of polyamines by transferring an aminopropyl group from decarboxylated SAM to putrescine. Homocysteine is joined to serine to form the intermediary cystathionine, which is transformed into cysteine and the intracellular messenger H2S.

Meanwhile, cysteine can be incorporated into the redox and antioxidant molecule glutathione (GSH) or can be converted into the osmolyte taurine. Hence, a condition in which ADO is increased beyond a threshold may accumulate SAH with the consequent interruption of the methylating reaction and the promotion of polyamine synthesis, as well as the reduction of homocysteine availability and the decrease in cystathionine and all its

). By the action of

xanthine as an intermediary with the capability to generate the anion superoxide (O2

Purinergic Signaling: A New Regulator of Ovarian Function

DOI: http://dx.doi.org/10.5772/intechopen.80756

Purines are part of important informational polymers, such as nucleic acids (DNA and RNA), and of various biomolecules with metabolic and functional significance such as bioenergetic factors (ATP and GTP), redox coenzymes (NAD (P)H/NAD(P)<sup>+</sup> , FMNH2/FMN+ , and FADH2/FAD<sup>+</sup> ), biochemical crossroad metabolites (coenzyme A), and signaling molecules (cyclic AMP, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate [NAADP]) and ligands for purinergic receptors. Caffeine and theobromine are naturally occurring purines in plants [5]. In this section, we will refer exclusively to ATP, adenosine (ADO), and other related molecules as factors in cellular communication and coordinators of metabolic networks (Figure 1).

Purinergic Signaling: A New Regulator of Ovarian Function DOI: http://dx.doi.org/10.5772/intechopen.80756

#### Figure 1.

squamous epithelial cells and is arrested in the diplotene phase of meiosis I. When it is recruited and enters the growing stage, granulosa cells change their shape from flat to cuboid, starting their proliferation; this entity is known as primary follicle. The granulosa layer continues proliferating to adopt a stratified epithelium; then, the theca layer surrounding the follicle emerges. This layer is innervated and vascularized conforming a secondary follicle. Inside the follicle, three to four cavities filled with fluid are formed; the cavities fuse and form the antrum. This stage is known as antral follicle. Antrum formation implicates the emergence of two granulosa populations: cumulus cells in contact with the oocyte and mural cells attached to the follicular theca. These follicles grow until reaching the preovulatory stage [1]. Changes in follicle development are orchestrated by diverse cellular mes-

Moreover, synthesis and secretion of ovarian steroid hormones are coordinated by the somatic components of the follicle; thus, cholesterol and acetate uptake from the blood by the theca layer allows the synthesis of androgens that are aromatized into estrogens in the granulosa cells [3]. This process is finely regulated by the

All these dynamic processes are organized at distinct levels by endocrine, nervous, and autocrine-paracrine mechanisms acting with high systematic precision. Recent findings suggest that purinergic signaling participates in the control of cellular process in the follicle cells, making it a new player in the network of signals regulating the cellular biology of ovarian physiology. Given that follicle cell types have defined roles and are typical for each follicular growth stage, receptor expression in each of these cell types suggests a potential physiological role; thus, we organized the information of this chapter to cover the role played by purinergic

From a chemical perspective, purines are defined as nitrogen-containing heterocyclic aromatic compounds formed by the fusion of a pyrimidine and an imidazole ring (in total, five carbon and four nitrogen atoms). The purine molecule can be associated with an amino group, such as adenine (6-amino purine), or with amino and keto groups, such as guanine (2-amino-6-oxy purine). The existence of functional groups that are weakly acidic next to a system of conjugated dienes favors the relocation of a proton along the molecule, allowing the formation of constitutional isomers called tautomers. The principal tautomeric equilibriums in purine molecules are amino (predominant)-imino forms and lactam (predomi-

Purines are part of important informational polymers, such as nucleic acids (DNA and RNA), and of various biomolecules with metabolic and functional significance such as bioenergetic factors (ATP and GTP), redox coenzymes (NAD

, and FADH2/FAD<sup>+</sup>

olites (coenzyme A), and signaling molecules (cyclic AMP, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate [NAADP]) and ligands for purinergic receptors. Caffeine and theobromine are naturally occurring purines in plants [5]. In this section, we will refer exclusively to ATP, adenosine (ADO), and other related molecules as factors in cellular communication and coordinators of

), biochemical crossroad metab-

coordinated action of intraovarian and endocrine components.

sengers (reviewed in [2]).

Adenosine Triphosphate in Health and Disease

receptors in ovarian physiology.

2. Purinergic system

nant)-lactim forms [4].

metabolic networks (Figure 1).

, FMNH2/FMN+

(P)H/NAD(P)<sup>+</sup>

26

Purines in cellular signaling and metabolic networks. The figure depicts two cells in communication using purines as signaling molecules. Released ATP is recognized by two types of receptors (P2X, ionotropic, and P2Y, metabotropic). ATP can be hydrolyzed by different ectonucleotidases (such as CD39 and CD73) to be transformed in less phosphorylated nucleotides (ADP and AMP) and eventually to the nucleoside adenosine (ADO). The receptors that mediate ADO's actions are all metabotropic. Thus, purinergic signaling is the result of the types of receptors present in the cellular system, as well as the variable proportion of adenine nucleotides and ADO that result from the enzymatic activities of the expressed ectonucleotidases. There are membrane transporters with the capacity to transfer ADO to the cellular inside. Within the cell, ADO can be transformed in multiple metabolites and act as modulator of strategic metabolic pathways. ADO is catabolized by adenosine deaminase (ADA) resulting in a set of purines that culminates with uric acid (a mild antioxidant) with xanthine as an intermediary with the capability to generate the anion superoxide (O2 ). By the action of adenosine kinase, ADO can turn into a nucleotide (AMP) that is a modulator of the AMPK, which is an important enzyme to regulate energy homeostasis. AMP can be further phosphorylated to ADP and ATP. Eventually, ATP can go to the extracellular space to fulfill its messenger role by exocytosis or by specialized channels (pannexin and connexin hemichannels). The proportion of adenine nucleotides (energy charge) is an important modulator of the equilibrium between catabolic and anabolic reactions. It has been reported that ADO is able to increase the cellular energy charge in the liver. When ADO binds homocysteine to form S-adenosylhomocysteine (SAH), it can also influence the transmethylation and transsulfuration pathways. SAH is usually formed when the methylating agent S-adenosylmethionine (SAM) transfers a methyl moiety to a given substrate. Interestingly, SAM can also participate in the synthesis of polyamines by transferring an aminopropyl group from decarboxylated SAM to putrescine. Homocysteine is joined to serine to form the intermediary cystathionine, which is transformed into cysteine and the intracellular messenger H2S. Meanwhile, cysteine can be incorporated into the redox and antioxidant molecule glutathione (GSH) or can be converted into the osmolyte taurine. Hence, a condition in which ADO is increased beyond a threshold may accumulate SAH with the consequent interruption of the methylating reaction and the promotion of polyamine synthesis, as well as the reduction of homocysteine availability and the decrease in cystathionine and all its derivatives.

Adenine-related molecules are ubiquitously present in all living beings. ATP (a nucleotide) and ADO (a nucleoside) are easily interconverted by a set of three phosphorylation/dephosphorylation steps. Interestingly, this purine conversion involves different mechanisms when it takes places either inside or outside the cellular milieu. Intermediates of these reactions are AMP and ADP nucleotides, whereas some metabolically important ADO derivatives are uric acid, S-adenosyl methionine (SAM), and S-adenosylhomocysteine (SAH). Various physiopathological events modulated by ATP and/or ADO have been reported, including sleep, immunity, tumorigenesis, platelet aggregation, vasodilation, inflammatory and hypoxic responses, and antioxidant status [6]. Overall, adenine nucleotides and ADO are interconnected signaling factors that activate specific membrane receptors and act as metabolic regulators that coordinate the equilibrium between anabolic and catabolic reactions [7].

means of the regulatory parameter known as energy charge (ATP + ½ADP/ ATP + ADP + AMP). In this context, ATP and AMP act as allosteric factors for a variety of enzymes that are important in the bioenergetic status of the cell: phosphofructokinase 1 (glycolysis), aspartate carbamoyltransferase (pyrimidine synthesis), and glycogen phosphorylase (glycogenolysis). Interestingly, ADO treatment is one of the few cases that can upregulate the energy charge in vivo [12]. Another example of how the AMP/ATP ratio influences the metabolic networks is AMP kinase (AMPK) activation. AMPK is a strategic kinase that modulates the fasting

Purinergic Signaling: A New Regulator of Ovarian Function

DOI: http://dx.doi.org/10.5772/intechopen.80756

response by phosphorylating and activating key catabolic enzymes [13].

tonin), and nucleic acids (during epigenesis and RNA processing). SAM is transformed into SAH, which is hydrolyzed into ADO and homocysteine. In this metabolic step, ADO levels can modulate the methylation process, as high ADO favors SAH synthesis, thus blocking methyl donation. SAM is also a substrate for polyamine synthesis. In the presence of serine, homocysteine is converted into cystathionine. Within the mitochondria, this intermediate is the precursor of both the

gasotransmitter H2S and the principal antioxidant agent, glutathione [16].

Extracellular and intracellular purine dynamics are interconnected, mainly through specialized protein transporters that allow ATP and ADO to transit

ATP exists at millimolar levels within the cell. It exits to the extracellular space, where it acts as a cellular messenger through two main paths: (1) exocytic, which involves secretion of vesicles mainly derived from Golgi and secretory ATPcontaining granules by means of Ca2+-dependent membrane depolarization and (2)

net flux of purines can be visualized: first, purine rings exiting as ATP and eventually purines returning to the cell interior as ADO. Indeed, this cycle involves the net efflux

The transformation among purine molecules inside and outside the cell has the potential of intricately regulating both purinergic signaling and metabolic control.


conductive, in which the ATP efflux is carried out by different ion channels: hexamers of connexin subunits, assemblies of pannexin subunits, volume-regulated anion channels, and maxi-anion channels [17]. As mentioned, ATP can lose its phosphates in the extracellular space and transform into ADO. In turn, ADO can return to the intracellular milieu by the action of two types of carriers; one is driven by a facilitated diffusion event (sensitive to dipyridamole), and the other mobilizes ADO

of phosphate as well as the net conversion of intracellular ATP into ADO.

2.3 Membrane transporters

throughout the plasma membrane.

by an active process regulated by the Na+

2.4 Integrative considerations

29

ADO is a crossroad metabolite; it can be turned into nucleotides (first in AMP by adenosine kinase), or it can originate active catabolites such as xanthine (a source of superoxide) and uric acid (a terminal metabolite and mild antioxidant). The production of ADO in the liver and its transport through the blood are controlled by the circadian timing system; eventually, these mechanisms allow the 24-h rhythmic presence of purine rings in the nervous system, which are necessary for the onset of sleeping [14]. ADO has also been used as a hepatoprotective and antitumoral agent [15].

ADO also plays a role in the transmethylation and transsulfuration pathways, with SAM as a central metabolite for both. Initially, ATP activates methionine, which in turn generates SAM, the main cellular methylating agent. Some principal methylated molecules are phospholipids (phosphatidylethanolamine turns into phosphatidylcholine), catecholamines (adrenaline turns into noradrenaline and serotonin into mela-

#### 2.1 Purinergic communication

ATP and ADO are well-known signaling molecules. Both purines have the capacity to promote a set of cellular responses by acting through specific membrane receptors, either by activating ionic conductance or by promoting the formation of second messengers. Two families of receptors for purine ligands have been characterized: (1) P1 or ADORA receptors, which are G-coupled metabotropic adenosine receptors and are classified as A1 and A3 (associated with adenylate cyclase inhibition and formation of IP3 and diacylglycerol) and as A2A and A2B (both activate adenylate cyclase), (2) P2 receptors, which recognize ATP as a principal ligand but also a variety of related compounds. P2 receptors are divided into ionotropic receptors (P2X1–7) and G-coupled metabotropic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11–P2Y14). In addition to ATP, P2Y receptors can be recognized by alternative ligands such as ADP, UTP, UDP, and UDP glucose [8].

Unlike other signal transduction systems, purinergic signaling involves the actions of two interconvertible ligands, ATP and ADO, within the same system. Interestingly, these ligands exert complementary or antagonistic actions on each other [9]. For example, epithelial-mesenchymal transition (EMT), a cellular plasticity process important in phenotypic programming of metastatic tumors, can be upregulated by ATP in SKOV-3 cells (from ovarian carcinoma) but downregulated by ADO [10]. This circumstance indicates that enzymes (ectonucleotidases) allowing the sequential conversion from ATP to ADO are a potential regulatory node that controls diverse cellular and physiological responses.

Four families of extracellular enzymes that transform purine nucleotides into ADO and phosphate are known: (1) ENTPD/CD39 (ectonucleoside triphosphate diphosphohydrolase) forms AMP from ATP/ADP; (2) ENPP (ectonucleotide pyrophosphatase) forms AMP from ATP/ADP/ADP-ribose; (3) alkaline phosphatase hydrolyzes different nucleotides to be transformed into ADO; and (4) NT5E (ecto-5′-nucleotidase) forms ADO from AMP [11]. It is expected that the combined and sequential activities of these enzymes in a given cell system result in a highly variable and potentially fine-tuned proportion of adenine nucleotides (ATP, ADP, and AMP) and ADO. Therefore, the physiological equilibrium between ATPergic and adenosinergic transmissions should be considered as a unique and emergent property of each cell system.

#### 2.2 Purine-related metabolites

Adenine purines are key elements in metabolic network control. The proportion of adenine nucleotides dictates the direction of anabolic and catabolic processes by

#### Purinergic Signaling: A New Regulator of Ovarian Function DOI: http://dx.doi.org/10.5772/intechopen.80756

Adenine-related molecules are ubiquitously present in all living beings. ATP (a nucleotide) and ADO (a nucleoside) are easily interconverted by a set of three phosphorylation/dephosphorylation steps. Interestingly, this purine conversion involves different mechanisms when it takes places either inside or outside the cellular milieu. Intermediates of these reactions are AMP and ADP nucleotides, whereas some metabolically important ADO derivatives are uric acid, S-adenosyl methionine (SAM), and S-adenosylhomocysteine (SAH). Various physiopathological events modulated by ATP and/or ADO have been reported, including sleep, immunity, tumorigenesis, platelet aggregation, vasodilation, inflammatory and hypoxic responses, and antioxidant status [6]. Overall, adenine nucleotides and ADO are interconnected signaling factors that activate specific membrane receptors and act as metabolic regulators that coordinate the equilibrium between anabolic

ATP and ADO are well-known signaling molecules. Both purines have the capacity to promote a set of cellular responses by acting through specific membrane receptors, either by activating ionic conductance or by promoting the formation of second messengers. Two families of receptors for purine ligands have been characterized: (1) P1 or ADORA receptors, which are G-coupled metabotropic adenosine receptors and are classified as A1 and A3 (associated with adenylate cyclase inhibition and formation of IP3 and diacylglycerol) and as A2A and A2B (both activate adenylate cyclase), (2) P2 receptors, which recognize ATP as a principal ligand but also a variety of related compounds. P2 receptors are divided into ionotropic receptors (P2X1–7) and G-coupled metabotropic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11–P2Y14). In addition to ATP, P2Y receptors can be recognized by alternative

Unlike other signal transduction systems, purinergic signaling involves the actions of two interconvertible ligands, ATP and ADO, within the same system. Interestingly, these ligands exert complementary or antagonistic actions on each other [9]. For example, epithelial-mesenchymal transition (EMT), a cellular plasticity process important in phenotypic programming of metastatic tumors, can be upregulated by ATP in SKOV-3 cells (from ovarian carcinoma) but downregulated by ADO [10]. This circumstance indicates that enzymes (ectonucleotidases) allowing the sequential conversion from ATP to ADO are a potential regulatory

Four families of extracellular enzymes that transform purine nucleotides into ADO and phosphate are known: (1) ENTPD/CD39 (ectonucleoside triphosphate diphosphohydrolase) forms AMP from ATP/ADP; (2) ENPP (ectonucleotide pyrophosphatase) forms AMP from ATP/ADP/ADP-ribose; (3) alkaline phosphatase hydrolyzes different nucleotides to be transformed into ADO; and (4) NT5E (ecto-5′-nucleotidase) forms ADO from AMP [11]. It is expected that the combined and sequential activities of these enzymes in a given cell system result in a highly variable and potentially fine-tuned proportion of adenine nucleotides (ATP, ADP, and AMP) and ADO. Therefore, the physiological equilibrium between ATPergic and adenosinergic transmissions should be considered as a unique and emergent

Adenine purines are key elements in metabolic network control. The proportion of adenine nucleotides dictates the direction of anabolic and catabolic processes by

and catabolic reactions [7].

2.1 Purinergic communication

Adenosine Triphosphate in Health and Disease

property of each cell system.

28

2.2 Purine-related metabolites

ligands such as ADP, UTP, UDP, and UDP glucose [8].

node that controls diverse cellular and physiological responses.

means of the regulatory parameter known as energy charge (ATP + ½ADP/ ATP + ADP + AMP). In this context, ATP and AMP act as allosteric factors for a variety of enzymes that are important in the bioenergetic status of the cell: phosphofructokinase 1 (glycolysis), aspartate carbamoyltransferase (pyrimidine synthesis), and glycogen phosphorylase (glycogenolysis). Interestingly, ADO treatment is one of the few cases that can upregulate the energy charge in vivo [12]. Another example of how the AMP/ATP ratio influences the metabolic networks is AMP kinase (AMPK) activation. AMPK is a strategic kinase that modulates the fasting response by phosphorylating and activating key catabolic enzymes [13].

ADO is a crossroad metabolite; it can be turned into nucleotides (first in AMP by adenosine kinase), or it can originate active catabolites such as xanthine (a source of superoxide) and uric acid (a terminal metabolite and mild antioxidant). The production of ADO in the liver and its transport through the blood are controlled by the circadian timing system; eventually, these mechanisms allow the 24-h rhythmic presence of purine rings in the nervous system, which are necessary for the onset of sleeping [14]. ADO has also been used as a hepatoprotective and antitumoral agent [15].

ADO also plays a role in the transmethylation and transsulfuration pathways, with SAM as a central metabolite for both. Initially, ATP activates methionine, which in turn generates SAM, the main cellular methylating agent. Some principal methylated molecules are phospholipids (phosphatidylethanolamine turns into phosphatidylcholine), catecholamines (adrenaline turns into noradrenaline and serotonin into melatonin), and nucleic acids (during epigenesis and RNA processing). SAM is transformed into SAH, which is hydrolyzed into ADO and homocysteine. In this metabolic step, ADO levels can modulate the methylation process, as high ADO favors SAH synthesis, thus blocking methyl donation. SAM is also a substrate for polyamine synthesis. In the presence of serine, homocysteine is converted into cystathionine. Within the mitochondria, this intermediate is the precursor of both the gasotransmitter H2S and the principal antioxidant agent, glutathione [16].

#### 2.3 Membrane transporters

Extracellular and intracellular purine dynamics are interconnected, mainly through specialized protein transporters that allow ATP and ADO to transit throughout the plasma membrane.

ATP exists at millimolar levels within the cell. It exits to the extracellular space, where it acts as a cellular messenger through two main paths: (1) exocytic, which involves secretion of vesicles mainly derived from Golgi and secretory ATPcontaining granules by means of Ca2+-dependent membrane depolarization and (2) conductive, in which the ATP efflux is carried out by different ion channels: hexamers of connexin subunits, assemblies of pannexin subunits, volume-regulated anion channels, and maxi-anion channels [17]. As mentioned, ATP can lose its phosphates in the extracellular space and transform into ADO. In turn, ADO can return to the intracellular milieu by the action of two types of carriers; one is driven by a facilitated diffusion event (sensitive to dipyridamole), and the other mobilizes ADO by an active process regulated by the Na+ -transmembrane gradient [18]. Overall, a net flux of purines can be visualized: first, purine rings exiting as ATP and eventually purines returning to the cell interior as ADO. Indeed, this cycle involves the net efflux of phosphate as well as the net conversion of intracellular ATP into ADO.

#### 2.4 Integrative considerations

The transformation among purine molecules inside and outside the cell has the potential of intricately regulating both purinergic signaling and metabolic control.

To propose a model of this interaction, the following considerations are necessary: (1) at least three compartments should be taken in account—extracellular, cytoplasmic, and mitochondrial. However, if there is segregation of receptor populations in different membrane domains (e.g., signalosomes), the extracellular compartment could be more complex. (2) It is important to know the principal metabolite and intermediate levels in the process; indeed, the concentrations of these factors are expected to fluctuate, but knowing average levels is necessary. For example, ATP is at [mM] in the intracellular milieu, but when it is released in the pericellular space, it changes from [nM] to [mM] [19]. (3) It is important to know which receptors are present in a given cell system, as well as their physical constants (Kd and Bmax). (4) It is convenient to have a clear idea of the activities and regulation of all ectonucleotidases and their corresponding carriers. (5) In the same context, determining the presence of purine metabolizing enzymes and kinetic constants (Km and Vmax) is required. (6) It is necessary to know the conformational status of all proteins involved in the purine cycle in order to detect potential allosteric modulation.

of Ca2+ ([Ca2+]i) was first investigated in human luteal cells and in porcine

Purinergic Signaling: A New Regulator of Ovarian Function

DOI: http://dx.doi.org/10.5772/intechopen.80756

stores [29].

(SERCA) [33].

31

granulosa cells; it was observed that nucleotides elicited an increment of [Ca2+]i in both cell types. Moreover, in luteal cells, these compounds also induced an increase in progesterone and estradiol secretion [27]. Consistently, in granulosa from hen preovulatory follicles, ATP, and other adenine nucleotides also induced an increment of [Ca2+]i; the pharmacological characterization of this response revealed that it was mediated by intracellular Ca2+ release and dihydropyridine-insensitive Ca2+ channels according to P2Y receptor activation [28]. In human granulosa cells, it was determined that ATP responses were dependent on Ca2+ released from intracellular

The molecular description of the P2Y2 receptor (then named P2U because of its sensitivity to UTP) in human granulosa-luteal cells (GLC) was made by Leung's group in British Columbia [30]. They detected the transcript of P2U receptor by Northern blot and reported the elevation of cAMP promoted by hCG. The stimulation of these cells with UTP/ATP induced an increment of [Ca2+]i associated with phospholipase C (PLC) activation. Downstream of this pathway, protein kinase C (PKC) was activated, and it negatively modulated the P2Y2-dependent [Ca2+]i response [31]. The molecular machinery and mechanism involved in the purineinduced increment of [Ca2+]i have been studied. ATP-induced Ca2+ release mediated by activation of PLC and inositol triphosphate (IP3) production. Indeed, IP3 and ryanodine receptor (RyR) expression in GLC has been demonstrated. The increment of ATP-induced [Ca2+]i was modulated by substances interfering with the activity of both RyR and IP3R, revealing an interplay between both receptors to amplify the purinergic [Ca2+]i signal [32]. Moreover, it was described that GLC expresses three isoforms of IP3R, RyR, and thapsigargin-sensitive Ca2+-ATPase

Electrophysiological and Ca2+ imaging studies in the GFSHR-17 cell line from granulosa determined that P2Y2 and P2Y4 receptor stimulation induces Ca2+ mobilization and hyperpolarization. Both responses were sensitive to the PLC inhibitor U73122 and to the IP3R antagonist 2-aminoethyl diphenyl; hyperpolarization was mediated by Cl channels, probably dependent on intracellular Ca2+ [34].

Moreover, it was shown that the P2Y2 (P2U) activation in human GLC induced a decrease in the LH-dependent cAMP levels; this antigonadotropic effect was mediated by PKCα activity [35]. In parallel, it was shown that P2Y2 stimulation also activated extracellular mitogen-regulated kinases (ERK) through a Gαq-dependent pathway; ERK activity was responsible for inhibiting LH-dependent production of progesterone induced by P2Y2 activation [36]. Further experiments revealed that phospho-ERK translocates to the nucleus and regulates cell proliferation by early growth-1 (egr-1) and c-raf-1 responses [37]. These data clearly show that the purinergic response mediated by P2Y2 in GLC can be an important modulator of

Expression of the P2Y6 receptor was observed in murine GLC. Its stimulation with the selective agonist UDP incremented cell viability and progesterone but did not affect estradiol production. This effect on steroidogenesis was concomitant with a negative regulation of enzymes corresponding to Δ4 steroidogenic pathway, CYP11A, 3β-HSD, and StAR. The effects were blocked by the antagonist MRS2578 before UTP stimulus [38]. These results suggested that purines acting through P2Y6

In human GLC, extracellular ATP promoted apoptosis by activating P2 receptors. These receptors elevated [Ca2+]i, which in turn activated Ca2+-dependent K+ channels, leading to membrane depolarization [39]; however, the specific receptor involved in this response was not identified. These observations were confirmed by a later study that demonstrated the participation of apoptotic marker caspase-3

gonadotropic actions and granulosa cell physiology.

regulate luteal body viability and steroidogenic function.

## 3. Purinergic signaling in the ovary

#### 3.1 Granulosa and luteal cells

Early studies analyzed the effects of ADO over the gonadotropin-induced cAMP production in granulosa and luteal cells. In rat and human granulosa cells, ADO incremented the accumulation of cAMP in response to follicle-stimulating hormone (FSH); similar effects were observed when human luteal cells were stimulated with human chorionic gonadotropin (hCG) or luteinizing hormone (LH), revealing the possibility that ADO is a gonadotropic modulator [20–23]. According to the dual role of ADO in metabolism and cell signaling, it was originally proposed that ADO effects were mediated by two mechanisms acting synergically: ADO translocation to the cytoplasm where the nucleoside can sustain the increment of cytosolic ATP and extracellular activation of specific membrane receptors [20].

In primary cultures of rat granulosa cells, incubation with ADO incremented intracellular ATP; dipyridamole blocked this effect, indicating ADO uptake through specific transporters [22]. The regulation of adenylate cyclase (AC) activity by ADO analogues was investigated in membranes isolated from the whole ovaries in follicular growth. It was observed that adenosinergic agonists incremented adenylate cyclase (AC) activity. Pharmacological approaches suggested that the effect of ADO analogues was mediated by the A2A receptor because it was strongly promoted by 5'-N-ethylcarboxamidoadenosine (NECA) and antagonized by 8-phenyltheophylline (8-PTH) [24]. Similar findings were observed in membranes from luteinized ovaries from superovulated rats induced by injection with pregnant mare serum gonadotropin (PMSG) and in homogenates of isolated cells from luteal bodies; moreover, it was shown that ADO analogues incremented progesterone synthesis. In these preparations, pharmacological evidence also indicated the participation of A2A receptor in the adenosinergic induction of cAMP accumulation, suggesting that ADO is a paracrine regulator of the luteal body's endocrine activity [25].

In addition, it has been shown that in ovine luteal cells, ADO increases the effect on luteotropins [hCG] as well as prostaglandins (E1 and E2) over progesterone production and inhibits the antigonadotropic and luteolytic effect of prostaglandin F2α (PGF2α) [20, 26]. Purinergic responses in ovarian cells were described before cloning P2 receptors. The effect of adenine nucleotides on cytosolic concentration

Purinergic Signaling: A New Regulator of Ovarian Function DOI: http://dx.doi.org/10.5772/intechopen.80756

To propose a model of this interaction, the following considerations are necessary: (1) at least three compartments should be taken in account—extracellular, cyto-

populations in different membrane domains (e.g., signalosomes), the extracellular compartment could be more complex. (2) It is important to know the principal metabolite and intermediate levels in the process; indeed, the concentrations of these factors are expected to fluctuate, but knowing average levels is necessary. For example, ATP is at [mM] in the intracellular milieu, but when it is released in the pericellular space, it changes from [nM] to [mM] [19]. (3) It is important to know which receptors are present in a given cell system, as well as their physical constants (Kd and Bmax). (4) It is convenient to have a clear idea of the activities and regulation of all ectonucleotidases and their corresponding carriers. (5) In the same context, determining the presence of purine metabolizing enzymes and kinetic constants (Km and Vmax) is required. (6) It is necessary to know the conformational status of all proteins involved in the purine cycle in order to detect potential

Early studies analyzed the effects of ADO over the gonadotropin-induced cAMP production in granulosa and luteal cells. In rat and human granulosa cells, ADO incremented the accumulation of cAMP in response to follicle-stimulating hormone (FSH); similar effects were observed when human luteal cells were stimulated with human chorionic gonadotropin (hCG) or luteinizing hormone (LH), revealing the possibility that ADO is a gonadotropic modulator [20–23]. According to the dual role of ADO in metabolism and cell signaling, it was originally proposed that ADO effects were mediated by two mechanisms acting synergically: ADO translocation to the cytoplasm where the nucleoside can sustain the increment of cytosolic ATP and

In primary cultures of rat granulosa cells, incubation with ADO incremented intracellular ATP; dipyridamole blocked this effect, indicating ADO uptake through specific transporters [22]. The regulation of adenylate cyclase (AC) activity by ADO analogues was investigated in membranes isolated from the whole ovaries in follicular growth. It was observed that adenosinergic agonists incremented adenylate cyclase (AC) activity. Pharmacological approaches suggested that the effect of ADO analogues was mediated by the A2A receptor because it was strongly promoted by 5'-N-ethylcarboxamidoadenosine (NECA) and antagonized by 8-phenyltheophylline (8-PTH) [24]. Similar findings were observed in membranes from

luteinized ovaries from superovulated rats induced by injection with pregnant mare serum gonadotropin (PMSG) and in homogenates of isolated cells from luteal bodies; moreover, it was shown that ADO analogues incremented progesterone synthesis. In these preparations, pharmacological evidence also indicated the

participation of A2A receptor in the adenosinergic induction of cAMP accumulation, suggesting that ADO is a paracrine regulator of the luteal body's endocrine

on luteotropins [hCG] as well as prostaglandins (E1 and E2) over progesterone production and inhibits the antigonadotropic and luteolytic effect of prostaglandin F2α (PGF2α) [20, 26]. Purinergic responses in ovarian cells were described before cloning P2 receptors. The effect of adenine nucleotides on cytosolic concentration

In addition, it has been shown that in ovine luteal cells, ADO increases the effect

plasmic, and mitochondrial. However, if there is segregation of receptor

allosteric modulation.

activity [25].

30

3. Purinergic signaling in the ovary

Adenosine Triphosphate in Health and Disease

extracellular activation of specific membrane receptors [20].

3.1 Granulosa and luteal cells

of Ca2+ ([Ca2+]i) was first investigated in human luteal cells and in porcine granulosa cells; it was observed that nucleotides elicited an increment of [Ca2+]i in both cell types. Moreover, in luteal cells, these compounds also induced an increase in progesterone and estradiol secretion [27]. Consistently, in granulosa from hen preovulatory follicles, ATP, and other adenine nucleotides also induced an increment of [Ca2+]i; the pharmacological characterization of this response revealed that it was mediated by intracellular Ca2+ release and dihydropyridine-insensitive Ca2+ channels according to P2Y receptor activation [28]. In human granulosa cells, it was determined that ATP responses were dependent on Ca2+ released from intracellular stores [29].

The molecular description of the P2Y2 receptor (then named P2U because of its sensitivity to UTP) in human granulosa-luteal cells (GLC) was made by Leung's group in British Columbia [30]. They detected the transcript of P2U receptor by Northern blot and reported the elevation of cAMP promoted by hCG. The stimulation of these cells with UTP/ATP induced an increment of [Ca2+]i associated with phospholipase C (PLC) activation. Downstream of this pathway, protein kinase C (PKC) was activated, and it negatively modulated the P2Y2-dependent [Ca2+]i response [31]. The molecular machinery and mechanism involved in the purineinduced increment of [Ca2+]i have been studied. ATP-induced Ca2+ release mediated by activation of PLC and inositol triphosphate (IP3) production. Indeed, IP3 and ryanodine receptor (RyR) expression in GLC has been demonstrated. The increment of ATP-induced [Ca2+]i was modulated by substances interfering with the activity of both RyR and IP3R, revealing an interplay between both receptors to amplify the purinergic [Ca2+]i signal [32]. Moreover, it was described that GLC expresses three isoforms of IP3R, RyR, and thapsigargin-sensitive Ca2+-ATPase (SERCA) [33].

Electrophysiological and Ca2+ imaging studies in the GFSHR-17 cell line from granulosa determined that P2Y2 and P2Y4 receptor stimulation induces Ca2+ mobilization and hyperpolarization. Both responses were sensitive to the PLC inhibitor U73122 and to the IP3R antagonist 2-aminoethyl diphenyl; hyperpolarization was mediated by Cl channels, probably dependent on intracellular Ca2+ [34].

Moreover, it was shown that the P2Y2 (P2U) activation in human GLC induced a decrease in the LH-dependent cAMP levels; this antigonadotropic effect was mediated by PKCα activity [35]. In parallel, it was shown that P2Y2 stimulation also activated extracellular mitogen-regulated kinases (ERK) through a Gαq-dependent pathway; ERK activity was responsible for inhibiting LH-dependent production of progesterone induced by P2Y2 activation [36]. Further experiments revealed that phospho-ERK translocates to the nucleus and regulates cell proliferation by early growth-1 (egr-1) and c-raf-1 responses [37]. These data clearly show that the purinergic response mediated by P2Y2 in GLC can be an important modulator of gonadotropic actions and granulosa cell physiology.

Expression of the P2Y6 receptor was observed in murine GLC. Its stimulation with the selective agonist UDP incremented cell viability and progesterone but did not affect estradiol production. This effect on steroidogenesis was concomitant with a negative regulation of enzymes corresponding to Δ4 steroidogenic pathway, CYP11A, 3β-HSD, and StAR. The effects were blocked by the antagonist MRS2578 before UTP stimulus [38]. These results suggested that purines acting through P2Y6 regulate luteal body viability and steroidogenic function.

In human GLC, extracellular ATP promoted apoptosis by activating P2 receptors. These receptors elevated [Ca2+]i, which in turn activated Ca2+-dependent K+ channels, leading to membrane depolarization [39]; however, the specific receptor involved in this response was not identified. These observations were confirmed by a later study that demonstrated the participation of apoptotic marker caspase-3

[40]. This effect was apparently not consistent with previous observations regarding ATP actions in GLC, but it could be explained by the differential sensitivity of distinct P2 receptors [41]. It is possible that extracellular concentration of ATP and the expression of P2 receptor determine the specific effect of purinergic stimulation.

Furthermore, an interaction between adenosine receptor A2 and P2Y receptors has been described in theca of Xenopus ovarian follicles. The authors suggested that this association took place when both the epithelial and theca cell layers of the

Collectively, these findings suggest that a tight regulation of purinergic expression and signaling must be in place for the theca cell layer to function properly and

The organized structure of the cumulus-enclosed oocyte (CEO) complex corresponds to a specialized GLC surrounding the oocyte. Cumulus cells secrete factors to regulate oocyte maturation and maintain meiotic arrest [56]. It was reported that porcine and murine follicular fluid contains purine compounds that presumably participate in CEO functions, suggesting that it could be an important signal to trigger physiological events [57, 56]. Until recently, purinergic receptors were identified and characterized in CEO, indicating that purinergic signaling partici-

When Eppig et al. discovered that the main components of follicular fluid were nucleotide-derived metabolites [60] and established a relationship with follicle maturation [56, 57], they hypothesized that the local purinergic metabolism in the ovarian fluid can be involved in oocyte maturation or may participate in other

In these studies, the concentration of nucleotide compounds in murine follicular

Eppig et al. also analyzed the same compounds in porcine follicular fluid; they identified that hypoxanthine at 1.4 mM is the major inhibitory component producing a transient inhibition [61]. This observation contrasted with that of other labo-

On the other hand, the cellular effects of purinergic ligands were studied by Ca2+ imaging and electrophysiological approaches. In 2002, two reports elucidated which purinergic receptor was expressed in CEO cells. The first report of P2Y expression in CEO began with an interesting observation that ATP could stimulate an intracellular Ca+2 transient. Experiments using the CEO complex and applying ATP or UTP to the extracellular solution induced a wave of Ca+2 mobilization from cumulus cells to the oocyte through gap junctions, suggesting that ATP was involved in oocyte maturation; moreover, they showed that the response involved the P2Y2 receptor. Since gonadotropin hormones, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) had no effect on Ca+2 changes [59], the authors concluded that ATP was the specific messenger to mediate calcium signals involved in oocyte matura-

In another report, the responses generated by a putative purinergic receptor expressed in the CEO were identified and characterized. Employing the voltage clamp technique with two electrodes, the authors observed depolarization

responses when extracellular ATP was applied. RT-PCR analysis revealed a product correspondent to the P2Y2 receptor, suggesting that calcium mobilization is dependent on this receptor. A detailed description of distinct currents generated from several ionic channels, such as Ca+2-dependent Cl current, voltage-dependent K<sup>+</sup>

concluded that both purinergic receptors and ionic channels were located in CEO

, was provided. The authors

currents, and a cationic current mainly driven by Na<sup>+</sup>

fluid was determined using high-performance liquid chromatography (HPLC). They identified two purine compounds: hypoxanthine and ADO, with concentrations in ranges of 2–4 and 0.3–0.7 mM, respectively. They also showed that these

purines affected the CEO by maintaining the meiotic arrest [56].

ratories which had failed to detect inhibitory activity in follicular fluid.

oocyte were intact [55].

3.3 Cumulus complex

communicate with neighboring cells.

Purinergic Signaling: A New Regulator of Ovarian Function

DOI: http://dx.doi.org/10.5772/intechopen.80756

pates in CEO physiology [58, 59].

aspects of follicular functions.

tion in the CEO complex.

33

Recently it was described that the P2X7 receptor is expressed in mouse luteal cells. Its activation with ATP or BzATP induced an antiproliferative effect by regulating the expression of cyclin D2 and cyclin E2, as well as the phosphorylation of mitogen-activated protein kinase p38 [42]. The result suggests a role for the P2X7 receptor in luteal body function.

The purinergic system is well represented in GLC. A set of purinergic receptors can modulate basic cellular processes such as proliferation, apoptosis, and steroidogenesis. Growing evidence indicates that purines are important regulators of GLC, but further studies are necessary to reinforce their role in ovarian physiology.

### 3.2 Theca cells

The theca cell layer is an enclosure of cells that surrounds the oocyte during folliculogenesis. It is crucial for maintaining the structural integrity of the follicle as well as for regulating nutrient influx to the avascular GCL [43, 44]. Theca is also the site for the synthesis of steroid hormones, specifically androgens (testosterone and dihydrotestosterone), from acetate or cholesterol into estrogens by granulosa cells in an LH-dependent manner [3]. In addition, theca is the only component of the follicle that is innervated by sympathetic and parasympathetic nervous systems, implicating that this layer functions as a complex integrator of endocrine and neural information [45].

When a primary follicle has one or two layers of granulosa cells, an outer granulosa cell layer differentiates into theca cells and, together with recruited theca precursor cells from the stroma, forms the theca cell layer surrounding the oocyte [44, 46]. Some pathophysiological reproduction-related conditions such as infertility or polycystic ovarian syndrome are often the result of dysfunctional activity of theca cells during ovulation and follicle development [47, 48].

The theca cell layer contacts the rich microvasculature system surrounding each follicle and integrates signals from autonomic innervation [49]. It has been demonstrated that ATP can be co-released with noradrenaline from terminals of the peripheral nervous system [50] and as a result of mechanical stress and changes in cell volume in the oocyte [51]; thus, ATP is a relevant modulator of cellular communication between the theca cell layer and surrounding oocyte cells.

Purinergic signaling has been described in female reproductive organs, and evidence has shown that ATP in the extracellular space participates in the physiological regulation of the ovary [52]. The first characterization of purinergic signaling in theca cells showed the functional expression, and activation of P2X7 receptors induced cell death, an important mechanism for the onset and physiological progression of follicle atresia [53]. P2X7 receptors have also been associated with the inhibition of luteal cell survival and proliferation, pointedly in small luteal cells, which have been suggested as theca-derived luteal cells [42].

On the other hand, there is also evidence of the expression of uridine triphosphate (UTP)-sensitive P2Y receptors in theca cells P2Y2 and P2Y6, but not P2Y4 [54]. In this system, stimulation of the expressed P2Y receptors with UTP in cultured theca cells induces the activation of mitogenic-signaling pathways that promote cell proliferation [54]. This finding is a relevant pathophysiological indication, since a slow but maintained proliferation takes place in polycystic ovarian syndrome [47].

Furthermore, an interaction between adenosine receptor A2 and P2Y receptors has been described in theca of Xenopus ovarian follicles. The authors suggested that this association took place when both the epithelial and theca cell layers of the oocyte were intact [55].

Collectively, these findings suggest that a tight regulation of purinergic expression and signaling must be in place for the theca cell layer to function properly and communicate with neighboring cells.
