Prostaglandin Biosynthesis and Action in the Ovary

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Prostaglandin Biosynthesis and Action in the Ovary JEAN SIROIS, DEREK BOERBOOM, AND KHAMPOUNE SAYASITH

which are named as the letters A through J. For instance, prostaglandin F (PGF) compounds have hydroxyls at the C-9 and C-11 positions, and prostaglandin E (PGE) compounds are characterized by a hydroxyl and a ketone group. Additional information in subscripts indicates the number of double bonds in the molecule, and the letter a denotes that the C-9 substituent is located behind the plane of the ring, as in PGF2a (see Figure 13.1) [2].

ABSTRACT The prostaglandins are a family of bioactive lipid molecules believed to be synthesized by every cell type in the body. The last three decades of biomedical research have shown them to function as mediators of a wide range of physiological and pathological processes, from renal hemodynamics and gastrointestinal cytoprotection to inflammation and tumorigenesis. In addition, some of the most important advances in the field of reproductive biology have resulted from the elucidation of the roles played by prostaglandins in the female reproductive tract. Notably, strong lines of pharmacological, biochemical, and genetic evidence now indicate that prostaglandins are crucial for proper ovulation, luteolysis, implantation, decidualization, and parturition. This chapter reviews fundamental aspects of prostaglandin synthesis and action and summarizes our current understanding of prostaglandin function in the ovary.

Biosynthetic Enzymes PHOSPHOLIPASES Arachidonic acid, the principal substrate for prostaglandin biosynthesis, is mainly stored in an esterified state at the sn2 position of cell membrane phospholipids [3]. The first regulated step involved in prostaglandin formation in all tissues involves the hydrolytic release of arachidonate (see Figure 13.1). This is believed to be principally mediated by members of the phospholipase A2 (PLA2) enzyme family. Originally isolated from pancreatic juices and snake venom, the PLA2 family now includes several structurally and functionally diverse enzymes that are involved in different biological processes [3, 4]. Among the PLA2 enzymes involved in prostaglandin biosynthesis is the group IV or cytosolic PLA2 (cPLA2). This enzyme rapidly translocates to the membrane fraction and binds phospholipids in response to receptor activation-induced rises in intracellular Ca2+ levels. Once in the membrane fraction, cPLA2 acts to release the arachidonate needed for the acute/early phase of prostaglandin synthesis [3, 4]. Whereas cPLA2 continues to function in late-phase prostaglandin formation and its expression can be induced by cytokines and mitogens, the type IIA enzyme (called soluble PLA2 or sPLA2) has been shown to be critical in situations where a priming or activation period is required [3, 4]. A current model proposes that in conditions where receptor activation is prolonged, sPLA2 is induced along with the downstream biosynthetic machinery (see later discussion), thereby permitting sustained prostaglandin synthesis [4]. Because sPLA2 is a secreted enzyme, its induction also creates a paracrine amplification loop

PROSTAGLANDINS AND THEIR RECEPTORS Prostaglandins: Structure and Nomenclature Prostaglandins are members of the eicosanoid biomolecule family, which also includes prostacyclin, thromboxane, leukotrienes, and lipoxins [1, 2]. They are derived from open-chain, 20-carbon polyunsaturated fatty acids, typically arachidonic acid. Whereas leukotrienes and lipoxins are formed directly from their parent fatty acid, prostaglandins, prostacyclin, and thromboxanes are derived from a cyclic endoperoxide intermediate, prostaglandin H2 (PGH2) (Figure 13.1). Various classes of prostaglandins can be synthesized from this precursor, and these are categorized into subfamilies according to specific structural criteria. Whereas all prostaglandins consist of a cyclopentane ring to which two side chains are attached at positions 8 and 12, it is the nature of the additional substituents on the ring that categorizes them into a particular series, The Ovary

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because its action on the surface of neighboring cells elicits additional prostaglandin synthesis [4]. PROSTAGLANDIN G/H SYNTHASES PGHS-1, PGHS-2, and PGHS-3. Following its release, arachidonate is converted to PGH2 by the action of prostaglandin G/H synthase (PGHS) (see Figure 13.1), which is situated on the luminal surface of the endoplasmic reticulum and the outer envelope of the nuclear membrane [5]. Numerous studies have demonstrated the existence of two distinct genes encoding isoforms of PGHS, named PGHS-1 and PGHS-2. Both enzymes possess a PGG2-synthetic cyclooxygenase activity that has resulted in their being colloquially referred to as cyclooxygenase-1 and -2 (or COX-1 and -2), but they are also responsible for the rapid conversion of PGG2 to PGH2 via a peroxidase activity [6]. Despite catalytic and structural similarities, PGHS-1 and -2 differ in most other respects, including gene structure and regulation, tissue distribution, and messenger ribonucleic acid (mRNA) stability [6, 7]. In almost all cases, PGHS-1 is considered to play a housekeeping role, because it is constitutively expressed at low levels in most tissues [7]. It is associated with the homeostatic levels of prostaglandin production required for the maintenance of blood perfusion in the kidney and gastric mucosa and for thromboxane synthesis in platelets [7]. PGHS-2 however is normally absent from most tissues but is readily inducible by hormones, cyclic adenosine monophosphate (cAMP), inflammatory factors, growth factors, tumor promoters, and cytokines in a variety of cell systems [6, 7]. Notable examples of PGHS-2 function in

PGF2α

FIGURE 13.1 The prostaglandin biosynthetic pathway. (Modified from Ref. [1], with permission from the American Physiological Society.)

physiological and pathological processes include kidney development; tumorigenesis; embryonic implantation; and mediation of inflammation, fever, and pain [7]. Recently, a third PGHS isoform (called PGHS-3 or COX-3) has been identified and shown to consist of an alternate splicing variant of the PGHS-1 gene in which intron 1 has been retained. This isoform is constitutively expressed at relatively high levels in the cerebral cortex and heart, is highly sensitive to acetaminophen, and is thought to be involved in sensing pain [8]. Nonsteroidal Antiinflammatory Drugs and Selective Cyclooxygenase Inhibitors. Although nonsteroidal antiinflammatory drugs (NSAIDs) have been widely used as pharmacological treatments for a variety of ailments for more than 100 years, it only become clear in the 1970s that they suppress inflammation by inhibiting PGHS activity [9, 10]. This discovery was followed by the elucidation of aspirin’s mechanism of action, which was found to irreversibly inactivate the cyclooxygenase active sites of PGHS-1 and -2 by acetylating them at a single serine residue [10, 11]. In large part because of this lack of PGHS isoform selectivity, the use of aspirin and other traditional NSAIDs often results in renal, gastrointestinal, and other deleterious side effects [10, 11]. The pharmaceutical industry was therefore prompted to develop a new class of COX-2 specific inhibitors (COXIBs), which aimed to improve the therapeutic profile of NSAIDS by targeting the pathological effects of PGHS-2 without disrupting the homeostatic functions of PGHS-1. Two COXIB drugs (celecoxib and rofecoxib) are now available on the U.S. market, with a second and third generation of COXIBs

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now in development [11]. Although widespread use of COXIBs has only just begun, early reports indicate significant reductions in NSAID-associated side effects [12], and new therapeutic applications are under evaluation for conditions such as cancer and Alzheimer’s disease [9, 11]. TERMINAL ENZYMES Subsequent to PGH2 formation by PGHS, specific prostaglandins are synthesized in given cell types according to the particular enzymatic machinery that they express (see Figure 13.1) [7]. Among the more extensively characterized prostaglandin-synthetic enzymes is PGD synthase (PGDS), of which two isoforms have been identified, the lipocalin-type PGDS (also known as beta-trace) and the hematopoietic PGDS [13]. Much like the PGHS enzymes, a constitutive and an induced isoform of PGE synthase have been identified. Microsomal PGE synthase (mPGES, also known as mGST-L1) is a glutathione-dependent enzyme of the MAPEG (membrane-associated proteins involved in eicosanoid and glutathione metabolism) enzyme family [2]. Whereas mPGES can be readily induced by interleukin-1b (IL-1b) and other cytokines, cytosolic PGE synthase (cPGES) expression generally remains basal in the presence of proinflammatory stimuli [2]. Interestingly, evidence is now accumulating that indicates a functional coupling of cPLA2 and PGHS-1 to cPGES as well as cPLA2 and PGHS-2 to mPGES [4]. This coupling could coordinate the activities of these enzymes and thereby provide a novel, subtle level of regulation of prostaglandin biosynthesis [4]. PGF synthase (PGFS) has been characterized most extensively in the bovine species, where “lung-type” and “liver-type” isoforms have been isolated. Both enzymes are members of the aldo-keto reductase superfamily and can catalyze a variety of biochemical reactions, although they differ in tissue distribution and kinetic properties [14, 15]. The enzyme 9-keto-PGE2 reductase (9K-PGR), which converts PGE2 into PGF2a, offers an alternative pathway for the biosynthesis of PGF2a [14]. Only one prostacyclin (PGI2) synthase (PGIS) gene has been characterized. A member of the cytochrome P450 family, PGIS localizes to the endoplasmic reticulum membrane where it presumably coordinates with PGHS to ensure efficient PGI2 synthesis [16, 17].

Prostaglandin Transporters Following their synthesis, prostaglandins are believed to diffuse freely toward the extracellular compartment to act in a paracrine and/or endocrine manner [18]. To permit their metabolic clearance after their interaction with cell surface receptors, prostaglandins must then perform a second passage through a plasma membrane so as to be inactivated (oxidized) by intracellular prostaglandin dehydrogenase. Unlike the initial efflux, this reuptake of prostaglandins is energy-dependent and requires a specific

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carrier mechanism. The first protein found to possess such an activity, the aptly named prostaglandin transporter (PGT), is expressed in a range of tissues and has been shown to coordinately regulate with PGHS-2 [18]. Consisting of 12 transmembrane spans, PGT is believed to mediate the reuptake of prostaglandins into various cell types by functioning as a lactate/prostaglandin exchanger [18]. More recently, additional mediators of prostaglandin transport have been identified that are phylogenetically related to PGT (including LST-1, moat1, and OAT-K2) and together form the organic anion transporting polypeptide (OATP) family. Furthermore, a separate family dubbed the organic anion transporters (OATs) also appears capable of catalyzing prostaglandin uptake [18]. Beyond its role in the clearance of prostaglandins, transport is now thought to play additional, subtle roles in prostaglandin signaling, such as modulating their interaction with cognate receptors by regulating their pericellular concentrations [18].

Prostaglandin Receptors Prostaglandins target specific cells by binding cognate cell surface receptors. To date, four receptor subtypes that bind PGE2 (EP1-EP4) have been characterized, two for PGD2 (DP1 and DP2) and one each for PGF2a and PGI2 (FP and IP, respectively) [19, 20]. In addition, multiple splice variants of EP1, EP3, and FP have been identified, creating additional levels of modulation of intracellular signaling [20]. With the exception of DP2, which is a chemoattractant receptor, all prostaglandin receptors are rhodopsin-type G-protein coupled receptors with seven transmembrane domains [19, 20]. On the basis of homology, physiological properties, and signal transduction mechanisms, these receptors have been subdivided into three groups [19, 21]. Group 1 receptors (IP, DP1, EP2, EP4), known as the relaxant receptors for their effects on smooth muscle, are linked to heterotrimeric G-proteins whose Gsa subunit stimulates adenylate cyclase to increase intracellular cAMP concentrations. The group 2 contractile receptors, which include EP1, FP, and the thromboxane A2 receptor, signal through Gaq and the phospholipase C pathway to increase intracellular Ca2+ levels. The EP3 subtype is the only member of the third group and is called an inhibitory receptor because it couples to the Gia family to decrease cAMP formation. The physiological functions of these receptors have recently been studied in a series of gene knockout experiments that have contributed to our understanding of prostaglandin receptor function [21, 22]. In addition to the classic, membrane-bound receptors, recent evidence now indicates that the peroxisomal proliferatoractivated receptor (PPAR) family of ligand-activated transcription factors can also function as eicosanoid receptors [19, 23]. Notably, PGI2-mediated activation of PPARd could be involved in such wide-ranging processes as embryo implantation, colorectal cancer, and apoptosis [23].

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However, rigorous proof of eicosanoids functioning as genuine PPAR ligands in vivo has yet to be obtained [19].

PROSTAGLANDINS AND THE OVARIAN FOLLICLE Prostaglandins in Preovulatory Follicles: 30 Years and Counting . . . The potential role of prostaglandins in ovarian follicular function was initially proposed in 1972 when indomethacin and aspirin were shown to block ovulation in rats and rabbits [24, 25]. These pioneering studies were followed by numerous investigations that confirmed the inhibitory effect of NSAIDs on ovulation in various species (for reviews, see [26–28]). Concerns were raised initially regarding whether indomethacin blocked ovulation directly at the ovarian level or indirectly via inhibition of gonadotropin secretion at the hypothalamo-pituitary level. However, the matter was resolved by several lines of investigation that showed, for example, that indomethacin does not block the preovulatory luteinizing hormone (LH) surge and that ovulation could be blocked by NSAIDs in an in vitro model using perfused ovaries [26–28]. Another line of evidence supporting the role of prostaglandins during the ovulatory process emerged when it became clear that the preovulatory LH surge causes a marked increase in concentrations of PGE2 and PGF2a in ovarian follicles just before ovulation [26–28]. This gonadotropin-dependent induction of prostaglandin synthesis in preovulatory follicles was documented in rodents, rabbits, pigs, sheep, primates, cows, and mares, with the granulosa cell layer generally recognized as the primary site of synthesis. However, the theca interna is also thought to be an important site of prostaglandin production in some species. The ability of indomethacin to block this preovulatory rise in follicular prostaglandins was seen as further evidence of the role of prostaglandins in ovulation. This concept was strengthened by studies in which systemic or intrafollicular administration of antisera to prostaglandins was shown to be antiovulatory in rabbits and mice [26, 28]. Although PGE2 is produced in larger amounts than PGF2a in several species, the relative importance of each prostaglandin during ovulation became the subject of controversy. The debate was fueled by conflicting results on their respective ability to restore ovulation in indomethacin-treated animals and the ability of PGE2 vs PGF2a antibodies to inhibit follicular rupture [27, 31]. A more fundamental dispute emerged when even the role of prostaglandins during ovulation was questioned because, for example, prostaglandins were unable to reverse the antiovulatory effect of indomethacin in some studies [27–30]. Although the precise basis for these contradictory

findings remains unclear, results from genetic studies in mice now provide convincing evidence for an obligatory role of prostaglandins during ovulation, with PGE2 serving as the principal actor in this species [31]. The preovulatory LH surge is the most potent physiological stimulator of ovarian prostaglandin synthesis and acts primarily via stimulation of an adenylate cyclase coupled membrane receptor. However, several other agonists acting at various stages of follicular development have been identified in ovarian cells. Gonadotropin-releasing hormone (Gn-RH) is a strong agonist of prostaglandin production in rodents and acts via specific phospholipase C-coupled receptors present on granulosa cells [32, 33]. Other stimulators include several growth factors, such as growth differentiation factor-9 (GDF-9), transforming growth factor-a (TGF-a), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), insulin-like growth factor 1 (IGF-1), as well as IGF binding protein-3 [34–38]. IL-1b, oxytocin, estradiol, and tumor necrosis factor-a (TNF-a) are also thought to be produced locally within the ovary and to stimulate prostaglandin synthesis [38–41]. In contrast, few negative regulators of follicular prostaglandin production have been identified. They include TGF-b [35] and progesterone that, by virtue of decreasing gonadotropin-stimulated PGE2 synthesis in rat preovulatory follicles, could act as an antiinflammatory steroid to limit the inflammatory reaction associated with the ovulatory process [42]. Numerous roles have been ascribed to prostaglandins during ovulation. They were proposed to be involved in inducing a local inflammatory reaction, acting as vasoactive agents and contributing to hyperemia, causing contraction of smooth muscle fibers in theca layers, affecting progesterone synthesis by granulosa cells, and regulating hyaluronan synthesis by cumulus cells [27, 29, 43]. More recently, they were also shown to mediate, at least in part, the gonadotropin-regulated and protein kinase C-dependent production of reactive oxygen species in preovulatory follicles [44]. The effect of prostaglandins on the enzymatic breakdown of the follicular wall, and more precisely on the regulation of plasminogen activator and follicular collagenases, has been extensively studied [29, 45]. Results from investigations in the early 1990s suggest that they would be important in mediating the gonadotropin-dependent induction of interstitial collagenolytic activity, responsible for the degradation of interstitial collagen present in the theca and tunica albuginea layers at the time of follicular rupture [45]. Indomethacin was shown to suppress the hCG-induced rise in interstitial collagenase but not that of collagenase IV responsible for the disintegration of the basement membrane, which provided a potential molecular basis for intraovarian ovulation observed in indomethacin-treated animals. Further evidence for such uncontrolled follicular collagenolytic activities in NSAID-treated animals was recently provided in meticulous histological studies in

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which follicular rupture was found to be abnormal but not inhibited [46]. Such a finding suggests that counting oocytes in the oviducts is not an accurate predictor of follicular rupture in indomethacin-treated animals [46].

Regulation of Follicular Phospholipases A2 The mobilization of arachidonic acid from cell membrane phospholipids by PLA2 is the initial step in prostaglandin biosynthesis, and cPLA2 and sPLA2 have been detected in ovarian follicles. In hen granulosa cells, cPLA2 is expressed at various stages of follicular development, with TGF-a stimulating and TGF-b inhibiting expression of the enzyme. Such reciprocal regulation of cPLA2 was proposed to modulate the prostaglandin synthetic capacity and mitogenic response of granulosa cells to growth factors during follicular development [47]. Studies in rats suggest that the expression cPLA2 and sPLA2 increases with advancing stages of follicular development, with both enzymes present in large mature follicles. Despite the marked increase in prostaglandin synthesis before ovulation, the expression of cPLA2 and sPLA2 was shown to remain relatively constant during the periovulatory period in vivo [48, 49]. However, studies in vitro using isolated granulosa cells or dispersed whole rat ovaries identified various agonists (IL-1b, human chorionic gonadotropin [hCG], follicle-stimulating hormone (FSH), and TGF-a) and antagonists (TGF-b and dexamethasone) of cPLA2 and sPLA2 expression [47, 50, 51]. The ability of a specific cPLA2 inhibitor to reduce ovarian PGE2 content and ovulation rate in rats suggests that the enzyme is involved in this process [48]. However, the modest reduction in ovulation rate observed in cPLA2-deficient mice indicates that the role of the enzyme, in contrast to COX-2, is limited [52].

Hormonal Regulation of Prostaglandin G/H Synthase Expression EXPRESSION OF PGHS-2 Induction of PGHS-2 in Rat Preovulatory Follicles. The regulation of PGHS enzymes in preovulatory follicles was first reported more than 15 years ago (for review, see [28]). Initial studies performed in rats showed that the induction of PGHS protein in vivo was tissue specific and gonadotropin dependent. Induced expression of PGHS was detected in follicles of preovulatory size but not in smaller antral and preantral follicles and was limited to the granulosa cell layer. The induction required an ovulatory dose of hCG and was transient in nature. Subsequent studies in vitro further established the ability of gonadotropins to induce PGHS enzyme in rat follicular cells. Unknowingly, the success of these early investigations depended on the ability of the anti-PGHS antisera to crossreact with a second PGHS isoform that would soon be discovered.

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The identification of two molecular weight variants of PGHS enzymes in the rat ovary was documented in the early 1990s [53]. They were shown to vary only slightly in size (69,000 vs 72,000 molecular weight), with only the larger isoform being induced by gonadotropins in rat granulosa cells during ovulation. Purification of this isoform by anionic exchange chromatography and size fractionation and sequencing of its N-terminus revealed that it represented a novel isoform of PGHS enzyme that was, at the time, called the inducible PGHS [54]. The characterization of this distinct PGHS protein coincided with the cloning of a new PGHS-related complementary deoxyribonucleic acid (cDNA), which collectively contributed to establish the identity of a novel PGHS isoform now called PGHS2. Subsequent Northern blot analyses using a PGHS-2 specific cDNA probe showed that the gonadotropindependent induction of PGHS-2 protein in rat preovulatory follicles was preceded by a transient induction of PGHS-2 transcript [55]. Regulation of Follicular PGHS-2 Expression in Other Species. A series of studies in different species showed that the molecular process of induction of PGHS-2 in granulosa cells before ovulation was highly conserved [28]. However, the time-course of PGHS-2 induction varied greatly among species and appeared directly related to length of the ovulatory process. In rats, a species with a short ovulatory process (12 to 14 hours post-hCG), the induction of PGHS-2 was rapid and occurred 2 to 4 hours post-hCG. In cattle, a species with a relatively long process (28 to 30 hours post-hCG), the induction of PGHS-2 was delayed and occurred only 18 hours post-hCG. In mares, a species with an even longer ovulatory process (39 to 42 hours post-hCG), the induction of PGHS-2 was further delayed to 30 hours post-hCG. Interestingly, the interval of time from PGHS-2 induction to follicular rupture appeared highly conserved (10 hours post-hCG in rats, cows, and mares), which suggests that the molecular control of PGHS-2 induction could act as a determinant of the species-specific length of the ovulatory process [56] (Figure 13.2). The induction of PGHS-2 during ovulation has been studied in mice, and results showed that the enzyme is induced not only in mural granulosa cells but also in cumulus cells surrounding the oocyte and that factor(s) produced by the oocyte could promote PGHS-2 expression [31, 57, 58]. Moreover, reports in mice and cattle suggest that the expression of PGHS-2 in cumulus cells may influence proper expansion of the cumulus cell-oocyte complexes (COC) and oocyte maturation [31, 58, 59]. A gonadotropin-dependent induction of PGHS-2 before ovulation has also been documented in monkeys [60]. In humans, although ethical reasons preclude the isolation of ovarian tissues at various time points, current reports demonstrate that PGHS-2 is present in granulosa cells obtained from follicular aspirates of women enrolled in in

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FIGURE 13.2 Relationship among PGHS-2 induction, time of ovulation, and length of the ovulatory process in rats (A), cows (B), and mares (C). (From Ref. [56], with permission from the Endocrine Society.)

Prostaglandins

PGF2α

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FIGURE 13.3 Proposed model for the regulation of PGHS-2 in granulosa cells and large luteal cells. PDD, Phorbol didecanoate. (Modified from Ref. [67], with permission from Elsevier, Inc.)

vitro fertilization programs [61]. The expression of PGHS2 following ovarian hyperstimulation has also been studied in cattle [62]. Results showed that, whereas the majority of follicles larger than 8 mm were PGHS-2 positive, an important proportion of follicles (24%) did not express PGHS-2. Interestingly, the proportion of PGHS-2 negative follicles was similar to the reported incidence of anovulatory follicles following ovarian hyperstimulation [62]. Molecular Control of PGHS-2 Gene Expression. The preovulatory LH surge is considered the primary regulator of PGHS-2 expression in preovulatory follicles, but other gonadotropins (FSH and hCG) and activators of the cAMP-dependent protein kinase (PKA) pathway have been shown to be equally effective [28]. Moreover, numerous additional agonists working through various signaling pathways have been implicated in the regulation of PGHS2 in ovarian cells, including GnRH, PGF2a, IL-1b, GDF-9, and reactive oxygen species [34, 35, 55, 63–65]. Recent elegant studies have revealed that the regulation of PGHS2 expression switches from a predominant PKA-dependent pathway in preovulatory follicles to a predominant PKC-dependent pathway in the corpus luteum (Figure 13.3) [66, 67]. Early investigations established that the preovulatory induction of PGHS-2 in granulosa cells was dependent on transcriptional events [68], which prompted an interest in characterizing the regulation of the PGHS-2 promoter. Initial studies showed that the proximal 200 base pairs (bp) of the rat PGHS-2 promoter were sufficient to confer basal and forskolin/gonadotropin inducible activities [69]. Several consensus cis-acting elements were identified in this region, including a C/EBP, an ATF/CRE, and an E-box element. Site-directed mutagenesis studies and electrophoretic mobility shift assays revealed that the E-box

element plays a crucial role in the regulation of the PGHS2 promoter in rat granulosa cells, and that upstream stimulatory factor (USF) transcription factors could bind to this element [70]. The subsequent characterization of the bovine PGHS-2 promoter identified similar elements and confirmed the central role played by the E-box in bovine granulosa cells [66, 71]. Interestingly, differences in the kinetics and nature of transcription factors binding to the rat and bovine PGHS-2 promoters were tentatively implicated in the rapid vs delayed induction of PGHS-2 [71]. Part of the molecular basis for the switch from PKA- to PKCdependence during bovine granulosa cell luteinization in vitro has also been elucidated at the promoter level [66]. Likewise, the induction of the ovine PGHS-2 promoter in large luteal cells was shown to be PKC-dependent and to involve primarily, but not exclusively, the E-box element [72]. EXPRESSION OF PGHS-1 In contrast to PGHS-2, the expression of PGHS-1 in the ovary has not received much attention, most likely because of the absence of regulation of this isoform in ovarian follicles [53, 60, 64]. PGHS-1 is either undetectable or constitutively expressed at low levels in follicles of different developmental stages, with theca interna serving as the predominant site of expression. Investigations in several species show that the dramatic increase in prostaglandin synthesis just before ovulation is PGHS-2-dependent and not associated with any regulation of PGHS-1. CHEMICAL AND GENETIC INACTIVATION OF PGHS In recent years the development of selective PGHS-2 inhibitors led to investigations that used these drugs to

demonstrate the role of the enzyme in the ovulatory process. NS-398 significantly reduced LH/hCG-stimulated production of PGE2 and the number of ovulations in vivo and in vitro in rats [73], whereas the highly PGHS-2 selective NSAID meloxicam was shown to inhibit ovulation in rabbits [74]. Likewise, the selective PGHS-2 inhibitor rofecoxib proved to delay follicular rupture in women but to have no effect on peripheral steroid profiles, signs reminiscent of the luteinized unruptured-follicle (LUF) syndrome [75]. Thus the use of these selective inhibitors and other NSAIDs is not recommended for women attempting to become pregnant because of their potential association with anovulatory and infertile cycles [76]. Gene-targeting studies have clearly demonstrated the obligatory role of PGHS-2, but not of PGHS-1, in the ovulatory process. PGHS-2-deficient female mice proved infertile because of failures in various reproductive processes, including a marked inhibition in ovulation [77]. Interestingly, the administration of PGE2 but not of PGF2a was able to restore ovulation in PGHS-2-deficient mice thus establishing the predominant role of PGE2 in mice [31].

Regulation of Terminal Prostaglandin Synthases Subsequent to the induction of PGHS-2, the expression of terminal enzymes is required for the synthesis of prostaglandins with biological functions. PGES catalyzes the conversion of PGH2 into PGE2 and, of the two PGES isoforms recently identified, only mPGES expression has thus far been described in the ovary [78–80]. In the bovine preovulatory follicle, there is a marked induction of mPGES transcript and protein just before ovulation in vivo, with the induction occurring predominantly in granulosa cells (Figure 13.4) [78]. Thus the dramatic increase in PGE2 synthesis in bovine follicles just before ovulation results from a concomitant induction of PGHS-2 and mPGES. Similar studies during the ovulatory process have not been performed in other species, but expression of mPGES has been detected in mouse follicular cells and oocytes [79, 80]. Ovulation is also accompanied by a marked increase in PGF2a synthesis [26–29], and yet the regulation of PGFS or 9K-PGR mRNAs and proteins has not been investigated in ovarian follicles. However, a significant increase in 9KPGR activity has been observed in ovine follicles just before ovulation [81]. Studies on the expression of PGIS showed that the enzyme is constitutively expressed in rat follicles with no significant changes during ovulation [82, 83]. Although the thecal layer is the predominant site of expression of the enzyme, low levels of PGIS are also detected in granulosa cells. Thus the reported rise in concentrations of PGI2 in preovulatory follicles of several species likely results from

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FIGURE 13.4 Regulation of PGES mRNA in bovine preovulatory follicles during hCG-induced ovulation or after onset of natural estrus. Semiquantitative RT-PCR/Southern blot analysis of total ribonucleic acid (RNA) extracted from bovine preovulatory follicles isolated 0, 6, 12, 18, and 24 hours post-hCG (A, B, C), or 0, 18, and 24 hours after onset of natural estrus (D, E, F). Samples were analyzed for PGES (A and D) and GAPDH (B and E; control gene) content. Graphs in C and F show relative changes in PGES transcripts (n = 2 to 4 follicles per time point). Bars with asterisk are significantly different from the 0 h time point (p < 0.05) (Modified from Ref. [78], with permission from the American Society for Biochemistry and Molecular Biology.)

conversion of increased PGH2 precursors (following PGHS-2 induction) but not from PGIS regulation. Lastly, there has been no report on the regulation of TXA synthase (TXAS) and PGD synthase (PGDS) in ovarian follicles, although both enzymes have been localized in the human corpus luteum [84]. However, a significant increase in TXA2 or its stable metabolite (TXB2) has been described in ovarian follicles before ovulation [85, 86], but platelet aggregates adhering to the follicular endothelial cells were considered as the predicted source of thromboxanes [85].

Prostaglandin Receptors Of all the prostaglandin receptors inactivated by genetargeting studies, only EP2-deficient mice were shown to exhibit a change in ovarian follicular phenotype. Female mice deficient in EP2 were shown to have a reduced litter size, but some controversy exists as to the precise nature of the defect. Whereas some reports identified impaired cumulus expansion and fertilization as the primary defects [87, 88], another study revealed that the reduced fertility was caused by severely impaired ovulation [89]. This

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apparent discrepancy among studies was attributed to the use of different genetic backgrounds and/or ages of mice (e.g., impaired ovulation was observed in adult but not in immature EP2-deficient mice) [89]. Studies in wild-type female mice show that the EP2 mRNA is expressed at low levels in thecal cells of preovulatory follicles before hCG treatment and is induced in cumulus cells 4 to 12 hours post-hCG in vivo [87]. The transcript has also been detected in cumulus and granulosa cells of other species [90, 91]. Studies on FP receptors in ovarian follicles indicate low or undetectable expression in granulosa cells before ovulation, but a marked induction of the transcript is observed after ovulation in luteal cells where it is thought to mediate PGF2a-induced luteolysis later in the cycle [92, 93]. Likewise, stimulation of bovine preovulatory granulosa cells or human granulosa-luteal cells with hCG, forskolin, and other PKA activators causes a marked increase in FP receptor expression in vitro. The fact that FP-deficient female mice are fertile supports the apparent limited role of this receptor in follicular development [94]. However, the central role of FP in parturition was demonstrated in these mice that were unable to deliver fetuses at term [95]. There has been no report on the expression of IP and TP receptors in ovarian follicles, and only a limited number of studies have investigated the presence and regulation of EP1, EP3, and EP4. EP1 mRNA has been detected in human granulosa-luteal cells obtained from follicular aspirates [95], but the transcript could not be detected in bovine COCs [91]. In contrast, although EP3 is present at low levels and is not regulated in granulosa and thecal cells of bovine follicles before ovulation [92], its transcript increases during maturation of COCs in vitro and is more highly expressed in COCs of better quality [91]. EP4 has been detected in freshly isolated human granulosa-luteal cells [89]. However, it should be noted that female mice deficient in IP, EP1, EP3, EP4, or TP are fertile, which suggests nonessential roles of these receptors in female reproduction [96].

PROSTAGLANDINS AND THE CORPUS LUTEUM Luteolysis REGULATION OF OVARIAN PGF2a SYNTHESIS ACTION Luteolysis, defined as a loss of function of the corpus luteum and/or its involution, is a key event in the normal ovarian cycle and in parturition in many species [97, 98]. Although the subject of controversy for many years, PGF2a is now widely accepted as the principal physiological luteolysin in many mammalian species and, according to species, can originate mainly from the uterus or the corpus AND

luteum itself [98]. The regulated synthesis of luteolytic PGF2a by the uterus has been extensively studied (for reviews, see [98–101]). The exact manner in which PGF2a synthesis by the corpus luteum is regulated remains somewhat more speculative, and various agents including progesterone, IL-1b, TNF-a, interferon g, and endothelin-1 (ET-1) have been proposed as potential modulators of PGF2a production [101]. In ruminants, PGF2a has been shown to upregulate the expression of PGHS-2 in luteal cells [63, 98, 101]. This has been proposed to create an autocrine-signaling loop that would amplify the effects of uterine PGF2a by stimulating high-level synthesis of PGF2a locally within the corpus luteum [97]. Whether or not this is required for luteolysis to take place has yet to be demonstrated. In humans, whereas it has been established that PGF2a levels in ovarian venous blood increase in the late luteal phase, underlying regulatory mechanisms have not been reported thus far [98]. Whether of ovarian or uterine origin, PGF2a acts on the corpus luteum by binding FP, which is expressed to varying degrees in subsets of luteal cells in a species-specific manner [97]. Predictably, FP expression increases during the late luteal phase in most species [97, 98]. Another mechanism by which PGF2a action may be modulated at the level of the receptor is via the expression of FP isoforms that differ in their intracellular signaling mechanisms and desensitization properties, which could result in the activation of additional signaling pathways or serve to regulate each other’s activity [97]. ROLES AND MECHANISMS OF PGF2a ACTION IN THE CORPUS LUTEUM Amplification of the Luteolytic Signal. Binding of PGF2a to FP at the luteal cell surface rapidly leads to the activation of phospholipase C, the release of intracellular Ca2+ from the endoplasmic reticulum, and ultimately to the activation of PKC isoforms [97]. In ruminants, this signaling pathway effectively amplifies the luteolytic uterine PGF2a signal by inducing PGHS-2 expression (as described earlier) but also by provoking the exocytosis of oxytocin granules [97, 98]. According to a current model based largely on experiments performed in sheep, oxytocin release by the corpus luteum occurs in response to lowlevel PGF2a release by the uterus, which itself is provoked by oxytocin secreted from the pituitary gland (Figure 13.5). This PGF2 binds FP in a putative high sensitivity state (HFPR) (see Figure 13.5), resulting in the release of luteal oxytocin that effectively supplements the oxytocin of pituitary origin, thereby amplifying the release of PGF2a by the uterus [98]. The functioning of these feedback loops presumably results in the eventual accumulation of levels of PGF2a high enough to activate FP in its low sensitivity state (LFPR) (see Figure 13.5) and thereby triggers luteolysis. In this manner the uterus can be viewed as a transducer of the luteolytic signal, converting central oxytocin secretion

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HFPR LFPR

Increase in frequency of central OT pulse generator

PGF2a (+)

OTR PGF2a (+)

FIGURE 13.5 Proposed model for the regulation of PGF2a during luteolysis in sheep. HFPR, High sensitivity state of the PGF2a receptor; LFPR, low sensitivity state of the PGF2a receptor; OT, oxytocin; OTR, oxytocin receptor; P, progesterone. (Modified from Ref. [98], with permission from the American Physiological Society.)

A

B

into the production of PGF2a, and the corpus luteum serves as an amplifier of the uterine signal that brings about its own demise [98]. In primates there is evidence suggesting that a similar process may occur but without use of the uterus as an intermediate. Indeed, components of the luteolytic signaling such as oxytocin, oxytocin receptor, PGF2a, and FP are present in the primate corpus luteum. Thus a central oxy-

FIGURE 13.6 Comparative models for the control of luteolysis in, A, ruminants and, B, primates. CL, Corpus luteum; OPG, oxytocin pulse generator; OT, oxytocin; OTR, oxytocin receptor; P, progesterone. (Modified from Ref. [98], with permission from the American Physiological Society.)

tocin signal could trigger a self-contained, ovarian autocrine/paracrine loop, with oxytocin triggering PGF2a release, resulting in local oxytocin secretion, and so on until luteolysis is initiated (Figure 13.6) [98]. Unlike ruminants, luteolysis in primates can therefore be regarded as an ovary-mediated event [98]. Functional Luteolysis. An initial event in luteolysis is a reduction in progesterone secretion by the corpus

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luteum. Although this precedes the actual involution of the tissue, the reduction of circulating progesterone levels mediates most of the physiological consequences of luteolysis and is thus referred to as functional luteolysis. Evidence now suggests that PGF2a can induce functional luteolysis by inhibiting the import of cholesterol via a decrease in the activity and mRNA levels of sterol carrier protein-2 [102], as well as by decreasing the expression of steroidogenic acute regulatory protein (StAR) [103, 104]. A similar PGF2a-induced reduction in luteal mRNA levels also occurs with 3b-hydroxysteroid dehydrogenase (3bHSD) [105]. Whereas these antisteroidogenic effects lie downstream of protein kinase C signaling, there is also evidence that other signaling pathways contribute to PGF2ainduced functional luteolysis. PGF2a-mediated rises in intracellular Ca2+ levels lead to the induction of the transcription factor nur77, which results in the induction of 20a-hydroxysteroid dehydrogenase [106, 107]. This enzyme then presumably interferes with progesterone action by converting it into an inactive metabolite. An intermediate step in this Ca2+-dependent PGF2a signaling pathway involves the activation of extracellular signal-regulated kinase (Erk), a mitogen-activated protein (MAP) kinase [106]. Interestingly, other recent reports have shown that Erk signaling can interfere with the ability of gonadotropins to upregulate StAR expression and progesterone secretion in ovarian cells [97]. Further studies are required to evaluate the importance of MAP kinase pathways in mediating the luteolytic effects of PGF2a, as well as to better define how they are activated by PGF2a in luteal cells. Recent studies have also shown that the endothelial cell-derived peptide endothelin-1 (ET-1) could be an important mediator of PGF2a-induced antisteroidogenic effects. ET-1 expression is induced by PGF2a and oxytocin, and it was shown to inhibit progesterone secretion by luteal cells in vitro [98]. Furthermore, ET-1 expression in the corpus luteum is elevated at the time of its spontaneous regression and can be rapidly induced by the administration of exogenous PGF2a [97, 98]. Although apparently insufficient by itself to induce luteolysis, ET-1 has been shown to cause premature regression of the corpus luteum when administered with a subluteolytic dose of PGF2a [108]. ET-1 is therefore generally believed to enhance or potentiate the antisteroidogenic effects of PGF2a in luteal cells. Structural Luteolysis. Structural luteolysis, defined as the involution of the corpus luteum, is a complex process involving a number of mediators and signaling pathways [97]. Whereas the role of PGF2a as a mammalian luteolysin is no longer contested, it remains somewhat unclear if it is directly involved in the death of luteal cells. For instance, in the ewe, the subset of luteal cells that express FP is not the first to die during PGF2a-induced luteolysis [97]. In addition, PGF2a appears unable to induce the death of

steroidogenic luteal cells in vitro, and the administration of subluteolytic doses of PGF2a in ewes can reduce progesterone secretion without causing an appreciable loss of luteal cells. It has been proposed that the principal role of PGF2a may be to mediate functional luteolysis, whereas its perceived role in the involution of the corpus luteum could in fact be a predominantly indirect effect, at least in certain species [97, 98]. Nonetheless, PGF2a has been shown to modulate several cellular and gene regulation events that could be involved in structural luteolysis [97, 98]. It was originally hypothesized that PGF2a could contribute to luteolysis via its vasoconstrictive properties by reducing ovarian blood flow. Although some initial experimental data seemed to support this, it is now accepted that functional luteolysis induced by PGF2a precedes any measurable changes in luteal blood flow. Furthermore, whether changes in blood flow are causal or consequential events in structural luteolysis, if they are mediated by PGF2a or even if they are required for involution to occur, remains unclear. More recently PGF2a was shown to induce the luteal expression of monocyte chemoattractant protein-1 (MCP-1). This presumably results in increased monocyte/ macrophage migration into the corpus luteum, where these cells could serve to phagocytose luteal cells and to produce other cytokines [98, 109]. In addition, PGF2a reduces the luteal expression of tissue inhibitor of metalloproteinase1 (TIMP-1), possibly leading to increased metalloproteinase activity associated with structural regression [110]. Another level at which PGF2a could participate in structural luteolysis is by regulating components of apoptotic signaling cascades, because apoptosis is now believed to be a central process in the involution of the corpus luteum [97]. Notably, caspase-3 has been shown to be required for proper luteal regression to occur, and caspase-3 activity is believed to be induced by PGF2a [111]. The exact roles and relative importance of PGF2a in mediating structural luteolysis therefore remain poorly defined but should become more clear as the underlying cellular and molecular processes mediating luteal involution are elucidated.

Luteotropic and Luteoprotective Prostaglandins Although much attention has been focused on the luteolytic properties of PGF2a, it has long been known that other prostaglandins, most notably PGE2 and prostacyclin, exert opposite effects. For instance, both stimulate the secretion of progesterone from luteal cells in vitro, and PGE2 can protect the corpus luteum from the luteolytic effects of PGF2a when they are administered simultaneously [98, 112]. The most intensely investigated effect of PGE2, however, is its luteoprotective action during pregnancy. Uterine PGE2 production increases during early pregnancy in species such as ruminants and pigs, and

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intrauterine infusions of PGE2 in these species can protect the corpus luteum against the luteolytic effects of endogenous or exogenous PGF2a [98]. Although uterine PGF2 production is not completely blocked during early pregnancy, the PGE2 : PGF2a ratio increases markedly when compared with the nongravid uterus at the end of the luteal phase, and it is this ratio rather than the absolute amount of PGE2 secretion that is most closely associated with the luteoprotective effects of PGE2 [98, 113]. Because of its luteotropic and luteoprotective properties, PGE2a is often regarded as a likely mediator of the maternal recognition of pregnancy in certain species. Consistent with this notion, compounds originating from the conceptus such as estrogen (in pigs) and interferon tau (in cows) appear able to increase the PGE2 : PGF2a production ratio by the endometrium [113, 114]. Despite these advances, however, basic principles such as the mechanism by which PGE2 exerts its luteoprotective effects have yet to be elucidated.

ACKNOWLEDGMENTS Studies from the authors’ laboratories were funded in part by the Canadian Institutes of Health Research (CIHR) Grant MT-13190 and Fonds pour la Formation de Chercheurs et l’Aide à la Recherche Grant 99-ER-3016. Jean Sirois is supported by a CIHR Investigator Award. Derek Boerboom is supported by a CIHR postdoctoral research fellowship.

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