Human Reproduction Update Advance Access originally published online on June 17, 2004
Human Reproduction Update 2004 10(5):373-385; doi:10.1093/humupd/dmh032
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cyclooxygenase-2 and its role in ovulation: a 2004 account
1 Centre de recherche en reproduction animale and Département de biomédecine vétérinaire and 2 Département de pathologie et microbiologie vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6
3 To whom correspondence should be addressed: Dr Jean Sirois, Faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6. Email: jean.sirois{at}umontreal.ca
 |
Abstract |
|---|
TOP
Abstract
IntroductionThe pre-ovulatory surge of gonadotrophins triggers a marked and obligatory increase in follicular prostaglandin synthesis prior to ovulation, and the cyclooxygenase (COX) enzyme is a key rate-limiting step in the biosynthesis of prostaglandins. In the early 1990s, the pre-ovulatory rise in follicular prostaglandin synthesis was shown to result from the selective induction of a novel COX isoform, now referred to as COX-2. Differences in the time-course of COX-2 induction in species with a short versus a long ovulatory process suggest that the enzyme could be a molecular determinant that sets the alarm of the mammalian ovulatory clock. Some of the fine molecular mechanisms involved in the transcriptional activation of the COX-2 gene in granulosa cells have also been elucidated. The binding of trans-activating upstream stimulatory factors (USF) to a consensus E-box cis-element in the proximal region of the promoter was shown to play a predominant role in COX-2 transcription. Studies showed that COX-2 expression could also serve as a valuable marker for follicular commitment to ovulation during hyperstimulatory cycles. This paper presents a comprehensive review of the events that led to the characterization of COX-2 in pre-ovulatory follicles, updates current concepts on the control of COX-2 expression in pre-ovulatory follicles, and addresses the consequences of COX-2 inhibition to women fertility and potential implications of COX-2 expression in ovarian cancer.
Key words: cyclooxygenase-2 / granulosa cells / ovary / ovulation / prostaglandins
|
Introduction |
|---|
The ovulatory process involves a complex series of biochemical and biophysical events that ultimately lead to the rupture of the pre-ovulatory follicle and the release of the maternal germ cell. The process has all the signs of an acute, self-controlled inflammatory reaction, including hyperaemia, edema, leukocyte extravasation, and induction of proteolytic and collagenolytic activities (Espey, 1980
; Espey and Lipner, 1994). Prostaglandins, which play a central role in inflammation, have been recognized as key mediators of ovulation for >30 years and, in retrospect, the history of this relationship can arbitrarily be divided into two major periods. During the first period, which spans from the early 1970s to the mid-1980s, radioimmunoassays and inhibitors of prostaglandin synthesis served as important experimental tools to establish the critical role of prostaglandins in follicular rupture, and results from these studies have been the subject of a number of reviews (Armstrong, 1981; Murdoch et al., 1993; Priddy and Killick, 1993; Tsafriri et al., 1993; Espey and Lipner, 1994). The second period, which spans from the late 1980s up to now, has taken advantage of the development of molecular biology techniques to unravel the regulation and role of key enzymes responsible for prostaglandin synthesis during ovulation. Undoubtedly, the discovery that the cyclooxygenase (COX) enzyme induced by the LH surge in granulosa cells prior to ovulation was a novel isoform (now known as COX-2) represented an important landmark in the field. This review presents a historical account of events that led to the characterization of COX-2 in pre-ovulatory follicles, and updates current concepts on the molecular control of follicular COX-2 expression during natural and hyperstimulatory cycles. The consequences of COX-2 inhibition to women's fertility, as well as the potential implication of COX-2 expression in ovarian cancer, are also presented. To begin, a brief overview of prostaglandin biosynthesis and COX enzymes is proposed; readers interested in a more comprehensive characterization are referred to excellent recent reviews (Garavito and Mulichak, 2003; Murakami and Kudo, 2004). |
Prostaglandin biosynthesis |
|---|
Prostaglandins are important mediators of a variety of biological and pathological processes, and have been implicated in a number of female reproductive functions, including ovulation, fertilization, luteolysis, implantation and parturition (Sirois et al., 2000
; Matsumoto et al., 2001a; Lim and Dey, 2002; Olson, 2003; Goff, 2004). Prostaglandins, along with prostacyclins (PGI) and thromboxanes (TX), are often referred to as prostanoids, which belong to a large family of biomolecules containing a 20-carbon backbone structure, the eicosanoids (Smith and Marnett, 1991; Funk, 2001). All prostanoids are derived from fatty acids stored in cellular membranes, and their biosynthesis can be divided into three phases: (i) release of the arachidonic acid from membrane phospholipids by the action of phospholipase A2 enzymes; (ii) conversion of arachidonic acid into an unstable prostaglandin intermediate, PGH2, by cyclooxygenase enzymes; and (iii) conversion of PGH2 into various biologically active prostanoids by cell-specific terminal synthases (Figure 1) (Smith and Marnett, 1991; Murakami and Kudo, 2004). Once released, prostanoids act on target cells via specific seven-transmembrane, G protein-coupled receptors or in some cases through interactions with nuclear receptors (Breyer et al., 2001; Wright et al., 2001; Lim and Dey, 2002; Helliwell et al., 2004).
|
|
Cyclooxygenases: structure and function |
|---|
The true rate-limiting step in prostanoid biosynthesis is believed to be under the control of the COX enzyme, also known as prostaglandin synthase, prostaglandin endoperoxide synthase or prostaglandin G/H synthase (abbreviated PGS, PGTS or PGHS). Because of its key role in generating potent inflammatory mediators, the enzyme is the primary target for a large group of non-steroidal anti-inflammatory drugs (NSAID) that includes aspirin, indomethacin, ibuprofen, celecoxib and rofecoxib (Vane, 2000
; FitzGerald, 2003). The enzyme exhibits two consecutive catalytic activities, a cyclooxygenase activity responsible for the bisoxygenation of arachidonic acid to PGG2, and a peroxidase activity involved in the reduction of PGG2 to PGH2 (Figure 1) (Garavito and Mulichak, 2003). To date, three COX enzymes with similar activities but derived from two distinct genes have been characterized. Cyclooxygenase-1
COX-1 is generally recognized as a constitutive enzyme involved in the biosynthesis of prostaglandins necessary for various homeostatic functions (Smith et al., 2000). The primary structure of COX-1 was first characterized in sheep and subsequently in a number of species (Merlie et al., 1988; DeWitt and Smith, 1988; Smith et al., 2000). The COX-1 gene is 
22 kilobases (kb) in length, contains 11 exons, maps to human chromosome 9q32-q33.3, and is transcribed as a 2.8 kb mRNA (Tanabe and Tohnai, 2002). The COX-1 transcript encodes a 600–602 amino acid protein (depending on species) that exists as a homodimer and as a haem-binding, integral membrane protein preferentially, but not exclusively, associated with the endoplasmic reticulum. The promoter region of the COX-1 gene does not possess a canonical TATA or CATT box but is GC rich, in keeping with features of a housekeeping gene (Tanabe and Tohnai, 2002).
Cyclooxygenase-2
COX-2 is normally undetectable in most tissues but can be induced by a variety of stimuli, and is often referred to as the inducible isoform involved in inflammatory and pathological processes (Smith et al., 2000). COX-2 cDNA's were first isolated as immediate–early genes expressed in virally transformed chicken embryo fibroblasts (Xie et al., 1991) and in mitogen-stimulated Swiss 3T3 cells (Kujubu et al., 1991), but the presence of a COX isoform distinct from COX-1 had been suspected prior to its cloning (Rosen et al., 1989). The human COX-2 gene is more compact than the COX-1 gene, being 8.3 kb in length and containing 10 exons, and maps to a distinct chromosome (chromosome 1q25.2-q25.3) (Tanabe and Tohnai, 2002). However, the COX-2 transcript is larger than COX-1, being 4.4 kb in length, primarily because of a relatively long 3'-untranslated region that also contains numerous AUUUA motifs involved in mRNA instability (Smith et al., 2000). The COX-2 transcript encodes a 604 amino acid protein that preferentially, but not exclusively, localizes to the perinuclear envelope, and that is highly conserved across species (Figure 2). Likewise, all important structural and catalytic domains involved in COX function are conserved in COX-1 and COX-2 (Figures 1 and 2) (Garavito and Mulichak, 2003). The promoter region of the COX-2 gene is quite distinct from that of COX-1, as it contains a TATA box and several cis-elements (NF-kB, Sp1, AP-2, C/EBP, CRE-ATF, E-box) often associated with early response genes (Tanabe and Tohnai, 2002; Murakami and Kudo, 2004).
|
Cyclooxygenase-3
A third COX isoform, named COX-3, has recently been characterized in dogs (Chandrasekharan et al., 2002). Canine COX-3 is not derived from a distinct gene but corresponds to an alternatively spliced COX-1 variant in which intron 1 (90 nulceotides) is retained and codes for an in-frame insertion of 30 amino acids. The presence of a putative COX-3 isoform has also been detected in mice and rats (Kis et al., 2003; Shaftel et al., 2003), but the expression of a functional COX-3 in humans is still debated since intron 1 in this species is 94 nucleotides long, which creates a frameshift that results in a protein sequence not related to COX-1 (Dinchuk et al., 2003; Schwab et al., 2003b; Simmons, 2003). The enzyme is sensitive to acetaminophen and highly expressed in the central nervous system, suggesting that inhibiting COX-3 may represent an important mechanism for controlling the synthesis of prostanoids mediating pain and fever (Schwab et al., 2003a).
|
Prostaglandins and the ovulatory process |
|---|
A potential relationship between prostaglandin biosynthesis and ovulation first emerged during the early 1970s (reviewed in Armstrong, 1981
; Espey and Lipner, 1994). This was an exciting period in the prostaglandin field as John Vane (1971) had just demonstrated that the inhibition of prostaglandin synthesis was the underlying mechanism of action of aspirin and other related NSAID. Indomethacin, another prostaglandin synthesis inhibitor, rapidly became a drug of choice to determine the putative role of prostaglandins in various biological processes.
The indomethacin-dependent inhibition of ovulation was initially reported in rats and rabbits (Armstrong and Grinwich, 1972; Orczyk and Behrman, 1972), but was subsequently observed in numerous species, including pigs, sheep, cows and humans (reviewed in Armstrong, 1981; Sirois et al., 2000). In these species, as well as in others, the LH surge causes a marked increase in concentrations of PGE2 and PGF2
in ovarian follicles just prior to ovulation, with granulosa cells generally thought to be the primary site of prostaglandin synthesis. The ability of indomethacin to act directly at the ovarian level and block the pre-ovulatory rise in follicular prostaglandins was quickly favoured as the underlying mechanism responsible for causing anovulation (Armstrong, 1981; Murdoch et al., 1993). The relationship between prostaglandins and ovulation was further strengthened by studies in which the administration of antisera to prostaglandins was shown to block follicular rupture in mice and rabbits (Armstrong et al., 1974; Lau et al., 1974), and by investigations in which prostaglandins were able to restore ovulation in indomethacin-treated animals (Tsafriri et al., 1973; Diaz-Infante et al., 1974; Wallach et al., 1975a; Downey and Ainsworth, 1980). However, it should be noted that a number of other studies in the 1970s and 1980s provided opposite results and questioned the obligatory role of prostaglandins during ovulation (reviewed in Murdoch et al., 1993; Espey and Lipner, 1994). The issue would be ultimately resolved in the 1990s by genetic studies in mice (see below).
|
From follicular prostaglandin synthase to COX-2 |
|---|
Different alternatives were proposed as potential biochemical mechanisms responsible for the increase in follicular prostaglandins mediated by the LH surge, including an increase in arachidonic acid release and/or a rise in prostaglandin synthase activity. While the work of Clark et al. (1978)
provided initial indirect evidence for the regulation of prostaglandin synthase activity, the ultimate demonstration of enzyme regulation came 10 years later. Prostaglandin endoperoxide synthase
The collective work of two groups provided in 1987 the first direct evidence that prostaglandin endoperoxide synthase, referred to as PG synthase or PGS in their studies, was up-regulated in follicles prior to ovulation (Curry et al., 1987; Hedin et al., 1987; Huslig et al., 1987). It is interesting to realize that this discovery was made at a time when only one COX had been characterized, and the success of their work unknowingly depended on the ability of the antibodies that were raised against ovine PGS purified from seminal vesicles (now known as COX-1) to cross-react with the PGS isoform induced in pre-ovulatory follicles (now known as COX-2). Using intact and hypophysectomized immature rats primed with estradiol and/or gonadotrophins as in vivo models of pre-ovulatory follicular development, Hedin et al. (1987) established by immunoblots and immunofluorescence that PGS was selectively and transiently induced in granulosa cells during the ovulatory process (Figure 3A). The induction was also stage-dependent, as low or negligible expression was observed in small antral follicles and in pre-ovulatory follicles isolated prior to hCG treatment. A marked increase in PG synthase protein was also documented by enzyme immunoassays in ovaries of immature, pregnant mare serum gonadotrophin-primed rats isolated 8 h after an ovulatory dose of hCG (Huslig et al., 1987). The physiological occurrence of this biochemical event was ultimately confirmed in pre-ovulatory follicles of adult rats exhibiting normal estrous cycles (Curry et al., 1990).
|
The mechanisms responsible for gonadotrophin-dependent induction of PGS in pre-ovulatory follicles were further investigated using various models in vitro, such as short-term incubations of isolated pre-ovulatory follicles and cultures of granulosa cells (Wong et al., 1989
). The pattern of PGS induction observed in vivo following hCG treatment was recapitulated in vitro by LH, FSH and forskolin. This agonist-dependent induction of PGS was not blocked after inhibition of PGS enzymatic activity with indomethacin, nor was it altered by addition of prostaglandins. However, it was abolished when transcriptional (-amanitin) or translational (cycloheximide) inhibitors were added to the culture media, consistent with results of previous studies that showed that LH- or cAMP-stimulated production of follicular PGE2 in vitro was dependent on transcription and translation (Clark et al., 1976; Zor et al., 1977). Attempts to relate the marked induction of PGS protein to a similar regulation in PGS transcript by Northern blots proved unsuccessful at the time, and understandably surprised the investigators (Wong et al., 1989). It would later become evident that the cDNA probe (now known as COX-1) available in these early studies was unable to hybridize with the isoform induced in pre-ovulatory follicles. However, this finding was perceived as an important discrepancy and raised initial suspicions as to the precise nature of the PGS isoform induced in granulosa cells. Distinct PGS molecular weight variants
The production of anti-PGS antibodies with distinct specificities led to the identification of two molecular weight variants differentially expressed in follicles and other tissues (Figure 3B) (Wong and Richards, 1991). One antibody recognized a 72 000 mol. wt isoform (PGS72) rapidly induced by LH in granulosa cells of pre-ovulatory follicles but not expressed in other ovarian and non-ovarian tissues tested. In contrast, another antibody identified the presence of a 69 000 mol. wt isoform (PGS69) constitutively expressed in small antral and pre-ovulatory follicles (primarily in thecal cells), unregulated by LH, and present in various non-ovarian tissues. While it remains not entirely clear how antibodies with such distinct specificities could be generated from a unique source of antigen [PGS purified from ovine seminal vesicles (again presumably COX-1)], this serendipitous finding provided the first evidence for the presence of two PGS variants in the rat ovary. Likewise, it helped to reconcile results of previous immunohistological studies identifying PGS expression in thecal cells (Curry et al., 1987, 1990).
Purification of a novel isoform of prostaglandin endoperoxide synthase: COX-2
To resolve rising concerns as to the precise nature of the PGS isoform induced by LH/hCG in granulosa cells of rat pre-ovulatory follicles, referred to as rPGSi for inducible form of rat PGS, the purification and characterization of the enzyme appeared as the most appropriate approach (Sirois and Richards, 1992). A granulosa cell extract was prepared from rat pre-ovulatory follicles isolated 6 h after an ovulatory dose of hCG, a time when the expression of the enzyme previously proved to be maximal (Hedin et al., 1987). The rPGSi enzyme was purified from the extract by a combination of anionic exchange chromatography (Mevkh et al., 1985) and size fractionation. Amino-terminal microsequencing analysis revealed that the first 26 residues of the rat protein was quite different (only 58–61% identity) from the corresponding region of the ovine and mouse PGS (DeWitt and Smith, 1988; Merlie et al., 1988; DeWitt et al., 1990), but highly similar (96% identity) to the deduced amino acid sequence of a new mouse PGS-related cDNA clone that had just been characterized (Figure 3C) (Kujubu et al., 1991). Thus, the purification and characterization of a new PGS protein in rat granulosa cells (Sirois and Richards, 1992), along with the cloning of a novel PGS-related clone in chicken (Xie et al., 1991) and mouse fibroblasts (Kujubu et al., 1991), contributed collectively to the identification of the second PGS enzyme. The isoform that was first characterized would be thereafter referred to as COX-1, and the novel isoform as COX-2. The availability of a COX-2 cDNA probe allowed the regulation of the transcript during the ovulatory process to be revisited, and results demonstrated that it was indeed markedly induced in granulosa cells by LH/hCG (Sirois et al., 1992).
|
Species-specific time-course of COX-2 induction |
|---|
Studies in rats revealed that the induction of COX-2 was very rapid, 2–4 h after hCG treatment, and preceded ovulation by
10 h (Figure 3D) (Sirois et al., 1992). It became of interest to determine whether the selective induction of COX-2 in granulosa cells was a molecular process conserved in other species. The bovine system was the first one tested, and results established that COX-2 was also induced in granulosa cells of pre-ovulatory follicles during hCG- or GnRH-induced ovulation (Sirois, 1994; Tsai et al., 1996), as well as following a natural endogenous LH surge (Liu et al., 1997). However, a striking difference was observed in the time-course of COX-2 induction in bovine follicles, as the induction was not rapid as in the rat but occurred only 18 h after hCG treatment (Figure 3D) (Sirois, 1994). Interestingly, the interval of time from COX-2 induction to follicular rupture, 10 h, was remarkably conserved in the bovine system (Figure 3D). The rapid versus delayed induction of COX-2 in species with a short (rat: 12–14 h post-hCG) versus a long (cattle: 28–30 h post-hCG) ovulatory process led to the hypothesis that the timing of COX-2 induction was involved in determining the species-specific length of the ovulatory process. This hypothesis was subsequently tested using the equine model, a species with a longer ovulatory process (39–42 h post-hCG). Results revealed that COX-2 induction was further delayed in this species and occurred only 30 h post-hCG, with follicular rupture again taking place 10 h later (Figure 3D) (Sirois and Doré, 1997; Boerboom and Sirois, 1998). Collectively, these results supported the concept that COX-2 could serve as a molecular determinant that sets the alarm of the mammalian ovulatory clock (Richards, 1997). Results from investigations in mice also agreed with the present model, as a rapid induction of COX-2 was also observed after hCG treatment (Joyce et al., 2001; Segi et al., 2003).
Studies on the regulation of COX-2 in human pre-ovulatory follicles have been limited. However, the transcript has been detected in granulosa cells and the protein measured in pre-ovulatory follicular fluid collected from women enrolled in an IVF programme (Narko et al., 1997; Tokuyama et al., 2001, 2003). Also, the pattern of COX-2 expression has been studied in the rhesus monkey, a primate model with an ovulatory process of 40 h (Duffy and Stouffer, 2001). A rise in COX-2 protein expression and in intrafollicular prostaglandin levels occurred 24 and 36 h post-hCG, respectively, which is consistent with results in other species. However, other aspects of COX-2 expression in primate follicles appeared unique, including the relatively early detection of COX-2 mRNA (12 h post-hCG) by RT–PCR and the localization of the protein in both granulosa and theca cell layers.
|
Control of follicular COX-2 expression |
|---|
While the pre-ovulatory gonadotrophin surge is recognized as the primary trigger of follicular COX-2 induction prior to ovulation, numerous agonists acting through distinct signalling pathways have been shown to regulate the expression of the enzyme in ovarian cells. Furthermore, unravelling the fine molecular mechanisms involved in the transcriptional activation of the COX-2 gene by gonadotrophins has also been the focus of a number of investigations, and noticeable progress has been made in this field.
Agonist-dependent regulation of COX-2 mRNA and protein
The use of hCG to mimic the endogenous LH surge should be regarded as the first model shown to induce follicular COX-2 prior to ovulation (Hedin et al., 1987; Huslig et al., 1987). Several subsequent experimental paradigms in vivo and in vitro revealed that LH and FSH, as well as other direct activators of the cyclic adenosine monophosphate-dependent protein kinase (PKA) pathway, were equally able to up-regulate COX-2 expression in pre-ovulatory granulosa cells (Wong et al., 1989; Wong and Richards, 1991; Liu et al., 1999). However, the LH surge-mediated induction of the enzyme was also shown to involve other signaling pathways, including calcium-dependent protein kinase (PKC) and tyrosine kinase-dependent pathways (Wong and Richards, 1992; Morris and Richards, 1993, 1995).
Apart from gonadotrophins, the list of molecules capable of regulating COX-2 in ovarian cells seems to be continuously expanding. The pattern of COX-2 induction by GnRH in rat pre-ovulatory follicles or granulosa cells in vitro was very similar to that observed with gonadotrophins (Wong and Richards, 1992). Transforming growth factor (TGF-) was also shown to induce COX-2 in hen pre-ovulatory granulosa cells (Li et al., 1996), whereas interleukin-1ß had a similar effect in human granulosa-luteal cells and rat granulosa cells (Narko et al., 1997; Ando et al., 1998; Saito et al., 2001). Likewise, stimulation of mouse granulosa cells with recombinant growth differentiation factor 9 (GDF-9), a factor exclusively produced by the oocyte, was shown to up-regulate COX-2, suggesting that oocyte-derived factors may contribute to increased prostaglandin synthesis during the ovulatory process (Elvin et al., 1999). Interestingly, the regulation of COX-2 appears to switch from a predominant PKA-dependent pathway in pre-ovulatory follicles to a predominant PKC-dependent pathway in the corpus luteum (Wu and Wiltbank, 2001, 2002). PGF2 and reactive oxygen species become strong inducers of COX-2 in luteal cells (Nakamura and Sakamoto, 2001; Tsai and Wiltbank, 2001).
In contrast to the impressive list of COX-2 agonists in ovarian cells, only a limited number of putative COX-2 repressors has been identified. TGF-ß significantly suppressed basal and TGF--induced COX-2 mRNA in hen granulosa cells (Li et al., 1996), whereas the work by Hedin and Eriksson (1997) revealed that progesterone was able to decrease LH-stimulated COX-2 expression in rat pre-ovulatory follicles in vitro.
Regulation of the COX-2 promoter in granulosa cells
The finding that the gonadotrophin-dependent induction of COX-2 in granulosa cells was dependent on transcriptional events (Wong et al., 1989) provided the rationale for cloning and studying the regulation of the COX-2 promoter. Initial studies revealed that the proximal region located within the first 150–200 base pairs upstream of the transcription start site of the rat and bovine COX-2 promoter was sufficient to confer basal and forskolin/gonadotrophin-inducible activities (Figure 4A) (Sirois and Richards, 1993; Sirois et al., 1993; Liu et al., 1999). A number of consensus cis-acting elements such as C/EBP, ATF/CRE and E-box elements were identified within this region, but electrophoretic mobility shift assays and site-directed mutagenesis studies revealed that the E-box element was playing a predominant role in both species in the regulation of the promoter in granulosa cells (Figure 4B) (Morris and Richards, 1996; Liu et al., 1999). Upstream stimulatory factor (USF)-1 and -2, transcription factors known to bind to the E-box, were detected in rat and bovine granulosa cells. Interestingly, the presence of an amino-terminal truncated form of USF-2 thought to repress gene expression was detected prior to hCG treatment only in bovine granulosa cells, and was proposed as a potential mechanism for the delayed induction of COX-2 in species with a long ovulatory process (Liu et al., 1999). The trans-activating capacity of USF on the COX-2 promoter in granulosa cells, as well as the ability of an amino-truncated USF-2 dominant negative mutant to repress promoter activation, were recently demonstrated experimentally (Figure 4C and D) (Sayasith et al., 2004). Moreover, critical interactions between USF proteins and the E-box element were shown to be conserved as the transcriptional regulation of COX-2 switches from PKA to PKC dependence during cellular luteinization (Wu and Wiltbank, 2002). Other studies revealed that the Il-1ß-regulated induction of COX-2 transcripts in whole ovarian dispersates was also dependent on promoter activation (Saito et al., 2001).
|
|
COX-2: a marker for ovulation during ovarian stimulation |
|---|
One undesirable outcome of ovarian stimulation (i.e. induction of multiple ovulation) protocols in cattle is the development of a proportion (up to 20%) of large follicles that fail to ovulate and that ultimately undergo atresia or become cystic (Laurincik et al., 1993
; Pruwantara et al., 1994).
To test the hypothesis that COX-2 could be used as a marker of follicular commitment to ovulation during ovarian stimulation, multiple follicular development was stimulated with exogenous FSH, ovulation was induced with hCG, and the pattern of COX-2 expression was characterized (Liu and Sirois, 1998). Results of this study showed that COX-2 expression 24 h after hCG was detected in 76% of follicles >8 mm, but the proportion (24%) of follicles not expressing the enzyme was very similar to the incidence of anovulatory follicles detected by ultrasonography (Laurincik et al., 1993). Interestingly, in contrast to COX-2-positive follicles, COX-2-negative follicles were not luteinized and contained a compact cumulus–oocyte complex, suggesting an apparent overall failure to respond to the gonadotrophin pre-ovulatory signal. Thus, COX-2 expression should provide a valuable marker to unravel the molecular basis behind the development of large anovulatory follicles during ovarian stimulation.
|
Inhibition of COX-2 action |
|---|
Considering the central role played by COX enzymes in the production of potent inflammatory mediators such as the prostaglandins, numerous inhibitors (i.e. NSAID) have been developed by the pharmaceutical industry, with several being used to study the role of prostaglandins during ovulation. In recent years, the generation of COX-2- and COX-1-deficient mice has also provided powerful tools to investigate the relative role of each enzyme in various physiological and pathological processes.
Non-selective and selective COX-2 inhibitors
Pharmaceutical drugs able to inhibit follicular COX-2 were actually used >20 years before the discovery of the enzyme. Indeed, the non-selective COX inhibitor indomethacin was administered in the 1970s in numerous species and was shown to block follicular rupture (reviewed in Armstrong, 1981; Murdoch et al., 1993; Priddy and Killick, 1993; Espey and Lipner, 1994). Other non-selective COX inhibitors such as aspirin and naproxen also impaired ovulation, at least in rabbits (Zanagnolo et al., 1996). Likewise, the administration of indomethacin blocked the ovulatory process in rhesus and marmoset monkeys (Wallach et al., 1975b; Maia et al., 1978; Duffy and Stouffer, 2002), and prevented or delayed ovulation in women (Killick and Elstein, 1987; Akil et al., 1996; Athanasiou et al., 1996; Nargund and Wei, 1996). However, in contrast to these studies, ibuprofen had apparently very limited, if any, effect in delaying follicular rupture in 12 women involved in a randomized clinical study (Uhler et al., 2001).
The 1990s brought the development of several selective COX-2 inhibitors that were tested for their effect on the ovulatory process. Celecoxib tended to reduce ovulation in mice (Reese et al., 2001), NS-398 impaired ovulation in vivo and in vitro in rats (Mikuni et al., 1998), and meloxicam blocked follicular rupture in rabbits (Salhab et al., 2001, 2003). A randomized double-blind study in women also showed that rofecoxib delayed follicular rupture without affecting peripheral hormone cyclicity (Pall et al., 2001). Collectively, these studies further underscored the role of COX-2 in ovulation.
Genetic inactivation of COX-2
The most compelling evidence of the importance of COX-2 in the ovulatory process came from genetic studies in which genes encoding COX-1 or COX-2 were inactivated (Dinchuk et al., 1995; Langenbach et al., 1995; Morham et al., 1995). While mice carrying a null mutation for COX-1 were fertile (Langenbach et al., 1995), mice deficient in COX-2 proved to be infertile because of multiple reproductive failures, including a marked anovulatory phenotype (Lim et al., 1997) that could be reversed with exogenous PGE2 (Davis et al., 1999). In general, genetic inactivation of COX-2 resulted in more severe effects on ovulation than pharmacological inhibition of the enzyme (Reese et al., 2001). Interestingly, a recent study showed that the anovulatory phenotype observed in COX-2-deficient mice produced in a C57BL/6J/129 background was not present in COX-2 null mice derived in a CD1 genetic background (Wang et al., 2004). In the latter group, the phenotypic rescue appeared related to the ability of COX-1 to replace a function generally attributed to COX-2 (Wang et al., 2004).
|
Implications for clinicians managing patients on COX-2 inhibitors |
|---|
NSAID are among the most widely prescribed drugs worldwide, and their use in women attempting to become pregnant has been associated with reversible female infertility (Akil et al., 1996
; Thylan, 1997; Mendonca et al., 2000; Norman, 2001; Deviere, 2002; Stone et al., 2002; Norman and Wu, 2004). It is thought that the use of selective or non-selective COX-2 inhibitors may cause a luteinized unruptured follicle (LUF) syndrome, a condition characterized by the ability of a normally growing pre-ovulatory follicle to luteinize, but not to rupture, in response to the LH surge, resulting in the entrapment of the oocyte and in infertile cycles. Moreover, results from animal studies indicate that inhibition of COX-2 also impairs cumulus expansion, fertilization, decidualization and implantation (Lim et al., 1997; Matsumoto et al., 2001a). Collectively, these findings clearly underscore the risk of taking COX-2 inhibitors while attempting to become pregnant. The potential adverse reproductive consequences of pharmacological inhibition of COX-2 action should therefore be considered given the central role played by the enzyme during ovulation and other reproductive processes, and avoiding NSAID usage during this critical period seems to be a logical recommendation to women consulting for infertility. |
COX-2 expression in ovarian cancer |
|---|
Ovarian cancer is the most common gynecological malignancy in western countries and represents one of the leading causes of death due to cancer in women (Jemal et al., 2004
). The majority of primary ovarian tumors is epithelial in origin and derives from the surface epithelium covering the ovary. Although the precise underlying mechanisms responsible for the development of epithelial ovarian cancer remain unclear, various hypotheses have been proposed (Fathalla, 1971; Cramer and Welch, 1983; Ness and Cottreau, 1999; Purdie et al., 2003). Among them, incessant ovulation (Fathalla, 1971; Purdie et al., 2003) and inflammation (Ness and Cottreau, 1999) appear best related to the subject of the present review. A number of factors that shorten the total duration of the women's ovulatory life (i.e. number of ovulations), such as parity, prolonged breast-feeding amenorrhoea and oral contraceptive use, have been associated with the greatest risk reduction for ovarian cancer. It is argued that incessant ovulation causes repeated minor trauma to the epithelial surface of the ovary, and that the inflammatory reaction and mediators (including prostaglandins) associated with the ovulatory process provide recurring oxidative stress that may be mutagenic (Ness and Cottreau, 1999; Purdie et al., 2003). Some evidence suggests that COX-2 could contribute to the early stages of cancer formation, as the enzyme was found to be overexpressed in pre-neoplastic changes of the ovarian surface epithelium (Roland et al., 2003). Borderline and benign tumours have also been reported to express COX-2 (Klimp et al., 2001; Matsumoto et al., 2001b). However, numerous biochemical and epidemiological studies investigating a potential link between COX-2 and ovarian cancer have yielded conflicting results. Several reports documented that COX-2 mRNA and/or protein were detectable in malignant ovarian tumours (Klimp et al., 2001; Matsumoto et al., 2001b; Landen et al., 2003; Li et al., 2004), and that the use of NSAID was associated with a decrease in the risk of ovarian cancer (Cramer et al., 1998; Akhmedkhanov et al., 2001; Moysich et al., 2001). COX-2 expression has been positively correlated with microvessel density of tumours, but negatively correlated with response to chemotherapy and patient survival (Denkert et al., 2002; Ferrandina et al., 2002; Ali-Fehmi et al., 2003). In contrast, other reports were unable to detect COX-2 expression in ovarian tumours (Doré et al., 1999; Gupta et al., 2003; Roland et al., 2003), or to find an association between NSAID use and cancer risk (Rosenberg et al., 2000; Tavani et al., 2000). The precise basis for these conflicting results remain unclear but differences in case selection have been proposed as a possible cause (Gupta et al., 2003), considering that agents used in standard chemotherapy for ovarian cancer can induce COX-2 expression (Subbaramaiah et al., 2000; Cassidy et al., 2002). Other possible reasons for the conflicting results include differences in detection methods, tissue processing and antibody used. Studies in vitro using several ovarian cancer cell lines also showed marked variability in levels of COX-2 expression among cell lines (Denkert et al., 2002; Munkarah et al., 2002; Gupta et al., 2003). |
Conclusions |
|---|
Much progress has been achieved in recent years on our understanding of the control of prostaglandin synthesis in pre-ovulatory follicles. Results from studies presented above have highlighted the central and obligatory role played by the gonodotrophin-dependent induction of COX-2 in ovarian follicles prior to ovulation. Even though COX-2 is generally considered as the rate-limiting enzyme in the prostaglandin biosynthetic pathway, attention will also need to be focused on terminal prostaglandin synthases since, for example, PGES was also shown to be regulated in bovine pre-ovulatory follicles (Filion et al., 2001
). The synthesis of biologically active prostaglandins such as PGE2 and PGF2 by follicular cells also raises questions regarding their precise roles during follicular rupture. Given their potential actions as local autocrine, paracrine and perhaps intracrine hormones, the fine molecular mechanisms of prostaglandin action remains a sizeable and exciting challenge.
Ultimately, the clinical implications of COX-2 inhibition to women's fertility should be fully appreciated, considering the obligatory role of the enzyme during the ovulatory process as well as its potential implication in fertilization and implantation. The use of non-selective and selective COX-2 inhibitors should generally be discouraged for women attempting to become pregnant (Norman and Wu, 2004). However, the value of COX-2 inhibition in pathological processes in which the enzyme has been detected such as ovarian cancer and ovarian endometriotic cysts (Ota et al., 2001; Fagotti et al., 2004) will require further investigations.
|
Acknowledgements |
|---|
Studies from the authors' laboratory were funded by Canadian Institutes of Health Research (CHIR) Grant MT-13190. J.Sirois is supported by a CIHR Investigator Award.
|
References |
|---|
Akhmedkhanov A, Toniolo P, Zeleniuch-Jacquotte A, Kato I, Koenig KL and Shore RE (2001) Aspirin and epithelial ovarian cancer. Prev Med 33, 682–687.[CrossRef][Web of Science][Medline]
Akil M, Amos RS and Stewart P (1996) Infertility may sometimes be associated with NSAID consumption. Br J Rheumatol 35, 76–78.
Ali-Fehmi R, Che M, Khalifeh I, Malone JM, Morris R, Lawrence WD and Munkarah AR (2003) The effect of cyclooxygenase-2 expression on tumor vascularity in advanced stage ovarian serous carcinoma. Cancer 98, 1423–1429.[CrossRef][Web of Science][Medline]
Ando M, Kol S, Kokia E, Ruutiainen-Altman K, Sirois J, Rohan RM, Payne DW and Adashi EY (1998) Rat ovarian prostaglandin endoperoxide synthase-1 and -2: periovulatory expression of granulosa cell-based interleukin-1-dependent enzymes. Endocrinology 139, 2501–2508.
Armstrong DT (1981) Prostaglandins and follicular functions. J Reprod Fertil 62, 283–291.
Armstrong DT and Grinwich DL (1972) Blockade of spontaneous and LH-induced ovulation in rats by indomethacin, an inhibitor of prostaglandin biosynthesis. Prostaglandins 1, 21–28.[CrossRef][Web of Science][Medline]
Armstrong DT, Grinwich DL, Moon YS and Zamecnik J (1974) Inhibition of ovulation in rabbits by intrafollicular injection of indomethacin and prostaglandin F antiserum. Life Sci 14, 129–140.[CrossRef][Web of Science][Medline]
Athanasiou S, Bourne TH, Khalid A, Okokon EV, Crayford TJ, Hagstrom HG, Campbell S and Collins WP (1996) Effects of indomethacin on follicular structure, vascularity, and function over the periovulatory period in women. Fertil Steril 65, 556–560.[Web of Science][Medline]
Boerboom D and Sirois J (1998) Molecular characterization of equine prostaglandin G/H synthase-2 and regulation of its messenger ribonucleic acid in preovulatory follicles. Endocrinology 139, 1662–1670.
Breyer RM, Bagdassarian CK, Myers SA and Breyer MD (2001) Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 41, 661–690.[CrossRef][Web of Science][Medline]
Cassidy PB, Moos PJ, Kelly RC and Fitzpatrick FA (2002) Cyclooxygenase-2 induction by paclitaxel, docetaxel, and taxane analogues in human monocytes and murine macrophages: structure-activity relationships and their implications. Clin Cancer Res 8, 846–855.
Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS and Simmons DL (2002) COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci USA 99, 13926–13931.
Clark MR, Marsh JM and LeMaire WJ (1976) The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles in vitro. Prostaglandins 12, 209–216.[CrossRef][Web of Science][Medline]
Clark MR, Marsh JM and LeMaire WJ (1978) Mechanism of luteinizing hormone regulation of prostaglandin synthesis in rat granulosa cells. J Biol Chem 253, 7757–7761.
Cramer DW and Welch WR (1983) Determinants of ovarian cancer risk II. Inferences regarding pathogenesis. J Natl Cancer Inst 71, 717–721.[Web of Science][Medline]
Cramer DW, Harlow BL, Titus-Ernstoff L, Bohlke K, Welch WR and Greenberg ER (1998) Over-the-counter analgesics and risk of ovarian cancer. Lancet 351, 104–107.[CrossRef][Web of Science][Medline]
Curry TE, Jr, Malik A and Clark MR (1987) Ovarian prostaglandin synthase: immunohistochemical localization in the rat. Am J Obstet Gynecol 157, 537–543.[Web of Science][Medline]
Curry TE, Jr, Bryant C, Haddix AC and Clark MR (1990) Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle. Biol Reprod 42, 307–316.[Abstract]
Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC and Langenbach R (1999) Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1ß. Endocrinology 140, 2685–2695.
Denkert C, Kobel M, Pest S, Koch I, Berger S, Schwabe M, Siegert A, Reles A, Klosterhlafen B and Hauptmann S (2002) Expression of cyclooxygenase-2 is an independent prognostic factor in human ovarian carcinoma. Am J Pathol 160, 893–903.
Deviere J (2002) Do selective cyclo-oxygenase inhibitors eliminate the adverse events associated with nonsteroidal anti-inflammatory drug therapy? Eur J Gastroenterol Hepatol (Suppl 1) 14, S29–S33.
DeWitt DL and Smith WL (1988) Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci USA 85, 1412–1416.
DeWitt DL, el-Harith EA, Kraemer SA, Andrews MJ, Yao EF, Armstrong RL and Smith WL (1990) The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. J Biol Chem 265, 5192–5198.
Diaz-Infante A, Jr, Wright KH and Wallach EE (1974) Effects of indomethacin and prostaglandin F2 on ovulation and ovarian contractility in the rabbit. Prostaglandins 5, 567–581.[CrossRef][Web of Science][Medline]
Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, Covington MB, Contel NR, Eng VM, Collins RJ, Czerniak PM et al. (1995) Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 378, 406–409.[CrossRef][Medline]
Dinchuk JE, Liu RQ and Trzaskos JM (2003) COX-3: in the wrong frame in mind. Immunol Lett 86, 121.[CrossRef][Web of Science][Medline]
Doré M, Côté LC, Mitchell A and Sirois J (1999) Expression of prostaglandin G/H synthase type 1, but not type 2, in human ovarian adenocarcinomas. J Histochem Cytochem 46, 77–84.
Downey BR and Ainsworth L (1980) Reversal of indomethacin blockade of ovulation in gilts by prostaglandins. Prostaglandins 19, 17–22.[CrossRef][Web of Science][Medline]
Duffy DM and Stouffer RL (2001) The ovulatory gonadotrophin surge stimulates cyclooxygenase expression and prostaglandin production by the monkey follicle. Mol Hum Reprod 7, 731–739.
Duffy DM and Stouffer RL (2002) Follicular administration of a cyclooxygenase inhibitor can prevent oocyte release without alteration of normal luteal function in rhesus monkeys. Hum Reprod 17, 2825–2831.
Elvin JA, Clark AT, Wang P, Wolfman NM and Matzuk MM (1999) Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocrinol 13, 1035–1048.
Espey LL (1980) Ovulation as an inflammatory response—a hypothesis. Biol Reprod 22, 73–106.[CrossRef][Web of Science][Medline]
Espey LL and Lipner H (1994) Ovulation. In Knobil E and Neill JD (eds) Physiology of Reproduction, vol 1. Raven Press, New York, pp 725–781.
Fagotti A, Ferrandina G, Fanfani F, Legge F, Lauriola L, Gessi M, Castelli P, Barbieri F, Minelli L and Scambia G (2004) Analysis of cyclooxygenase-2 (COX-2) expression in different sites of endometriosis and correlation with clinico-pathological parameters. Hum Reprod 19, 393–397.
Fathalla MF (1971) Incessant ovulation—a factor in ovarian neoplasia? Lancet 2, 163.[CrossRef][Web of Science][Medline]
Ferrandina G, Lauriola L, Zannoni GF, Fagotti A, Fanfani F, Legge F, Maggiano N, Gessi M, Mancuso S, Ranelletti FO et al. (2002) Increased cyclooxygenase-2 (COX-2) expression is associated with chemotherapy resistance and outcome in ovarian cancer patients. Ann Oncol 13, 1205–1211.
Filion F, Bouchard N, Goff AK, Lussier JG and Sirois J (2001) Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo. J Biol Chem 276, 34323–34330.
FitzGerald GA (2003) COX-2 and beyond: approaches to prostaglandin inhibition in human disease. Nat Rev Drug Discov 2, 879–890.[CrossRef][Web of Science][Medline]
Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294, 1871–1875.
Garavito RM and Mulichak AM (2003) The structure of mammalian cyclooxygenases. Annu Rev Biophys Biomol Struct 32, 183–206.[CrossRef][Web of Science][Medline]
Goff AK (2004) Steroid hormone modulation of prostaglandin secretion in the ruminant endometrium during the estrous cycle. Biol Reprod [E-pub ahead of print].
Gupta RA, Tejada LV, Tong BJ, Das SK, Morrow JD, Dey SK and DuBois RN (2003) Cyclooxygenase-1 is overexpressed and promotes angiogenic growth factor production in ovarian cancer. Cancer Res 63, 906–911.
Hedin L and Eriksson A (1997) Prostaglandin synthesis is suppressed by progesterone in rat preovulatory follicles in vitro. Prostaglandins 53, 91–106.[CrossRef][Web of Science][Medline]
Hedin L, Gaddy-Kurten D, Kurten R, DeWitt DL, Smith WL and Richards JS (1987) Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation. Endocrinology 121, 722–731.
Helliwell RJ, Berry EB, O'Carroll SJ and Mitchell MD (2004) Nuclear prostaglandin receptors: role in pregnancy and parturition? Prostagl Leukot Essent Fatty Acids 70, 149–165.[CrossRef][Web of Science][Medline]
Huslig RL, Malik A and Clark MR (1987) Human chorionic gonadotropin stimulation of immunoreactive prostaglandin synthase in the rat ovary. Mol Cell Endocrinol 50, 237–246.[CrossRef][Web of Science][Medline]
Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ and Thun MJ (2004) Cancer statistics 2004. CA Cancer J Clin 54, 8–29.
Joyce IM, Pendola FL, O'Brien M and Eppig JJ (2001) Regulation of prostaglandin-endoperoxide synthase 2 messenger ribonucleic acid expression in mouse granulosa cells during ovulation. Endocrinology 142, 3187–3197.
Killick S and Elstein M (1987) Pharmacologic production of luteinized unruptured follicles by prostaglandin synthetase inhibitors. Fertil Steril 47, 773–777.[Web of Science][Medline]
Kis B, Snipes JA, Isse T, Nagy K and Busija DW (2003) Putative cyclooxygenase-3 expression in rat brain cells. J Cereb Blood Flow Metab 23, 1287–1292.[CrossRef][Web of Science][Medline]
Klimp AH, Hollema H, Kempinga C, van der Zee AGJ, de Vries AGE and Daemen T (2001) Expression of cyclooxygenase-2 and inducible nitric oxide synthase in human ovarian tumors and tumor-associated macrophages. Cancer Res 61, 7305–7309.
Kujubu DA, Fletcher BS, Varnum BC, Lim RW and Herschman HR (1991) TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 266, 12866–12872.
Landen CN, Mathur SP, Richardson MS and Creasman WT (2003) Expression of cyclooxgenase-2 in cervical, endometrial, and ovarian malignancies. Am J Obstet Gynecol 188, 1174–1176.[CrossRef][Web of Science][Medline]
Langenbach R, Morham SG, Tiano HF, Loftin CD, Ghanayem BI, Chulada PC, Mahler JF, Lee CA, Goulding EH, Kluckman KD et al. (1995) Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 83, 483–492.[CrossRef][Web of Science][Medline]
Lau IF, Saksena SK and Chang MC (1974) Prostaglandins F and ovulation in mice. J Reprod Fertil 40, 467–469.
Laurincik J, Oberfranc M, Hyttel P, Grafenau P, Tomanek M and Pivko J (1993) Characterization of the periovulatory period in superovulated heifers. Theriogenology 39, 537–544.
Li J, Simmons DL and Tsang BK (1996) Regulation of hen granulosa cell prostaglandin production by transforming growth factors during follicular development: involvement of cyclooxygenase II. Endocrinology 137, 2522–2529.[Abstract]
Li S, Miner K, Fannin R, Barrett JC and Davis BJ (2004) Cyclooxygenase-1 and 2 in normal and malignant human ovarian epithelium. Gynecol Oncol 92, 622–627.[CrossRef][Web of Science][Medline]
Lim H and Dey SK (2002) A novel pathway of prostacyclin signaling-hanging out with nuclear receptors. Endocrinology 143, 3207–3210.
Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM and Dey SK (1997) Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91, 197–208.[CrossRef][Web of Science][Medline]
Liu J and Sirois J (1998) Follicle size-dependent induction of prostaglandin G/H synthase-2 during superovulation in cattle. Biol Reprod 58, 1527–1532.
Liu J, Carriere PD, Dore M and Sirois J (1997) Prostaglandin G/H synthase-2 is expressed in bovine preovulatory follicles after the endogenous surge of luteinizing hormone. Biol Reprod 57, 1524–1531.[Abstract]
Liu J, Antaya M, Boerboom D, Lussier JG, Silversides DW and Sirois J (1999) The delayed activation of the prostaglandin G/H synthase-2 promoter in bovine granulosa cells is associated with down-regulation of truncated upstream stimulatory factor-2. J Biol Chem 274, 35037–35045.
Maia H, Jr, Barbosa I and Coutinho EM (1978) Inhibition of ovulation in marmoset monkeys by indomethacin. Fertil Steril 29, 565–570.[Web of Science][Medline]
Matsumoto H, Ma W, Smalley W, Trzaskos J, Breyer RM and Dey SK (2001a) Diversification of cyclooxygenase-2-derived prostaglandins in ovulation and implantation. Biol Reprod 64, 1557–1565.
Matsumoto Y, Ishiko O, Deguchi M, Nakagawa E and Ogita S (2001b) Cyclooxygenase-2 expression in normal ovaries and epithelial ovarian neoplasms. Int J Mol Med 8, 31–36.[Web of Science][Medline]
Mendonca LL, Khamashta MA, Nelson-Piercy C, Hunt BJ and Hughes GR (2000) Non-steroidal anti-inflammatory drugs as a possible cause for reversible infertility. Rheumatology (Oxf) 39, 880–882.
Merlie JP, Fagan D, Mudd J and Needleman P (1988) Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem 263, 3550–3553.
Mevkh AT, Sud'ina GF, Golub NB and Varfolomeev SD (1985) Purification of prostaglandin H synthetase and a fluorometric assay for its activity. Anal Biochem 150, 91–96.[CrossRef][Web of Science][Medline]
Mikuni M, Pall M, Peterson CM, Peterson CA, Hellberg P, Brannstrom M, Richards JS and Hedin L (1998) The selective prostaglandin endoperoxide synthase-2 inhibitor, NS-398, reduces prostaglandin production and ovulation in vivo and in vitro in the rat. Biol Reprod 59, 1077–1083.
Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, Jennette JC, Mahler JF, Kluckman KD, Ledford A, Lee CA et al. (1995) Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 83, 473–482.[CrossRef][Web of Science][Medline]
Morris JK and Richards JS (1993) Hormone induction of luteinization and prostaglandin endoperoxide synthase-2 involves multiple cellular signaling pathways. Endocrinology 133, 770–779.
Morris JK and Richards JS (1995) Luteinizing hormone induces prostaglandin endoperoxide synthase-2 and luteinization in vitro by A-kinase and C-kinase pathways. Endocrinology 136, 1549–1558.[Abstract]
Morris JK and Richards JS (1996) An E-box region within the prostaglandin endoperoxide synthase-2 (PGS-2) promoter is required for transcription in rat ovarian granulosa cells. J Biol Chem 271, 16633–16643.
Moysich KB, Mettlin C, Piver MS, Natarajan N, Menezes RJ and Swede H (2001) Regular use of analgesic drugs and ovarian cancer risk. Cancer Epidemiol Biomark Prev 10, 903–906.
Munkarah AR, Morris R, Baumann P, Deppe G, Malone J, Diamond MP and Saed GM (2002) Effects of prostaglandin E2 on proliferation and apoptosis of epithelial ovarian cancer cells. J Soc Gynecol Invest 9, 168–173.[Web of Science][Medline]
Murakami M and Kudo I (2004) Recent advances in molecular biology and physiology of the prostaglandin E2-biosynthetic pathway. Prog Lipid Res 43, 3–35.[CrossRef][Web of Science][Medline]
Murdoch WJ, Hansen TR and McPherson LA (1993) A review—role of eicosanoids in vertebrate ovulation. Prostaglandins 46, 85–115.[CrossRef][Web of Science][Medline]
Nakamura T and Sakamoto K (2001) Reactive oxygen species up-regulates cyclooxygenase-2, p53, and Bax mRNA expression in bovine luteal cells. Biochem Biophys Res Commun 284, 203–210.[CrossRef][Web of Science][Medline]
Nargund G and Wei CC (1996) Successful planned delay of ovulation for one week with indomethacin. J Assist Reprod Genet 13, 683–684.[CrossRef][Web of Science][Medline]
Narko K, Ritvos O and Ristimaki A (1997) Induction of cyclooxygenase-2 and prostaglandin F2 receptor expression by interleukin-1ß in cultured human granulosa-luteal cells. Endocrinology 138, 3638–3644.
Ness RB and Cottreau C (1999) Possible role of ovarian epithelial inflammation in ovarian cancer. J Natl Cancer Inst 91, 1459–1467.
Norman RJ (2001) Reproductive consequences of COX-2 inhibition. Lancet 358, 1287–1288.[CrossRef][Web of Science][Medline]
Norman RJ and Wu R (2004) The potential danger of COX-2 inhibitors. Fertil Steril 81, 493–494.[CrossRef][Web of Science][Medline]
Olson DM (2003) The role of prostaglandins in the initiation of parturition. Best Pract Res Clin Obstet Gynaecol 17, 717–730.[CrossRef][Medline]
Orczyk GP and Behrman HR (1972) Ovulation blockade by aspirin or indomethacin—in vivo evidence for a role of prostaglandin in gonadotrophin secretion. Prostaglandins 1, 3–20.[CrossRef][Web of Science][Medline]
Ota H, Igarashi S, Sasaki M and Tanaka T (2001) Distribution of cyclooxygenase-2 in eutopic and ectopic endometrium in endometriosis and adenomyosis. Hum Reprod 16, 561–566.
Pall M, Friden BE and Brannstrom M (2001) Induction of delayed follicular rupture in the human by the selective COX-2 inhibitor rofecoxib: a randomized double-blind study. Hum Reprod 16, 1323–1328.
Priddy AR and Killick SR (1993) Eicosanoids and ovulation. Prostagl Leukot Essent Fatty Acids 49, 827–831.[CrossRef][Web of Science][Medline]
Pruwantara B, Callesen H and Greve T (1994) Characteristics of ovulations in superovulated cattle. Anim Reprod Sci 37, 1–5.
Purdie DM, Bain CJ, Siskind V, Webb PM and Green AC (2003) Ovulation and risk of epithelial ovarian cancer. Int J Cancer 104, 228–232.[CrossRef][Web of Science][Medline]
Reese J, Zhao X, Ma WG, Brown N, Maziasz TJ and Dey SK (2001) Comparative analysis of pharmacologic and/or genetic disruption of cyclooxygenase-1 and cyclooxygenase-2 function in female reproduction in mice. Endocrinology 142, 3198–3206.
Richards JS (1997) Sounding the alarm—does induction of prostaglandin endoperoxide synthase-2 control the mammalian ovulatory clock? Endocrinology 138, 4047–4048.
Roland IH, Yang WL, Yang DH, Daly MB, Ozols RF, Hamilton TC, Lynch HT, Godwin AK and Xu XX (2003) Loss of surface and cyst epithelial basement membranes and preneoplastic morphologic changes in prophylactic oophorectomies. Cancer 98, 2607–2623.[CrossRef][Web of Science][Medline]
Rosen GD, Birkenmeier TM, Raz A and Holtzman MJ (1989) Identification of a cyclooxygenase-related gene and its potential role in prostaglandin formation. Biochem Biophys Res Commun 164, 1358–1365.[CrossRef][Web of Science][Medline]
Rosenberg L, Palmer JR, Rao RS, Coogan PF, Strom BL, Zauber AG, Stolley PD and Shapiro S (2000) A case–control study of analgesic use and ovarian cancer. Cancer Epidemiol Biomark Prev 9, 933–937.
Saito J, Ando M, Sussman D, Negishi H, King G and Adashi EY (2001) Interleukin 1 upregulates ovarian prostaglandin endoperoxide synthase-2 expression: evidence for prostaglandin-dependent/ceramide-independent transcriptional stimulation and for message stabilization. Biol Reprod 65, 1759–1765.
Salhab AS, Gharaibeh MN, Shomaf MS and Amro BI (2001) Meloxicam inhibits rabbit ovulation. Contraception 63, 329–333.[CrossRef][Web of Science][Medline]
Salhab AS, Amro BI and Shomaf MS (2003) Further investigation on meloxicam contraceptivity in female rabbits: luteinizing unruptured follicles, a microscopic evidence. Contraception 67, 485–489.[CrossRef][Web of Science][Medline]
Sayasith K, Bouchard N, Sawadogo M, Lussier JG and Sirois J (2004) Molecular characterization and role of bovine upstream stimulatory factor 1 and 2 in the regulation of the prostaglandin G/H synthase-2 promoter in granulosa cells. J Biol Chem 279, 6327–6336.
Schwab JM, Schluesener HJ, Meyermann R and Serhan CN (2003a) COX-3 the enzyme and the concept: steps towards highly specialized pathways and precision therapeutics? Prostaglandins Leukot Essent Fatty Acids 69, 339–343.[CrossRef][Web of Science][Medline]
Schwab JM, Beiter T, Linder JU, Laufer S, Schulz JE, Meyermann R and Schluesener HJ (2003b) COX-3—a virtual pain target in humans? FASEB J 17, 2174–2175.
Segi E, Haraguchi K, Sugimoto Y, Tsuji M, Tsunekawa H, Tamba S, Tsuboi K, Tanaka S and Ichikawa A (2003) Expression of messenger RNA for prostaglandin E receptor subtypes EP4/EP2 and cyclooxygenase isozymes in mouse periovulatory follicles and oviducts during superovulation. Biol Reprod 68, 804–811.
Shaftel SS, Olschowka JA, Hurley SD, Moore AH and O'Banion MK (2003) COX-3: a splice variant of cyclooxygenase-1 in mouse neural tissue and cells. Brain Res Mol Brain Res 119, 213–215.[Medline]
Simmons DL (2003) Variants of cyclooxygenase-1 and their roles in medicine. Thromb Res 110, 265–268.[CrossRef][Web of Science][Medline]
Sirois J (1994) Induction of prostaglandin endoperoxide synthase-2 by human chorionic gonadotropin in bovine preovulatory follicles in vivo. Endocrinology 135, 841–848.[Abstract]
Sirois J and Doré M (1997) The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process. Endocrinology 138, 4427–4434.
Sirois J and Richards JS (1992) Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles. J Biol Chem 267, 6382–6388.
Sirois J and Richards JS (1993) Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBPb promoter element. J Biol Chem 268, 21931–21938.
Sirois J, Simmons DL and Richards JS (1992) Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles Induction in vivo and in vitro. J Biol Chem 267, 11586–11592.
Sirois J, Levy LO, Simmons DL and Richards JS (1993) Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells Identification of functional and protein-binding regions. J Biol Chem 268, 12199–12206.
Sirois J, Liu J, Boerboom D and Antaya M (2000) Prostaglandins and ovulation: from indomethacin to PGHS-2 knockout. In Adashi EY (ed) Ovulation: Evolving Scientific and Clinical Concepts. Springer, New York, pp 208–220.
Smith WL and Marnett LJ (1991) Prostaglandin endoperoxide synthase: structure and catalysis. Biochim Biophys Acta 1083, 1–17.[Medline]
Smith WL, DeWitt DL and Garavito RM (2000) Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 69, 145–182.[CrossRef][Web of Science][Medline]
Stone S, Khamashta MA and Nelson-Piercy C (2002) Nonsteroidal anti-inflammatory drugs and reversible female infertility: is there a link? Drug Safety 25, 545–551.[CrossRef][Web of Science][Medline]
Subbaramaiah K, Hart JC, Norton L and Dannenberg AJ (2000) Microtubule-interfering agents stimulate the transcription of cyclooxygenase-2. Evidence for involvement of ERK1/2 and p38 mitogen-activated protein kinase pathways. J Biol Chem 275, 14838–14845.
Tanabe T and Tohnai N (2002) Cyclooxygenase isozymes and their gene structures and expression. Prostagl Other Lipid Mediat 68-69, 95–114.
Tavani A, Gallus S, La Vecchia C, Conti E, Montella M and Franceschi S (2000) Aspirin and ovarian cancer: an Italian case–control study. Ann Oncol 11, 1171–1173.
Thylan S (1997) NSAIDs and fertility. Br J Rheumatol 36, 145–146.
Tokuyama O, Nakamura Y, Muso A, Honda K, Ishiko O and Ogita S (2001) Expression and distribution of cyclooxygenase-2 in human periovulatory ovary. Int J Mol Med 8, 603–606.[Web of Science][Medline]
Tokuyama O, Nakamura Y, Musoh A, Honda K, Ozaki K and Ishiko O (2003) Expression and distribution of cyclooxygenase-2 in human ovary during follicular development. Osaka City Med J 49, 39–47.[Medline]
Tsafriri A, Koch Y and Lindner HR (1973) Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2. Prostaglandins 3, 461–467.[CrossRef][Web of Science][Medline]
Tsafriri A, Chun SY and Reich R (1993) Follicular rupture and ovulation. In Adashi EY and Leung PCK (eds) Ovulation. Raven Press, New York, pp 227–244.
Tsai SJ and Wiltbank MC (2001) Differential effects of prostaglandin F2 on in vitro luteinized bovine granulosa cells. Reproduction 122, 245–253.[Abstract]
Tsai SJ, Wiltbank MC and Bodensteiner KJ (1996) Distinct mechanisms regulate induction of messenger ribonucleic acid for prostaglandin (PG) G/H synthase-2, PGE (EP3) receptor, and PGF2a receptor in bovine preovulatory follicles. Endocrinology 137, 3348–3355.[Abstract]
Uhler ML, Hsu JW, Fisher SG and Zinaman MJ (2001) The effect of nonsteroidal anti-inflammatory drugs on ovulation: a prospective, randomized clinical trial. Fertil Steril 76, 957–961.[CrossRef][Web of Science][Medline]
Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 231, 232–235.[Web of Science][Medline]
Vane JR (2000) Aspirin and other anti-inflammatory drugs. Thorax 55(Suppl 2), S3–S9.
Wallach EE, Bronson R, Hamada Y, Wright KH and Stevens VC (1975a) Effectiveness of prostaglandin F2 in restoration of HMG-HCG induced ovulation in indomethacin-treated rhesus monkeys. Prostaglandins 10, 129–138.[Web of Science][Medline]
Wallach EE, Cruz A, Hunt J, Wright KH and Stevens VC (1975b) The effect of indomethacin of HMG-HCG induced ovulation in the rhesus monkey. Prostaglandins 9, 645–658.[CrossRef][Web of Science][Medline]
Wang H, Ma WG, Tejada L, Zhang H, Morrow JD, Das SK and Dey SK (2004) Rescue of female infertility from the loss of cyclooxygenase-2 by compensatory upregulation of cyclooxygenase-1 is a function of genetic background. J Biol Chem 279, 10649–10658.
Wong WY and Richards JS (1991) Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary. Mol Endocrinol 5, 1269–1279.
Wong WY and Richards JS (1992) Induction of prostaglandin H synthase in rat preovulatory follicles by gonadotropin-releasing hormone. Endocrinology 130, 3512–3521.
Wong WY, DeWitt DL, Smith WL and Richards JS (1989) Rapid induction of prostaglandin endoperoxide synthase in rat preovulatory follicles by luteinizing hormone and cAMP is blocked by inhibitors of transcription and translation. Mol Endocrinol 3, 1714–1723.
Wright DH, Abran D, Bhattacharya M, Hou X, Bernier SG, Bouayad A, Fouron JC, Vazquez-Tello A, Beauchamp MH, Clyman RI et al. (2001) Prostanoid receptors: ontogeny and implications in vascular physiology. Am J Physiol Regul Integr Comp Physiol 281, R1343–R1360.
Wu YL and Wiltbank MC (2001) Differential regulation of prostaglandin endoperoxide synthase-2 transcription in ovine granulosa and large luteal cells. Prostaglandins Other Lipid Mediat 65, 103–116.[CrossRef][Web of Science][Medline]
Wu YL and Wiltbank MC (2002) Transcriptional regulation of the cyclooxygenase-2 gene changes from protein kinase (PK) A- to PKC-dependence after luteinization of granulosa cells. Biol Reprod 66, 1505–1514.
Xie WL, Chipman JG, Robertson DL, Erikson RL and Simmons DL (1991) Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 88, 2692–2696.
Zanagnolo V, Dharmarajan AM, Endo K and Wallach EE (1996) Effects of acetylsalicylic acid (aspirin) and naproxen sodium (naproxen) on ovulation, prostaglandin, and progesterone production in the rabbit. Fertil Steril 65, 1036–1043.[Web of Science][Medline]
Zor U, Strulovici B, Nimrod A and Lindner HR (1977) Stimulation by cyclic nucleotides of prostaglandin E production in isolated graafian follicles. Prostaglandins 14, 947–959.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  F. Gaytan, M. Gaytan, J. M. Castellano, M. Romero, J. Roa, B. Aparicio, N. Garrido, J. E. Sanchez-Criado, R. P. Millar, A. Pellicer, et al. KiSS-1 in the mammalian ovary: distribution of kisspeptin in human and marmoset and alterations in KiSS-1 mRNA levels in a rat model of ovulatory dysfunction Am J Physiol Endocrinol Metab, March 1, 2009; 296(3): E520 - E531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kurusu, M. Jinno, H. Ehara, T. Yonezawa, and M. Kawaminami Inhibition of ovulation by a lipoxygenase inhibitor involves reduced cyclooxygenase-2 expression and prostaglandin E2 production in gonadotropin-primed immature rats Reproduction, January 1, 2009; 137(1): 59 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Krishnaswamy, P. Chapdelaine, J. P. Tremblay, and M. A. Fortier Development and Characterization of a Simian Virus 40 Immortalized Bovine Endometrial Stromal Cell Line Endocrinology, January 1, 2009; 150(1): 485 - 491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Markosyan and D. M. Duffy Prostaglandin E2 Acts via Multiple Receptors to Regulate Plasminogen-Dependent Proteolysis in the Primate Periovulatory Follicle Endocrinology, January 1, 2009; 150(1): 435 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Klipper, E. Tatz, T. Kisliouk, I. Vlodavsky, U. Moallem, D. Schams, Y. Lavon, D. Wolfenson, and R. Meidan Induction of Heparanase in Bovine Granulosa Cells by Luteinizing Hormone: Possible Role during the Ovulatory Process Endocrinology, January 1, 2009; 150(1): 413 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M Seneda, M. Godmann, B. D Murphy, S. Kimmins, and V. Bordignon Developmental regulation of histone H3 methylation at lysine 4 in the porcine ovary Reproduction, June 1, 2008; 135(6): 829 - 838. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Rae, O. Gubbay, A. Kostogiannou, D. Price, H. O. D. Critchley, and S. G. Hillier Thyroid Hormone Signaling in Human Ovarian Surface Epithelial Cells J. Clin. Endocrinol. Metab., January 1, 2007; 92(1): 322 - 327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.-M. Tsai, T.-F. Chan, Y. Chang, P.-H. Chiang, C.-Y. Chuang, C.-Y. Long, C.-Y. Chai, and J.-N. Lee Leptin Suppresses Human Chorionic Gonadotropin-Induced Cyclooxygenase-2 Expression and Prostaglandin Production in Cultured Human Granulose Luteal Cells Reproductive Sciences, December 1, 2006; 13(8): 551 - 557. [Abstract] [PDF]
|
|
|
|
 |
|
 Y.-J. Kang, B. A. Wingerd, T. Arakawa, and W. L. Smith Cyclooxygenase-2 Gene Transcription in a Macrophage Model of Inflammation J. Immunol., December 1, 2006; 177(11): 8111 - 8122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Markosyan, B. L. Dozier, F. A. Lattanzio, and D. M. Duffy Primate Granulosa Cell Response via Prostaglandin E2 Receptors Increases Late in the Periovulatory Interval Biol Reprod, December 1, 2006; 75(6): 868 - 876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ledoux, D. B. Campos, F. L. Lopes, M. Dobias-Goff, M.-F. Palin, and B. D. Murphy Adiponectin Induces Periovulatory Changes in Ovarian Follicular Cells Endocrinology, November 1, 2006; 147(11): 5178 - 5186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Gaytan, C Bellido, C Morales, J E Sanchez-Criado, and F Gaytan Effects of selective inhibition of cyclooxygenase and lipooxygenase pathways in follicle rupture and ovulation in the rat. Reproduction, October 1, 2006; 132(4): 571 - 577. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Bridges, C. M. Komar, and J. E. Fortune Gonadotropin-Induced Expression of Messenger Ribonucleic Acid for Cyclooxygenase-2 and Production of Prostaglandins E and F2{alpha} in Bovine Preovulatory Follicles Are Regulated by the Progesterone Receptor Endocrinology, October 1, 2006; 147(10): 4713 - 4722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Ben-Ami, S. Freimann, L. Armon, A. Dantes, D. Strassburger, S. Friedler, A. Raziel, R. Seger, R. Ron-El, and A. Amsterdam PGE2 up-regulates EGF-like growth factor biosynthesis in human granulosa cells: new insights into the coordination between PGE2 and LH in ovulation Mol. Hum. Reprod., October 1, 2006; 12(10): 593 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Frungieri, S. I. Gonzalez-Calvar, F. Parborell, M. Albrecht, A. Mayerhofer, and R. S. Calandra Cyclooxygenase-2 and Prostaglandin F2{alpha} in Syrian Hamster Leydig Cells: Inhibitory Role on Luteinizing Hormone/Human Chorionic Gonadotropin-Stimulated Testosterone Production Endocrinology, September 1, 2006; 147(9): 4476 - 4485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Diouf, K. Sayasith, R. Lefebvre, D. W. Silversides, J. Sirois, and J. G. Lussier Expression of Phospholipase A2 Group IVA (PLA2G4A) Is Upregulated by Human Chorionic Gonadotropin in Bovine Granulosa Cells of Ovulatory Follicles Biol Reprod, June 1, 2006; 74(6): 1096 - 1103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B Lohrke, T Viergutz, and B Kruger Polar phospholipids from bovine endogenously oxidized low density lipoprotein interfere with follicular thecal function J. Mol. Endocrinol., December 1, 2005; 35(3): 531 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sayasith, J. G. Lussier, and J. Sirois Role of Upstream Stimulatory Factor Phosphorylation in the Regulation of the Prostaglandin G/H Synthase-2 Promoter in Granulosa Cells J. Biol. Chem., August 12, 2005; 280(32): 28885 - 28893. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||